Microbiology

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GOVERNMENT OF TAMIL NADU

MICROBIOLOGY

HIGHER SECONDARY FIRST YEAR

Untouchability is Inhuman and a Crime

A publication under Free Textbook Programme of Government of Tamil Nadu

Department of School Education

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Government of Tamil Nadu

First Edition - 2018

NOT FOR SALE

Content Creation

The wise possess all

State Council of Educational Research and Training © SCERT 2018

Printing & Publishing

Tamil NaduTextbook and Educational Services Corporation www.textbooksonline.tn.nic.in

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Contents

Chapter 1 Introduction to Microbiology ...... 01 Chapter 2 Microscopy ...... 15 Chapter 3 Stains and Staining Methods ...... 25 Chapter 4 Sterilization ...... 40 Chapter 5 Cultivation of Microorganisms ...... 53 Chapter 6 Microbial Nutrition and Growth ...... 72 Chapter 7 Morphology of Bacteria ...... 88 Chapter 8 Microbial Taxonomy ...... 113 Chapter 9 Environmental Microbiology ...... 124 Chapter 10 Soil Microbiology ...... 152 Chapter 11 Agricultural Microbiology...... 165 Chapter 12 Medical Microbiology ...... 189 Chapter 13 Immunology ...... 227 Chapter 14 Microbial Genetics ...... 280

E-Book Assessment Digi-Link

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HOW TO USE THE BOOK ?

Chapter Outline Presents a complete overview of the chapter

Learning Goals to transform the classroom processes into Objectives: learner centric with a list of bench marks

Amazing facts, Rhetorical questions to lead students to biological inquiry

Activity Directions are provided to students to conduct activities in order to explore, enrich the concept.

Infographics Visual representation of the lesson to enrich learning .

To motivate the students to further explore the content digitally and take them to virtual world

ICT To enhance digital Science skills among students

Glossary Explanation of scientific terms

Evaluation Assess students to pause, think and check their understanding

Career corner List of professions particular to that chapter

References List of related books for further details of the topic

Web links List of digital resources

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Career Opportunities for Microbiologists

Microbiologists are biological scientists microbiologists study the relationship who study about organisms that are between microorganisms and disease generally so small and can only be seen establishment. with a microscope. These microorganisms Immunology – Those who work on include bacteria, algae, yeasts, fungi, immune system related work are called protozoa, viruses, and other microscopic immunologists. Immunologist study forms of life. Some microbiologists the body’s defensive responses to specialize in one type of microorganism. microorganisms. They learn how our For example, bacteriologists concentrate immune system protects our body during on bacteria and virologists study viruses. the infection. They suggest possible ways Microbiologists isolate and make cultures to increase our immunity. It is one of the of microorganisms, identify their fastest growing areas in science. characteristics, and observe their reactions Microbial Ecology - The microbial to chemicals and other kinds of stimuli. interactions with living and non-living They also study how microorganisms matters of the environmental habitats is develop and reproduce as well as their referred to as microbial ecology. Microbial distribution in nature. ecologists study the contributions of microorganisms to the cycling of various The Scope of Microbiology (Course nutrients or elements. The ecologists are Benefit / Advantages) employed in reducing the pollution of the The whole ecosystem depends on bacterial environment which is the burning issue in activities. The modern microbiology is a all metro cities. They work on employing large discipline with different specialities. microorganisms in bioremediation to Microbiology has a great impact on reduce pollution effects. fields such as medicine, agriculture, food Food and Dairy Microbiology – Some sciences, ecology, genetics, biochemistry of our foods are actually the by-products and molecular biology. There are many of microbial growth. Example: Cheese is possible avenues of advancement for produced by the growth of microorganisms microbiologists. such as Leuconostoc citrovorum and Medical Microbiology – Medical Streptococcus lactis. Yoghurt results microbiologists are involved in from the growth of bacteria such as identifying the microorganisms causing Lactobacillus bulgaricus and Streptococcus the infectious diseases. They work on thermophilus in milk. The leavening of identifying the pathogens and assist the bread is accomplished by Saccharomyces medical practitioners for prescribing the cerevisiae (Baker’s yeast). Main work of apt antibiotics in right dosages. They the food and dairy microbiologists in the also study the ways in which the micro food industry is to prevent contamination organisms cause the infection. Medical during processing and the transmission

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of food borne diseases. Microbiologists and do research. Microbiologists can are currently employed in all food and become directors of research in medical dairy processing industries. They are also centres, private firms, or government employed in Mineral water companies to agencies. Those who hold a teaching check the quality of water. and research position in a university can Agricultural Microbiology – It advance to the rank of full professor. They is concerned with the impact of can also make significant discoveries in microorganisms on agriculture. Most their research and gain the recognition bacteria and fungi live saprophytically on of other microbiologists. Many scientists dead and organic matter of the soil. They consider this to be the highest form of decompose the complex organic matter advancement. into simpler form making it available Microbial Genetics and Molecular for the soil microorganisms. Thus they Biology – The use of micro organisms form an important constituent of soil has been very helpful in understanding called humus. Certain microbes increase the functions of the genes. Microbial the fertility of the soil by converting the geneticists play an important role in atmospheric nitrogen into ammonia, applied microbiology by producing new nitrites and nitrates. This is brought about microbial strains that are more efficient by the microbes such as Nitrosomonas, in synthesizing useful products. Genetic Nitrobacter and Rhizobium sp. Agricultural techniques are used to test substances microbiologists try to combat plant for their ability to cause cancer. diseases that attack commercial food Microbiologists are in greater demand crops and they also work on methods to in genetic engineering companies and increase soil fertility and crop yields. research units. Industrial Microbiology – Biomining – Microbes are used in Microorganisms are used to make extracting valuable metals like uranium products such as antibiotics, vaccines, from rocks. Thiobacillus ferrooxidans steroids, alcohols, vitamins, amino acids unlocks energy from inorganic compounds and enzymes. Some important drugs are like iron sulphide. During this process, it synthesized by microorganisms such as produces sulphuric acid and iron sulphate. streptomycin, penicillin, chloramphenicol, The use of micro organisms in mining has tetracycline. Industrial microbiologists considerably reduced the cost of mining work on improving the strains that to 75%. Microbiologists involved in produce the industrially important Biomining research are highly paid in the products and thereby increase the yield. Government sector. The Research and Development (R&D) Medical coding – Medical coding units in the industries provide various job is the transformation of healthcare opportunities to microbiologists. diagnosis, procedures, medical services Directors of Research Units and and equipment into universal medical Universities – Many microbiologists alphanumeric codes. The diagnoses work for universities, where they teach and procedure codes are taken from

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medical record documentation, such which conduct selection through entrance as transcription of physician’s notes, examination. Candidates can choose any laboratory and radiologic results. Medical of the specialization streams in order to coding jobs are assigned to Life Science, choose courses related to Microbiology Paramedical and Medical Graduates and • Agricultural Microbiology Post Graduates. • Food microbiology Editor in Scientific Journals – Editing, • Medical Microbiology proof reading in scientific journals, handle manuscripts on topics ranging • Pharmaceutical Microbiology from Zoology, Biology, Plants and Animal • Microbial Genetics sciences are few assignments that could • Environmental Microbiology be accomplished by microbiologists. • Aero Microbiology Microbiologists review the research articles that are to be published in reputed • Microbial Physiology National and International Journals. Different Courses in Microbiology They are employed as Editors and Associate Editors of Scientific Publishing • Bachelor of Science in Microbiology Companies. • Bachelor of Science in Microbiology Pharma companies – A microbiologist in and Microbial Technology a pharmaceutical company is a member • Bachelor of Science in Clinical of quality department. The role of the Microbiology microbiologist is to ensure the quality of • Bachelor of Science in Medical raw materials before they are processed Microbiology in the production area, monitor the microbiological quality of environment • Bachelor of Science in Industrial and water and validate the test methods Microbiology used in testing the finished products from • Bachelor of Arts in Microbiology microbiological perspective. • Diploma Courses in Microbiology • Post Graduate Diploma in Marine Eligibility Criteria for Undergraduate Microbiology level courses in Microbiology • M.Sc in Microbiology In order to apply for under graduate level • M.Sc in Applied Microbiology courses in Microbiology, candidates should complete 12th class. It is important to opt • M.Sc in Microbial Genetics and Physics, Chemistry and Biology subjects in Bioinformatics 12th class to join for Microbiology courses. Candidates need to score good percentage Universities offering Courses in of marks in 12th class as the selection Microbiology process for undergraduate level courses • Indian Institute of Technology in this stream will be based on the marks • Banaras Hindu University scored. There are certain top universities

VII

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• Aligarh Muslim University • Indian Institute of Science • University of Mumbai • Amity University • Vinayaka Mission University • Kurukshetra University • Mahatma Gandhi University

Para Medical Courses and certificate courses in Tamilnadu Government Medical Colleges

1 Year Certificate Courses Courses Educational Age limit Qualification Cardio Sonography Technician ECG/ Tread Mill Technician Pump Technician Cardiac Catheterisation Lab Technician Emergency Care Technician Pass in H.Sc. Dialysis Technician with physics, Chemistry, Anaesthesia Technician Should complete 17 yrs Botany & Should not exceed 32 yrs Theatre Technician Zoology (or) Orthopaedic Technician Biology and Audiometry Technician Microbiology Hearing Language and Speech Technician Clinical, Therapeutic, Nutrition & Food Ser- vice Management Technician E.C.G/E.M.G Course Technician Multipurpose Hospital Worker Course Pass in SSLC

2 Years Diploma Courses Courses Educational Age limit Qualification Dental Mechanic(Male) Pass in H.Sc. with Dental Hygienist (Female) Should complete physics, Chemistry, Diploma in Medical Lan Rechnology (Dmlt) 17 yrs Botany & Zoology Diploma in Radio Diagnosis Technology (Drdt) Should not exceed (or) Biology and Diploma in Radio Therapy Technology (Drtt) 32 yrs Microbiology Diploma in Optometry

VIII

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Medical Record Science Courses Educational Qualification Age limit Diploma in Medical Record Pass in H.Sc. with physics, Should complete 17 yrs Technician (Six Months) Chemistry, Botany & Zoology Should not exceed 32 yrs (or) Biology and Microbiology

Job Prospects similarities between microbiology Candidates who have studied courses and biotechnology, the career options related to Microbiology have good scope available for professionals in the field for jobs in different sectors. Candidates of biotechnology are applicable to the can take up jobs in private sectors professionals in the field of microbiology mainly in pharmaceutical companies, as well. research firms. Candidates can get in Central Government jobs after M.Sc to roles like Medical Microbiologists, Microbiology Agricultural Microbiologists, and Marine Microbiologists. Candidates can join Post Graduates of Microbiology can find for teaching jobs as well. Jobs are also plenty of job opportunities in the Central available in public sector after doing under Government sector. Several vacancies graduate or post graduate level courses in are available for them in the research Microbiology. Job opportunities occur institutes run by Central Government. in government controlled development These graduates can apply for Scientist, laboratories, chemical industries, Research Assistant, Technical Assistant, hospitals, food industry, pharmaceutical Field Assistant or Project Assistant companies. Apart from this, candidates posts in these institutes whenever can also try for jobs abroad. Candidates vacancies are available. Institute of Liver who attain good experience in this field and Biliary Sciences, New Delhi offers will get higher salary packages in jobs. Microbiologist job for these graduates. They can apply for this post when Career Prospects after completion of the notification gets published in the B.Sc Microbiology course newspaper or website. Staff Selection Candidates, who have completed Commission conducts Combined their B.Sc Microbiology, can become Graduate Level Exam for recruiting microbiologists and there is wide range graduates to various departments in of employment opportunities available the Government. Those who have for microbiologists. They can find job completed M.Sc Microbiology can apply placement in research laboratories and for this exam, if they are interested to research organizations in public sector work in the Government sector. There and private sector. They can also find are many laboratories working under job placement in pharmaceutical firms, the supervision of Council of Scientific chemical firms. Since there is many and Industrial Research (CSIR). M.Sc

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Microbiology graduates can apply The Future of Microbiology for various posts available for them Microbiology has a clearer mission than in these laboratories. There are also other scientific disciplines. It is confident of vacancies available for these graduates its value because of its practical significance. in Government hospitals. The following brief list will give us some idea of what the future may hold: Teaching Profession in Government Sector after M.Sc Microbiology • Everyday microbes are changing its nature (mutation) and new diseases are Candidates who want to work in the emerging. Microbiologists will have to teaching field after M.Sc Microbiology can respond to these threats. apply to various colleges or universities. An M. Phil / Ph.D degree is required for • Microbiologists must find ways to stop these graduates to apply for these posts. the spread of established infectious They also need to clear NET exam so as to diseases. be eligible for teaching posts available in various universities. • Microbial diversity is another area requiring considerable research.

Microbiology in India • Much work needs to be done on There are number of Institutes engaged in microorganisms living in extreme microbiological research in our country. environmental conditions. The The Indian Institute of Petroleum, discovery of new microorganisms may Dehradun; Tata Energy Research Institute, lead to further advances in industrial Delhi and National Chemical Laboratory, processes and enhanced environmental Pune have worked on microbial dewaxing control. of heavier petroleum fractions. The Institutes has also played a vital role on the • The genomes of many micro organisms area of microbial enhanced oil recovery already have been sequenced, and and production of biosurfactants. National many more will be determined in the Institute of Nutrition, Hyderabad and coming years. National Institute of Occupational Health, • Microorganisms are essential partners Ahmedabad have already completed a long with higher organisms in symbiotic time plan on monitoring and surveillance relationships. Greater knowledge of food contaminants hazards in India of symbiotic relationships can help while genome analysis and synthetic improve our appreciation of the living gene design for modulation of genome world. It also will lead to improvements expression invivo was carried out by the in the health of plants, livestock and scientists of Indian Institute of Science, humans. Bangalore.

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Chapter 1 Microbiology includes the study of Introduction to Microbiology Bacteria Algae Chapter Outline

1.1 Groups of Microorganisms Protozoa 1.2 Contributors to Microbiology

1.3 Branches of Microbiology Viruses Fungi

Microorganisms - Bacteria, Fungi, Algae, Protozoa and Viruses - have been around for at least 3,500 million years.Microbes affect every aspect of life on earth. They have an amazing variety of shapes and sizes. They can exist in a wide range of habitats.

Science knows no country, because knowledge belongs to humanity, and is the torch which illuminates the world. Louis Pasteur

Learning Objectives

After studying this chapter the student developments in biotechnology, genetic will be able, engineering and nanotechnology have • To know the features of microorgan- placed Microbiology in the limelight. isms. Microorganisms provide the model for interdisciplinary research and for studying • To know the contributions of fundamental life processes. There is different scientists. growing recognition of microorganisms • To know the branches of and their potential in many applied Microbiology. areas like Environmental science, Agriculture, Food and Pharmaceutical Microbiology is one of the fascinating industries. The uses of microorganisms fields of science. Microorganisms and their are becoming increasingly attractive. activities are the major concerns of society Some microorganisms are beneficial both nationally and internationally. The to us and we cannot live without them.

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However microorganisms can be harmful usually multicellular. They range in size in many ways and bring about undesirable and shape from single celled microscopic changes. These microorganisms can cause yeasts to giant multicellular mushrooms diseases that can make us sick or even kill and puffballs. us. Although much more is known today Example: Aspergillus niger, Agaricus about microbial life than ever before, the bisporus vast majority of this invisible world remains Protozoa: They are unicellular unexplored. Microbiologists continue to eukaryotic organisms. Their role in nature identify new ways that microbes benefit are varied. The best known protozoa cause and threaten humans. disease in human beings and animals. Microbiology is the study of living Example: Giardia lamblia, Plasmodium organisms of microscopic size, which vivax include bacteria, fungi, algae, protozoa, and viruses. Microbiology is concerned Algae: They range from unicellular, with form, structure, reproduction, colonial to multicellular forms. All algal physiology, metabolism, and classification cell contain chlorophyll and are capable of microorganisms. It includes the study of of photosynthesis. They are found most commonly in aquatic environments and • their distribution in nature, damp soil. • their relationship to each other and to Example: Spirogyra, Chlamydomonas other living organisms, Viruses: In the study of Microbiology, • their effects on human beings, animals we encounter “organisms” which may and plants, represent the borderline of life. Viruses • their abilities to make physical and are simpler in structure and composition chemical changes in our environment, than other living cells. A virus is made • their reaction to physical and chemical up of nucleic acids and proteins. Viruses agents. are obligate parasites. They grow only within an appropriate host cell (plant, 1.1 Groups of Microorganisms animal, humans or microbe). They cannot multiply outside a host cell. There are many kinds of microorganisms present in the universe. They are broadly Example: HIV, Rabies virus classified into the following groups. Bacteria: They are unicellular prokaryotic organisms or simple association of similar cells. Cell multiplication usually Prions are infectious happen by binary fission. agents composed Example: Escherichia coli, Bacillus entirely of protein subtilis material. Creutzfeldt– Jacob Disease (CJD) is one of the human Fungi: They are eukaryotic organisms prion diseases. which is devoid of chlorophyll. They are

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1.2 Contributors to Microbiology Many scientists contributed to the science of Microbiology from the 17th century to the present day. Some prominent microbiologists who have made significant contribution to the study of microorganisms are given below:

1.2.1 Antony Van Leeuwenhoek Antony Van Leeuwenhoek (1632-1723) of Holland (Figure 1.1) developed microscopes. He was a Dutch merchant and a skilled lens Figure 1.1: maker. He made a variety of lenses with Antony Van Leeuwenhoek magnifying power 50-300X. Antony Van Leeuwenhoek wrote He was the first person to invent simple many letters. He wrote them in microscope. It has a single biconvex lens with a Dutch, the only language that he magnification of about 200X (Figure 1.2). His knew. These letters, described his microscopes resolved bodies with diameters complete scientific output. Antony measuring below 1micron. He examined Van Leeuwenhoek in a letter dated water, mud, saliva and found living 12th June 1716, wrote “... my work, organisms. He called these microorganisms which I’ve done for a long time, was as Animalcules (little animals). Bacteria like not pursued in order to gain the praise cocci, bacilli and spirochetes were recognized. I now enjoy, but chiefly from a craving He proposed that the size of bacteria is one after knowledge, which I notice resides sixth the diameter of Red Blood Cells. in me more than in most other men. He observed the growth of bacteria in And therewithal, whenever I found out infusions. The existence of spermatozoa anything remarkable, I have thought and RBC was revealed by him. Animal it my duty to put down my discovery histology was established by him. He on paper, so that all ingenious people described capillary circulation and added might be informed thereof”. a new dimension to Biology. All kinds of unicellular microorganisms were accurately described by him including human oral Contribution to science as a chemist microbial flora. He is commonly known as Louis Pasteur was working with tartaric the ‘Father of Microbiology’. acid crystals. He could pick up the dextro and levo rotatory crystals by seeing the 1.2.2 Louis Pasteur (1822-1895) morphology of the crystals. Later he was Louis Pasteur was a French chemist and a called to solve some of the problems crystallographer (Figure 1.3). His greatest in fermentation industry and turned contribution to microbiology made him to his attention to biological process of be the ‘Father of Modern Microbiology’. fermentation.

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Lens Sample Observer’s eye Mounting pin

Focusing screws

Figure 1.2: Leeuwenhoek’s Microscope

theory of spontaneous generation. He strongly supported theory of Biogenesis (life orginates from pre-existing life forms). To prove this he carried out several experiments. Pasteur poured meat infusions into flasks and then drew the top of each flask into a long curved neck that would admit air but not dust (Figure 1.4). He found that if the infusions were heated, they remained sterile (free from any growth) until they were exposed to dust. After opening them on a dusty Figure 1.3: Louis Pasteur (1822-1895) road and resealing them, he demonstrated the Contribution to Microbiology growth of microorganisms in all the flasks. The unopened flasks were sterile. Thus he disproved To wine industry the theory of spontaneous generation. Louis Pasteur discovered alcohol production from grape juice was due to Pasteurization yeast. The presence or contamination Louis Pasteur used heat to destroy undesirable of rod shaped bacteria resulted in large microbes in fruit juices. He employed 62.8°C amounts of lactic acid production in wine. (145°F) for 30 mins to kill microbes. This He also found that microorganisms in process is called Pasteurization which is fermented fruits and grains, resulting in commonly used in distillaries and dairy alcohol production. He coined the term industry. “fermentation”. Discovery of diseases Pasteur disproved spontaneous generation Louis Pasteur found that Pebrine disease Spontaneous generation states that life could in silk worm was caused by a protozoan arise spontaneously from inanimate (non-living) parasite. He suggested that Pebrine disease materials (Abiogenesis). Pasteur disproved the could be eliminated by using only healthy,

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Wait

Boil No growth

Wait Break Boil neck Microbial growth

Figure 1.4: Pasteur’s swan neck flask experiment disease free silk worms. Wool Sorter’s disease an individual against the disease. He was named as “Anthrax” by him. He isolated developed vaccines for anthrax and rabies. Bacillus anthracis from the blood of infected 1.2.3 Edward Jenner (1749-1823) animals. Chicken cholera bacterium was also isolated by Louis Pasteur using pure In ancient observation, persons who had culture. suffered from a specific disease such as small pox (causative agent of small pox He proved that many is varicella virus) or mumps, resisted the diseases were caused by infection on subsequent exposures. They the presence of foreign rarely contracted these infections for second microorganisms (Germ time. Edward Jenner, a country doctor in theory of disease). England noted a pustular disease on the He discovered various hooves of horses called the grease. This was infection causing microorganisms such carried by farm workers to the nipples of as Staphylococcus, Streptococcus and cows (cow pox). This was again carried by Pneumococcus. milk maids. They got inflamed spots on the Vaccination hands and wrists. The people who got this Pasteur found out that bacteria could cow pox were protected from small pox. be attenuated by growing them in He reported that 16 farm workers who had unnatural conditions. He coined the term recovered from cow pox (causative agent of “attenuation”. It is a process wherein cow pox is vaccinia virus) were resistant to bacteria lose their virulence due to small pox infection. repeated subculturing under laboratory He took the material (pus) from the cow conditions. He used attenuated cultures as pox and inoculated into the cut of 8 year vaccines for immunizing and protecting old boy on 14th May 1796 (Figure 1.5). Two

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months later Jenner inoculated the same He modified Ziehl-Neelsen Acid Fast boy with material taken from small pox staining procedure which was introduced by patients. This was a dangerous but accepted Ehrlich. He devised solid medium to grow procedure at that time. This procedure was microorganism. He developed powerful called variolation. The boy was protected method to isolate the microorganisms in against small pox. His exposure to the mild pure culture from diseased tissue. He also cow pox disease had made him immune perfected the techniques of identification of to the small pox disease. In this manner the isolated bacteria. Jenner began the Science of Immunology, He introduced Koch’s thread method the study of the body’s response to foreign to find out the efficacy of disinfectants. substances. Edward Jenner was regarded as He established certain rules that must be the ‘Father of Immunology’. followed to establish a cause and effect relationship between a microorganism and a disease. They are known as Koch’s Postulates. He also described the Koch’s Phenomenon. He was regarded as the ‘Father of Medical Microbiology’.

Infobits

Koch’s Thread Method Figure 1.5: Dr. Edward Jenner Robert Koch carried out systematic performing his first vaccination (1796) experiments on disinfection, using pure cultures of bacteria. By means of 1.2.4 Robert Koch (1843-1910) his Thread Method, he investigated the Robert Koch was a effect on anthrax spores of the popular German physician disinfectants at that time. Koch’s Thread and microbiologist Method also called as carrier test. A (Figure 1.6). He carrier such as silk is contaminated by was the founder submerging in a liquid culture of the of Modern Bacillus anthracis, a test organism. The Bacteriology. Robert carrier is further dried and immersed Koch discovered in the disinfectant solution for a given Bacillus anthracis exposure time. Thereafter the thread is (Anthrax bacillus), Figure 1.6: cultured in a nutrient broth. No growth Mycobacterium Robert Koch after incubation indicated that the tuberculosis, and (1843-1910) product (disinfectant) is active. Vibrio cholerae. For the first time he showed the evidence that a specific germ (Anthrax Koch’s Postulates bacillus) was the cause of a specific disease Four criteria were established by Robert (splenic fever in sheep) and introduced Koch to identify the causative agents of an scientific approach in Microbiology. infectious disease. These include

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1. A specific organisms can always be 4. It is possible to recover the organism in found in association with a given pure culture from the experimentally disease. If we take typhoid as an infected animals and it is observed to example it is caused by a bacterium be the same as originally inoculated Salmonella typhi. pathogen. Figure 1.7 explains the 2. The organism can be isolated and Koch’s postulates. grown in pure culture in the laboratory. Limitations Salmonella typhi are grown in soild Some organisms have not yet been grown media under laboratory conditions. in artificial culture media 3. The pure culture will produce the Example: Mycobacterium leprae and diseases when inoculated into a Treponema pallidum. susceptible animal. Modern addition to Koch’s Postulates Almost all the pathogenic organisms produce the same disease in experimental Today we recognize additional criteria of animals. Usually rats, mice, rabbits or causal relation between a microorganism guinea pigs are used as experimental and a disease. The important one is animals. Pneumococci produce the demonstration of abnormally high pneumonia in animals. Salmonella species concentration of specific circulating do not produce typhoid fever in rat, antibodies to the organism in the infected mice or rabbit. So chimpanzee is taken host or the presence of abnormally as experimental animal and it produces high degree of specific immunity or fever in chimpanzee. hypersensitivity to the infecting agent in

Koch postulates

Postulate 1 The same microorganisms are present in every case of the disease.

Anthrax bacilli Postulate 2 The microoraganisms are isolated from the tissues of a dead animal, and a pure culture is prepared. Postulate 4 The identical microorganisms are isolated and recultivated from the tissue specimens of the experimental animal.

Postulate 3 Microorganisms from the pure culture are inoculated into a healthy , susceptible animal. The disease is reproduced.

Figure 1.7: Koch’s postulates for infectious diseases

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a recently recovered host. In addition to culture techniques, serological techniques are also used for diagnosis of diseases.

Usefulness of Koch’s Postulates • It is useful in determining pathogenic organisms. • To differentiate the pathogenic and nonpathogenic microorganism. • For the classification of organisms.

• To detect the susceptibility or resistance Figure 1.9: Original culture plate on of the laboratory animals. which the observation of action of penicillin was made by Alexander Fleming 1.2.5 Joseph Lister(1827-1912) inhibited (Figure 1.9). He also showed Joseph Lister was that the culture filtrate of mold inhibited a British surgeon the growth of Staphylococcus aureus. He (Figure 1.8). He called this substance Penicillin, which found out that acted on Gram positive bacteria. For microorganisms the discovery of this antibiotic Fleming were responsible for (Figure 1.10), Florey and Chain got Nobel wound infections. He Prize in 1945. Penicillin eventually came developed a system into use during world war II as a result of antiseptic surgery. Figure 1.8: of the work of a team of scientists led by He used bandages Joseph Lister Howard Florey of the University of Oxford. soaked in phenol (1827-1912) solution to prevent wound infection. He sterilized instruments by heat and sprayed diluted phenol over surgical area and prevented contamination of wounds. He was the first person to isolate bacteria in pure culture using liquid culture. Thus, he was considered as co-founder Figure 1.10: Alexander Fleming (1881-1955) of Medical Microbiology with Koch, who later isolated bacteria on solid media. Alexander Fleming, 1.2.6 Alexander Fleming (1881-1955) the discoverer of penicillin warned He was a British Bacteriologist. He about the possibility observed a mold (Penicillium notatum) of antibiotic resistant bacteria due to growing on a plate of Staphylococcus antibiotics misuse, as early as in 1920s. aureus. The growth of Staphylococcus aureus around the mold colony was

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1.2.7 Selman Abraham Waksman griseus which is not required for its (1888-1973) growth but may help it to compete with Waksman was from Rutger University, other bacteria for food and space in the USA (Figure 1.11). His research was environment. Streptomycin is used in largely on soil microorganisms. He showed the treatment of tuberculosis. Waksman antimicrobial activity of streptomyces that got Nobel Prize in 1952. for his work on led to the discovery of Streptomycin and Streptomycin several other antibiotics. Antibiotics are usually not effective for sore throats and common colds. They are commonly caused by viruses rather than bacteria. Taking antibiotics for such illnesses is considered more harmful than beneficial.

Figure 1.11: Selman Abraham Waksman (1888-1973) 1.3 Branches of Microbiology Waksman and his co-workers isolated Microbiology can be classified into Pure and Actinomycin in 1940, Streptothrecin in 1942, Applied Microbiology. Pure Microbiology Streptomycin in 1943, and Neomycin in 1949. is classified based on taxonomical and Streptomycin is produced by integrative characteristics. Table 1.1 shows Streptomyces griseus. It is a secondary various branches of microbiology. metabolite produced by Streptomyces

Table 1.1: Branches of Microbiology

Based on Taxonomical characteristics Bacteriology The study of bacteria Mycology The study of fungi Protozoology The study of protozoa Based on Taxonomical characteristics Phycology (or algology) The study of algae Parasitology The study of parasites Immunology The study of the immune system Virology The study of viruses Nematology The study of the nematodes

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Based on integrative characteristics

Microbial Cytology The study of microscopic and sub microscopic details of microorganisms Microbial Physiology The study of biochemical functions of microbial cell. It also includes the study of microbial growth, microbial metabolism and microbial cell structure Microbial Ecology The study of relationship between microorganisms and their environment Microbial Genetics The study of gene are organisation and regulation in microbes in relation to their cellular functions. Cellular Microbiology A discipline bridging microbiology and cell biology

Evolutionary Microbiology The study of the evolution of microbes

Microbial Taxonomy The study of naming and classification of microorganisms

Microbial Systematics The study of the diversity and genetic relationship of microorganisms Systems Microbiology A discipline bridging systems biology and microbiology Generation Microbiology The study of microorganisms which have the same characters as their parents Molecular Microbiology The study of the molecular principles of physiological processes in microorganisms Nano Microbiology The study of microorganisms at nano level Exo Microbiology (or Astro The study of microorganisms in outer space Microbiology) Biological Warfare The study of microorganisms used in weapon industries Applied microbiology Medical Microbiology The study of the pathogenic microbes and the role of microbes in human illness. Includes the study of microbial pathogenesis and Epidemiology and is related to the study of disease, Pathology and Immunology Pharmaceutical The study of microorganisms that are related to the Microbiology production of antibiotics, enzymes, vitamins, vaccines, and other pharmaceutical products

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Industrial Microbiology The study of exploitation of microbes for use in industrial processes. Examples include industrial fermentation and waste water treatment. This field also includes brewing, an important application of microbiology Microbial Biotechnology The study of manipulation of microorganisms at the genetic and molecular level to generate useful products Food Microbiology and The study of microorganisms in food spoilage, foodborne Dairy Microbiology illness and food production.

Summary Student Activity Microbiology is the study of microorganisms that includes bacteria, 1. Want to see spontaneous fungi,algae,protozoa and viruses. Many generation of life? scientists contributed to the science of Take chicken soup or meat soup microbiology. boil it in a bottle. Keep it over Antony Van Leuwenhoek made the shadow of your window/or in simple microscope. For the first time, a open place with mouth open. Antony Van Leuwenhoek described the Observe for a week. You will microorganisms. Louis Pasteur disproved see maggots (worms) growing. the theory of spontaneous generation. Observe and record your findings. Germ theory of disease came from the 2. For you to enjoy-like Antony Van work of Pasteur and Robert Koch. Vaccines Leeuwenhoek !! for Anthrax and rabies was developed Get a palmist lens, see through it by Pasteur. Direct relationship between a paper print. You will see letter the suspected pathogen and disease was becomes big, bigger, and at one established by Koch’s postulates. Koch point it is no longer magnifying developed the technique of pure culture the letter. A simple convex lens is on solid medium. Joseph lister developed magnifying things. Leeuweenhoek antiseptic surgery. Alexander Fleming used such lens only. (as seen discovered Penicillin. Waksman showed above) You know useful and antimicrobian activity that led to the useless magnification. discovery of Streptomycin and other antibiotics. The branches of microbiology can be classified into pure and applied microbiology. Pure microbiology is classified based on taxonomical and integrated characteristics. Microbiology has got vast areas open for job opportunities.

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ICT CORNER Microbiology

Lets meet our micro friends

STEPS: • Use the URL or scan the QR code to open ‘NPTEL’ page. • Click ‘History’ and ‘Scope of Microbiology’ to know the history of microbiology. • Select history of microbiology and click ‘Start Course’ at the bottom. • Select ‘Members of the Microbial world’ to know about it.

Step1 Step2 Step3

URL: http://nptel.ac.in/courses/102103015/41#

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Evaluation c. Streptomyces griseus Multiple choice questions d. Penicillium mornefii 1. Theory of spontaneous generation 7. Which of the following antibiotics was discovered by Waksman? was disproved by whom? a. Streptomycin a. Robert Koch b. Neomycin b. Edward Jenner c. Actinomycin c. Louis Pasteur d. All the above d. All of them 2. Which of the following did Edward Answer the following Jenner used to protect the boy against 1. Name the causative agent of cow pox small pox? and small pox. a. Cow pox material 2. Explain the method of Edward Jenner b. Small pox material used to protect people against small c. Both the above pox. d. Rabbit pox 3. List two organisms that do not obey 3. Among the following scientists, who Koch’s postulates. discovered solid medium? 4. Give the usefulness of Koch’s a. Louis Pasteur postulates. b. Edward Jenner 5. What are the modern additions to c. Robert Koch Koch’s postulates? d. None of them 6. List the contribution of Alexander 4. Which of the following organisms Fleming. does not obey Koch’s postulates? 7. What is the theory of spontaneous a. Cow pox virus generation? b. Small pox virus 8. How was spontaneous generation c. Treponema pallidum theory disproved? d. M.Tuberculosis 9. Highlight the contribution of 5. Who modified Ziehl-Neelsen staining Waksman. technique? 10. State the characteristics of a. Louis Pasteur streptomycin. b. Robert Koch 11. Give a list of contribution of Louis c. Ziehl-Neelsen Pasteur to wine industry. d. All the above 12. Explain Koch’s postulates? 6. Which of the following fungi grow on 13. Describe the microscope made by Alexander Fleming’s plate? Antony Van Leeuwenhoek. a. Penicillium chrysogenum 14. What are the contributions of Antony b. Penicillium notatum Van Leeuwenhoek to microbiology?

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Chapter 2 Microscopy

Chapter Outline

2.1 Historical Background 2.2 Principles of Microscopy 2.3 Bright Field Microscope 2.4 Dark Field Microscope

Microorganisms are very small and cannot be viewed by human eye. The microscope helps in observing the microbial world which exists in a wide range of sizes. The prokaryotes (bacteria and archae) are smaller (~ 0.4-10μm) and the eukaryotes are larger (~ or >10μm). The word microscope is derived from the Latin word micro, which means small, and the Greek word skopos means to look at.

Learning Objectives Robert Hooke, built compound microscopes with multiple lenses. In 17th century, Dutch spectacle maker Zaccharias After studying this chapter the student Janssen is given the credit for making first will be able, compound microscope. However, the early • To know the properties of light and compound microscopes were poor in quality. lens. In 1830, Joseph Jackson Lister (the father of Joseph Lister who practised antiseptic surgery) • To know the science of image made significant development which resulted formation in brightfield microscopy. in the invention of modern compound • To understand the design of light microscope used in microbiology today. microscope. • To learn and compare the principle, 2.2 Principles of Microscopy instrumentation and working of All kind of microscopes use visible light brightfield and darkfield microscopy. to observe specimens. Light has a number of properties that affect our ability to visualise objects. 2.1 Historical Background 2.2.1 Properties of Light Antony Van Leeuwenhoek (1632-1723) was the first person to use a simple Light is a part of the wide spectrum of microscope with one lens similar to a electromagnetic radiation from the sun. It magnifying glass. The lens is capable of is a form of energy. The most important 50X to 300X magnification. property of light is wavelength (the length

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of light ray) (Figure 2.1). light waves or light particles. The combined properties of particle and One wavelength Wave Crest wave enable light to interact with an object in several different ways like transmission, absorption, reflection, refraction, diffraction and scattering (Figure 2.3). Wave trough 2.2.2 Lenses and its Properties Figure 2.1: Wavelength-the distance between two adjacent crests or two Lenses are optical devices which focus adjacent troughs of the wave and denoted or disperse a light beam by means of by greek letter (λ) refraction. A simple lens consists of a single piece of transparent material. The sun produces a continuous spectrum Light rays from a distant source are of electromagnetic radiation with waves of focused at the focal point F. The focal various lengths (Figure 2.2). Radiation of point lies at a distance f (focal length) longer wavelength includes Infrared (IR) from the lens’ centre (Figure 2.4). and radiowaves, the shorter wavelengths include Ultra Violet (UV) rays and X-rays. The physical behaviour of light can be caterigorised as either light rays,

Increasing wavelength Increasing frequency (wavelength) nm nm -6 -2 1×10 10 nm 400 nm 700 nm 1 nm 10 cm 100 km 1×10 X-rays UV IR Microwave Radio and TV

400 nm Visible light 700 nm Figure 2.2: The electromagnetic spectrum-White light is a combination of all colours of visible spectrum

Transmission Reflection Refraction

Diffraction Absorption Scattering

Figure 2.3: Interaction of light with matter

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by Ernst Abbe, and is defined by the The larger the numerical aperture following expression the better the resolving power. It is

f important to illuminate the specimens properly to have higher resolution. The concave mirror in the microscope creates a narrow cone of light and has a F D (Diameter small numerical aperture. However, the of lens) resolution can be improved with a sub stage condenser. A wide cone of light through the slide and into the objective lens increases the numerical aperture Numerical Aperture (NA) =n × sin(θ) there by improves the resolution of the microscope. n = the refractive index of the medium between the specimen and objective; Types of microscopes θ = half aperture angle or collection angle In order to view microorganism and of the objective. (the maximum half angle microbial structures of different sizes we of the cone of light that can enter or exit require different kinds of microscopes. the lens). • Light microscopes resolve images with the help of light. The specimen is viewed Infobits as dark object against a light background in bright field microscope. Dark field The smallest cells on the planet are microscope uses a special condenser some forms of Mycoplasma with and the specimen appears light against dimensions of 0.2 to 0.3 μm, which is a black background. The other types within the limit of resolution of light of mircoscopes are Phase contrast and microscopes. Tiny cells that look like Fluorescence microscope. dwarf bacteria but are 10 times smaller • Electron microscope uses a beam of than Mycoplasma and 100 times electrons instead of light. Electrons pass smaller than the average bacterial cell through the specimen and form a two are called nanobacteria or nanobes dimensional image in Transmission Electron Microscope (TEM). Electrons (Greek nanos means one billionth). are reflected from the specimen and produce a three dimensional image in The resolving power of a light Scanning Electron Microscope (SEM). microscope depends on the wavelength of light used and the NA of the objective 2.3 Bright Field Microscope lens. The numerical aperture of a lens can The most commonly used microscope be increased by for general laboratory observations is the standard bright field microscope (Figure 2.6). • Increasing the size of the lens It contains the following components opening and/or • Increasing the refractive index of • A mirror or an electric illuminator is the material between the lens and the light source which is located at the the specimen. base of the microscope.

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Figure 2.6: Bright field Microscope

• There are two focusing knobs, the which is many times larger than the real fine and the coarse adjustment knobs image. This magnification occurs when light which are located on the arm. These rays from an illuminator (light source), pass are used to move either the stage or the through a condenser which has lenses that nosepiece to focus the image. direct the light rays through the specimen. The • Mechanical stage is positioned about light rays then pass into objective lens (the lens half way up the arm, which allows closest to the specimen). The image is again precise contact on moving the slide. magnified by the ocular lens or the eyepiece. • The substage condenser is mounted (Figure 2.7). within or beneath the stage and focuses a cone of light on the slide. In • Magnification is the process of the simpler microscope, its position is enlarging the image of the specimen fixed where as in advanced microscope and can be calculated by multiplying it can be adjusted vertically. the objective lens magnification power The upper part of microscope arm holds by ocular lens magnification power. the body assembly. The nose piece and one or Representative magnification values for more eyepieces or oculars are attached to it. a 10X ocular are: The body assembly contains series of mirrors Scanning objective (4X) × (10X) = 40X and prisms so that the barrel holding the magnification eyepiece may be tilted for viewing. Three or five Low power objective (10X) × (10X) = objectives with different magnification power 100X magnification are fixed to the nosepiece and can be rotated High dry objective (40X) × (10X) = 400X to the position beneath the body assembly. In magnification bright field microscopy; the specimen is viewed Oil immersion objective (100X) × (10X) against a bright background. The details of the = 1000X magnification image are defined by the surrounding light. A series of finely ground lenses forms an image

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Path of light rays (Bottom to top) to eye Oil Immersion

Ocular lens enlarges primary image formed Oil immersion lens is designed to be in by objective lenses. Prism that direct contact with oil placed on the cover directs rays to ocular lens slip. An oil immersion lens has a short focal length and hence there is a short working distance between the objective lens and the specimen. Immersion oil has

Objective lenses (those closest a refractive index closer to that of glass to specimen) form the primary image. Most compound light than the refractive index of air, so the use microscopes have several. of oil increases the cone of light that enters the objective lens. Figure 2.8 explains Stage supports the working principle of oil immersion microscope slide objective lens.

Condenser lenses focus light rays through specimen. HOTS

• What are the two ways by which Illuminator the resolving power of microscope can be enhanced? • What are the advantages of the low-power objective over the oil immersion objective for viewing Figure 2.7: The path of light in light fungi or algae? Microscopes • What will happen if water is used instead of immersion oil under a 100X objective lens?

Microscope Microscope objective enses objective

efracted nrefracted light rays light rays lost to lens enter lens Immersion oil lass cover slip lass cover slip

Slide Slide

Specimen ight source Specimen ight source (a) ithout immersion oil (b) ith immersion oil Figure 2.8: Oil Immersion Objective Working Principle

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2.4 Dark Field Microscope The disc blocks direct entry of light to the objective lens. The light rays reflected off This is used for examining live the specimen enter the objective lens and microorganisms which are invisible in in the absence of direct background light, light microscope and cannot be stained the specimen appears light against a dark by standard methods. It can be used to background (Figure 2.9). The microbes study samples which can get distorted by are visualized as halos of bright light staining and cannot be identified further. against the darkness, as stars are observed The distinct feature is the dark field against the night sky (Figure 2.10). condenser that contains an opaque disc.

Objective

Specimen

Abbe condenser

(a)

Dark-fields stop

(b) Figure 2.9: Dark Field Microscopy. The simplest way to convert a microscope to dark field microscope is to place. (a) a dark field stop underneath (b) the condenser lens system

Infobits

Compound microscope (also known as light microscope) produces a mono (2D) image and stereo microscope produces stereo (3D) image. ‘Upright’ life science microscopes are the most numerous of all microscopes. An inverted microscope is the kind of microscope that views objects from an inverted position. Digital microscopes are becoming widespread. These provide Figure 2.10: Dark field observation of simple image and are convenient for bacteria Treponema pallidum specimen electronic image capturing. from a patient with Syphilis

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ICT CORNER SEM

Lets focus with SEM

STEPS: • Use the URL or scan the QR code to reach ‘myscope outreach’ interactive page. • Click ‘The Scanning Electron microscope’ under ‘Basic’ menu to know about its parts and function. • Follow the successive steps that lead to describe the n nuances of SEM. • Select ‘Let’s Zoom in’ under the activity to menu and explore the SEM stimulations.

Step1 Step2 Step3

URL: http://myscopeoutreach.org

Summary Evaluation The microscope is a tool to study small Multiple choice questions microscopic life forms. Zaccharias 1. The first compound Janssen is given the credit for making first microscope was invented by compound microscope. Light microscopy a. Robert Hook has undergone a renaissance during the later b. Anton von Leewenhoek years of the 20th century and early stages of c. Kepler and Galileo 21st century. d. Zaccharias Janssen There are two main types of microscopes (i) Light microscope and (ii) 2. All the following are components of compound Electron microscope. Light microscope microscope except makes use of light and Electron microscope a. Stage clips b. Fine adjustment knob uses the electrons.

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c. Electron gun background d. Binocular eye piece d. Image formation is without use of light 3. Resolving power of an instrument can be Answer the following increased by 1. What is the importance of microscopy in a. Using an illumination of longer wavelength microbiology? and by decreasing the NA 2. Write down the names of different types of b. Using an illumination of longer wavelength microscopes. and by increasing the NA c. Using an illumination of shorter wavelength 3. What principle defines an object as and by increasing the NA “microscope”? d. Using an illumination of shorter wavelength 4. What happens to light rays when they and by decreasing the NA interact with an object? 4. The resolving power of unaided human eye is 5. Elucidate the lens function in image a. 1 cm formation. b. 100 μm 6. Define the characteristics of resolution, c. 200 μm magnification and numerical aperture. d. 400 μm 7. How do eukaryotic and prokaryotic 5. Which of the following is false about dark field cells differ in appearance under the light microscopy? microscope? 8. Trace the pathway of light in brightfield a. Adding disc called “stop” to the condenser microscopy. will make bright field to darkfield 9. Elaborate the role of condenser and image b. The stop disc prevents the entry of light from formation in dark field microscope. the central field and object is illuminated 10. Differentiate between Bright field and Dark with beam of light field microscopy. c. The light gets reflected from the sides of the specimen and appears bright in dark

Student Activity Experiment and enjoy…… Imaging Properties of a Simple Lens Objective: In this experiment you will observe and measure the imaging properties of a simple lens. Apparatus: You will need a good lens (magnifying glass), a flashlight, a viewing screen (tri-folded white copy paper), a meter stick and perhaps some modeling clay to hold things in place. Set all these things on a flat table about 1 meter wide in an area where the lighting can be dimmed.

Flashlight Lens Screen

Objecct Image

o i

Meter stick

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Chapter 3 Stains and Staining Methods

Chapter Outline

3.1 Techniques in Observing Microorganisms 3.2 Purpose of Staining 3.3 Stains 3.4 Principle of Staining 3.5 Preparation of Materials for Staining 3.6 Simple Staining Method 3.7 Differential Staining 3.8 Special Staining – Endospore Staining 3.9 Commonly used Stains and its Unstained and stained Lactobacillus sp. in curd. Applications Lactobacillus is a genus of bacteria which can convert lactose in milk into lactic acid by means of fermentation. Staining is used to visualize microbial cells under a microscope.

Learning Objectives

After studying this chapter the student • To describe the procedure of simple, will be able, Gram’s and endospore staining • To appreciate the need for staining. methods. • To differentiate between an acidic • To describe the appearance of dye and a basic dye and understand Gram positive and Gram negative the principle of staining. cells after each step of Gram staining procedure. • To classify organisms based on staining reaction and differentiate • To know the importance of Gram between simple and differential staining and endospore staining in stains. diagnosing and identifying bacteria. • To know smear preparation and heat • To learn a few staining solutions and fixation. names of bacteria.

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Have you ever thought of observing the using a match stick. A drop of culture microorganisms present in rain water when (liquid containing microorganisms) is you play? Have you ever wondered how milk placed on a cover slip. The cavity slide turns into curd and which microorganisms is placed upside down on the cover are involved? It is clearly understood from slip and inverted such that the drop is previous unit that microorganisms can be seen hanging (Figure 3.1b). only under microscopes. But microorganisms Since microbial cells are colourless do not show much of its structural details and transparent, observation of under the light microscope due to lack of microorganisms in wet preparation by contrast and poor resolution. To improve the bright field microscope is difficult. But, visibility of these tiny living organisms, stains dark-field and phase contrast microscopes and staining methods are of great use. give contrast and make structures within 3.1 Techniques for Observing the cells to appear clear. Therefore, these Microorganism microscopes are useful for examination of unstained preparation. A considerable amount of information can be gained by careful microscopic examination of microorganisms. There are two general 3.1.2 Examination of Stained techniques used in the preparation of Preparation microbial specimens to observe them under Staining enables better visualization of microscope. First technique employs the microorganisms under a microscope. unstained preparation of living cells and Microscopic examination of stained second one employs stained preparations of cells helps to reveal the size, shape killed microorganisms. and arrangement of microbial cells. Microbial cell staining is important in the 3.1.1 Examination of Unstained identification of infectious pathogens. Preparation Living microorganisms can be examined 3.2 Purpose of Staining directly by wet mount or by hanging drop preparations. Both the techniques are Staining is very useful for the following very useful in determining size, shape reasons: and motility of the microorganisms. The • To make the microscopic spirochetes (spiral bacteria) are normally semi transparent microbial cell visible. examined in wet preparation through Dark- field microscope. Some cell inclusion bodies • To reveal the size and shape of such as vacuoles and spores can be readily microorganisms. observed even without staining. • To demonstrate the presence of internal • A wet mount is made by keeping a drop and external structures of microbial of liquid containing microorganisms cells. (culture) on a microscope slide and • To distinguish between different types placing a cover slip over the drop. of microorganisms. (Figure 3.1a) • To produce specific chemical and • A hanging drop mount is made by using physical reactions. a cover slip and a cavity slide. Vaseline • To preserve the stained microorganisms is applied on each of the four corner as specimen slide. of the cover slip or around the cavity

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er sli

lass sli e

a Li i c ntaining micr rganism

In c lati n l

anging r

acterial c lt re

ait slie

er sli b Figure 3.1: a) Wet mount and b) Hanging drop preparation

3.3 Stains contain not only a chromophore group but also another group known as auxochrome Stains are dyes used to increase colour contrast. that imparts the property of electrolytic Dye is a coloured organic compound that dissociation. Auxochrome gives salt adheres to microbial cells, giving colour to the forming properties to the compound. cell. Today several stains and staining procedures are available to study the morphological details Hence, each stain or dye is composed of various microorganisms. The process of of three components: imparting colour to the microbial cell is known (i) Benzene ring: It is the basic as staining. colourless structural component Stains are organic compounds of a stain or dye. containing chromophore and auxochrome groups linked to benzene ring. (ii) Chromophore: It is the functional A chromophore group imparts colour group that gives colour. to the compound. Compounds of benzene (iii) Auxochrome: It is the group that containing chromophore radicals are gives ionic properties to the stain. called chromogens. Such a compound, The term stain and dye are not the even though it is coloured, is not a dye. In same. The basic differences between dye order for a compound to be a dye, it must and stain are given in Table 3.1.

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Table 3.1: Difference between dyes and • A majority of stains used in stains. microbiology are the synthetic type and manufactured from Aniline. For Dyes Stains example, Crystal violet, Safranin, Dyes are a Stains are Methylene blue and Acid fuchsin. colouring agents colouring 2. On the basis of chemical behavior, agents used used for general dyes are classified as acidic, basic and purposes. for biological purposes. neutral. • An acidic dye is one in which Dyes are the Stains are the colour bearing ion, the textile colouring pure. They are chromophore, is an anion. agents that are prepared with prepared with lesser greater care and • A basic dye is one in which the colour specification and bearing ion, the chromophore, is a they may contain specification. impurities. cation. • A neutral dye is a complex salt of a dye acid with a dye base. 3.3.1 Classification of Stains Acid dyes generally combine more strongly 1. On the basis of origin, stains can be with cytoplasmic (basic) elements of the cell, classified as natural and synthetic. and basic dyes combine best with nucleic (i) Natural stains: acid (acidic) elements of the cell. Table 3.2 • These stains are obtained directly shows the chemical characteristics of a stain from natural products. For example, or dye. Haematoxylin is obtained from the heartwood of a tree (Haematoxylon 3.4 Principle of Staining campechianum). Positive Staining • The natural stains are used mainly In positive staining, the surface of the for histological purposes. bacterial cell takes on the colour of the (ii) Synthetic stains: stain. When basic stain is applied, there is an attraction between the negatively • These are artificially produced charged cell surface and positively charged mainly from coal tar products and chromophore, which leads to staining of hence popularly called coal-tar dyes. the cell (Figure 3.2).

Stain (C )

egatively charged Stain containing cell surface positively charged chromophore

Stained bacterial cell Figure 3.2: Positive staining

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Table 3.2: Chemical characteristic of stain or dye

Acid stain Basic stain Neutral stain Chromogen of acidic stain Chromogen or coloured It is a complex salt of dye is negatively charged, so it is part of basic stain is acid with dye base. also known as anionic stain. positively charged, so it is also known as cationic stain. Used to stain the positively Used to stain negatively It stains both positive and charged component of charged component of negative charged components microbial cell. microbial cell. of microbial cell. Example: Eosin, Nigrosin, Example: Methylene blue, Example: Giemsa stain, India ink, Acid fuchsin, Safranin, Malachite green, Leishmanstain. Congo red. Basic fuchsin, Crystal violet

Infobits

On the basis of demonstrating the living or non-living status of microorganisms, some stains are classified as vital stains. These stains differentiate between living and non-living microbial cells. For example, Tryphan blue selectively colour dead tissues or cells. Certain stains will give a different colour to the cell inclusion bodies from its original colour. Such stains are called metachromatic stains. Metachromatic granules of Corynebacterium diphtheriae contain polymerized inorganic polyphosphate responsible for metachromasia with Toluidine blue or Methylene blue.

Negative Staining In negative staining, the background is The background gets stained and the coloured and bacteria remains colourless. cell remains colourless. This technique It is because the acidic dyes are repelled by is useful for revealing the cell shape, size the negatively charged bacterial surface. and demonstrating capsule (Figure 3.3).

Stain (C )

egatively charged Stain containing cell surface positively charged chromophore

Stained bacterial cell

Figure 3.3: Negative staining egative staining

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3.5 Preparation of Materials for Staining Heat fixation The essential steps in the preparation of In this method the slide is gently heated by materials to be observed are passed through a flame (Figure 3.5). Heat fixation will preserve the overall morphology 1) Preparation of smear of the cell without destroying the internal 2) Fixation structures. 3) Application of one or more staining solutions 3.5.1 Preparation of Smear Smears can be made from liquid or solid cultures or from clinical specimens. Smear is prepared by placing a loopful of culture on a clear glass slide with an inoculation loop. The culture is spread on the glass slide so as to form a thin film. This film is allowed to air dry (Figure 3.4). Figure 3.5: Fixation of smear by passing slide gently through the flame Step 1: Place the liquid on the slide Chemical fixation It involves the use of chemical fixative to protect the fine cellular structures of delicate microorganisms. For this purpose, Ethanol, Acetic acid, Formaldehyde, Glutaraldehyde and Mercuric chloride are Step 2: usually used.

3.5.3 Bacterial Staining Methods Add the microbes to the Different staining methods are employed liquid and spread over to study the bacterial morphology and the slide to identify bacteria. Some methods are Step 3: used for general purposes and others are Air dry or heat gently. when dry used for special purposes. There are three brieﬂy heat ﬁx the cells to the slide categories of staining methods, they are: Figure 3.4: Preparation of smear

3.5.2 Fixation Robert Hooke was the first to describe Fixation kills the microorganisms and attaches them to the slide. This prevents washing away the appearance of of microorganism in further steps of staining stained objects under procedure. It also preserves various parts of light microscope. microorganisms in their natural state with Professor Joseph Von Gerlach of only minimal distortion. The two fixation Germany was the first to use stain in methods that are used to fix microbial cells are heat fixation and chemical fixation. histology.

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i) Simple staining method iii) Special staining method. ii) Differential staining method Different types of bacterial staining methods are summarized in Flowchart 3.1

Staining Methods

Simple staining Special staining Differenial staining (For examination of shape, (For visualising the (For differentiating size and arrangement of bacterial external and bacterial groups) bacterial cells) internal structure)

Grams staining Acid Fast staining To distinguish To distinguish Acid Fast bacteria Gram positive and such as Mycobacterium sp from Gram negative bacteria Non-Acid Fast bacteria

Endospore staining Flagella staining Metachromatic Capsule staining Demonstrates Demonstrates the staining Demonstrates the spore structure in presence and Demonstrates presence of capsules bacteria. Example: arrangement of flagella. the presence of surrounding the cells Schaeffer Fulton Example: Silver nitrate granules. Example: using nigrosin stain method staining method Albert staining

Flowchart 3.1: Types of Bacterial Staining methods

3.6 Simple Staining Method In Simple Staining method only one stain is used. Stain is applied to the smear in one application. The fixed smear on the glass slide is flooded with a staining solution for about one minute. The solution is then washed off with water and the slide is blot dried. The stained slide is examined under a microscope (Figure 3.6). The cells stain uniformly. The simple stains used by the microbiologists for routine purposes are dilute solutions of Methylene blue, Crystal Figure 3.6: Simple stain – Micrococcus violet, Safranin and Carbol fuchsin. sp. stained with Methylene blue

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they are mixed and applied in one application. Mycobacterium lep- These procedures show differences between rae which causes the cells or parts of a cell and can be used for leprosy is an uncul- of identification. The two most important turable bacterium. It differential stains used by bacteriologists is primarily diagnosed by using a spe- are Gram stain and Acid Fast stain. The cial bacteriological stain called Acid differences between simple and differential staining are shown in Table 3.3. Fast stain. 3.7.1 Gram’s Staining Method The Gram’s stain technique was developed by Danish Bacteriologist Hans Christian Gram in 1884. It is one of the most useful staining methods because it classifies bacteria into two large groups namely Gram positive and Gram negative. In this method, the fixed Mycobacterium leprae (Acid Fast bacterial smear is subjected to staining bacilli) stained with modified Ziehl reagents in the order of sequence listed Neelson stain. below:

Methylene blue is more frequently used than Crystal violet any other stain in Bacteriology. It is used for the (Primary stain) rapid survey of bacterial population of milk. It is also used for the diagnosis of Diphtheria. Iodine This stain is incorporated along with Eosin (Mordant) in Lactose Agar to distinguish Escherichia coli from other fecal bacteria in contaminated water. Alcohol/Acetone 3.7 Differential Staining (Decolourising agent) In this method more than one stain is employed. In some method the stains are Basic fuschsin/Safranin applied separately, while in other method (Counter stain)

Table 3.3: Differences between Simple and Differential Staining Simple staining Differential staining 1. This method uses only one stain. This method uses more than one stain. 2. It imparts only one colour to all It imparts two or more different colours bacterial cells. to bacterial cells. 3. It reveals the size, shape and It reveals the size, shape and arrangement. arrangement of bacterial cells. In addition, it differentiates two groups of bacteria. Example: Methylene blue staining Example: 1. Gram’s staining method method. 2. Acid Fast staining method

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The organisms that retain the colour of 3.7.2 Procedure of Gram’s Staining the primary stain are called Gram positive Gram’s Staining comprises of four steps: and those that do not retain the primary stain when decolorised and take on the Step 1: A heat fixed smear colour of the counter stain are called is covered with a basic Gram negative. violet dye, Example: Crystal violet. This stain imparts Mordants: Mordants are not dyes. They its colour to all cells. It is are important to increase the biological referred to as a primary specimen’s affinity for a dye. Some stains stain, since it is applied first. never stain the cells or its components unless treated with a mordant. The mordant Step 2: After a short time, the slide is becomes attached to a cell or its components washed off and the smear is covered with and then combines with the stain to form an iodine, a mordant. At this stage both insoluble colour complex. Gram positive and Gram negative bacteria

Ste Micr sc ic a earance hemical reacti n cell in cell all

ram siti e ram nagati e

licati n r stal i let rimar stain

licati n I ine M r ant

lc h l ash ec l ri ati n

licati n Sa ranin nter stain

O ter membrane etiglen ell membrane Figure 3.7: Steps, micrograph and chemical reaction of Gram Stained Bacteria

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appear dark violet. and Gram negative bacteria. In addition, Step 3: Next, the slide is decolurized with Gram negative bacteria contain a layer of alcohol or an acetone alcohol solution. lipo polysaccharide (consists of lipids and This solution is a decolurizing agent, polysaccharide) as part of their cell wall. which removes the primary stain from the When Crystal violet and subsequently cells of some species but not from others. Iodine is applied to both Gram positive and Gram negative cells, the two combine Step 4: The slide is immediately washed after to form CV-I complex. decolurization and the slide is then counter stained with basic fuchsin or safranin, a The cell wall of Gram positive bacteria basic red dye. The smear is washed again, with lower lipid content get dehydrated blot dried and examined under microscope during alcohol treatment. The pore (Figure 3.7). size decreases and the permeability is reduced. Thus, the CV-I complex cannot be extracted and the cells remain violet. 3.7.3 Principle of Gram’s Staining The exact mechanism of action of The alcohol treatment of Gram negative this staining technique is not clearly bacteria extracts the lipid which results understood. However, the most acceptable in increased porosity or permeability explanations are associated with the of the cell wall. Thus, the crystal violet structure and composition of the cell wall. iodine [CV-I] complex is extracted and the bacteria are decolorized. These cells The cell wall of Gram positive bacteria subsequently take on the colour of the have a thicker peptidoglycan (consists counter stain basic fuchsin or safranin of disaccharides and amino acids) than and appears red to pink. Gram negative bacteria. Figure 3.8 depicts the cell wall of Gram positive

ram siti e ram negati e

O ter membrane etiglcan etiglcan

ell membrane ell membrane Figure 3.8: Cell wall of Gram positive and Gram negative Bacteria

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Infobits HOTS There are several modifications of 1. If the iodine step were omitted Gram’s Stain in the Gram’s staining procedure, • Kopeloff and Beerman’s what colour would you expect modification. Gram positive and Gram negative • Jensen’s modification. bacteria to stain? • Weigert’s modification. a. Gram positive : pink and • Preston and Morell’s Gram negative : purple modification. b. Gram positive : purple and Gram negative : pink medical technology, the Gram’s staining c. Gram positive : purple and remains an important, inexpensive and unbeatable tool in the identification of Gram negative : purple pathogens. d. Gram positive : pink and Examination of Gram stained organisms Gram negative : pink usually provides the basis for classifying, 2. In a Gram’s staining method, a identifying and characterizing bacteria. step could be omitted and still Gram staining of clinical specimens, however allow differentiation between provides only a preliminary indication of Gram positive and Gram negative the identity of the etiological agent (the organism causing the disease). Gram nature cells. Name the step. of common pathogenic bacteria is given in Table 3.4. 3.7.4 Importance of Gram Staining Gram stains of clinical specimens or This century old staining method still of growth on culture plates are especially remains as the universal basis for bacterial important in determining the most classification and identification. Even effective antibiotic for the ill patients who with today’s elaborate and expensive required immediate therapy.

Prof. Hans Christian Gram had died of pneumonia, discovered that (September 13, 1853-November 14, 1938) certain stains were preferentially taken up and retained by bacterial cells. Gram was a modest man, and in his initial publication he remarked, “I have therefore published the method, although I am aware that as yet it is very defective and imperfect; but it is hoped that also in the hands of other investigators it will turn out to be useful”. Dr. Gram used Bismarck brown instead of Safranin. It was a few years later, In 1884, Prof. Hans Christian Gram while German pathologist Carl Weigert (1845- examining lung tissue from patients who 1904), added the final step of staining with Safranin.

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Table 3.4: Gram nature of common pathogenic bacteria Gram positive bacteria Gram negative bacteria Cocci Staphylococcus aureus, Streptococcus Neisseria gonorrhoeae pyogenes Rods(bacilli) Mycobacterium tuberculosis, Escherichia coli, Shigella Bacillus anthracis, Corynebacterium Salmonella, Pseudomonas diphtheriae, Clostridium tetani aeruginosa Spirochaetes Leptospira, Treponema

3.8 Special Staining – Endospore stained, the spore tends to retain the dye Staining even after treatment with decolorizing agents. The most commonly used endospore Endospores are highly resistant structures staining procedure is the Schaeffer Fulton produced by some bacteria during endospore staining method. Malachite unfavourable environment conditions. green, the primary stain, is applied to a Endospore formation is a distinguishing heat fixed smear and heated to steaming feature of aerobic genera Bacillus and for about 5 minutes. Heat helps the stain anaerobic genera Clostridium. The to penetrate the endospore wall. Then the size, shape and position of the spore preparation is washed for about 30 seconds (Figure 3.9) are relatively constant with water. Next safranin, a counterstain is characteristics of a given species and applied to the smear to stain the portions of are important in identifying the species the cell other than endospores. within genera. The position of spore in the cell may be terminal, central or sub- In a properly prepared smear, the terminal. Figure 3.9 shows the position of endospores appear green within red cells spores in a vegetative cell. (Figure 3.10). Endospores are highly refractive. They can be detected under the light microscope when unstained, but cannot be differentiated from inclusions of stored material without a special stain. Terminal Central Subterminal spores Spores Spores Figure 3.9: Position of spore in a vegetative cell. Endospores cannot be stained by ordinary methods, such as simple staining and Gram staining, because the dyes do not penetrate the wall of the endospore. If simple stains are used, the vegetative body of the bacillus is deeply coloured, whereas the spore is unstained and appears as a clear area in the organism. Figure 3.10: Schaeffer Fulton Endospore staining method- spores stained green By vigorous staining procedure, the and vegetative cell stained pink dye can be introduced into the spore. Once

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Common Bacteria with their Gram reactions

Gram positive bacilli (rod) Corynebacterium s Clostridium s Listeria s Gram positive cocci Bacillus s Staphylococcus Streptococcus Pneumococci

Gram negative diplococci Gram nagative bacilli (rod) Neisseria s Klebsiella s Gram negative coccobacilli Escherichia. coli Haemophilus s Pseudomonas s Brucella s Vibrio s

3.9 Commonly used Stains and its Summary Applications Staining makes microscopic semi transparent Lactophenol cotton blue stain is the most bacterial cell visible. It is a substance that widely used for staining and observing adheres to a cell and impart colour. On the fungi. Giemsa stain is a Romanowsky stain, basis of the chemical composition, stains widely used in microbiology laboratory for or dyes are classified as acidic, basic and staining of blood and blood parasites like neutral. Staining techniques are classified malarial protozoans. Calcofluor white stain as simple, differential and special. Simple is commonly used stain to directly detect staining uses a single dye and can help to the fungal elements in tissues and in culture. identify the shape and size of an organism. Acridine orange stain is used to confirm Differential staining use more than one the presence of bacteria in blood cultures dye to distinguish between structures in when Gram stain results are difficult to a cell or different types of cells. The Gram interpret using light microscopy. The stain stain procedure divides bacteria into Gram binds to nucleic acid and stains them. It is also positive and Gram negative bacteria. used for the detection of cell wall deficient Specialized staining such as endospore bacteria example Mycoplasma. Fluorochrome staining is used to detect the presence of stains such as auramine-rhodamine stains are endospores in bacteria. readily available to detect the bacteria in the specimens through Fluorescent microscopy.

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Evaluation b. Crystal violet, iodine, alcohol, safranin Multiple choice questions c. Methylene blue, alcohol, iodine, 1. An dye has negative charge. safranin a. Basic d. Crystal violet, alcohol, iodine, b. Acidic safranin c. Neutral d. None 9. The Schaeffer-Fulton endospore staining 2. stain is incorporated with usually shows Eosin in Lactose agar to distinguish a. Spore green within pink cells typical Escherichia coli in contaminated b. Spores pink within green cells water. a. Crystal violet b. Acid fuchsin c. Colourless spores within pink cells c. Methylene blue d. Safranin d. Colourless spores within 3. Which of the following is not an anionic green cells dye? a. Safranin b. Eosin Answer the following c. Rose Bengal d. Acid fuchsin 1. Define stain. 4. Christian Gram discovered a staining 2. Give examples for basic stain. technique to differentiate the bacteria of similar morphology in the year. 3. Why heat fixation is important? a. 1857 b. 1880 c. 1884 d. 1881 4. What are endospores? 5. Distinguish between a dye and a stain. 5. Which of the following is used for negative staining of microbial cells? 6. List out few gram positive bacteria. a. Nigrosin and Acid fuchsin 7. What is the purpose of a counterstain/ b. Rose Bengal and malachite green decolorizer in the gram stain? c. Safranin and Eosin 8. Fill in the following table regarding the d. Nigrosin and Indian Ink gram stain. 6. is used as a mordant Appearance after this step of in Gram staining techniques. gram staining a. Iodine Steps Gram Gram b. Crystal violet positive cells negative cells c. Methylene blue Crystal d. Safranin violet 7. Which of the following pairs is Iodine mismatched? Alcohol a. Capsule-negative stain Safranin b. Cell arrangement-simple stain c. Cell size-albert stain 9. What is meant by negative staining? d. Gram stain-bacterial 10. What are the uses of staining? identification 11. Differentiate simple and differential stain. 8. The order of reagents in the gram 12. What are acidic stains? Give examples. staining reactions are: a. Safranin, alcohol, methylene blue, 13. Why do basic dyes stain bacterial cells? iodine Why won’t acidic dyes stain bacterial cells?

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14. For what purpose would you use each 16. How will you appreciate the need of of the following? staining? a. Simple stain 17. Classify staining technique based on b. Negative stain their purpose. c. Acid- fast stain 18. Explain the principle of grams d. Gram stain staining. 15. The gram stain has been described 19. Diagrammatically explain Gram’s as the most important stain for staining procedure. microbiologist. Explain why? 20. How to visualise an endospore.

ICT CORNER Gram Staining of Bacteria

Know the Gram Staining process

STEPS: • Use the URL OR Scan the QR code to reach ‘Virtual Interactive bacteriology laboratory’. • Click ‘module’ and select ‘steps’ and read the procedure to follow. • Select ‘start’ to enter the ‘Gram Stain’ process and follow the procedure. • Leave the slide to dry and heat fix with Bunsen burner and view under microscope OBSERVATIONS : • Select other examples and record your observation on Gram +ve and Gram –ve bacterial stains.

Step1 Step2 Step3 Step4

URL: https://www.cellsalive.com/toc_micro.htm

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Chapter 4 Sterilization

Chapter Outline

4.1 Need for Sterilization 4.2 Methods of Sterilization 4.3 Physical Methods of Sterilization 4.4 Sterilization by Heat 4.5 Radiation 4.6 Filtration The inoculation loop is sterilized with flame or any other heat source, until it becomes red hot before and after each use.

Learning Objectives

After studying this chapter the student The process of sterilization is used in will be able, Microbiology • To understand the concepts of • for preventing contamination by sterilization to maintain aseptic extraneous organisms conditions. • in surgery for maintaining asepsis • To compare the effectiveness of dry • in food and drug manufacture for heat (red heat, flamimg, incineration, ensuring safety from contaminating hot air oven), and moist heat (boiling, organisms autoclaving, pasteurization). The choice of methods of sterilization • To learn the uses of pasteurization depend on the purpose for which it is in the field of food industry. carried out: the material to be sterilized and • To describe the role of radiation in the nature of the microorganisms that are to killing pathogens. be removed or destroyed. • To describe how separation of microorganism is achieved through filtration. As early as the stone Microorganisms are ubiquitous. They can age, humans used contaminate, infect or decay inorganic physical methods of microbial control and organic matter. Hence, it becomes to preserve foods, necessary to kill or remove them from like drying (desiccation) and salting materials or from areas around us. This (osmotic pressure). is the objective of sterilization.

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Sterilization is defined as the process 4.2 Methods of Sterilization of complete removal or destruction of all Growth and multiplication of forms of microbial life, including vegetative microorganisms can be controlled by cells and their spores. removing, killing or inhibiting them using various physical or chemical agents. 4.1 Need for Sterilization The aim of all sterilization strategies is to kill 4.3 Physical Methods of Sterilization or remove the unwanted microorganisms. The various physical methods of sterilization In certain cases, microbes are regarded are given in flowchart 4.1 as potential pathogens and therefore it is essential to eliminate these forms 4.4 Sterilization by Heat (vegetative and spores) of microbial life. Heat is the most rapid and best method of All microbiological techniques require sterilization. It is the method of choice that appropriate and adequate sterilization. the material to be sterilized is stable enough to withstand the required temperature necessary Sterilization of culture media, to kill the microbes. The time needed for containers and instruments is essential sterilization depends on the initial number in microbiological work for isolation of organisms present, type of materials to be and maintenance of microorganisms. In sterilized (hence washed and cleaned items surgery and medicine, the sterilization are easier to sterilize than dirty ones) and also of instruments, drugs and other supplies on the temperature used. Spores need higher is important for the prevention of temperatures while vegetative bacteria can be infection. destroyed at lower temperatures.

Physical methods

Heat Radiation Filtration

• Depth filter • Membrane filter Dry heat Moist heat Non ionizing Ionizing • Air filter

• Red heat • Temperature • Infrared • X-rays • Flaming below 100°C • Ultraviolet • Gamma rays • Incineration • Temperature • Hot air oven at 100°C • Temperature above 100°C

Flowchart 4.1: Physical Methods of Sterilization

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a) Red heat Infobits Inoculating wires, points of forceps and searing spatulas are sterilized by holding Heating process in canning was first them in the flame of a bunsen burner until used by Nicholas Appert in 1890. He they are seen to be red hot. described a safe means of preserving all kinds of food substances in containers b) Flaming or in cans. Appert is known as father This method is used for sterilizing scalpels, of cannning. needles, mouths of culture tubes, slides and cover slips. It involves passing the article through the bunsen flame without allowing Heat resistance varies among different it to become red hot. microorganisms. These differences can be expressed in terms of thermal death point. c) Incineration Thermal Death Point (TDP) is the lowest This is an excellent method for destroying temperature at which all the microorganisms materials such as contaminated clothes, in a particular liquid suspension will be cotton wool stoppers, animal carcasses and killed in 10 minutes. pathological materials. It involves burning of materials in incinerators. Another factor to be considered in sterilization is the duration of time d) Hot air oven required. This is expressed as Thermal This is the most widely used method of Death Time (TDT). TDT is the minimal sterilization using dry heat. The oven is time required for all microorganism in a usually heated by electricity and it has a particular liquid culture to be killed at a thermostat that maintains the chamber air given temperature. Both TDP and TDT constantly at the chosen temperature. are useful guidelines that indicate the It has a fan or turbo-blower to assist degree of treatment required to kill a given the circulation of air and to ensure rapid, population of bacteria. uniform heating of the load. In Hot Air Decimal Reduction Time (DRT) is Oven, the air is heated at a temperature related to bacterial heat resistance. DRT of 160oC for one hour. Figure 4.1 shows is the time, in minutes, in which 90% of a laboratory hot air oven. population of microorganism at a given temperature will be killed. Heat is employed either as dry heat or moist heat. ouble jac et chamber

4.4.1 Sterilization by Dry Heat Dry heat is frequently used for the hermometer sterilization of glassware and laboratory Shelves equipments. In dry heat sterilization, emperature indicator microbial cells are apparently killed by valve oxidation of their constituents and protein denaturation. Dry heat is applied in the following ways: ouble jac et door Figure 4.1: Hot Air Oven

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This is the best method of sterilizing dry harmful microorganisms. It should be glass ware such as test tubes, petri dishes, noted that pasteurization process kills flasks, pipettes and instruments such as only vegetative cells but not the spores. forceps, scalpels and scissors. It is also used Pasteurization named in honour of its to sterilize some pharmaceutical products developer Louis Pasteur. Table 4.1 gives such as liquid paraffin, dusting powder, fats comparison between Sterilization and and grease. Pasteurization. Quality control of dry heat sterilization: Raw milk can The spores of a nontoxigenic strain of harbour dangerous Clostridium tetani are used to test the microorganisms, efficiency of dry heat sterlization. such as Salmonella, 4.4.2 Sterilization by Moist Heat Escherichia coli and Moist heat kills microorganisms primarily by Listeria, that can pose serious health the coagulation of proteins (denaturation), risk, and children are particularly which is caused by breakage of the hydrogen susceptible to the potential infection of bonds that hold the proteins in three unpasteurized or raw milk dimensional structure. There are three methods employed in Pasteurization can be done in the moist heat sterilization. following methods, • Temperature below 100°C. • Low Temperature Holding Method (LTH) • Temperature at 100°C. In this method milk, beer and fruit • Temperature above 100°C. juices are maintained at 62.8°C for 30 minutes. a) Temperature below 100°C: • High Temperature Short Time Pasteurization Method (HTST) The process of heating a liquid food Products are held at 72°C for or beverage either at 62.8°C for 30 15 seconds. minutes or 72°C for 15 seconds to enhance their shelf life and destroy • Ultra High Temperature (UHT)

Table 4.1: Comparison between Sterilization and Pasteurization Sterilization Pasteurization Sterilized products have a longer shelf life Pasteurized products have shorter shelf life Discovered by Nicolas Appert Discovered by Louis Pasteur Eliminates all forms of microorganisms Eliminates pathogenic microorganisms only Can be accomplished in many ways Can be accomplished with heat Applied in food industry, medical, Mainly applied in food industry surgery and packaging

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Milk can be treated at 141°C for in autoclave is 121°C at 15 lbs (pounds) 2 seconds (This method employ pressure for 15 minutes (Figure 4. 2a & b). temperature above 100°C). Autoclaving is used in sterilizing culture b) Temperature at 100°C: media, instruments, dressings, applicators, solutions, syringes, transfusion equipment, i) Water at 100°C (Boiling): pharmaceutical products, aqueous solutions Boiling is one of the moist heat and numerous other items that can sterilization methods. It kills vegetative withstand high temperatures and pressures. forms of bacterial pathogens, almost all The same principle of autoclaving applies viruses and fungi (including their spores) for the common household pressure cooker within 10 minutes, usually much faster. used for cooking food. • Most vegetative bacteria will die Factors influencing sterilization by heat: in 5-10 minutes when immersed in boiling water, but some spores Sterilization by heat depends upon will survive at this temperature for various factors such as time, temperature several hours. employed, number of microorganisms, spores and nature of material to be • Articles sterilized by this method sterilized. cannot be stored for a long time. Quality control of moist heat sterilization: ii) Steaming at 100°C (Tyndallization): To check the efficiency of moist heat It is a process discovered by John Tyndall sterlization, the indicator commonly used is th in 19 century for sterilizing substances to the paper strips containing spores of Bacillus kill the spores of bacteria. The process of sterothermophilus. exposure of materials to steam at 100°C for 20 min for three consecutive days is 4.5 Radiation known as tyndallization. First exposure Radiation is commonly kills all the vegetative forms and in the employed for sterilizing intervals between heating, the remaining heat sensitive materials spores germinate into vegetative forms such as disposable which are killed on subsequent heating. plastic products and Tyndallization is also called fractional materials that cannot sterilization or intermittent boiling. withstand moisture. The most effective type of radiation to c) Temperature above 100°C: sterilize or reduce the microbial burden Moist heat sterilization can be carried in the substance is through the use of out at temperature above 100°C in electromagnetic radiations. Figure 4.3 order to destroy bacterial endospores. shows different types of electromagnetic This requires the use of saturated steam radiations. Radiation has various effects under pressure. This is achieved using on cells, depending on its wavelength, autoclave. intensity and duration of explosure Autoclave (Flowchart 4.2). Radiation that kills microorganism is of two types namely Sterilization using an autoclave is most ionizing and nonionizing. effective when the organisms are either contacted by the steam directly or contained a) Non-ionizing radiation in a small volume of aqueous liquid Infra-red rays and ultra-violet rays are non (primarily water). The temperature used ionizing radiation.

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i) Infra-red radiation Radiation These are electromagnetic rays with wavelengths longer than those of visible light. These are low energy type. It kills microorganisms by oxidation of molecules Non ionizing Ionizing radiation as a result of heat generated. Infrared radiation radiation is used for rapid mass sterilization of pre-packed items such as syringes and catheters. • Infrared radiation • X-Rays • UV radiation • Gamma Rays Flowchart 4.2: Radiation

elease valve Pressure gauge

Safety valve Cover Cover tightening nuts Body

ac et Buc et Stand

eating electrode eg

Figure 4.2: (a) Laboratory autoclave (b) Components of autoclave

Types of radiation in the electromagnetic spectrum Non-ionizing Ionizing

Radio Infrared Ultraviolet Extremely Type of radiation low frequency Microwave x-ray Gamma rays Visible light

Non-thermal Thermal Optical Broken bonds

Effects Induces low Induces high Excites Damages currents currents electrons DNA

??? Heating Photo- chemical effects Source Static Power AM FM radio Microwave Heat tanning Medical ﬁeld line ladio TV oven lamp booth x-rays Figure 4.3: Types of radiation in electromagnetic spectrum

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ii) Ultra-violet radiation Cobalt 60 source is used in the cold The ultraviolet (UV) portion of the sterilization of antibiotics, hormones, electromagnetic spectrum includes sutures and plastic disposables supplied all radiations from 150-3900A°, UV such as syringes and in pasteurization of radiation around 2600A° is most lethal meat. to microorganisms. UV has a very little 4.6 Filtration ability to penetrate matter. Thus, only Filtration is an effective and reasonably the microorganisms on the surface of an economical method of sterilization. It is used object, exposed directly to the ultraviolet to sterilize heat-sensitive fluids, and air. It is light are susceptible to destruction. UV particularly useful for solutions containing radiations are used to sterilize operation toxins, enzymes, drug, serum and sugars. theaters, laboratories and entry ways. Sugar solutions used for the cultivation of b) Ionizing radiation microorganisms tend to caramelise during Ionizing radiations (X-rays, Gamma rays autoclaving and so they are best sterilized by and Cosmic rays) are an excellent sterilizing filtration. Filtration is also used extensively in agents and they penetrate deep into the beer and wine industries. Filters with known objects. These radiations do not produce pore sizes which are sufficiently small to hold heat on the surface of materials. Hence, back bacteria are employed. Recently filters sterilization using ionizing radiations is that can remove viruses are also available. referred as cold sterilization. It will destroy Filtration is an excellent way to remove bacterial endospores and vegetative cells, the microbial population from solution both Prokaryotic and Eukaryotic; however containing heat sensitive material. ionizing radiation is not always effective There are two types of filters namely against viruses. Gamma radiation from (Figure 4.4): i) Membrane filter (surface filtration) and Deinococcus radiodu- rans is an extremo- ii) Depth filter philic bacterium. It is one of the most Membrane filters radiation-resistant or- Membrane filtration is used for preparing ganisms known. heat-labile culture media components. It

Figure 4.4: Principle of filtration

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is also useful in removing bacteria from random paths through the filter that trap heat-sensitive pharmaceutical products many particles. Depth filter are made up and biological solutions. of diatomaceous earth (Berkefeld filters) Membrane filters are made up of which are used as water purifiers. Examples either cellulose acetate, cellulose nitrate, of types of depth filters (Figure 4.6) polycarbonate, polyvinylidene fluoride or contains unglazed porcelain (Chamberl other synthetic porous materials. These filters and filters) and asbestos (Seitz Filter). remove microorganisms by screening them Air filtration out, such as a sieve separates large sand particles from small ones. Membranes with pore size of Air also can be sterilized by filtration. 0.2μm in diameter are used to remove most vegetative cells but not viruses. These filters HOTS are used to sterilize pharmaceutical products, ophthalmic solutions, culture media, oils, Give a reasonable method of sterilization antibiotics, and other heat sensitive solutions for the following. (Figure 4.5a, b & c). 1. Operation theatre 2. Serum 3. Pot Depth filters of soil 4. Plastic Petri Dishes 5. Rubber Depth filters are the oldest type of filters gloves 6. Disposable syringes. 7. Metal and they consist of overlapping layers of instruments 8. Flask of nutrient agar fibrous sheets of paper, asbestos or glass 9. Milk 10. Papers with spores. fibers. The overlapping fibers create

Membrane trans erre Sam le t be t cltre meim iltere b Membrane ilter LM m retains cells

ac m

Inc bati n

l nies

a c Figure 4.5: (a) Membrane filter apparatus (b) Light microscope image of microorganism filtered through membrane filter (c) Membrane filters showing microbial colonies on culture media

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Figure 4.6: Types of depth filters

Outside Safety glass haust vie screen P fiter Blo er Supply P filter ight

igh velocity air barrier

Figure 4.7: Laminar air flow

The air is freed from infection by passing size. Some operation theaters and rooms it through High Efficiency Particle occupied by burn patients receive filtered Arrester (HEPA) filter. Laminar air flow air to lower the numbers of airborne biological safety cabinets are one of the microbes. HEPA filters remove almost all most important air filtration systems microorganisms above 0.3μm in diameter. (Figure 4.7). It employes HEPA filters Various physical methods of sterlization which remove 99.97% of 0.33μm particles is summarized in Table 4.2

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Table 4.2: Physical methods used to control microbial growth Mechanism Preferred for Method of action Comment sterilizing Heat 1 Dry heat a. Direct Burning Very effective method of Inoculating loops flaming contaminants sterilization to ashes b. Incineration Burning to Very effective method of Paper cups, ashes sterilization contaminated dressings, animal carcasses, bags, and wipes c. Hot –air Oxidation Very effective method of Empty glassware, sterilization sterilization, but requires instruments, needles, temperature of 160°C for and glass syringes about 1 hour

2 Moist heat a. Boiling or Protein Kills vegetative bacterial Dishes, basins, pitchers, flowing denaturation and fungal pathogens various equipment steam and almost all viruses within 10 min; less effective on endospores b. Autoclaving Protein Very effective method Microbiological media, denaturation of sterilization; at about solutions, linens, 15 lbs of pressure (121°C), utensils, dressings, all vegetative cells and equipment, and other their endospores are items that can withstand killed in about 15 min temperature and presure c. Pasteurization Protein Heat treatment for milk Milk, cream, and denaturation (72°C for about 15 sec) certain alcoholic that kills all pathogens beverages(beer and and most nonpathogens wine) 3 Radiation a. Ionizing Destruction Not widespread in Used for sterilizing of DNA routine sterilization pharmaceuticals and medical and dental supplies b. Nonionizing Damage to Radiation not very Control of closed DNA penetrating (non environment with UV penetrating)

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Summary c. Hot-air sterilization Physical methods of microbial control d. Pasteurization include heat, radiation, drying and filtration. 2. Which of the following is most effective Heat is the most widely used method for sterilizing mattresses and plastic of microbial control. It is used in both Petri dishes? forms: moist and dry. The thermal death time (TDT) is the time required to kill all a. Chlorine microbes at a specific temperature. The b. Ethylene oxide thermal death point (TDP) is the lowest c. Autoclaving temperature at which all microbes are killed d. Nonionizing radiation in a specified duration of time. 3. Which of the following cannot be used Autoclaving, or steam sterilization, is to sterilize a heat labile solution stored the process by which steam is heated under in a plastic container? pressure to sterilize a wide range of materials in a comparatively short time. a. Gamma radiation Dry heat kills the microorganisms under b. Ethylene oxide specified time and temperature. Dry heat c. Nonionizing radiation is applied in the following ways: Red heat, d. Autoclaving incineration and Hot air oven. 4. kills organisms by coagulation Ionizing radiation (cold sterilization) by and denaturing their proteins X rays and gamma rays is used to sterilize medical products and meat. It damages DNA a. Dry heat and cell organelles by producing disruptive b. Moist heat ions. Ultraviolet light, or nonionizing c. Both a & b radiation, has limited penetrating ability. It d. None of the above is therefore restricted to sterilize suface of the materials. 5. In which method, temperature of Decontamination by filtration removes 160°C for 1 hour is employed? microbes from heat sensitive liquids and a. Red heat circulating air. The pore size of the filter b. Infrared radiation determines what kinds of microbes are c. Hot air oven removed. d. Flaming Evaluation 6. Which of the following temperature and Multiple choice questions time are employed in autoclave for 1. Which of the sterilization of materials? following does not kill a. 16 lbs 120°C for 18 mints endospores? b. 18 lbs 180°C for 20 mints a. Autoclaving c. 22 lbs 170°C for 35 mints b. Incineration d. 15 lbs 121°C for 15 mints

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7. Wavelength used for the absorption of 8. How do you sterilize heat sensitive UV spectrum is materials? a. 4000A° 9. Define Tyndallization. b. 2600A° 10. Describe the sterilization in an c. 20A° autoclave. 11. Explain the methods of sterilization d. None of the above by dry heat. Answer the following 12. Explain the methods of radiation. 1. Define Pasteurization. Student Activity 2. What is Incineration? 1. Collect samples of raw milk 3. Define membrane filters? (unpasteurized) and boiled milk, 4. What is Sterilization? place them in open containers 5. Explain the principle moist heat separately. Observe the changes sterilization? after a few hours in both and infer. 6. Differentiate the mechanism of 2. Making a working model of operation employed in autoclave and depth and membrane filters and hot air oven. demonstrating their uses. 7. Discuss ionizing radiation.

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Chapter 5 Cultivation of Microorganisms

Chapter Outline

5.1 Significance of Culturing Microorganisms 5.2 Bacteriological Media and its Types 5.3 Pure Culture 5.4 Growth and Colony Characteristics of Bacteria and Fungi The microorganisms on the handprint of an eight-year-old boy. After incubation the plates showed coloured colonies of bacteria and fungi as you see above. The cultivation of microorganisms under laboratory condition makes the microscopic cells to grow and form individual colonies macroscopically.

Learning Objectives in environment as pathogens and normal microflora. Excellent supporting factors After studying this chapter the student are available in nature for microorganisms will be able, to survive in the environment. This • To know the importance of bacterial leads to microbial proliferation as an media for growth of microorganisms. extended community in nature. The term To understand various types ‘cultivation of microorganisms’ means of media for differentiation and growing microorganisms in the laboratory diagnosis of important pathogenic with ample supply of specific nutrients microorganisms. (Figure 5.1). Obligate intracellular parasites like viruses, Rickettsias and • To know pure culture techniques Chlamydias are cultivated within living • To understand the methods involved cells. in isolating pure culture of bacteria, Survival and growth of microorganisms which includes pour plate, spread depend upon the favourable growth plate and streak plate. environment. Laboratory cultivation • To differentiate the growth plays a crucial role in the isolation, characteristics of bacteria and fungi. identification and classification of microorganisms. Cultivation of bacteria Microorganisms are omnipresent and they and fungi by artificial formulated medium exist in soil, air, water, spoiled food, decayed is one of the important milestones in the animal and plant residues. They are found history of Microbiology.

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Robert Koch devised the solid medium understand the nutritional requirements (by using gelatin) to grow and isolate the of that microorganism and then supply microorganisms. the essential nutrients in proper form and proportions in culture medium. 5.1 Significance of Culturing Flowchart 5.1 describes the types of media. Microorganisms A common bacteriological medium has Carbon and Nitrogen sources along with • To isolate microorganisms from any buffering agents. Most of the media are samples prepared using dehydrated components. • To study the morphology and The basic components are peptone, beef biochemical characteristics of extract, meat extract, yeast extract and agar microorganisms (Table 5.1). • To maintain the stock culture Table 5.1: Common ingredients of a • To identify disease causing culture media microorganisms • To study the role of microorganisms S.No Ingredients Source of in the production of industrially Peptone Carbon, important products a. (protein nitrogen, hydrolysates) energy 5.2 Bacteriological Media and its Types Beef extract Aminoacids, Generally microorganisms occur as mixed b. (Extract of vitamins, culture in nature. Human beings, animal lean beef) minerals bodies and other natural resources harbour Yeast extract Vitamin B, microbes in mixed population. By using c. (Brewer’s Carbon, appropriate media, microorganisms can yeast) Nitrogen be grown separately in pure form and can Solidifying d. Agar be studied. For successful cultivation of agent a given microorganism, it is necessary to

Types of Media

Physical Chemical Special purpose

* Liquid * Synthetic * Basal * Anaerobic * Semi-solid * Non-synthetic * Enriched * Transport * Solid * Selective * Antibiotic * Differential sensitive Flowchart 5.1: Types of media

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Uses of agar: pectin from the Agar. It is used in Molecular • It is one of the principle ingredients in the Biology laboratory for the separation of preparation of solid or semisolid media. DNA molecules by gel electrophoresis. • It is used as a solidifying agent in culture medium. Agar was first described for use in Microbiology • It is extracted from certain seaweeds in 1882 by the German belonging to genera of red algae like microbiologist Walther Gelidium and Gracilaria (Figure 5.1). Hesse, an assistant working in Rober Koch’s laboratory, as suggested by his wife Fannie Hesse. A cheap substitute for agar in microbial culture media is Guar gum, which can be used for the isolation and maintenance of thermophiles.

HOTS

Why is agar preferred to gelatin as a Figure 5.1: Gelidium – Red algae solidifying agent in culture media? • It is a sulphated polymer mainly consisting of D-galactose. 5.2.1 Physical Nature of Agar Medium • Agar is a highly preferred solidifying The concentration of agar plays a major agent because it does not affect the role in determining the consistency growth of microorganisms. Agar is also of the medium. A medium with agar used in the food and pharmaceutical concentration of 2% or greater is said to be a industries. solid medium and that of 0.5% is said to be • The purified form of the agar is called a semisolid medium (jelly like appearance). Agarose. It is prepared by removing the Tabel 5.2 lists the concentration of agar in

Table 5.2: Concentration of agar in media Nature of Medium Concentration Example Uses Solid 2% Nutrient agar To isolate microorganisms on petridish and forming agar slant Semisolid 0.5% SIM (Sulphur Indole Agar stab to observe Motility medium) motility Liquid 0% Nutrient broth To observe biochemical reaction.

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media. However liquid media (broth) does not contain agar. Figure 5.2 shows types of Infobits media depending on physical nature. Veggitone is a vegetable based product containing peptones. It is made from raw materials such as peas and fungal proteins that are digested using fungal and bacterial enzymes.

5.2.3 Special Purpose Medium i) Basal medium This medium promotes the growth of many types of microorganisms which do not require any special nutrient supplement. It is a routine laboratory medium with Carbon and Nitrogen sources along Figure 5.2: Solid, liquid and with some minerals. Example: Nutrient semi-solid media Agar or Nutrient Broth. It is also called general purpose medium. It is used for 5.2.2 Chemical Nature of Medium subculturing the pathogens. It is a non- • Synthetic medium selective medium, which is designed to support the growth of a wide spectrum of Chemically defined synthetic Medium heterotrophic organisms. (Figure 5.3) is used for various experiments. This medium is prepared exclusively from pure substances with known chemical composition and concentrations. This is widely used in research to find the type of compound metabolized by the experimental organism.

• Non-synthetic medium The medium in which the exact chemical composition and the concentration of each Figure 5.3: Growth of bacteria on ingredient is not certainly known is called Nutrient agar non-synthetic medium. In this medium, crude materials such as meat extract, yeast ii) Enriched medium extract, various sugars, molasses and corn In enriched medium, substances like blood, steep broth are used. This supports the egg or serum are added along with the growth of a variety of microorganisms. It basal medium. It is used to grow fastidious is otherwise called as complex medium. organisms that are very particular in their nutritional needs. Fastidious organisms

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have elaborate requirements of specific 7% Sodium chloride that inhibits the nutrients like vitamins and growth growth of other bacterial population promoting subtances and or not easily but allows the growth of Staphylococci pleased or satisfied by ordinary nutrients (Figure 5.5). Moreover it has Phenol available in nature. Example: Blood agar red dye to indicate acid production. is used to identify haemolytic bacteria Staphylococcus utilizes Mannitol and (Figure 5.4) and Chocolate agar used to produces acid which changes the colour identify Neisseria gonorrhoeae. of the Phenol red indicator to yellow. Salmonella-Shigella (SS) agar is selective for Salmonella (Figure 5.6).

Figure 5.4: Blood Agar showing alpha, beta & gamma – haemolytic colonies Figure 5.5: Growth of Staphylococcus aureus on Mannitol salt agar In 1919, James Brown used blood agar as diagnostic medium to study the haemolytic patterns of bacteria. iii) Selective medium Selective medium contains one or more agents (selective components) that inhibit unwanted organisms but allow the desired organisms to grow. Growth of unwanted microbes is suppressed by adding bile salts, antibiotics and dyes. Example: Mannitol salt agar is selective Figure 5.6: Growth of Salmonella for Staphylococci. This medium contains on SS agar

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It is nothing short of amazing and humbling fact that even after 120 years of trying to grow microbes in the laboratory, we have succeeded in culturing only 0.1% of the microorganisms around us. iv) Differential medium Differential medium distinguishes between different groups of bacteria and permit Figure 5.8: Growth of lactose fermenting bacteria on EMB Medium tentative identification of microorganisms based on their biological charaterstictics as Eosin Methylene Blue (EMB) agar they cause a visible change in the medium. medium is also a differential medium. It We can differentiate haemolytic and is used to differentiate lactose fermentors non-haemolytic patterns of bacteria using from non-lactose fermentors. It has lactose blood agar. Differential medium is otherwise sugar and two dyes namely Eosin –Y and called indicator medium as it distinguishes Methylene blue. These dyes act as inhibitory one organism from another growing on the agent towards Gram positive bacteria. same plate by the formation of pigments due Example: Lactose fermentors such as faecal to its biochemical and physiological nature. Escherichia coli show metallic sheen and Example: MacConkey agar medium has non lactose fermentors such as Enterococcus neutral red dye. Lactose fermentors form do not show metallic sheen. (Figure 5.8). pink coloured colonies and non fermentors form colourless translucent colonies on it Lactose fermentation (Figure 5.7).

Acidic pH (pH drops below 6.8)

Makes Eosin-Y and Methylene blue to form a complex

Inhibits Gram positive bacteria

Figure 5.7: Growth of microorganisms on MacConkey agar (Lactose fermenting Colonies of lactose fermentors has bacterial colonies appears pink) metallic sheen

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vi) Antibiotic sensitivity medium Chromogenic Antibiotic sensitivity medium is a medium is used microbiological growth medium that is for the simple commonly used for antibiotic sensitivity and fast detection testing. Example: Muller- Hinton agar of transformed bacteria by using medium. It is a non-selective and non- chromogenic substrates. The differential medium. It allows the growth chromogenic mixture contains of most type of microorganisms. It contains substrates such as Salmon-GAL, X-GAL. starch which absorbs toxins released from Certain bacterial enzymes cleave the bacteria. Hence toxins do not interfere chromogenic substrate resulting in the with antibiotics. Agar concentration of coloured colonies. 1.7% is used in this media which allows better diffusion of antibiotics (Figure 5.9).

HOTS

Why is EMB medium called a selective, differential as well as complex medium? v) Enrichment medium Enrichment medium is a liquid medium. It is used to grow a particular microorganism that is present in much smaller number Figure 5.9: Antibiotic sensitivity on along with others present in sufficiently Muller Hinton agar large numbers. An enrichment medium vii) Anaerobic medium provides nutrients and environmental Anaerobic medium is a medium used for conditions that favour the growth of a the cultivation of anaerobes, Example: desired microorganisms. It is used to culture i) Robertson cooked meat medium: This microorganisms present in soil or faecal is used for the isolation of Clostridium samples that are very small in number. ii) Thioglycolate broth: In this medium Example: Selenite F Broth is used to isolate Sodium thioglycollate is used as a reducing Salmonella typhi present in low density in agent which maintain a low Oxygen tension faecal sample. It is cultured in an enrichment by removing the molecular Oxygen from medium containing Selenium. Selenium the environment. supports the growth of the desired organism and increase it to detectable levels compared viii) Transport medium to intestinal flora. Sodium selenite inhibits Transport medium is used for the many species of Gram positive and Gram temporary storage of specimens that are negative bacteria including Enterococci and being transported to the laboratory for coliforms. cultivation. It maintains the viability of all

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organisms in the specimen without altering 5.3 Pure Culture their concentration. It mainly contains In nature, microorganisms usually exist as buffers and salts. Example: Stuart’s transport complex multispecies community. A single medium that lacks Carbon, Nitrogen and species has to be characterized in order organic growth factors. Other examples of to know the morphology, pathogenicity transport media are Cary Blair and Amies. and molecular genomic pattern of the organism. For characterizing a species we Infobits have to isolate the organisms in pure form. Viral Transport Medium is used to carry Pure culture or axenic culture is a culture a specimen containing viruses. Universal containing only one type of organism. Transport Viral Medium (UTVM). The descendents of a single organism in This liquid medium is stable at room pure culture is called a strain. A strain temperature. It is used for collection, forms a single colony. Colony is a cluster of transport, and maintenance and long microorganisms in which all the characters term freeze storage of viruses. of the family remain same. With the advent of the pure culture techniques many Exceptions in cultivation of microbes in microorganisms are being identified. artifical medium 5.3.1 Methods Employed in the Some bacteria like Mycobacterium leprae Isolation of Microorganisms and Treponema pallidum cannot be cultivated in artificial medium. Though there are many methods designed for isolation of microorganisms, ix) Media used for isolation of fungi pour plate method, spread plate method Apart from the bacteriological media, and streak plate method are widely used fungal media are used to study fungal in the field of Microbiology. morphology pigmentation and sporulation. Sabouraud’s Dextrose Agar (SDA) is used i) Pour plate method as a common medium to isolate fungus. • It is the used for the isolation and There are several other important fungal counting of colony forming bacteria in media used for fungal cultivation. Examples the specified sample. Niger Seed Agar and Potato Dextrose Agar • In this technique a sample is diluted HOTS several times to reduce the density of the microbial population. 1. Which medium is used to carry • A very small amount of diluted sample the sample when the sick person is (1ml or 0.1ml) is mixed with the molten unable to come to the laboratory? agar at a temperature of 45°C. 2. Give a special medium to check • The mixture is poured into the sterile the growth of anerobes in a burn petridish (In 1887, Juluis Richard Petri, wound infection with dead tissues. a worker in Koch’s laboratory, designed the Petriplate.) in an aseptic condition

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Infobits

Nowadays media are available as contact plates, agar strips, media cassettes, contact slide and settle plates which are used for microbial air monitoring and compressed gas lines in food and beverage production plants. These media are also used for the enumeration of typical food contaminants such as coliforms, yeast and molds. Colour coded MC-MEDIA pads are available for rapid and convenient microbial testing for Escherichia coli, yeast, mold, coliform and aerobes.

and plates are incubated at a specific ii) Reduced growth of obligate aerobes temperature for a given period of time. in the depth of agar. • Plates are incubated in an inverted iii) Colonies embedded within the manner. agar are much smaller than that of • After incubation, the colonies are formed surface and may be confluent or in a discrete pattern both on the surface invisible. of agar and also embedded within the Basic five ‘I’ steps medium. one should follow • Pour plate can be also used to deter- in culturing micro mine the number of cells in a popula- organisms tion.(Figure 5.10) a. Inoculation Disadvantages of pour plate method b. Incubation i) Loss of viability of heat sensitive c. Isolation organisms coming into contact with d. Inspection hot agar. e. Identification

M lten acterial agar S s ensi n at

Mi Inc bate

l nies gr n s r ace an ithin agar Figure 5.10: Pour plate method

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ii) Spread plate method iii) Streak plate technique • Spread plate method is an easy and • The streak plate direct method of isolating a pure technique is one of the culture. most commonly used • In this technique a specified amount methods for isolating of diluted inoculum (0.1ml or less) pure culture of bacteria. of microbial culture is seeded on agar • In this method, a plate. loopful of inoculum from a sample • After inoculation of the sample on the is taken and it is streaked across the agar medium, the inoculum is evenly surface of the sterile solid medium. spread on the surface with the help of • Different streaking patterns can be a sterile glass L rod (a bent glass rod) used to separate individual bacterial • Microorganisms are evenly distributed cell on the agar surface. in the entire surface of agar. • After the first sector is streaked the inoculated loop is sterilized and • The dispersed microorganisms develop inoculum for the second sector is into isolated colonies. obtained from the first sector. • In this method, the plates are incubated • Similar process is followed for streaking at a specified temperature for a given the further areas in the sectors. period of time. • Since the inoculum is serially diluted • After incubation the plates are observed during streaking patterns the dilution for the growth of discrete colonies. gradient is established across the • The number of colonies are equal to surface of the medium. the number of viable organism. This • After streaking, plates are incubated method can be used to count the at a specific temperature for a given microbial population (Figure 5.11). period of time.

S rea late meth

Sam le mL re S rea sam le e enl acterial c l nies nt sli meim er the s r ace gr n the s r ace the me i m acterial il ti n

Inc bati n

Figure 5.11: Spread plate method

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• After incubation, plates are observed 5.4 Growth and Colony Characteristics for growth of colonies (based on the of Bacteria and Fungi streaking pattern and density of culture In the previous section we have learned growth of microbes are abundant in the various types of media and specific the first sector in comparison with purpose of each medium. Morphology the formation of separated discrete is the basic criteria for the isolation, colonies in the fourth sector of the identification and classification of agar medium). microorganisms. Colony characteristics • Each isolated colony is assumed to be are the basic tool in the field of taxonomy. grown from a single bacteria and thus Bacteria grow in both solid and represent a clone of pure culture. liquid medium, but identification will • Successful isolation depends on spatial be easy on the solid medium. In solid separation of single cells (Figure 5.12). medium bacteria form colonies. In liquid medium growth of bacteria are generally Infobits not distinctive because there is uniform turbidity or sediment at the bottom or Micro manipulator: It is a device used pellicle is formed on the surface. along with a microscope to pick a single Some basic attributes such as shape, bacterial cell from a mixed culture. size, colour, pigmentation, texture, elevation It has micropipette or microprobe and margin of the bacterial colony in the so that a single cell can be picked up. growth medium are explained below.

AB C

ea c n l ent gr th iscrete c l nies Initial Sec n set inclm streaks

ea gr th hir set F rth set Inc bati n streaks streaks Light gr th

D E Figure 5.12: Steps in streak plate isolation method

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orm

Punctiform Circular ilamentous Irregular hi oid Spindle (lens)

levation

lat aised Conve Pulvinate mbonate

Margin

ntire ndulate ilamentous obate rose Curled (even) ( avy) (lobes) (serrated) Figure 5.13: Colony morphology of bacteria

5.4.1 Colony Morphology of Bacteria be dry, moist, mucoid, brittle (dry breaks on Solid Media apart), viscid (sticks to loop, hard to get Shape: The shape of colony may be off), viscous, or butyrous (buttery). circular, irregular, filamentous, rhizoid. Opacity of the bacterial Colony: Colonies Elevation: It is the side view of the colony. may exhibit different optical density. It may It may be flat, raised, umbonate (having be transparent (clear), opaque (not clear), a knobby protuberance) crateriform, translucent (almost clear), or iridescent convex pulvinate (cushion shaped) (changing colour in reflected light). Margin: The margin of the bacterial colony Colony Odour: Some bacteria produce a may be entire (smooth) irregular, undulate characteristic smell, which sometimes helps (ovary), lobate, curled, filiform. The in identifying the bacteria. Actinomycetes irregular shape of the colony give irregular produce an earthy odour which is quite margin (Figure 5.13). often experienced after rain. Many fungi produce fruity smell while Escherichia coli Colony Size: The diameter of the colony produce a faecal odour. is measured in millimeter. It is described in relative terms such as pinpoint, small, Smooth colonies medium and large. of Streptococcus Appearance of colony on the surface: The pneumoniae are bacterial colonies are frequently shiny/ usually virulent, where smooth in appearance. Colonies may be as rough colonies are non-virulent. But veined, rough, dull, wrinkled, or glistening. in Mycobacterium tuberculosis colonies Texture of the colony: Texture means with rough surface indicates a good consistency of the bacterial growth. It may factor of virulence.

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Colony Colour: Many bacteria develop colonies which are pigmented.(Table 5.3) Certain water soluble Some bacteria produce and retain water pigments are fluorescent insoluble pigments and the colonies in nature Example: Py- appear coloured by taking the pigment overdin. Agar medium intracellularly (Figure 5.14). But some around the colonies glows white or blue bacteria produce water soluble pigment green when exposed to ultraviolet light. which diffuse into the surrounding agar. Example: Pyocyanin pigment of 5.4.2 Nature of bacterial growth in Pseudomonas aeruginosa is a water soluble liquid medium pigment and give blue colour to the 1. If the entire broth appears milky and medium. cloudy it is called turbid. 2. If deposit of cells are present at the bottom of the tube, the term sediment is used. 3. If the bacterial growth forms a continous or interrupted sheet over the broth it is called pellicle (Figure 5.15).

5.4.3 Growth and Colony Characteristics of Fungi Fungi are eukaryotic organisms. They exist in both unicellular-yeast like form and in filamentous multicellular hyphae or mold Figure 5.14: Pigmentation of bacterial colonies on culture medium form and some are dimorphic. Generally

Figure 5.15: Microbial growth in Liquid medium

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Table 5.3: Pigmentation of chromogenic bacteria Bacteria Pigment colour Serratia marcescens Red Staphylococcus aureus Golden yellow Micrococcus luteus Yellow Pseudomonas aeruginosa Green fungi prefer to grow in the acidic medium. • Growth and colony characteristics of Sabourad Dextrose Agar (SDA) plates and yeast Candida Potato Agar plates are used for general Yeasts are grown on Sabourad cultivation of fungi. The acidic nature of Dextrose Agar aerobically. Yeasts SDA agar reduce the growth of bacteria. grow as typical pasty colonies and The characters to be noticed in colony give out yeasty odour. The colony of fungi are colour of the surface and morphology varies with different reverse of the colony, texture of the surface yeasts. Yeasts colonies generally have (powdery, granular, ecolly, cottony, velvety smooth texture and are larger than or glabrous), the topography (elevation, bacterial colonies on SDA medium folding, margin) and the rate of growth. (Figure 5.16a).

Infobits • Growth and Colony characteristics of mold Mucor Dimorphic fungi are fungi that can exist in both mold and yeast form The genus Mucor is typically coloured depending on environmental and white to brown or grey and is fast physiological conditions. Example: growing. Older colonies become grey Histoplasma capsulatum, a human to brown due to the development of pathogen, grows as a mold form at spores. (Figure 5.16b). room temperature and as a yeast form at human body temperature.

Figure 5.16: Fungal growth on Sabouraud Dextrose Agar media a) yeast growth b) mold growth 66

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ICT CORNER Streak Plate Technique

Isolation of pure culture of bacteria

STEPS: • Use the URL or scan the QR code to reach ‘Virtual Interactive Bacteriology Laboratory. • Click module and read the description and steps. • Do the streak plating process from the top left part to the bottom left order. • Heat and cool the loop between each steps.

Step1 Step2 Step3

Step4

URL: http://learn.chm.msu.edu/vibl/content/ streakplate.html

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Summary c. Basal media In natural environments microogran- d. General purpose media isms exist as mixed cultures. Survival and 2. is an example growth of microorganisms depends upon for differential media the availability of favourable growth envi- a. Blood agar ronment. Cultivation of microorganisms b. EMB agar in the laboratory plays an important role c. Both a and b in isolation, identification and classifica- d. None tion of microorganisms. A medium is an 3. A medium in which precise environment which supplies the nutrients ingredients are clearly defined. necessary for the growth of the microorganisms. Various kinds of media have a. Synthetic medium. been prepared to satisfy the need of the b. Non synthetic medium. microorganism to be isolated as a pure c. Complex medium culture. Based on the physical, chemical d. Natural medium and special purposes, media are classified 4. A microbial inoculum of faecal and are used to identify a particular or- specimen is subjected to isolation ganism from a clinical specimen or envi- of typhoid bacilli species. Which ronment. In Microbiology there are many medium can be used to select the methods used for isolation of microor- bacilli? ganisms. The methods commonly used a. Selective medium for isolation are pour plate, spread plate b. Basal medium method and streak plate method. c. Enriched medium The growth of organisms on media d. Differential medium is a basic criteria in the isolation, identification, and classification of 5. is the method microorganisms. Colony characterization in which inoculum is not placed over of both bacteria and fungi highly depends the surface of agar plate. upon the nutrients, temperature and pH. a. Pour plate method b. Spread plate method Evaluation c. Streak plate method Multiple choice questions d. All the above 1. In a culture, the 6. In perfect isolation of pure colonies, desired organism which method will be the most is low in number successful one? when compared with unwanted a. Pour plate method microorganism. Which media can be b. Pour and spread plate method used to isolate the desired organism? c. Streak plate method a. Selective media d. All the above b. Enriched media

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7. Name the method in which the c. Change in the colony pattern of inoculums is mixed with the molten both A and B agar medium in the test tube and d. No change poured into the sterile petridish 12. If a plate observing for colony a. Pour plate method morphology is subjected to b. Spread plate method contamination, what will happen to c. Streak plate the colony? d. All the above a. Growth will be clear 8. The culture with only one type of b. Growth will not be clear organism in the colony is called c. Growth will be either clear or disturbed. a. Pure culture d. None of the above b. Mixed culture 13. If agar seeded plates exposed c. Semi mixed culture to atmosphere are incubated at room temperature, which colony d. Contaminated culture morphology will be predominantly 9. Identify the reason for the meager present in the plate? growth of aerobic colonies in pour plate isolation method a. Bacteria b. Fungi a. Less oxygen availability c. Virus b. More oxygen availability d. None of the above c. Carbon-di-oxide availability 14. If chromogenic bacteria produce d. None of the above intracellular water insoluble pigment, 10. If a microbial inoculum is with more it will stain contaminations, which method will be used for isolation? a. Growth of a colony b. Agar medium a. Spread plate method c. Both a and b b. Pour plate method d. None of the above c. Streak plate method 15. If the water soluble pigment of the d. All the above pigmented bacteria diffuses into the 11. The plate has a culture of A and B with medium, definite circular morphology If A is producing an inhibitory substance a. Medium gets pigmented towards B, what will happen to the b. Colony get stained colony morphology of B? c. Both a and b a. Change in the colony pattern of A d. None of the above b. Change in the colony pattern of a B

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Answer the following 14. Why is agar mainly used as a 1. Define semisolid media with an solidifying agent even though other example. solidifying agents are available? 2. State basal media with an example. 15. How do you differentiate enrichment medium from selective medium? 3. What is synthetic medium? Give suitable example. 16. Give a list of pigment producing bacteria. 4. State a few aspects of enrichment medium. 17. Explain the opacity of a bacterial colony. 5. State 3 fungal media used for isolation of fungi. 18. Explain the special purpose media in detail (any 5). 6. Define pure culture. 19. Why should we use a streak plate to 7. How do you differentiate pure culture grow a bacterium rather than on agar from mixed culture? medium slant or in broth medium? 8. Why are the colonies growth on 20. Expain the colony morphology of surface in pour plate method are quite bacteria with diagrams. larger than those within the medium? 9. Why is it important to invert the Student Activity petridish during incubation? 10. State the various forms in the appea- 1. The student will list out the rance of the colony. Name the pigments substances which contain agar in produced by Pseudomonas aeroginosa. their routine life and the role of agar in it. 11. Colony characteristics will be studied and identified clearly by using the 2. Students will prepare chart/scrap nutrient agar medium in agar rather book containing pictures of different than agar slant. Why? types of media and colony types of bacteria and fungi. 12. Write about the elevation of the bacterial colony? 3. Collect decayed/spoilt food for macroscopic observation. 13. Explain streak plate/pour plate/spread plate method.

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Chapter 6 Microbial Nutrition and Growth

Chapter Outline

6.1 Microbial Nutrients 6.2 Nutrient Requirement of Microorganisms 6.3 Nutritional Types of Microorganisms 6.4 Photosynthesis 6.5 Microbial Growth Mold is a type of fungi that grows on food and other organic matters. It breaks down the complex substances into 6.6 Measurement of Microbial Growth simpler ones and extracts nutrient for its growth from them.

Learning Objectives acquire energy from various organic and After studying this chapter the student inorganic compounds, light and CO2. The will be able, requirement of energy depends on their need and metabolic ability. • To know the essential nutrients required by bacterial cell. 6.2 Nutrient Requirement of • To differentiate between macronu- Microorganisms trients and micronutrients. • To describe an organism based on Microorganisms requires macronutrients, the sources of carbon and energy. micronutrients and growth factors, for their growth. These nutrients help in constructing • To compare the photosynthesis the cellular components like proteins, process in plant, algae and bacteria. nucleic acids and lipids. • To understand the phases of growth in bacterial growth curve. Macronutrients • To know the methods of counting Elements that are required in large amounts bacteria. are called macronutrients. Nitrogen (N), Carbon (C), Oxygen (O), Hydrogen (H), 6.1 Microbial Nutrition Sulphur (S) and Phosphorus (P), Potassium (K), Calcium (Ca), Magnesium (Mg) and All living organisms on this planet require energy for the normal functioning, growth Iron (Fe) are macroelements. and reproduction. Likewise, microorganisms

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Nitrogen is needed for the synthesis of Micronutrients amino acids, nucleotides like purines and Nutrients that are needed in trace quantities pyrimidines which are part of nucleic acids are called micronutrients. Example: Zinc (DNA and RNA). (Zn), Molybdenum (Mo), Cobalt (Co), Phosphorus is a part of phospholipids, Manganese (Mn). nucleotides like ATP and phosphodiester bonds of nucleic acids. Besides macro and micronutrients, Carbon, Hydrogen and Oxygen are the some microorganisms need growth factors backbone of all organic macromolecules like amino acids, purines and pyrimidines like peptidoglycan, proteins and lipids and and vitamins. Example: Biotin is required by nucleic acids. Leuconostoc sp and folic acid is required by Sulphur is needed for the synthesis Enterococcus faecalis. of thiamin, biotin, and aminoacids like cysteine and methionine. HOTS Potassium, Calcium, Magnesium and Iron exist as cations in the cell. These Is there a microbe that can grow in element plays vital role in the metabolic a medium that contains only the activity of microorganisms. Potassium (K+) following compounds in water: calcium carbonate, magnesium nitrate, ferrous is needed for the activity of many enzymes chloride, zinc sulphate and glucose. Example: Pyruvate Kinase. Defend your answer. Calcium (Ca2+) is involved in the heat resistance of bacterial endospores. 6.3 Nutritional Types of Magnesium (Mg2+) binds with ATP Microorganisms and serves as a cofactor of enzymes like Microorganisms can be classified into hexokinase. nutritional classes based on how they satisfy Iron (Fe2+ or Fe3+) is present in the requirements of carbon, energy and cytochromes and act as cofactors for electrons for their growth and nutrition. cytochrome oxidase, catalase and peroxidase. Based on the carbon source, microorganisms are able to utilize, they are Diatoms (A group classified into Autotrophs and Heterotrophs. of algae) need silicon Autotrophs: These are organisms that to construct their beautiful cell walls. utilize CO2 as their sole source of carbon. Heterotrophs: These are organisms that use preformed organic substances from other organisms as their carbon source. Based on energy source, microorganisms are classified into Phototrophs and Chemotrophs.

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Phototrophs: These are organisms that utilize light (radiant energy) as their energy source. Chemotrophs: These are organisms that obtain energy by oxidation of organic or inorganic compounds. Microorganisms are classified into Lithotrophs and Organotrophs based on the source from which they extract electrons. Lithotrophs are organisms that use reduced inorganic substances as their Figure 6.1: Microscopic view of Cyanobacteria electron source whereas Organotrophs obtain electrons from organic compounds 2. Photoheterotrophs: These organisms (Table 6.1). make use of light as energy source and organic compounds as electron and All microorganisms fall into any one carbon source. Example: Purple and of the four nutritional classes based on Green Non sulphur bacteria their primary source of carbon, energy and electrons. 3. Chemoautotrophs: These are ecologically important microorganisms. 1. Photoautotrophs: Eukaryotic algae, They oxidize inorganic compounds Cyanobacteria (Blue Green Algae) like nitrate, iron and sulphur to obtain (Figure 6.1) and Purple and Green energy and electrons. Sulphur bacteria belong to this class. 4. Chemoheterotrophs: These organisms They are capable of using light energy use organic compounds to satisfy their and have carbondioxide as the sole needs of energy, electron and carbon. source of carbon. (Table 6.2)

Table 6.1: Classification of microorganism based on carbon, energy and electron sources Carbon, Energy and Electron sources Carbon sources

Autotrophs CO2 as sole carbon source Heterotrophs Organic substances from other organisms Energy sources Phototroph Light energy Chemotrophs Chemical energy source (Organic or Inorganic) Electron sources Lithotrophs Reduced inorganic substances Organotrophs Organic compounds

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a. Blood lake in Texas-the blood red colour is due to the excess presence of purple sulphur bacteria. b. Giant tube worms seen in deep sea hydrothermal vents survive on nutrients given by chemolithotrophic bacteria.

a) b)

6.4 Photosynthesis Eukaryotes (plants and algae) and Photosynthesis is a process prokaryotes (cyanobacteria and purple, of capturing light energy and green bacteria) are capable of carrying out converting into chemical photosynthesis. Cyanobacteria perform energy. The chemical photosynthesis in a similar manner to energy produced in the plants. form of ATP and NADPH is used to synthesise organic Process of photosynthesis in compounds (carbohydrates); to be used as cyanobacteria food. This ability makes photosynthesis, a The process of Photosynthesis is divided significant process taking place on earth. into

Table 6.2: Nutritional classes of Microorganisms Nutritional class Energy/Electron/Carbon source Organisms Photoautotrophs Light energy Cyanobacteria, Purple and Inorganic e- donor Green sulphur Bacteria

CO2 Photoheterotrophs Light energy Purple and Green Organic e- donor Nonsulfur bacteria Organic carbon source Chemoautotrophs Inorganic chemical compounds as Nitrifying bacteria, Iron energy source bacteria Inorganic e- donor

CO2 Chemoheterotrophs Organic compounds as energy, Most pathogenic bacteria, electron and carbon source. fungi and protozoa.

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1. Light reaction and gets excited, the electron passes 2. Dark reaction through pheophytin, plastaquinone, cytochromes and plastacyanin to be In light reaction, light energy is captured donated to the oxidised P700 in order by photosynthetic pigments and converted to replenish the lost electron. It is into chemical energy (ATP and NADPH). during this process, ATP (Adenosine Tri In dark reaction, the chemical energy phosphate) is generated (Figure 6.2). is used to fix CO2 to construct organic Oxidised P680 obtains electron when compounds (carbohydrates); to be used water is split (Photolysis) into oxygen as food. This reaction is also called CO2 atoms (1/2 O2) and hydrogen ions fixation or Calvin’scycle. (2H+) which results in evolution of Light reactions in cyanobacteria oxygen. Hence it is called oxygenic • Photosynthetic pigments photosynthesis. All photosynthetic organisms contain Light pigments to observe light. Chlorophyll CO2 + H2O (CH2O)n+ O2 is the main pigment involved in the absorption of light. In cyanobacteria, This process of electron flow where chlorophyll a is the predominant two photosystems are involved in the pigment present and it absorbs red light generation of ATP is called non cyclic at a wavelength of 665nm. Cyanobacteria photophosphorylation. also contain accessory pigments like phycobiliprotein (Phycoerythrin and Photosynthesis in Bacteria phycocyanin) which help to absorb light There are four groups of photosynthetic at a broader wavelength (470-630nm) bacteria. They are green sulphur bacteria and makes photosynthesis more efficient. (Example: Chlorobium) and green non These pigments are located in the sulphur bacteria (Example: Chloroflexus) cytoplasmic membrane of cyanobacteria. purple sulphur bacteria (Example: • Photosystems Chromatium) and purple non sulphur The pigment molecules are arranged in bacteria (Example: Rhodospirillum). These highly organized arrays called reaction photosynthetic bacteria can fix atmospheric center or Photosystems. Cyanobacteria and CO2 in a similar fashion like cyanobacteria green plants have two photosystems namely but using only one photosystem and using Photosystem I (P700) and Photosystem II H2S as the electron donor instead of H2O. (P680) through which the electrons, excited Process of photosynthesis in bacteria by the capture of photons, flow. The electron transport system in purple When P700 (Photosystem I) absorbs and green bacteria consists of only one energy and gets excited, the excited Photosystem PSI (P870). They do not electron is transferred through a series possess photosystem II. When P870 gets of proteins like ferrodoxins and are excited upon capture of light energy, it donates the electron to bacteriopheophytin. eventually used to reduce NADP+ to + Electrons flow through quinones and produce NADPH . (When a compound cytochromes and are reverted back to P870. accepts electrons, it is said to be This process is cyclic (since the electron reduced). When P680 absorbs light

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P700* Membrane bound iron sulfur proteins + + Ferre doxin2NADPH + 2H P680* Pheophytin Light – Plastoquinone 2e 2e– NADP reductase Cytochrome b6f complex providesThis energy electron for transport chemiosmotic chain – Light 2e Plastocyanin

H O synthesis of ATP 2 2NADPH – 2e P700 2e– Oxygen evolving complex P680 ATP

Direction of increasing energy electron Photosystem I

1/20 +2H+ 2 Photosystem II

Figure 6.2: Simplified scheme of the light reactions of photosynthesis excited from P870 comes back to P870) e- acceptor and generates ATP. A reversed electron flow operates in purple bacteria to reduce e– + NAD to NADH. Electrons are extracted NADP NADPH from external electron donors like hydrogen ATP sulphide, hydrogen, elemental sulphur and organic compounds to synthesise NADH. – e ADP+P Since H2O is not used as electron donor, oxygen is not evolved which explains the anoxygenic nature of the organisms involved (Figure 6.3). The sulphur evolved PS I during this reaction is deposited as sulphur globules either outside or inside the cells. H2S 2e + 2H+ + S CO2 + H2S Æ (CH2O)n + S. Table 6.3 compares the photosystheic process in Figure 6.3: Green sulphur and purple plants, algae and bacteria. sulphur bacteria that use sulphides as electron donors 6.5 Microbial Growth HOTS In bacteria, growth can be defined as an increase in cellular constituents. Growth 1. What will be the electron flow results in increase of cell number. sequence of noncyclic and cyclic When bacteria are cultivated in liquid photo phosphorylation? medium and are grown as batch culture 2. Chemical energy produced in (Growth occurring in a single batch of photosynthesis is either ATP medium with no fresh medium provided), NADPH or ATP NADH. Why? cell multiplication happens till all the

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Table 6.3: Photosynthetic process in plants, algae and bacteria

Green plants and Purple and Green algae Cyanobacteria bacteria Primary pigment Chlorophyll a and b Chlorophyll a Bacteriochlorophyll Accessory pigments Carotenoids Phycocyanin - Photosystem Both Both Photosystem I Only Photosystem I Photosystem I and II and II present present present

Electron donors H2OH2OH2S

Oxygen evolution O2 is evolved O2 is evolved S is evolved Oxygenic Oxygenic Non Oxygenic nutrients are exhausted. After sometime, medium. The growth rate is constant during nutrient concentrations decline and the exponential phase. The organism divides bacterial cells begin to die. This growth and doubles in number at regular intervals. pattern can be plotted in a graph as the The growth curve rises smoothly logarithm of viable cells versus incubation 3. Stationary Phase time (Figure 6.4). The growth curve has four distinct phases. As the nutrients get depleted, the cell growth stops and the growth curve become 1. Lag phase horizontal. The total number of viable cells 2. Logrithmic phase/Exponential phase remains constant which is due to a balance between cell division and cell death. 3. Stationary phase 4. Death phase 4. Death Phase Nutrient deprivation and build up of 1. Lag Phase wastes lead to the decline in cell numbers. When bacteria are introduced into fresh The microbial population dies rapidly and medium, no immediate cell multiplication logarithmically and the growth curve also and increase in cell numbers occur. stops down. The cell prepares itself for cell division Batch culture by synthesizing cell components and increase in cell mass. Since there is a lag It is the growth of microorganisms in a fixed in cell division, this phase is called lag volume of culture medium in which nutrient phase. supply is not renewed and wastes are not removed. It is a closed system. This can be used to study the various growth phases of 2. Logrithmic Phase/Exponential microorganisms. Phase During this phase, microorganisms rapidly Continuous culture divide and grow at a maximal rate possible A continuous culture is an open system utilizing all the nutrients present in the with constant volume to which fresh

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Stationary phase

Logarithmic Death growth (decline) phase phase

Lag phase Logarithm of viable cells

0510 Time (hr.)

Number of viable and Few or no cells Viable cells Nonviable cells nonviable cells in population Figure 6.4: Bacterial growth curve showing phases of growth in laboratory conditions medium is added and utilized (spent) rate can be controlled by adjusting the medium are removed continuously at dilution rate and cell density is controlled a constant rate. A microbial culture by modifying the concentration of the remains in exponential state for longer limiting nutrient (Figure 6.5). periods, for days and even weeks. This enables the researcher to learn about the Turbidostat physiological processes and enzymatic This type of continuous culture system activities of organisms. has a photocell that measures the turbidity of the culture vessel. This There are two ways by which continuous automatically regulates the flow rate culture is operated. of the culture medium. Turbidostat a) Chemostat does not contain limiting nutrients b) Turbidostat (Figure 6.6). Chemostat 6.5.1 Factors Influencing Growth The chemostat operates so that the sterile The growth and activities of nutrient medium enters the culture vessel microorganisms are greatly influenced at the same rate as the spent medium is by the physical and chemical conditions removed. The chemostat can control of their environment. Among all growth rate and cell density simultaneously factors, four key factors play major and independently of each other. Two roles in controlling the growth of factors play an important role in achieving microorganisms. They are this dilution rate and concentration of the limiting nutrient (a carbon or a nitrogen 1. Temperature source like sugars or aminoacids). Growth 2. pH

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Fresh me i m eser ir sterile me i m

ntr l al e

al e c ntr lling ir Fl mei m s l ir ilter O tlet r lt re s ent essel meim Light s rce h t cell

ece tacle rbi star Figure 6.5: The Chemostat Figure 6.6: The Turbidostat

3. Water activity • Mesophiles 4. Oxygen • Thermophiles 1. Temperature • Hyperthermophiles Temperature is one of the most important Psychrophiles environmental factor affecting the A psychrophile can be defined as an growth and survival of microorganisms. organism with an optimal growth Temperature can affect microorganisms temperature of 15°C, maximum growth because the enzyme catalysed reactions temperature of 20°C and a minimum are sensitive to fluctuations in growth temperature at 0°C. These temperature. organisms are found in polar regions like For every microorganism, there is a Arctic and Antarctic oceans. They are minimum temperature below which no rapidly killed as the temperature rises because the cellular constituents start to growth occurs, an optimum temperature at leak due to cell membrane disruption. Some which growth is most rapid, and a maximum examples of psychrophiles are Moritella, temperature above which no growth occur. Photobacterium and Pseudomonas. These three temperatures are called cardinal temperatures. Psychrotolerant Organisms that can grow at 0°C, but Temperature classes of microorganisms have temperature optimum growth Microorganisms are broadly distinguished temperature range of 20°C-40°C are called into four groups in relation to their psychrotolerant. temperature optima. Mesophiles • Psychrophiles These are microorganisms that grow in optimum temperature between 20-45°C,

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Infobits Snow alga – Chlam- ydomonas nivalis Taq polymerase, a DNA polymerase grows within the snow enzyme which is of great applied and its brilliant red importance used in DNA amplification. coloured spores are It is isolated from Thermus aquaticus, a responsible for the formation of pink thermophile. snow. 2. pH pH is defined as the negative logarithm of the hydrogen ion concentration. pH scale extends from pH 0.0 to pH 14.0 and each exchange of 1 pH unit represents a 10 fold change in hydrogen ion concentration. pH greatly influences microbial growth. Each organism has a definite pH range and well defined pH growth optimum. Most natural environments HOTS have pH values between 5 and 9. Organisms are classified into Why do unopened pasteurized milk Acidophiles, Neutrophiles and Alkalophiles spoil even under refrigeration? based on their optimum growth pH. Acidophiles are organisms that grow they have a temperature minimum of best at low pH (0.0–5.5) Example: Most 15-20°C and a maximum temperature fungi, bacteria like Acidithiobacillus, of 45°C. All human pathogens are mesophiles. Archaebacteria like Sulfolobus and Thermoplasma. Thermophiles Neutrophiles are organisms that grow Organisms whose growth temperature well at an optimum pH between 5.5 and 8.0. optimum is between 55-65°C are called thermophiles. They have minimum Most bacteria and protozoa are neutrophiles. growth temperature of 45°C. These Organisms that prefer to grow at pH organisms are found in compost stacks, between 8.5-11.5 are called alkalophiles. hot water lines and hot springs. They These microorganisms are typically found contain enzymes that are heat stable and in soda lakes and high carbonate soils. protein synthesis systems function well at Example: Bacillus firmus. high temperature. Hyperthermophiles 3. Water Activity and Osmosis

Organisms whose growth optimum Water activity, (aw) is the ratio of vapour temperature is above 80°C are called pressure of the solution to the vapour hyperthermophiles. These are mostly pressure of pure water (aw values vary bacteria and archaebacteria. They between 0 and 1). Water activity is inversely are found in boiling hot springs and related to osmotic pressure. Organisms hydrothermal vents on seafloor. that can grow in low aw values are called osmotolerant. Example: Staphylococus

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not required for their growth. Example: Crenation: Streptococcus pyogenes. Shriveling of cytoplasm in the cell is called (5). Facultative anaerobes can grow either crenation. This effect under oxic or anoxic conditions: Example: helps to preserve some foods. Escherichia coli. (Figure 6.7) 6.6 Measurement of Growth aureus . Different methods are employed for Only a few organisms are capable of measuring the cell growth of microorganisms. tolerating high salt concentration and still Cell growth is indicated by increase in the growing optimally in low water activity. Such number of cells or increase in weight of cell organisms are called halophiles. Halophiles mass. There are direct and indirect methods can grow in 1-15% Sodium chloride (NaCl) of measuring microbial growth. concentrations. Organisms that can grow in 1. Direct Measurements very salty environments are called extreme halophiles. (They can grow in 15-30%) NaCl Total count and viable count are the two concentration. Example: Halobacterium. methods widely employed to count cell numbers. 4. Oxygen Total count: Most of the microorganisms require oxygen The total number of cells in a population for their optimal growth but some of them can be measured by counting a sample survive very well in total absence of oxygen under the microscope. This is called and are killed when exposed to air. direct microscopic count. This is done Based on their need and tolerance for by using a specialized counting chamber called Petroff Hausser chamber which is oxygen, microorganisms are classified into a specially designed slide with a grid. The the following types. liquid sample is placed on the grid which 2 (1) Obligate aerobes exhibit growth only at has a total area of 1mm and divided into 25 large squares. The number of cells in large full oxygen level (21% O2 on air) because O2 is needed for their respiration and metabolic square is counted and the total number of activities Example: Micrococcus, most Algae, cells is calculated by multiplying it with a Fungi and Protozoa. conversion factor based on the volume of the chamber (Figure 6.8). (2) Microaerophiles are aerobes that Advantages require oxygen at levels lower than that of air. Example: Azospirillum, Campylobacter, This is a quick method of estimating cell Treponema numbers. Disadvantages (3) Obligate anaerobes does not require oxygen for their respiration and growth. This 1. Dead cells are also counted group cannot tolerate O2 and are killed in its 2. Special microscopes like phase contrast presence. Example: Methanogens, Clostridium. microscope are needed if unstained samples are used. (4) Aerotolerant anaerobes can grow 3. Small cells are difficult to count in the presence of oxygen though O2 is

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Obligate Obligate Facultative Aerotolerant aerobes anaerobes anaerobes anaerobes Microaerophiles

ABCDE Figure 6.7: The effect of oxygen on the growth of various types of bacteria

Count cells in this square 1.00 mm

1.00 mm 0.05 mm

0.25 mm 1.00 mm

(a) (b) Figure 6.8: (a) Petroff-Hausser counting chamber (b) Microscopic observation of bacterial cells 2. Viable Count sterile agar medium, using a sterile spreader. A viable cell is one that is able to divide and The total number of colonies appearing on form a visible colony on the nutrient media. the plate after incubation represents the total Viable cells are counted by methods pour number of viable cells in the culture. plate and spread plate. 3. Measurement of Cell Mass Pour plate method A cell suspension appears turbid or cloudy In this method, a known volume (0.1 or due to active cell growth. When light 1.0ml) of the culture is pipetted into a sterile is passed through this cell suspension, petri plate, then molten nutrient medium is microbial cells scatter light striking them. poured over and incubated. Colonies will As the concentration of cells and turbidity appear throughout the agar medium and are increases, more light is scattered and less light counted to obtain viable count. is transmitted through the suspension. The amount of unscattered light can be measured Spread plate method using a spectrophotometer, the values of which In this method, a known volume of the culture are indirectly related to cell numbers. (0.1ml) is plated and spread over solidified

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ICT CORNER Culture Media

Preparation of Bacteriological Media

STEPS: • Use the URL or scan the QR code to reach ‘Virtual Interactive Bacterial Laboratory’. • Click module at the bottom and read the description and steps. • Follow the steps and open activities under’ Common Bacteriologic Media’ one by one and explore it. • Record your observation of Differential Media. Click examples and record the specimen suitable for particular media

Step1 Step2

Step3 Step4

URL: http://learn.chm.msu.edu/vibl/content/ differential.html

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Summary 2. Magnesium is needed a. For cell wall synthesis Microorganisms need macro and micronutrients for their growth. Based b. As cofactor for enzymes on the energy source, organisms c. For photosynthesis are grouped into Phototrophs and d. For protein synthesis Chemotrophs. Based on carbon source, 3. One of the following is an example they are classified into autotrophs and heterotrophs. Organisms are grouped into for chemoautotroph lithotrophs and organotrophs based on a. For cell wall synthesis their electron source. The four nutritional b. As cofactor for enzymes classes of microbes are photoautotrophs, c. For photosynthesis Photoheterotrophs, chemoautotrophs d. For protein synthesis and chemoheterotrophs. 4. The phase of growth in which the Cyanobacteria are prokaryotes that can growth rate is equal to the death rate perform photosynthesis. Chlorophyll is is the pigment needed to capture light energy a. Stationary phase (photons). In cyanobacteria and green b. Death phase plants, non cyclic photophosphorylation c. Exponential phase takes place to generate ATP and NADPH d. Lag phase during photosynthesis whereas cyclic 5. Organisms that are capable of growing photophosphorylation takes place in purple in 0°C are called and green bacteria involving only one a. Thermophiles Photosystem (PS I). b. Hyper thermophiles In a batch culture, bacteria show a c. Barophiles characteristic growth pattern which consists d. Psychrophiles of lag phase, log phase and stationary phase 6. Halophiles are organisms that can and decline phase. In a chemostat, cultures grow in can be maintained in an exponential phase a. Low water activity for long periods. The most important factors b. High salt concentration affecting microbial growth are temperature, c. Low temperature pH and oxygen level. Total count and viable d. High pH count are the two widely used methods to measure cell numbers. 7. An example of microaerophilic organism is Evaluation a. Bacillus b. Azospirillum c. Pseudomonas d. Escherichia.coli Multiple choice questions 8. The specialized chamber used for the 1. An example of photoautotroph counting of microbial cells is a. Cyanobacteria a. Haemocytometer b. Algae b. Counting chamber c. Green plants c. Petroff Hauss chamber d. All of the above d. Counting slide

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Answer the following 15. Explain the classification of microbes based on their nutrition. If H S is toxic 1. Give notes on the nutritional classes of 2 to living organisms, how do purple and microorganisms. green bacteria survive and use H2S in 2. Classify microorganisms based on such environments. energy and carbon source. 16. Describe the photosystems of 3. What are light and dark reactions in cyanobacteria. photosynthesis? 17. Draw a schematic representation 4. What is bacteriochlorophyll? Give of Z scheme of non cyclic its role. photophosporylation. 5. Define chemoautotroph. 18. Compare photosynthesis between 6. Define photosynthesis. plants, cyanobacteria and purple green 7. Give examples of photosynthetic bacteria. bacteria. 19. Explain the principle and uses of 8. What do mean by cardinal temperature? chemostat and turbidostat with 9. Give notes on photosynthetic pigments. diagrams. 10. What are halophiles? 20. Describe the classification of microorganism based on their oxygen 11. Give reason for the ability of requirement. thermophiles to grow in high temperatures. 21. Explain the principle and uses of chemostat and turbidostat with 12. How bacterial cells are counted using diagrams. counting chamber? 22. Explain the relation of osmosis to water 13. Classify microorganisms based on their activity. temperature requirement. 23. Define growth. Explain the phases of 14. Describe the role of macro and growth of bacteria with neat diagram. micronutrients in microorganisms. How do you think bacteria acquire their nutrients from their environment?

Student Activity • Expose a container with water to sunlight for a week. Observe the growth of cyanobacteria on water which explains the photoautotrophic mode of nutrition. • Store a loaf of bread for a week after the expiry date. You can observe the growth of fungi/molds which demonstrates the mode of nutrition of chemoheterotrophs. • Collect rusted iron pipes which contain chemolithotrophic Thiobacillus sp which can oxidize iron for their nutrients. • Place two bowls of cooked rice/vegetables–one inside the refrigerator at 6°C, and another at room temperature at 30-35°C. Give reasons for the quick spoilage of the rice stored at 30-35°C. Check the pH of milk using a pH paper.

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Chapter 7 Morphology of Bacteria

Chapter Outline

7.1 Bacterial Size, Shape and Arrangement 7.2 Structures External to Cell Wall of Bacteria 7.3 Cell Envelope of Bacteria 7.4 Structures Internal to Cell Membrane The distinction between prokaryotes and eukaryotes of Bacteria is considered to be the most important distinction 7.5 Eukaryotic Cell among groups of organisms. Eukaryotic cells contain membrane bound organelles, such as mitochondria, while Structure prokaryotic cells do not.

Learning Objectives • To differentiate between Gram After studying this chapter the student positive and Gram negative bacteria. will be able, • To know the structures and functions • To know the size, shape and internal to cell membrane. arrangement of bacteria. • To differentiate between prokaryotic • To list a few examples of bacteria and eukaryotic cell structure. with their shapes. • To understand and describe the role of Living organisms are differentiated from the structures external to the cell wall. non living matter by their (1) ability to reproduce (2) ability to ingest or assimilate • To understand the structure, function food and metabolize them for energy and arrangement of bacterial and growth (3) ability to excrete waste flagella. products (4) ability to react to changes • To describe the role of capsule, slime in their environment (irritability) and layer, pili, flagella and fimbriae in a (5) susceptibility to mutation. The living prokaryotic cell. organisms include a variety of micro and • To describe the structure and function macro organisms of different size, shape, of cell wall, outer membrane and cell morphology and behaviour. They include membrane. tiny bacteria, protozoans, worms, plants • To know the significance of Cell and animals. Envelope.

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Generalized structure of a bacterium Infolding of plasma Capsule Cell wall membrane DNA coiled into nucleoid

Basal body

Flagellum

Ribosomes Cytoplasmic inclusion Cytoplasm Pili Plasma membrane Figure 7.1: Generalized structure of a bacterium

Bacteria, cyanobacteria (blue green made until the development of electron algae) microalgae, protozoa, yeasts and microscope, which depicted the internal fungi represent the microorganisms. structure of these organisms. The absence Prokaryotes are organisms with primitive of membrane bound internal structures in type of nucleus lacking a well defined bacteria and their presence in fungi, algae, membrane (Figure 7.1). The nuclear protozoa, plant and animal cells was taken material is a DNA molecule in prokaryotes as criterion to differentiate prokaryotes and compared to chromosomes of higher eukaryotes. organisms. Eukaryotes are organisms 7.1 Size, Shape and Arrangement of with cells having true nuclei enclosed in Bacteria a nuclear membrane and are structurally more complex than prokaryotes. There 7.1.1 Size of Bacteria exists varying degree of localization of Bacteria are minute living bodies and cellular functions in eukaryotes that occur represent one of the lowest orders of living in distinct membrane bound intracellular cells. The determination of size of the organelles like nuclei, mitochondria, different forms is originally carried out chloroplasts. The cells of living organisms by comparison with known RBC. A more are either prokaryotic or eukaryotic in accurate estimation is now obtained by nature and there is not any intermediate the use of a special micrometer eye-piece, condition. The size, shape, morphology containing a graduated scale. The unit of and the internal cellular organizations are measurement of bacteria is called micron different in these two groups. (μ or μm). 1 micron is equal to 1 thousand Satisfactory criteria to differentiate of millimeter. Resolution of unaided eye is bacteria, fungi and algae could not be 200μm. The size of bacteria is constant but

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pm nm nm nm μm μm μm μm

ight Microscope

lectron Microscope

Proteins

ipids Small Organelles tom Molecules irus Bacteria u aryotic Cells Figure 7.2: Metric unit of measurement depends upon environmental and growth 1 metre (m) = 1000mm (millimeter) condition. Medically important bacteria 1mm (10-3m) = 1000 μm (micrometer) ranges from 0.2 – 1.5 μm in diameter and 1 μm (10-6m) = 1000nm (nanometer) 3-5μm in length (Figure 7.2). 1nm (10-9m) = 1000pm (picometer) 1A0 (10-10m) (angstrom)

Infobits

The smallest bacteria is Mycoplasma genitalium, which has a diameter of 200-300nm. The largest and longest bacterium is Thiomargarita namibiensis (750μm) found in the ocean sediments in the continental shelf of Namibia. They are large enough to be visible to the naked eye. The previously known largest bacterial cell Epulopiscium fishelsoni is found only in the intestinal tract of certain topical fish over 500μm long. Epulopiscium means “guest at the table of fish”.

Epulopiscium fishelsoni Mycoplasma genitalium

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7.1.2 Cell Shape and Arrangement of and spirochetes (agent of syphilis). Bacteria Although these two are similar in shape The shape of a bacterium is governed by spirochetes are flexible in nature. Spiral its rigid cell wall. Typical bacterial cells bacteria are far too thin to be seen with are spherical (called cocci), straight rods the standard Brightfield microscope (called bacilli) and helically curved rods but are readily observed by Darkfield (called spiral). These shapes are constant microscope (Figure 7.3). for the particular species or genus but there Filamentous bacteria are bacterial cells that are pleomorphic in nature. They exhibit a variety of shapes. Bacteria tend to form long strands composed of many cells. In these cases, • Cocci appear in several characteristic an occasional single cell may be seen after arrangements, depending on the it breaks away from a long filament. These plane of cellular division and whether organisms resemble the threadlike strands daughter cells remain together with of fungi but their internal structure is the parents even after cell division. The typical of bacteria. Filamentous soil bacteria cells may occurs in pairs (diplococci), include Streptomyces species. in groups of four (tetracocci), in clusters (Staphylococcus), in a bead like chain Pleomorphic bacteria (Streptococci) or in cuboidal arrangement A few bacteria lack rigid cell walls, and their of cells (Sarcinae). flexible plasma membrane allows them to • Bacilli are rod shaped organism change shape. These are called pleomorphic (Singular, bacillus = stick) usually bacteria (pleo-more; morph-form). ranging between 1 and 10 μm in length. Example: Mycoplasma. Some bacilli are so short and stumpy that they appear ovoid and are referred to as 7.2 Structures External to Cell Wall of Bacetria coccobacilli. Bacilli are not arranged in patterns as complex as those of cocci 7.2.1 Appendages and mostly occur as singles or in pairs Flagella (diplobacilli, Example: Bacillus subtilis) Flagella (singular flagellum) are threadlike, or in the form of chains (Streptobacilli). long, thin helical filaments measuring 0.01- Some form trichomes, which are similar 0.02nm in diameter. These appendages to chains. In other Bacilli such as extend outward from the plasma Corynebacterium diphtheria the cells membrane and cell wall. Flagella are so are lined side by side like matchsticks thin that they cannot be observed directly (pallisade arrangement). Some bacilli with a bright field microscope, but must be are curved into a form resembling a stained with special techniques (example: comma. These cells are called vibrios as Fontana’s silver staining technique) that in Vibrio cholera. increase their thickness. The detailed • Spiral bacteria: They are divided into structure of a flagellum can only be seen in two groups, spirilla (singular spirillum) the electron microscope.

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The bacterial flagellum is composed 2. In lateral arrangement, flagella are of three parts: a basal body (associated arranged randomly all over the with the cytoplasmic membrane and cell surface of the cell. Bacteria with wall), a short hook and a helical filament lateral flagellar arrangement are (which is usually several times as long as called peritrichous. (Table 7.1) the cell). Filament is external to cell wall Various types of mobility are observed and is connected to the hook at cell surface; based on the arrangement of the the hook and basal body are embedded in flagella. Serpentine motility is seen with the cell envelope (Figure 7.4). Hook and Salmonella, darting motility with Vibrio filament are composed of protein subunits and tumbling motility with Listeria called as flagellin. monocytogenes. Some bacteria like Cytophaga One can generalize that all spirilla, about exhibit a gliding motility, which is slow half of the bacilli and a small number of sinuous flexing motion. This occurs when cocci are flagellated. Some bacteria do not the cells come in contact with solid surface. have flagella. Flagella vary both in number Some bacteria have the ability to move and arrangement on the cell surface. Flagella toward or away from chemical substance. are arranged generally in two patterns. This movement is called chemotaxis. 1. In polar arrangement, the flagella Positive chemotaxis is the movement of a are attached at one or both ends of cell in the direction of a favorable chemical the cell. Bacteria with polar flagellar stimulus (usually a nutrient). Negative arrangement are further classified chemotaxis is the movement away from into monotrichous, lophotrichous, a chemical substance (usually harmful and amphitrichous. compound). Some photosynthetic bacteria exhibit phototaxis, movement in response to light rather than chemicals.

lagellum

ilament

Outer membrane

Basal Body od

Peptidoglycan portion of cell all Figure 7.4: Structure of bacterial flagella

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Table 7.1: Arrangement of bacterial flagella Structure Flagella type Example Monotrichous(single Vibrio cholera flagella on one side)

Lophotrichous(tuft of Pseudomonas fluorescens flagella on one end)

Amphitrichous(single or Aquaspirillum serpens tuft on both ends)

Peritrichous(flagella Salmonella typhi throughout the cells)

in certain species of Gram negative bacteria. HOTS Pili play no role in motility. Pili originate from the plasma membrane and are made up A. If a bacterium loses its flagella, of a special protein called pilin (Figure 7.5). does it survive? Pili play a major role in human infection B. If you remove the cell wall from a by allowing pathogenic bacteria to attach flagellated bacterium, the organism to epithelial cells lining the respiratory, loses the ability to move. Explain. intestinal or genitourinary tracts. This attachment prevents the bacteria being The presence of motility is one piece of washed away by body fluids, thus helps in information used to identify a pathogen in establishment of infection. One specialized the laboratory. One way to detect motility is type of pilus (sex pilus) helps in the transfer to stab a tiny mass of cells into soft (semi solid) of genetic material between the bacterial medium in a test tube. Growth spreading cells. This process is called conjugation. rapidly through the entire medium is Fimbriae indicative of motility. Alternatively, cells can Fimbriae (singular: fimbria) is another term be observed microscopically by a hanging used for short pili that occur in great number drop method. around the cell. They enable bacteria to Pili attach to surfaces and to each other, so that Pili (singular pilus) are straight, short and the bacteria form clumps or films called thin and more numerous than flagella pellicles on the surface of liquid in which they around the cell. They can be observed only are growing. Fimbriae are found in Gram by electron microscopy. They are found only positive as well as in Gram negative bacteria.

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Flagell m Fimbriae Se il s

Flagella Fimbriae

il s

Figure 7.5: Structures of pili and fimbriae

Table 7.2 compares the pili and fimbriae. homopolysaccharide (made up of a single kind of sugar) or heteropolysaccharide 7.2.2 Extracellular Polymeric Substance (made up of several kinds of sugars). These (EPS) are synthesized from sugars within the cell, Many bacteria secrete high molecular transported and polymerized outside the weight polymers that adhere to the exterior cell. The capsule of some bacteria is made of the cell wall to form a capsule or slime of polypeptides. The capsule ofBacillus layer. Glycocalyx is often used to refer to any anthracis has polymer of D-glutamic polysaccharide material outside the cell wall. acid. Capsules are highly impermeable. Capsules and slime layer are considered to Capsules can be demonstrated using be glycocalyxes (Table 7.3). special staining technique utilizing Indian ink or with Nigrosin stain. The presence of Capsules capsule in fresh isolates gives a moist and Some bacterial cells are surrounded by a shiny appearance to the bacterial colonies viscous substance forming a covering layer on an agar medium. Capsular material or envelope around the cell wall called is antigenic and may be demonstrated by capsule (Figure 7.6). Capsule is usually serological methods. made up of polysaccharide. It may be

Table 7.2: Comparison of pili and fimbriae Characteristics Pili Fimbriae Appearance Hair like, straight Tiny bristle like fibers arising from the appendages. surface of bacterial cell. Length Longer than fimbriae Shorter than pili Numbers per cell 1-10/cell 200-400/cell Presence Present only in Gram Present in both Gram positive and negative bacteria Gram negative bacteria Made -up of Pilin protein Fimbrillin protein

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(Figure 7.8). Within the shealth, the bacteria Function: are capable of growth and division. Aquatic • It provides mechanical support. bacteria mostly form sheath. Examples • In a few bacteria, shealth is of sheathed bacteria include Leptothrix strengthened by the deposition of discophora (also known as iron bacteria), ferric and manganese hydroxides. Sphaerotilus and Clonothrix. Prosthecae They are semi rigid extensions of cell wall and cell membrane. Some bacteria may contain more than one prosthecae (Figure 7.9). Aerobic bacteria in fresh water and marine environment possess prosthecae. Some of the Bacterial cels prosthecate bacteria are Caulobacter, Stellar, Prosthecobacter and Hyphomicrobium. Sheath Figure 7.8: Sheathed bacterium

Infobits

Biofilms:

Microbial adhesion to animate or inanimate l c cal slime surfaces can be mediated by polysaccharides capsules or slime. These adherence polymers are collectively called as adhesions. Microorganism atheter s r ace tend to adhere to any surface and the layer they produce is called Biofilm. Biofilm can be harmful ell cl ster or beneficial to humans. Biofilm formation is a critical issue for almost all surfaces in health care and food preparation settings. Biofilms may form on a wide variety of surfaces, including living tissues, medical devices, industrial or portable water piping system, etc., Biofilm formation is a multi-step process starting with attachment to a surface, then formation of three dimensional structure and finally ending with maturation and detachment. During biofilm formation many species of bacteria are able to communicate with one another through specific mechanism called quorum sensing.

First c l nies l c cal ells sticks Organic s r ace t c ating c ating

S r ace s cells i i e the rm a iti nal micr rganisms are attache t ense mat b n e t gether e el ing ilm an create a mat re b stick e tracell lar e sits c mm nit ith cm le ncti ns l c cal slime

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Function: oldfast • Stalk helps in attachment of cells to solid surface. Prosthecae 7.3 Cell Envelope of Bacteria lagellum Cell envelope is an external covering that lies outside the cytoplasm. It is composed of two or three basic layers: the cell wall, S armer cell the cell membrane and in some bacteria the outer membrane. Figure 7.9: Prosthecate bacteria 7.3.1 Structure of Prokaryotic Cell Wall Function: Prokaryotic cells almost always are • Prosthecae increase surface area for bounded by a chemically complex cell wall. absorption of nutrients from the dilute Cell wall lies beneath the external structures aquatic environment. (capsules, sheaths and flagella). Cell wall • Helps in adhesion. lies external to the plasma membrane • Some prosthecae develop bud at the tip (cell membrane). Cell wall of eubacteria and helps in asexual reproduction. is made up of peptidoglycan or murein, whereas that of Archaeobacteria is composed Stalk of proteins, glycoproteins or polysaccharides. A few genera such as Methanobacterium, It is a nonliving ribbon like tubular structure. have cell walls composed of pseudomurein, It is formed by excretory product of bacteria. a polymer whose structure superficially Some of the stalked bacteria are Gallionella, resemble eubacteria peptidoglycan of Planctomyces ( Figure 7.10). eubacteria but differs markedly in chemical composition.(Note: Ordinary or typical lagellum bacteria are sometimes called eubacteria to distinguish them from the phylogenetically distinct group known as archaeobacteria). Separating daughter Peptidoglycan is a cross linked polymer cell of enormous strength and rigidity. It is a polymer composed of many identical subunits (Figure 7.11). Peptidoglycan differs Stal somewhat in composition and structure oldfast from one species to another, but it is basically a polymer of N-acetylglucosamine(NAG), N-acetylmuramic acid(NAM), L-alanine, D-alanine, D-glutamate, and a diamino acid Figure 7.10: Stalked bacteria (LL- or meso-diaminopimelic acid, L-lysine, L-ornithine, or L- diaminobutyric acid).

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O l sacchari e re l sacchari e

Lii

O ter Membrane

Li r tein

eri lasm e ti gl can e ti gl can

ell Membrane ell Membrane

Membrane r teins Membrane r teins ell en el e ram siti e ell en el e ram siti e bacteria bacteria

Figure 7.11: Cell envelope of Gram positive and Gram negative bacteria

Cell wall may contain other substances membrane and the outer membrane but in addition to peptidoglycan. For instance, no cell wall. Staphylococcus aureus and Streptococcus Functions of cell wall fecalis contain teichoic acids (polymer of acidic polysaccharides) covalently • It gives shape to bacteria like a bicycle linked to peptidoglycan. Cell wall of Gram tyre that maintains the necessary shape positive bacteria contain very little lipid and prevents the more delicate inner but Mycobacterium and Corynebacterium tube (the cytoplasmic membrane) cell walls are rich in mycolic acid (or Cord from bursting when it is expanded. factor) which make them acid fast. When • It protects bacteria from osmotic stained, the cells cannot be decolorized lysis in dilute solutions (hypotonic easily despite treatment with dilute acids. environment). Mycoplasma lack cell wall. • It protects cell from toxic substances. Protoplast is a bacterial cell consisting of cell material bound by a cytoplasmic HOTS membrane. Spheroplast is a bacterial cell with How do bacteria maintain their shape? two membranes namely the cytoplasmic

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7.3.2 Structure of Outer Membrane Special protein channels called porins Eubacteria and Archaeobacteria (Gram span the membrane. The points of contact positive and Gram negative) differ with between outermembrane and cytoplasmic respect to their cell walls. Gram negative membrane are known as adhesions. Outer cell walls are more complex. An outer membrane is anchored to peptidoglycan membrane surrounds a thin underlying layer by means of Braun’s lipoprotein. layer of peptidoglycan (Table 7.4). Periplasmic space between the cell Outer membrane is bilayered, consisting membrane and the outer membrane. mainly of phospholipids, proteins and Functions of outer membrane lipopolysaccharide (LPS). • It serves as an impermeable barrier to LPS is composed of three parts which prevent the escape of important enzymes are covalently linked to each other. They are (such as those involved in cell wall 1. Lipid A which is firmly embedded in the growth) from the periplasmic space. membrane, • It serves as a barrier to various external 2. Core polysaccharide that is located at chemicals and enzymes that could the membrane surface and damage the cell. For example, the walls of many Gram positive bacteria can be 3. Polysaccharide O antigens that extend easily destroyed by treatment with an like whiskers from the membrane surface enzyme called lysozyme, which selectively into the surrounding medium

Table 7.4: Difference between Gram positive and Gram negative bacteria Gram positive bacteria Gram negative bacteria Gram reaction The bacteria that retain the The bacteria that cannot retain colour of the primary stain the primary stain but takes on (crystal violet) are Gram the colour of the counterstain positive safranin are called Gram negative Cell wall The cell wall is thick The cell wall is thin (8-12nm (20-30nm thick) thick) Peptidoglycan layer Thick (multilayered) Thin (single layered) LPS content None High Lipopolysaccharide Periplasmic space Absent Present Outer membrane Absent Present Lipid and Low (acid fast bacteria High due to the presence of outer lipoprotein content have lipids linked to membrane peptidoglycan) Teichoic acids Present in many Absent Example: Streptococcus, Staphylococcus, Escherichia coli, Pseudomonas, Corynebacterium, Bacillus, Haemophilus, Salmonella, Clostridium Shigella.

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dissolves peptidoglycan. However, Gram such as energy reactions, nutrient negative bacteria are refractory to this processing and synthesis. enzyme because large protein molecules • It regulates transport, the passage of of enzyme cannot penetrate the outer nutrients into the cell and the discharge membrane. Only when outer membrane of wastes. It is a selectively permeable is damaged the enzyme can penetrate. membrane. • Porins allow the smaller molecules, • It is also involved in secretion or such as amino acids, monosaccharides discharge of a metabolic product into to pass across. extracellular environment. • Adhesions are export sites for newly • Cell membrane is an important site synthesised LPS and porins, and are for a number of metabolic activities. sites at which pili and flagella are Most enzymes of respiration and ATP made. synthesis reside in the cell membrane since prokaryotes lack mitochondria. 7.3.3 Structure of Cytoplasmic Membrane Significance of cell envelope • It has toxic properties (Example: LPS) Immediately beneath the cell wall is the cytoplasmic membrane also known as • It stimulates antibody production by plasma membrane or cell membrane. It is immune system composed of phospholipids and proteins. • The cell walls of many pathogens have The phospholipids form a bilayer. Integral components that contribute to their proteins are embedded within this pathogenicity. Example mycolic acids of bilayer. Surface proteins or peripheral Mycobacterium tuberculosis proteins are loosely attached to the • Cell wall is a site of action of several bilayer. The lipid matrix of the membrane antibiotics. has fluidity, allowing the components to • Many of the serological properties of move around laterally. In eubacteria, the Gram negative bacteria are attributable phospholipids are phosphoglycerides, in to O antigens; they can also serve as which straight chain fatty acids are ester receptors for bacteriophage attachment. linked to glycerol. In archaeobacteria, the lipids are polyisoprenoid branched-chain 7.4 Structures Internal to Cell lipids, in which long-chain branched Membrane of Bacteria alcohols (phytanols) are ether linked to Cytoplasm is called as the internal matrix glycerol. of the cell inside the cell membrane. Its Functions of the cell membrane major component is water (70-80%). It also • Prokaryotes do not have intracellular contains proteins carbohydrates, lipids, membrane bound organelles as present inorganic ions, and certain low molecular in eukaryotic organelles. Thus cell weight compounds. Inorganic ions are membrane provides a site for functions present in much higher concentrations in cytoplasm than in most media.

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Cytoplasm is thick, aqueous, semi- Nucleus transparent and elastic. The major structures The nuclear area has the hereditary in the cytoplasm of prokaryotes are nucleoid material of most bacteria. It contains a (containing DNA), ribosomes and reserve single, circular, long, continuous, thread deposits called inclusions. Prokaryotic like double stranded DNA called the cytoplasm lacks certain features of bacterial chromosome. Some bacteria eukaryotic cytoplasm such as a cytoskeleton with linear chromosome also exist. It and cytoplasmic streaming. carries the information required for the cells structure and function. They are not Ribosomes surrounded by a nuclear envelope and All living cells contain ribosomes. They are devoid of highly conserved histone are the sites of protein synthesis. High proteins. The nuclear area can be spherical, number of ribosomes represents the high elongated or dumbbell shaped. In actively rate of protein synthesis. Prokaryotic growing bacteria, as much as 20% of the ribosomes are freely found in the cell volume is occupied by DNA, because cytoplasm, whereas eukaryotic ribosomes such cells presynthesize nuclear material are attached to the cell membrane. for future cells. The chromosome is Prokaryotic ribosomes consists of protein attached to the cell membrane. Proteins in and a type of RNA called ribosomal RNA. the plasma membrane are believed to be They are smaller and less dense than the responsible for the replication of the DNA eukaryotic ribosomes. The ribosomes and segregation of the new chromosomes of prokaryotes are 70S where as that of to daughter cells in cell division. eukaryote are 80S (Figure 7.12).

r kar tic ib s me kar tic ib s me

S S S S S s b nit S s b nit S

S S

S S S S s b nit s b nit

Figure 7.12: Prokaryotic and Eukaryotic Ribosomes

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Plasmids Endospores: Apart from the bacterial chromosome, Some species of bacteria produce bacteria also contain small circular, double metabolically dormant structures called stranded DNA molecules called plasmids spores. They are highly durable and (Figure 7.13). Plasmids are self replicating dehydrated resting bodies produced inside extra chromosomal genetic elements. the cells. They are formed by bacteria only Plasmids may carry genes for activities when there is lack of water or depletion such as antibiotic resistance and tolerance of essential nutrients in the environment. to toxic metals. Examples: Fertility plasmid Endospores are coated with a specific (F plasmid), Resistance plasmid (R plasmid) chemical compound diaminopimelic acid. It and colicin plasmid (Col plasmid). binds with the Calcium and forms Calcium dipicolinate which removes the water from acterial lasmi s it and makes the spore resistant to extreme conditions Example: Bacillus anthracis and Clostridium tetani possess endospores. Mesosomes Generally prokaryotes do not have cytoplasmic organelles like mitochondria and chloroplast. Figure 7.13: Plasmids in Prokaryotes It contains mesosome as their organelle. They are the invaginations of the cell membrane Molecular Chaperones and they are in the form of tubules, vesicles or They are the helper proteins which recognize lamellae. They are seen in both Gram positive the newly formed polypeptides and fold and Gram negative bacteria, generally more in them into their proper shape of secondary Gram positive bacteria. They are located next and tertiary structure. Many chaperones to the septa or cross walls in dividing bacteria are involved in proper folding of bacteria. (Figure 7.14). They may be involved in cell They were first identified in Escherichia coli wall formation during division or play a role mutant. Example: Heat shock proteins are in chromosome replication and distribution produced in Escherichia coli cells subjected to daughter cells. If they are located near to the to live at high temperatures, or in any other surface they are called peripheral mesosomes stressful unfavorable conditions. and if they are located deep into the cytoplasm they are called central mesosomes. Inclusions ell all The cytoplasm of prokaryotic cells has several kinds of reserve deposits known lasma as inclusions. Cells may accumulate Membrane Messme certain nutrients when they are plentiful Sac and use them when they are deficient.

Some inclusions are common to a wide b les variety of bacteria whereas others are limited to certain species (Table 7.5). Figure 7.14: Bacterial Mesosome

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Table 7.5: Different types of inclusion bodies in bacteria

Type of inclusion Example of organisms bodies possessing Significance Polyhydroxybutyrate Bacillus Reserve of Carbon and energy (PHB) megaterium sources. Sudan dye is used to observe lipid inclusions Polyphosphate Corynebacterium Reserve of phosphate (volutin granules) or diphtheriae metachromatic granules Sulphur globules Phototrophic bacteria Elemental sulphur, reserve of Like purple and green electrons in phototrophs. Reserve of Sulphur bacteria energy source in lithotrophs Example: Thiobacillus Gas vesicles Aquatic bacteria, They are protein shells filled with Cyanobacterium gases. They provide buoyancy and keep the cells floating in vertical water column Parasporal crystals Genus Bacillus It is a proteinaceous compound, It is toxic to certain insects Magnetosomes Aquaspirillum They are like intracellular chains of magnetotacticum magnetite particles. They help the bacteria to swim to nutrient rich sediments. It protects the cell against

H2O2 accumulation Carboxysomes Photosynthetic They contain the enzyme Ribulose Bacteria, cyanobacteria 1-5 bisphosphate carboxylase which Autotrophic bacteria is involved in Carbon dioxide fixation during photosynthesis Phycobilisomes or Cyanobacteria They have a long polypeptide with cyanophycin Granules equal proportion of Arginine and Aspartic acid. They store Nitrogen Chlorosomes Green bacteria They contain bacteriocholorophyll pigments which are involved in bacterial photosynthesis

HOTS

Why are endospores so difficult to destory?

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7.5 Eukaryotic Cell Structure It is primarily the structure of cell walls and membranes, and the absence As mentioned earlier, eukaryotic of organelles (specialized cellular organisms include algae, protozoa, fungi, structures that have specific functions), higher plants and animals. The eukaryotic that distinguish prokaryotes from cell is typically larger and structurally eukaryotes (Table 7.6). more complex than the prokaryotic cell (Flowchart 7.1). The general, eukaryotic microbial cells have a cytoplasmic membrane, nucleus, Prokaryotes and Eukaryotes are mitochondria, endoplasmic reticulum, chemically similar, in the sense that Golgi apparatus, vacuoles, cytoskeleton, they both contain nucleic acids, and glycocalyx. A cell wall, locomotor proteins, lipids, and carbohydrates appendages and chloroplasts are found only (Figure 7.15). They use the same kinds in some groups. The structure and functions of chemical reactions to metabolize of the eukaryotic cells are discussed in food, build proteins, and store energy. (Table 7.7).

Eukaryotic Cell Structure

External Structures Cell Envelope Internal Structures

Appendages • Cell Wall Nucleus • Flagella • Cytoplasmic Organelles • Cilia membrane • Endoplasmic Glycocalyx reticulum • Capsule • Golgi apparatus • Slime layer • Ribosome • Lysosomes • Mitochondria • Chloroplasts Cytoskeleton • Microtubules • Microfilaments

Flowchart 7.1: Eukaryotic Cell Structure

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Table 7.6: Differences between prokaryotic and eukaryotic cell (Continued)

10 Chromosome Single circular Multiple linear chromosomes (DNA) chromosome, lacks with histone arrangement histone 11 Cell division Binary fission Mitosis 12 Sexual No meiosis (transfer Involves meiosis recombination of DNA fragments only)

Table 7.7: Functions of Eukaryotic organelles Eukaryotic organelles Functions Plasma membrane Mechanical cell boundary, selectively permeable barrier with transport systems, mediates cell to cell interactions and adhesion to surfaces, secretion Cytoplasmic matrix Environment for other organelles, location of many metabolic processes

Microfilaments, intermediate Cell structure and movements from the cytoskeleton filaments, and Microtubules.

Endoplasmic reticulum Transport of materials, protein and lipid synthesis Ribosome Proteins synthesis Golgi apparatus Packaging and secretion of materials for various purposes, lysosome formation Lysosomes Intracelluar digestion Mitochondria Energy production through use of the tricarboxylic acid cycle, electron transport, oxidative hosphorylation, and other path ways Chloroplasts Photosynthesis, trapping light energy and formation of

carbohydrate from CO2 and water Nucleus Repository for genetic information, control center for cell Cell wall and pellicle Strengthen and give shape to the cell Cilia and flagella Cell attachment and Cell movement Vacuole Temporary storage and transport, digestion (food vacuoles), water balance(contractile vacuole)

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Different Shapes of Bacteria

Staphylococcus aureus Streptococcus pyo enes Streptococcus pneumoniae

Klebsiella pneumoniae Vibrio cholerae

Escherichia coli Salmonella

Bordetella pertussis Corynebacterium diphtheriae Helicobacter pylori

Clostridium botulinum Clostridium tetani Neisseria onorrhoeae reponema pallidum

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ICT CORNER

Bacteria

Know the various shapes of Bacteria

STEPS: • Use the URL or scan the QR code to download the Bacteria interactive educational VR 3D app. • Select sphere, rod and spiral to observe the structure of bacteria shapes. • Select ‘structure’ tab and note the internal structure of bacteria. • Click cell wall and note the difference between different shapes.

Step1 Step2 Step3

URL: https://play.google.com/store/apps/ details?id=com.rendernet.bacteria&hl=en

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Summary in all functions. The genetic material of bacteria is DNA and the genes Most prokaryotes have one of three are arranged on larger, circular general shapes coccus (round), bacillus chromosomes. Additional genes are (rod), or spiral, based on the configuration carried on plasmid. Bacterial ribosomes of the cell wall. Two types of spiral cells are dispersed is the cytoplasm in chains are spirochetes and spirlla. Shape and and are also embedded in the cell arrangement of cells are key factors for membrane. describing prokaryotes. Arrangements of cells are based on the number of planes in Bacteria may store nutrients in their which a given species divides. cytoplasm, in form of inclusions. Inclusion vary in structure and the materials that are Cocci can divide in many planes to stored. A few bacteria produce dormant form pairs, chains, packets, or clusters. bodies called endospores, which are the Bacilli divide only in the transverse plane. hardiest of all life forms, survival for If they remain attached, they form chains or hundreds or thousands of years. The genus palisades. Bacillus and Clostridium are spore forming Some bacterial cell walls are covered deadly pathogens. by capsules or slime which protect the cell Eukaryotes are cells with nucleus from phagocytosis, drying and nutrient loss. and membrane bound organelles. Cell Fimbriae and Pili are involved in attachment structures common to most eukaryotes and in transfer of genetic information are the cell membrane, nucleus, vacuoles, between bacterial cells. Flagella are involved mitochondria, endoplasmic reticulum, in cell motility. golgi apparatus and a cytoskeleton. Cell The cell envelope is the complex wall, chloroplast and locomotory organelles boundary structure surrounding a are present in some eukaryote groups. bacterial cell. In Gram negative bacteria, the envelope consists of outer membrane, Evaluation the cell wall, and the cell membrane. Multiple choice questions Gram positive bacteria have only cell wall and cell membrane. Gram positive bacteria 1. The arrangement of flagella on cell have thick murein and teichoic acid. The surface can sometimes help in the cell walls of Gram negative bacteria are identification of an organism for thinner and have wide periplasmic space. example, Escherichia coli to has The outer membrane of Gram negative flagella throughout the cell surface cells contains LPS toxic to mammalian that is referred to as. hosts. The cell membrane is typically a. Lophotrichous composed of phospholipids and proteins, b. Monotrichous and it performs many metabolic functions c. Peritrichous as well as transport activities. d. None of the above The cytoplasm of bacterial cell serve as a solvent for material used

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2. The movement of an organism toward 8. The components of cell wall which or away from a chemical substance in the outer membrane of enteric Gram its environmentis called. negative bacteria to murein layer a. Tracking a. Lignic acid b. Chemotaxis b. Pseudomurein c. Murein c. Tumbling d. Teichoicacid d. Tumbling of none of the above. 9. Endotoxin present in 3. Bacterial cell wall is composed of a. Outer membrane a. Lipid b. Plasma membrane b. Murein c. Murein d. All the above. c. Cellulose 10. has fluidity d. Chitin a. Cell wall 4. Cell wall shows b. Cell membrane a. Semipermeability c. Outer membrane b. Complete permeability d. All the above c. Differential permeability 11. The organelle of prokaryote involved in active cell division. d. Impermeability a. Mesosomes 5. Gram negative differ from Gram b. Mitochondria positive in having c. Ribosomes a. Thick wall d. Endoplasmic reticulum b. Absence of wall lipids 12. The metachromaticgranules are seen c. Complete wall in the bacteria a. Escherichia coli d. Simple wall b. Corynebacterium diptheriae 6. Lipopolysaccharide is found in cell c. Pseudomonas aeroginosa wall of d. Bacillus antharcis a. Gram positive bacteria 13. The extra chromosomal DNA is called b. Gram negative bacteria a. Plasmid c. Both Gram positive and Gram b. Episome negative d. Algae c. Nucleus 7. Cell wall of archaeobateria contain d. Nucleoid a. Peptidoglycan 14. The siderophores has high affinity b. Murein towards. c. Pseudomurein a. Iron d. All the above b. Magnesium c. Chloride d. Copper

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15. The molecular chaperones are 12. List functions of cell wall involved in a. Cell membrane a. Folding of proteins b. Outer membrane b. Folding of carbohydrates 13. Discuss why a cell lyses without cell wall c. Folding of lipids 14. Give the significance of cell d. Folding of fatty aids envelope. 15. What is the role of siderophores? Answer the following 16. Differentiate between capsule and 1. What is Glycocalyx? slime/pili and fimbriae. 2. What is a capsule? What are its 17. Diagrammatically explain structure functions? of cell wall. 3. What is a pilus, what is its function? 18. Differentiate Gram positive and Gram negative bacteria. 4. What is chemotaxis? 19. Explain any five cytoplasmic inclusions 5. Explain the arrangement of flagella 20. Differentiate between Prokaryotes in bacteria with example. and Eukaryotes. 6. Define carboxysomes 7. State volutin granules Student Activity 8. What is called magnetosomes? 1. Students will prepare clay model 9. What is the role of ribosomes of bacteria. involved in protein synthesis? 2. Students will collect the pictures of different Eukaryotic 10. Define periplasmic space. microorganisms. 11. What is LPS composed of?

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A Chapter 8 t king m s stem Linnae s Microbial lantae nimalia Taxonomy i e king m s stem hittaker M nera r tista F ngi lantae nimalia Chapter Outline si king m s stem ese rchae r tista 8.1 Diversity of Living Organisms is bacteria bacteria F ngi lantae nimalia Fascinating three main s stem ese

8.2 Binomial Nomenclature acteria rchaea kar a

8.3 Whittaker’s System of Classification The correct identification of micro organisms is of 8.4 Taxonomic Systems fundamental importance to microbial systematists as well as to scientists involved in many other areas of applied 8.5 The Three Domain System research and industry (Example: agriculture, clinical 8.6 The Past and Present Status of microbiology and food production). Bacterial Taxonomy

Learning Objectives the characteristics of different organisms to identify and group them. To understand After studying this chapter the student life, it is essential to understand taxonomy. will be able, The method of grouping related organisms is the basis of classification (Figure 8.1). • To understand the concept of The objectives of taxonomy are: taxonomy, taxon and phylogeny. • To appreciate the contribution of • To establish the criteria for identifying Linnaeus and Whittaker. organisms • To learn the characteristics of • To arrange related organisms into Kingdom Monera, Protista, Fungi, groups Plantae and Animalia. • To provide evolutionary information • To know some special methods used of the organism in classification of microorganisms. The system of naming living organism is called Nomenclature. 8.1 Diversity of Living Organisms is Fascinating Classification The branch of science which deals with the classification, nomenclature and identification of all living organisms is called Taxonomy. (Greek taxis means arrangements and nomos means law or to Identification Nomenclature distribute). Because of large number and great diversity of organisms, biologists use Figure 8.1: The three facets of taxonomy

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8.2 Binomial Nomenclature A variant strain that differ physiologically and biologically from other Swedish botanist Carolus Linnaeus in strains in a particular species is called as 1735 introduced a formal system of “Biovar”. Variations in a species is biological classification which divided all living in nature. One biovar in a species may organism into two kingdoms-Animalia grow on sucrose, while another cannot. If and Plantae. He introduced “two name” the biovars are very similar except for one system, the first name, genus and second property, they belong to the same genus name species. The name often gives and species, though vary in biological information on something special about growth properties. it. Taxa (the basic taxonomic group) are constructed from strains which are The strain that differ morphologically successions of cultures derived from are called as Morphovar or Morphotypes. an initial colony. The basic taxonomic Serovars or Serotypes are those strains group is called the species (a collection that differ in their antigenic properties. of strains having similar characteristics). It refers to immunological variations in a The special bacterial strain which is species. An example of differing serovars the permanent reference specimen for is Salmonella. Cell surface of Salmonella the species is called the “type strain” varies slightly from one serovar to (Figure 8.2). another. Because of this cell surface change, a person who has been infected HOTS by or become resistant or immune to one serovar will not be immune to a second Why is type strain refered as the most type, because the immune system cannot important strain in a bacterial species? recognize a similar bacterium with a new surface cover.

Figure 8.2: Taxonomic hierarchy-an example of hierarchy in microbial taxonomy

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8.3 Whittaker’s System of Classification Infobits It is the five kingdom classification. In The Microbial Type Culture the 20th century, advances in cell biology Collection and Gene Bank (MTCC), a and interest in evolutionary biology national facility established in 1986 is led scientists to question the two or funded jointly by the Department of three-kingdom classification schemes. In Biotechnology (DBT) and the Council 1969, Robert H. Whittaker proposed a of Scientific and Industrial Research system which recognizes five kingdoms of (CSIR), Government of India. The living things: Monera (Bacteria), Protista, MTCC, housed at the Institute of Fungi, Plantae and Animalia (Table 8.1). Microbial Technology (IMTECH), Whittaker’s system of classification is Chandigarh, has established itself based on 1) complexity of cell structure as a distinguished culture collection 2) mode of nutrition 3) body organization centre for microbial resources in 4) phylogenetic or evolutionary relationship. India. It is an affiliate member of Monera: This kingdom includes the World Federation for Culture all prokaryotic organisms. Unicellular Collections (WFCC) and is registered microorganism such as Mycoplasma, with the World Data Centre for Bacteria, Actinomycetes and Cyanobacteria Microorganisms (WDCM). The main are grouped under kingdom Monera. objectives of this national facility are to act as a depository, to supply authentic Phylogeny is microbial cultures and to provide the evolutionary related services to the scientists history of organisms working in research institutions, that refer to the universities and industries. relationship between life forms.

Table 8.1 Properties of the five kingdoms Kingdom Monera Protista Fungi Plantae Animalia Cell type Prokaryotic Eukaryotic Eukaryotic Eukaryotic Eukaryotic Cell unicellular unicellular Multicellular Multicellular Multicellular organization and unicellular Cell Wall Present in most Present in Present Present Absent some absent in others Nutritional Phototrophic, Heterotrophic Heterotrophic Phototrophic Heterotrophic heterotrophicor and Class chemoautotrophic phototrophic Mode of Absorptive Absorptive or Absorptive Mostly Mostly nutrition ingestive Absorptive ingestive

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organisms have typical eukaryotic cell Infobits organization. Hints of life: Fungi: This kingdom includes non green, non photosynthetic eukaryotic fungi. The Precambrian was the age molds, mushroom, toad stools, puffballs of microorganisms. They were and bracket fungi are grouped under this macroscopically expressed in a kingdom. They are multicellular and consist colonial structure called stromatolite. of specialized eukaryotic cells arranged in a It is a layer produced by live or fossilized filamentous form. mats of photosynthetic prokaryotes (cyanobacteria) associated with warm Plantae: It includes all multicellular lagoons or hot springs. The ancient plants of land and water. They use stromalite belongs to anoxygenic photosynthesis to synthesize their organic phototrophic filamentous bacteria molecules. and modern stromalite belongs to Animalia: This kingdom includes all oxygenic photo trophic cyanobacteria. multicellular eukaryotic animals. They are also referred to as Metazoans. Animals Protista: This kingdom includes ingest their food through one of any eukaryotic unicellular Protozoans, slime ingestion portal and then use digestive molds and algae. The kingdom is made enzymes to break food particles into up of more than 250000 species. These absorbable fragments (Figure 8.3).

kar tic kar tic kar tic m lticell tar m lticell tar m lticell lar h t s nth si ingest abs rb n nm tile m tile n nm tile se al se al se al

nimals lants F ngi

kar tic nicell lar r m lticell lar abs rb ingest r h t s nthesi e r tists se al an ase al

r kar tic nicell lar abs rb r h t s nthesi e m tile r nnm tile M nera ase al

Figure 8.3: Whittaker’s Five Kingdom classification

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8.4 Taxonomic Systems are subject to rapid change due to change in environment. In classifying prokaryotes, Classical Taxonomy metabolic reactions, genetic relatedness Classical taxonomy is a method of and other specialized properties are used classification based on morphology, (Figure 8.4). physiology, biochemical and ecological characteristics of the microorganisms. HOTS • Morphological Characteristics: The structural characteristics are the usual If two microorganisms have an tools which help in classification. Cell identical mol% G+C value for their morphology gives little information DNA, are they necessarily related? about phylogenetic relationship. The Explain first step in identification of bacteria is If two micro organisms have very differential staining. different mol% G+C values for their • Physiological and metabolic DNA, are they necessarily unrelated? characteristics: These characteristics Explain are useful because they are directly related to nature and activity of microbial Numerical Taxonomy enzymes and transport protein. Since The objective classification system proteins are gene products, analysis deals with the grouping by numerical of these characteristics provides an methods of taxonomic units based indirect comparison of microbial on their character and does not use genomes. subjective evaluation of their properties. • Biochemical characteristics: Enzy- To be more objective about grouping matic activities are widely used to bacteria, the scientists determine many differentiate bacteria. Bacteria can characteristics (usually 100 to 200) be separated into separate species by for each strain studied, giving equal various biochemical tests. Example: weightage for each character. Then Carbohydrate fermentation ability of by using computer %similarity is bacteria. calculated (%S of each strain to every • Ecological characteristics: Many other strain). For any two strains, this is properties are ecological in nature NS %S 5 since they alter the relation of NS 1 ND microorganism to their environment. where, NS is the number of Microorganisms living in various parts characteristics that are the same (positive of the human body markedly differ or negative) for the two strains, and ND from one another and from those is the number of characteristics that are growing in freshwater, terrestrial and different. Those strains having a high marine environments. %S to each other are placed into groups; Prokaryotes have only a few structural and those groups having a high %S to characteristics and these characteristics each other are in turn placed into larger 117

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is ersi n il ti n e tracti n

n selecti e S r gene am li icati n Selecti e agars ith ni ersal rimers

Inc bate n er a r riate l ning artial atm s heric c n iti ns se encing r ari s times Search r hmlg ln c nt in atabase

nstr cti n seci ic I enti icati n scheme r bes r sbse ent anal sis Figure 8.4: General scheme for classification and identification in microbial taxonomy groups. Numerical taxonomy also yields Molecular Taxonomy classification that has a high degree of Molecular techniques in stability and predictability. the field of biology has helped to understand In Numerical Taxonomy, genetic relationship which was defined by between the numbers Peter H.A.Sneath and of different taxonomic Robert Sokal, each categories. characteristic is given equal weightage DNA and protein sequencing, and it is converted into numerical form immunological methods, DNA-DNA or and compared by means of a computer. DNA-RNA hybridization methods are Atleast 50 and preferably several very helpful in studying different species. hundred characterictics are compared. The data or information from such The presence and absence of studies are used to construct phylogenetic selected characters in the group of tree (a branching diagram showing the organism is calculated by simple evolutionary relationship among various matching coefficient (SSM), called biological species based on similarities Jaccard coefficient. and difference in their physical or genetic characteristics).

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A classification technique that is identification & taxonomic classification widely used is DNA base composition of microbial species. which is expressed as the percentage of Guanine plus Cytosine (G+C). It is 8.5 The Three Domain System a fixed property that reveals the degree This system of classification was introduced of species relatedness. Ribosomal by C.Woese, O. Kandler and M.L.Wheelis, RNA sequencing is used to determine is an evolutionary model of phylogeny the diversity of organisms and the based on cells rRNA sequences(differences phylogenetic relationship. Basically in the sequences of nucleotides) studies. ribosomes consists of two subunits, each They grouped all living organisms into of which is composed of protein and a three domains: Bacteria, Archaea and type of RNA. Specific base sequences Eukaryota (Figure 8.5). called as signature sequences are found Bacteria and Archaea are two different in all groups of organisms. These unique groups of prokaryotes. The domain DNA sequences are 5-10 bases long and Bacteria comprise the vast majority found in 16s rRNA location and unique to of prokaryotes. The domain Archaea major groups of prokaryotic organisms. contains prokaryotes that live mostly Nucleic acid based detection methods in extreme environments. The domain help in the detection of genomic materials. Eukaryota contains living organisms that The 16s rRNA gene sequencing has been includes Kingdom Protista, Kingdom established as the “gold standard” for Fungi, Kingdom Plantae and Kingdom

Figure 8.5: A phylogenetic tree based on rRNA analysis. Organisms are classified into three domains: Bacteria, Archaea and Eukaryotes as proposed by Carl Woese et al.

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Animalia. This system of classification is 8.6 The Past and Present State of currently accepted by most biologist. Bacterial Taxonomy The three domain system is based The first classification scheme for on the current state of knowledge. As bacteria was published in 1773 based on knowledge of organisms increase in the morphological characteristics. One of the future, classification will undoubtedly unique, broadscope and widely accepted continue to change. classification scheme was published in 1927 by David Bergey & colleagues is Bergey’s Manual of Determinative Infobits Bacteriology. The Bergey’s Manual of Systematic It provides identification schemes for Bacteriology’s first edition was identifying Bacteria and Archae based on published initially in four volumes. their morphology, differential staining Volume 1 included Gram negative and biochemical tests. Whereas in 1984, a bacteria of general, medical or industrial more detailed work was entitled. Bergey’s importance, Volume 2 included manual of Systematic Bacteriology Gram positive bacteria other than provides information on Bacteria and actinomycetes, Volume 3 included Archaea based on rRNA sequencing. Cyanobacteria, Archaebacteria and The classification in Bergey’s Manual is remaining Gram negative bacteria and accepted by the most microbiologists Volume 4 included Actinomycetes. as the best consensus for prokaryotic taxonomy. The current grouping edition 2 (2012) has five volumes based on 16S The present classification scheme based rRNA sequencing: on genetic relatedness has more practical value. This is expected to provide greater Volume 1 (2001) includes stability and predictability. It would lead Archaea and the deeply branching and to improved identification schemes, and phototrophic Bacteria. to aid our understanding of the origin of Volume 2 (2005) includes Proteobac- present day genera and species. teria. Volume 3 (2009) includes Firmicutes. Summary Volume 4 (2011) includes The branch of science which deals with Bacteroidetes, Spirochaetes, Tenericutes the classification, nomenclature and (Mollicutes), Acidobacteria, identification of all living organisms is called Fibrobacteres, Fusobacteria, taxonomy. The system of naming living Dictyoglomi, Gemmatimonadetes, organisms is called as nomenclature. Carolus Lentisphaerae, Verrucomicrobia, Linnaeus divided all living organisms into Chlamydiae, and Planctomycetes. two kingdoms- Animalia and Plantae. Volume 5 (in two parts) (2012) He introduced Binomial Nomenclature includes Actinobacteria. for naming living organisms. Whittaker

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proposed five kingdom classification based linked to its environment and the on various properties of living organisms. correlation has to be established by Currently accepted classification proposed description of environmental parameters by Woese, Kandler and Wheelis is the three whenever sampling is carried out. It is domain classification. Modern developments also important to study the phenotypic of sequencing technologies and recognition characteristics and link them to the of rDNA sequences are of now cornerstone observations obtained from genotyping for identification purposes. techniques. The link between habitat Overall, it is important to recognize and diversity then becomes easier to that microbial diversity is very much understand for future studies.

Student Activity The student must understand the characteristics of each domain under the five kingdom classification and fill in the chart below.

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Evaluation 5. Binomial nomenclature means writing the name of microorganism Multiple choice questions in two words is 1. Which of the following is a reason- a. Order and family able representation of phylogenetic b. Family and genus diversity? c. Species and variety a. The Chain of Being d. Genus and species b. The Ladder of Life c. The 5-Kingdom Tree Answer the following d. The 3-Domain Tree 1. Define: Taxonomy and what is the 2. Microorganisms belonging to the here inter related parts of taxonomy? same are expected to have the most 2. Define: Classification, Nomenclature characteristics in common with and Identification. eachother. a. Order 3. What is meant by binomial system? b. Species 4. Who developed the Bionomial system in the year? c. Family 5. What is taxonomic rank and why are d. Kingdom we using this? 3. What was the first and most useful 6. What is the difference between microscopic tests for classifying biovars, serovars and morphovars? bacteria? a. Gramstain 7. What is type strain and why it is called as type strain? b. Flagellar stain 8. Write down the techniques which c. Simple stain are used to identify the taxonomic d. Capsular stain characters of an organism? 4. Which of the following is the 9. Explain in detail about the molecular arrangement of organisum into characteristics which are used to groups or taxa? identify the taxonomic orders? a. Nomenclature 10. What are the five kingdom classifica- b. Identification tions? c. Systematics d. Classification

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Chapter 9 Environmental Microbiology

Chapter Outline

9.1 Interrelationship with other Fields 9.2 Air Microbiology 9.3 Microbiology of Water 9.4 Water Pollution and Microbial Microorganisms are found in every corner of the earth Contamination from miles below the surface to boiling hot springs to Antartic 9.5 Sewage Treatment ice. Every ecosystem on earth contains microorganisms thay occupy unique niches based on their specific metabolic 9.6 Recycling of Treated Sewage properties. 9.7 Composting 9.8 Biogas Production

Learning Objectives biotic and abiotic environments. Microorganisms in the environment After studying this chapter the student are diverse in origin and ubiquitous. will be able, Environmental Microbiology involves the study of the applied effects of • To gain knowledge of various layers of microorganisms on the environment and atmosphere and micro fauna of air. on human activity, health and welfare. • To understand air pollution and air It was in the 1970s that a new area of borne diseases. Microbiology emerged and developed into • To learn water borne diseases and the field of Environmental Microbiology. water treatment procedures. The initial focus was on water quality • To know Eutrophication. pathogens in the environment in the • To know composting techniques. context of public health safety. The • To gain knowledge of biogas developing field of environmental production. microbiology expanded to several other areas of applied research. These include microbial interactions with chemical Environmental Microbiology is the field pollutants in the environment and the of science that examines the relationship use of microorganisms for resource between microorganisms and their production and resource recovery.

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Chemical pollutants in soil and 9.1 Interrelationship with ground water have pronounced effects on other Fields human population. The cost of cleanup Since environmental microorganisms or remediation of the contaminated affect so many aspects of life and are easily sites is too high. The overall objective of transported between environments, this chapter is to define the important the field of Environmental Microbiology microbes involved in environmental interfaces with a number of subspecialities. microbiology, the nature of the different It includes soil, aquatic, aeromicrobiology, possible environments in which the as well as bioremediation, Water microbes are situated, the methodologies quality, Occupational health, Infection used to monitor the microbes and their control, Food safety and Industrial activities and finally the possible effects of Microbiology. the microbes on human activities.

Hazardous waste Bioremediation Aeromicrobiology Aquatic Microbiology

Biotechnology Water Quality

Environmental Microbiology Soil Diagnostic Microbiology Microbiology

Industrial Microbiology Food safety

Occupational health/ infection control

Modern environmental microbiology discovery and identification of new has a much wider scope. There are microbes and their products that may many different fields which recognises have practical application for protection the problems in various fileds of of environment and human health and Environmental Microbiology. It includes commercial applications.

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9.2 Air Microbiology Microorganisms are frequently found in the lower portion of the troposphere, Earth’s environment is endowed with where they are dispersed by the air atmosphere, hydrosphere, lithosphere and currents. Most of the microbes present biosphere. In the 1930s, F.C. Meier coined in the troposphere are either spore the term “Aerobiology”. Air is a natural formers or microbes that are easily resource and is fundamental to human life dispersed in air. The stratosphere has a as it makes breathing possible. Its universal temperature range of -80°C to -10°C. presence and requirement for the survival The temperature in the stratosphere can of human beings make air an important reach a maximum of several thousand environmental factor. Air contains degrees. Microorganisms are not found significant number of microorganisms. It in the upper regions of the atmosphere also acts as a medium for the transmission because of the temperature extremes, of microorganisms including bacteria, scarcity of available oxygen, absence viruses, fungi, yeast and protozoa. Air borne of nutrients and moisture, and low transmission is one of the important atmospheric pressure. The relatively low modes for the transmission of infectious humidity in the atmosphere (especially diseases. Aeromicrobiology is the study of during daylight hours) and UV rays from air borne microorganisms. the sun, limit the types and number of Infobits microorganisms that are able to survive in this part of the biosphere.

Miquel (1883) and Carnally & s here colleagues (1887) carried out the herm s here most systematic studies in airborne Mes s here km Strat s here microorganisms. km r s here km km 9.2.1 Layers of Atmosphere km Earth’s atmosphere is divided into five major layers (Figure 9.1) they are: • Exosphere : 700 to 10,000 km Figure 9.1: Diagram showing layers of • Thermosphere : 80 to 700 km atmosphere • Mesosphere : 50 to 80 km • Stratosphere : 12 to 50 km 9.2.2 Composition of Air • Troposphere : 0 to 12 km The air in our atmosphere is composed The air in the troposphere, the of different gaseous molecules. The most layer close to the earth is constantly in common gases are nitrogen (78%), CO2 motion and the temperature decreases (0.034%) oxygen (21%) argon (1%) and with increasing altitude, reaching a other molecules in trace level are present low of -57°C at the apex of this region. in the atmosphere.

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Table 9.1: Composition of Air Outdoor Microflora Gas Symbol Content The air in the exterior environment is called outdoor air and the microbes that Nitrogen N2 78.084% reside there are called outdoor microflora. Oxygen O2 20.947% 99.99% Argon Ar 0.934% Example: Bacillus, Aspergillus. Carbon CO 0.033% 2 9.2.4 Sources of Microorganisms in Air dioxide Neon Ne 18.20 ppm Air is not a natural environment for Helium He 5.20 ppm microorganisms as it doesn’t contain enough moisture and nutrients to support Krypton Kr 1.10 ppm their growth and reproduction. Soil Sulphur SO2 1.00 ppm microorganisms when disturbed by the dioxide blow of wind, gets liberated into air and CH 2.00 ppm Methane 4 remain suspended there for a long period. Hydrogen H2 0.50 ppm Man-made actions like digging, N O 0.50 ppm Nitrogen 2 ploughing the soil may also release oxide soil borne microbes into the air. Xenon Xe 0.09 ppm Microorganisms found in water may also

Ozone O3 0.07 ppm enter air by wind action or tidal action

Nitrogen NO2 0.02 ppm in the form of droplets or aerosols. Air dioxide currents may bring the microorganism

Iodine I2 0.01 ppm from plant or animal surfaces into air. Commensal as well as pathogenic flora 9.2.3 Microflora of Air of the human beings may enter the air by Human beings and animals are continuously activities like coughing, sneezing, talking and inhaling the microbes present in the air that laughing. The microorganisms are discharged cause various infectious diseases. Most of out in different forms of particles which are the respiratory tract infections are acquired grouped on the basis of their relative size by inhaling the air pathogen. The microflora and moisture content. These are aerosols, of air is studied under two categories such as droplets, droplet nuclei and infectious dust. indoor and outdoor microflora. Aerosols Indoor microflora An aerosols are mixture of small liquid or The air found inside the closed solid particles dispresed in air. Aerosols can environment (building/ room) is referred be natural or anthropogenic. Example: Dust as indoor air and the microbes present in and smoke. this region is called indoor microflora. Droplets Example: Staphylococcus, Bacillus, Droplets are formed by sneezing, coughing Penicillium. and talking which consists of saliva and

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mucus (Figure 9.2). Droplets are relatively droplet nuclei. These are 1-4μm in size. large, about 10μm or more in size, and They can remain in air for longer period they tend to settle rapidly in still air. When and transmit various infectious airborne inhaled these droplets are trapped on the diseases. moist surfaces of the respiratory tract. Infectious Dust Thus, the droplets containing pathogenic microorganisms may be a source of Large aerosol droplets settle out rapidly infectious disease. from air on to various surfaces and get dried. Nasal and throat discharges from patient can also contaminate surfaces and become dry. Microorganisms can survive for longer period in dust. This creates a significant hazard, especially in hospital areas.

9.2.5 Air Borne Diseases Many microbial diseases are transmitted through the air (Table 9.2). The incidence of diseases caused by airborne transmission Figure 9.2: Droplets released during sneezing can be reduced by covering one’s nose and mouth during coughing or sneezing and by Droplet nuclei the use of face masks. Small droplets in a warm, dry atmosphere tend to evaporate rapidly and become

Table 9.2: Important airborne human diseases and causative agents (pathogens) Human Diseases Pathogens Bacterial diseases Pulmonary tuberculosis Mycobacterium tuberculosis Pneumonia Klebsiella pneumoniae Streptococcal respiratory infections Streptococcus pyogenes Fungal diseases Aspergillosis Aspergillus fumigatus Cryptococcosis Cryptococcus neoformans Viral diseases Influenza Influenza Virus Common cold Picorna Virus Protozoal diseases Pneumocystosis Pneumocystis carinii

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Nosocomial infection Hospital acquired infection are also known as a nosocomial infection. It is acquired in a hospital or other health care facility. Infection is spread to the susceptible patient in the clinical setting by various means, one of them being air droplets. The infection can originate from another infected patient, staff, or in some cases, the source of the infection cannot be determined. The most common pathogens that cause nosocomial infections are Staphylococcus aureus, Pseudomonas aeruginosa, Figure 9.3: Settle plate technique and Escherichia coli. One of the common showing bacterial and fungal growth nosocomial infections is respiratory pneumonia. Choice of the medium depends upon the kind of microorganisms to 9.2.6 Enumeration of Microorganisms be enumerated. For bacterial isolation in Air Nutrient agar and for fungal isolation There are several methods adopted Sabourauds dextrose agar (SDA) can be to enumerate microorganisms in air. used. The most important methods are solid During sampling it is better to keep the impingement and liquid impingement, plates about one meter above the ground filtration, sedimentation, centrifugation level. Then the plates are uncovered in the electrostatic precipitation. Many tools selected position for the required period have been designed for the collection of time. Immediately after exposure for the of air samples. Choosing an appropriate given period of time the plates are closed sampling device is based on many factors, with the lids. Then the plates are incubated such as availability, cost, volume of air to for 24 hrs at 37°C for aerobic bacteria and be sampled, sampling efficiency and the for 2 days at room temperature (27°C) for environmental conditions under which fungi. Enumeration results are expressed sampling will be conducted. One of the as numbers of viable organisms per unit methods is Settle plate technique, where area of air colony forming unit (CFU/ the microorganism carrying particles mm3). are allowed to settle onto the medium (solid impingement) for a given period HOTS of time and incubated at the optimum temperature (Figure 9.3). It works under Can microorganisms grow in clouds? the principle of gravitational force.

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9.3 Microbiology of Water 9.3.2 Estuaries Aquatic microbiology is the study A partly enclosed coastal body of water of microorganisms and microbial in which fresh water is mixed with seawater communities in water environments. is called an estuary. As a consequence Aquatic environments occupy more than of this geographical location, estuaries 70% of the earth’s surface. Water is essential are of salinity levels that range from less for life and one of the most important than 0.5 to 2.5 grams of dissolved salts natural resource. Thus, protection and per 100 grams of water at their mouths, preservation of aquatic environments are where the water flows into the marine vital for the continuation of life. There are environment. Estuaries are ecologically two kinds of water found on earth: sensitive environments and serve as habitats for fish and wide variety of marine 1. Salt water (97%) life. There is an increasing concern about 2. Fresh water (3%) the quality of life in estuaries seriously damaged by human impact, including 9.3.1 Salt Water Microflora over development and pollution from Salt water contains a significant level of industrial and waste water discharges. The dissolved salts. The major bodies of salt bacterial population in estuaries consists of water are oceans, seas, estuaries, and Pseudomonas, Flavobacterium, and Vibrio, certain salt water lakes. Average salinity as well as enteric organisms. The quantities of ocean is 3.5 grams per 100 grams and types of organisms vary, and depend of water. The pH of salt water remains on the tide, rainfall, salinity, depth and relatively constant at a range of 8.3 to 8.5. temperature. Most of the bacteria found The temperature of seawater fluctuates in water runoff come from animal and depending on location, season and depth.

The hot, sulphur-rich, acidic pool of yellow stone national park (U.S.) is home to many Archaebacteria. The colour differences in the pool result from the different communities of microbes that are able to thrive at extreme water temperatures. Pyrolobus fumarii is a unique Archaebacteria, which is hyperthermophilic that can grow at the temperature of 113°C. Some Archaebacteria live in thousands of miles deep in ocean near superheated volcanic vents.

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Salt resh Brac ish

iver

stuary

Ocean

Figure 9.4: Estuary fowl fecal matter deposited on the ground. Sometimes overflow from sewage systems Barophiles are contributes to these higher number of bacteria that thrive bacteria in the water. Microbiota are deep in the ocean the primary producers in the aquatic and live at pressures ecosystem since they play a major of 400-700 atmosphere but die at role in food chain in water. Drifting 1 atmosphere. A strain MT-41, microbial life of aquatic environment a bacteria is isolated from the marine is called plankton. It is composed of sediments in the Mariana Trench near phytoplankton and zooplankton. The the Phillippines where the depth exceeds bottom region of the water harbours 10 kilometres. This strain has optimum largest number of microorganisms growth at a pressure of 709 bar. called as benthic microorganisms. Microorganisms have been found at all 9.3.3 Freshwater Microflora depths and at all latitudes within the The study of fresh water ecosystem is ocean. Microorganisms are abundant near referred to as Limnology. The major shore regions, where organic pollution bodies of fresh water are rivers, streams, as well as microbial pollution from the swamps, marshes and lakes. The fresh surrounding environment occurs. water ecosystem is divided into lentic Algae are common in these environ- (still water) and lotic (flowing water) ments. Because of their dependence on ecosystems. Lentic ecosystem is divided light as a source of energy, algae occur into zones based on light penetration and primarily in the upper strata of the temperature. They are littoral, limnetic oceans and seas. Although they constitute and profundal zones (Figure 9.5). a vital part of the food chain in the Most lakes are surrounded by rooted marine environments, they also can be a vegetation in a large littoral zone along nuisance and threat to other forms of life. the shore. Light penetrates the shallow Nutrient levels in such environments can littoral and open-water limnetic zone but significantly increase as a result of sewage is unable to reach the profundal zone in plant discharges. Under such conditions the deep portions of many lakes. algal blooms are common.

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As lake vegetation and animal life impart an earthy odour and taste to river decompose, their organic matter provides water. Rivers also receive high concentration a source of nutrients. Lakes that have of bacteria and agricultural chemicals very high concentrations of nutrients through surface runoff water from adjoining (particularly nitrogen and phosphorus) soil during heavy rains and irrigation. In are termed eutrophic and have low oxygen many countries, rivers are heavily polluted concentrations because of extensive with sewage bacteria, especially Escherichia microbial decomposition of organic matter. coli, Enterobacter fecalis, Proteus vulgaris In comparison, the lakes that receive small and other intestinal bacteria. amounts of nutrients and are nutrient Infobits sparse are oligotrophic. Caulobacter grows well in oligotrophic lakes. Red Tides Red tide is a common name for a The common microorganisms phenomenon known as an algal found in fresh water are Pseudomonas, bloom which is caused by a species of Flavobacterium, Serratia, Dinoflagellates (Gymnodinium) and the Chromobacterium, Micrococcus, bloom takes on a red colour due to the Aeromonas and Alcaligenes. presence of photosynthetic pigments As these waters reach the surface or with the production of toxins. The most conspicuous effects of red tides are inland bodies of water, they become associated with mortalities of marine contaminated with different types of species microorganisms. Rivers flowing water (lotic) in close contact with the soil may contain large numbers of soil bacteria (Bacillus, Actinomyces and Streptomyces), Fungi (Polyphagus, Penicillium and Aspergillus), and algae (Microcystis and Nostoc). These microorganisms frequently

ittoral one

imnectic one

Profundal one

Benthic one

Figure 9.5: Light penetration zones of a freshwater lake

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9.3.4 Eutrophication eutrophication is algal blooms. The addition of excess quantities of • When a bloom occurs, the river, lake nutrients to aquatic ecosystems termed or ocean becomes covered with algae, eutrophication often cause damage on which is usually bright green. the communities involved. The sudden • Blue green algae like Microcystis, influx of abundant nutrients along Anabaena, Gonyaulax and other with warm temperatures encourages a Dinoflagellates produce toxins which heavy surface growth of cyanobacteria are poisonous to aquatic lives such as and algae called a bloom (Figure 9.6). fish. This heavy matt of biomass effectively • Algal toxin may also contaminate the shuts off the oxygen supply to the lakes drinking water supply. below. The oxygen content below the • Plankton blooms of green alga create surface is further depleted by aerobic problems of O supply in the water. heterotrophs that actively decompose 2 the organic matter. The lack of oxygen • Musty tastes and odors in drinking greatly disturbs the ecological balance of water are the other effects of the community. It causes massive death Eutrophication. of strict aerobes. Fish invertebrates and • Excessive growth of aquatic weeds anaerobic or facultative microbes will which impair fishing, swimming, survive. Eutrophication is an enrichment boating, shell fish production. of water by nutrients, especially nitrogen • Irrigation canals may become clogged. and phosphorus which are maintained at high levels that causes structural changes Control measures to the eco system. Five different methods have been suggested to control eutrophication they are, Effects of eutrophication 1. Ecological management (control the • Eutrophication can have serious, long flow of nutrients into natural wastes) term effect. The most notable effect of

Figure 9.6: Algal blooms in a polluted eutrophic lake

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2. Advanced treatments Example: the quality of biological life in water. Among Phosphate removal is effected by these are pH, temperature, dissolved oxygen precipitation with lime. concentration and salinity. 3. Chemical algicides Example: copper Potable Water sulphate Clean water free from odour, disagreeable 4. Biological algicides Example: Bacteria taste, harmful chemicals, turbidity and 5. Destratification Example: physical/ microorganisms is called potable water, mixing / forced aeration. which is safe to drink and can use for food preparation without risk of health 9.4 Water Pollution and Microbial problems. Contamination Biological oxygen demand (BOD) Water is polluted by both natural as well as Biological oxygen demand is one of the man-made activities. Polluted water is one common parameter to monitor water quality which consists of undesirable substances and purity. BOD is the amount of dissolved rendering it unfit for drinking and domestic oxygen needed by aerobic organisms to use. break down organic material present in a Sources of water pollutants given water sample at certain temperature 1. Industrial waste over a specific period of time. The amount 2. Sewage waste of decomposable organic material in 3. Mining activities sewage is measured by the biochemical 4. Marine dumping oxygen demand, or BOD. The BOD of the 5. Accidental oil leakage water sample is determined by aerating it, 6. Burning of fossil fuels measuring the amount of oxygen in sample 7. Chemical fertilizers and pesticides before incubation, placing the sample 8. Radio-active waste in a sealed container (BOD bottle) and incubating the container for five days at 20°C. The most prevalent biological During this five day period, microorganisms contaminants in water are microbes, in the water grow and oxidize any organic particularly bacteria and viruses. Most of the materials in it. After incubation period, the bacteria carried by storm runoff originate BOD of the water can be from animal fecal matter. Studies have determined by measuring shown that during storms, the water that the quantity of residual drains off the land and into sewage systems oxygen in the container. also carries large quantity of bacteria and BOD of drinking water chemicals. The chemicals include pesticides should be below 3ppm or applied to lawns, chemical wastes near 3mg/litre. industrial plants, and organic matter deposited o the ground by different sources. Indicator Microorganisms In addition to the chemical and biological Indicator organism are frequently used contaminants, physical properties also affect to monitor bacterial contamination of

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Infobits

Microbes at work: One of the major environmental problems today is hydrocarbon contamination resulting from the activities related to the Petrochemical Iindustry. Accidental releases of petroleum products are of particular concern in the environment. Hydrocarbon components have been known to belong to the family of carcinogens and neurotoxic organic pollutants. Petroleum-based products are the major source of energy for industry and daily life. Leaks and accidental spills occur regularly during the exploration, production, refining, transport, and storage of petroleum and petroleum products. There are the two main approaches to oil spill bioremediation: (a) Bioaugmentation, in which known oil-degrading bacteria are added to supplement the existing microbial population, and (b) Biostimulation, in which the growth of indigenous oil degraders is stimulated by the addition of nutrients or other growth-limiting cosubstrates. Bacteria like Pseudomonas putida and Alcanivorax borkumens are the most active agents in petroleum degradation, and they work as primary degraders of spilled oil in environment. Bioremediation is a potential source for clean and green environment.

he Marine acteri m lcani ora ee s n il

Sea ater lcani ora secretes nat ral em lsi iers hich hel t break il r lets

Oil r let

i im lcani ora at il ater interace nan metres a millimetre

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water. These indicator organisms provide drainage of contaminated wastewater a representative index of the water into surface water or groundwater. The contamination by pathogenic microbes. wastewater treatment is a major element The indicator organisms generally used in of water pollution control. water quality monitoring are those that are Sewage associated with the gastrointestinal tract and fecal matter. The most common group of indicator organisms used in water quality monitoring are the coliforms, bacteria that are Gram negative, aerobic or facultative anaerobic, non-spore forming rods that Water (99.9%) Solid (0.1%) ferment lactose with gas production within 48 hours at 35°C. Examples of coliforms are Escherichia coli, Enterobacter aerogenes and Klebsiella pneumonia. Two analytical procedures were followed to check the Organic Inorganic presence of coliforms in water. They are Most (70%) (30%) Probable Number (MPN) and Membrane Filteration (MF) technique. The number Goal of Sewage Treatment of coliforms per 100ml of water sample is estimated to find the quality of water and its • To convert waste water into a reusable suitability for drinking purposes. In addition resources. to coliforms, coli phages, Clostridia and • To reduce the spread of pathogenic human enteric viruses are also monitored in microorganisms drinking water. • To avoid health hazards while swimming and boating in the water. Waterborne diseases • To prevent the development of Waterborne diseases are posing a serious objectionable colours and tastes threat to health (Table 9.3). The predominant method of wastewater disposal in large cities and towns is discharge 9.5 Sewage Treatment into a body of surface water. Suburban Wastewater treatments also called sewage and rural areas rely more on subsurface treatment which removes the impurities disposal. In either case, wastewater must from wastewater, or sewage, before be purified or treated to some degree in disposal into natural bodies of water. In order to protect both public health and broad terms, water is said to be polluted water quality. Suspended particulates and when it contains enough impurities to biodegradable organics must be removed make it unfit for a particular use, such as to varying extents. Pathogenic bacteria drinking, swimming, or fishing. Water must be destroyed. It may also be necessary pollution is caused primarily by the to remove nitrates, phosphates (plant

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HOTS

List the major oil spills that occurred recently in India and other countries. Is it practically possible to clean up these oil spills using bacteria?

Table 9.3: Waterborne diseases Waterborne diseases Causative agent Symptoms Bacterial diseases Enteric fever Salmonella typhi Fever & enlargement of spleen Cholera Vibrio cholerae Vomiting & watery diarrhea Leptospirosis (Weils Leptospira interrogans High fever, red eyes, muscle pain and Disease) vomiting Viral diseases Infectious Hepatitis Hepatitis A Jaundice, vomiting & abdominal pain Gastroenteritis Rotavirus Diarrhoea, vomiting Poliomyelitis Coxsackie Virus Head ache, neck stiffness, flaccid paralysis. Protozoan diseases Giardiasis Giardia lamblia Chronic diarrhoea, abdominal cramp, fatigue & weight loss Amoebiasis Entamoeba histolytica Stomach pain, bloody stools, fever Meningoencephalitis Naegleria fowleri Ulceration, watery, bloody diarrhoea Treamatode disease Schistosomiasis Schistosoma Drowsy, confusion, head ache, stiff neck, Diarrhoea

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nutrients) to neutralize or remove the sewage through screens (Figure 9.8) industrial wastes and toxic chemicals. The and grit chambers. The screened water degree to which wastewater must be treated is then sent to settling tanks or basin, varies, depending on local environmental where the suspended solids are allowed to conditions and governmental standards. settle as primary sludge. Materials such as oil or grease, which float on the surface, are removed with a skimmer. The liquid Sewage Treatment Methods Sewage treatment defined as an artificial process in which sewage is subjected to remove / alter its constituents to render it less offensive. There are three levels of wastewater treatment (Figure 9.7): primary, secondary, and tertiary (or advanced). 1. Primary treatment 2. Secondary treatment 3. Tertiary / Final treatment Primary treatment Primary treatment removes about 60 percent of total suspended solids and about 35 percent of BOD; dissolved impurities are not removed. It is the physical method which remove large floating and suspended solids from sewage water. Example: papers, leaves, bottles, rocks, pieces of metal or Figure 9.8: Bar Screening in Sewage wood. These objects are removed by passing Treatment

Preliminary Primary Secondary stage ertiary stage stage ( ctivated sludge process) stage

rit Primary ertiary Bar screens eration tan Secondary isinfection chamber clarifier clarifier filter one

ospital influent inal efluent ( a sa age) ir for disposal ecycle sludge

Sludge Primary sludge digestion Secondary sludge tan

Sludge disposal Figure 9.7: Schematic diagram of waste water treatment

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wastewater remaining in the settling tank or recycled into the tank as an inoculums to basin is then ready for secondary treatment. continue the process. The remaining floc The fluid from primary treatment is called is further treated or removed for burial or primary effluent. incineration. Secondary treatment Oxidation ponds (Figure 9.10) are Secondary treatment removes more than used in some communities to handle small 85 percent of both suspended solids and loads of sewage. Small communities and BOD. Secondary treatment involves isolated areas frequently use oxidation the oxidation of the primary effluent by ponds for treatment of waste water. microorganisms. The common types Sewage is channeled into an initial pond processes used are: where the sludge settles out. The liquid portion of the sewage is then pumped 1. Trickling filter process into an adjacent series of ponds where 2. Activated Sludge process aeration allows bacterial growth and 3. Oxidation ponds degradation of organic matter. These secondary ponds often are seeded with A trickling filter consists of a large tank algae which provide oxygen for the or basin filled with a bed of crushed stone, growth of aerobic, heterotrophic bacteria. gravel, slag or other porous material. Sewage is sprayed in a fine mist over the Tertiary treatment rocks. As the sewage trickles through the Tertiary processes can remove 99 percent bed, organic matter clings to the rocks, of all the impurities from sewage. where it is digested by heterotrophic Although effluents from secondary microorganisms (Figure 9.9). The treatment have a low BOD, they may microorganisms are contained within contain eutrophication inducing salts a biofilm which are produced by slime (Phosphorus and Nitrogen compounds), forming bacteria such as Zooglea and the organic and inorganic suspended solids, organic matter, is oxidized to gases and and poorly biodegradable organic inorganic products. materials. Advanced or tertiary treatment In the activated sludge process sewage process are designed to reduce or eliminate is mixed with a slime forming bacteria these materials depends more on physical (Zooglea) in a large aeration tank. As the and chemical processes than biological mixture is aerated, large flocs, or clumps, processes. For phosphorus elimination the form. These clumps contain not only phosphates are converted to poorly soluble the original slime forming bacteria but aluminum, calcium or iron compounds also large population of heterotrophs, and removed by precipitation. Nitrogen which oxidize organic matter within in sewage effluent is removed primarily these clumps. In this system wastewater through nitrification by microorganisms. is continuously pumped into the tank The extent of nitrification during and the treated water is removed into a tertiary treatment depends on adequate holding tank and the flocs are allowed treatment plant designed and the proper to settle. The settled floc material is

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Sprin ler

ilter

eed pipe ir ilter support Outlet Collection

Figure 9.9: Trickling Filter in sewage treatment removal of sludge so that these bacteria replenishing a ground water basin. Water are grown under optimal conditions. recycling offers resource and financial savings. Otherwise, large amounts of nitrogenous Recycled water for landscape irrigation compounds may escape tertiary treatment requires less treatment than recycled water and released in the effluent. Suspended for drinking purpose. Recycled water can solids are eliminated in sewage through satisfy most water demands, as long as it is filtration or sedimentation. Poorly adequately treated to ensure water quality biodegradable substances can be removed appropriate for the use. by the use of specialized microorganisms The residue that accumulates in sewage capable of using them as substrates. treatment plants is called sludge. Treatment Chlorine is frequently added to tertiary and disposal of sewage sludge are major treated effluent to kill any remaining factors in the design and operation of microorganism. all wastewater treatment plants. Two basic goals of treating sludge before final 9.6 Recycling of Treated Sewage disposal are to reduce its volume and to Water recycling is reusing treated stabilize the organic materials. Stabilized wastewater for beneficial purposes such sludge does not have an offensive odour as agricultural and landscape irrigation, and can be handled without causing a industrial processes, toilet flushing, and nuisance or health hazard.

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Sun Carbon O ygen dio ide

ind action

erobic one

lgae O Bacteria CO PO

naerobic one

Organics Organic acids etc. CO C

Figure 9.10: Oxidation Ponds

Sludge Digestion digestion is mediated by microbes. Sludge Treatment of sewage sludge may include digestion is a biological process in which a combination of thickening, digestion, organic solids are decomposed into stable and dewatering processes. Among these substances. Digestion reduces the total mass of solids, destroys pathogens, and makes it

Septic tank is a small scale anaerobic treatment process. It is commonly used in rural areas. It is simple, inexpensive and satisfactory if properly operated. The septic tank consists of an underground sedimentation container into which sewage from a home enter and is retained for a short time. The organic matter in the sewage settles to the bottom of the tank where it is covered by a thin organic film that excludes oxygen. Anaerobic bacteria in the sediment digests the organic matter into simpler chemical compounds and gases. The gases are then discharged through a vent in the tank. Liquids in the tank rise and overflow through an outlet pipe and are distribute in the surrounding soil. As the water trickles through the soil any remaining organic matter is decomposed by aerobic prokaryotes. Septic tank should not be located near water supplies because not all bacterial pathogens are removed by this treatment. Undigested solids in the bottom of the septic tanks must be periodically removed.

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easier to dewater or dry the sludge. Digested its value as a soil conditioner and fertilizer. sludge is inoffensive, having the appearance Since sludge may contain toxic industrial and characteristics of a rich potting soil. chemicals, it is not spread on land where Most large sewage treatment plants use a crops are grown for human consumption. two-stage digestion system in which organics When a suitable site for land disposal is are metabolized by bacteria anaerobically (in not available, as in urban areas, sludge may the absence of oxygen). In the first stage, the be incinerated. Incineration completely sludge, thickened to a dry solids (DS) content evaporates the moisture and converts the of about 5%, is heated and mixed in a closed organic solids into inert ash. The ash must be tank for several days. Acid forming bacteria disposed of, but the reduced volume makes hydrolyze large molecules such as proteins disposal more economical. Air pollution and lipids, breaking them into smaller water- control is a very important consideration soluble molecules, and then ferment those when sewage sludge is incinerated. smaller molecules into various fatty acids. Appropriate air-cleaning devices such as The sludge then flows into a second tank, scrubbers and filters must be used. where the dissolved matter is converted by other bacteria into biogas, a mixture of carbon Benefits of Sewage Treatment dioxide and methane. Methane is combustible • Water recycling has proven to be and is used as a fuel to heat the first digestion effective and successful in creating a tank as well as to generate electricity for the new and reliable water supply, while plant. not compromising on public health. Sludge digestion may also take place • Water recycling can help us find ways aerobically The sludge is vigorously to decrease the diversion of water from aerated in an open tank for about 20 days. sensitive ecosystems Methane gas is not formed in this process. • Water users can supplement their Digested sewage sludge is usually demands by using recycled water. dewatered before disposal. Sludge-drying • Decreases wastewater discharges beds provide the simplest method of • Reduces and prevents water pollution dewatering. A digested sludge slurry is • Recycled water can be further used in spread on an open bed of sand and allowed Thermal power plant (for cooling), to remain until dry. Drying takes place by Municipal use, Irrigation and a combination of evaporation and gravity Agricultural use. drainage through the sand. Infobits Sludge Disposal There is a huge business opportunity The final destination of treated sewage in finding ways to use these waste sludge usually is the land. Dewatered dumps for productive purposes - energy, sludge can be buried underground in organic fertilizer. This requires methods a sanitary landfill. It also may be spread on of dealing with old waste that has been accumulating over the years as opposed to agricultural land in order to make use of new/fresh waste.

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9.7 Composting other resistant materials. It is important that the material be thoroughly mixed Compost is organic matter that has been and kept aerated during this stage. decomposed and recycled as a fertilizer and Example: Bacillus stearothermophilus soil amendment. It is a mass of rotted organic matter made from waste. Example: garbage, 3. Curing stage - The temperature paper, sugarcane trash, paddy straw, aquatic decreases during this final stage and weeds, other agricultural waste. the material being composted is recolonized by mesophillic organisms, Composting is a natural process in which which often produce plant-growth aerobic and anaerobic microorganisms stimulating compounds. decomposes organic matter into valuable manure called as compost. The primary The humification of organic material objective of composting is to convert an is characterized by an increase in unstable material into stable end product concentration of humic acids approximately (Figure 9.11). from 4 to 12 percent, and decreases during the composting process.

ater h acinth se age sl ge etc What should you

Micr rganisms erati n compost? acteria ngi gen etc m sting When selecting mate- O a r r cess rials for your compost O eat pile avoid the following:

Finishe c m st • Wastes that attract pests ms • Diseased / insect ridden plants Figure 9.11: Composting process • Non-biodegradable things The humification of organic material occurs in three stages 1. Mesophilic stage - Mesophilic is the initial stage of decomposition, lasting for about a week, during which sugars and other simple carbohydrates are rapidly metabolized. This is an exothermic process and may cause an increase in temperature by 40°C. Example: Bacillus subtilis Compost bed types 2. Thermophilic stage - Thermophilic is 1. Pit method the second stage, lasting for about two 2. Heap method weeks, during which the temperature may rise to about 50 to 75°C. Such a drastic Pit method increase in temperature is accompanied The compost pits dug in soil with by the decomposition of cellulose and dimension of 3.5m u 2.5m u 1.5m

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Figure 9.12: Pit method Figure 9.13: Heap method (LuBuH). The pits are filled layer by layer are spread in the cattle shed to serve as using green plants and animal excreta. bedding. Trenches are dug with dimension The layering is repeated until the pit is of 10ft u 6ft u 2ft. filled. Finally a layer of mud is plastered Dry wastes with cattle dung and soil on the top of the pit (Figure 9.12). are added in ratio of 4: 2: 1 up to 2 inches layer in composting pit. A moisture Heap method level of about 40-50% is ideal for good In regions with heavy rainfall, the compost composting. Odour and insect problems may be prepared in heaps above the ground can be controlled by covering the piles level and protected by a shed. The pile is with a layer of soil or wood chips. made with dimension of 2m u2m u1.5m The heap is left undisturbed for about 8 (LuBuH) (Figure 9.13). to 9 months. Turning the pile for every 15 Methods of compost preparation days is important for coplete composting 1. Indore method because pile needs a periodic influx of O2. Plant residues, weeds, sugarcane leaves, 2. Bangalore method grass, wood ashes, animal dung, and water Indore method urine soaked mud can also be used as raw This method was developed at Indore, materials for this type of composting. India. In this method organic wastes

Two types of microbes which help in composting process are

Aerobes Anaerobes

which decompose organic matter which decompose organic matter matter in the presence of oxygen in the absence of oxygen

Example: Bacillus subtilis Example: Clostridium thermocellum

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Bangalore method partial sterilization and detoxifies • This method was developed at pollutants. Bangalore, India. It is recommended • Compost material is principally used for as a satisfactory method for disposal the reclamation of drastically disturbed. of town wastes and night soil. Example: mined soil, landscaping and • The compost pits dug in soil with agriculture. dimension of 4.5m u 2.5m u 90cm • Compost finds unrestricted application (LuBuH) in parks and gardens for ornamental • In the Bangalore method of composting, plants, in land reclamation and dry waste material of 25cm thick is highway beautification projects. spread in a pit and a thick suspension 9.8 Biogas Production of cow dung in water is sprinkled over for moistening. Worldwide energy consumption and • A thin layer of dry waste is laid over demand are growing up since past 50 years. the moistened layer With the growth of population, demand for energy is also increasing leading to • The pit is filled alternatively with an uneven supply and distribution of dry layer of material and cow dung resources. Therefore, the requirement of suspension till it rises 0.5m above the sustainable and eco friendly energy in India ground level and plastered with wet to satisfy the energy demand is inevitable. mud and left undisturbed for about Along with the source of sustainable green 4-6 months or till required. energy, biogas production is an alternative • This method saves labour cost because way to produce clean energy through solid there is no need of turning & regular waste management. sprinkling of water. Biogas is a type of renewable energy that Benefits of compost can be produced from decomposition of • Compost improves the quality of soil animal and plant waste. It is composed of 50– hence called as a soil conditioner. 75% methane, 25–50% carbon dioxide, 0–10% nitrogen, 0–3% hydrogen sulphide, 0–1% • Compost contains a variety of the basic hydrogen and traces of other gases. The term nutrients required for healthy growth “anaerobic” suggests that the process occurs of the plant. in the absence of free oxygen and produces • Nitrogen, phosphorous, potassium and CH through decomposition of waste in nature certain micronutrients viz, manganese, 4 and reduces environmental pollution. copper, iron and zinc are found in compost. Biogas generating technology is a favorable dual purpose technology at • The composted product is safe and easy present since used as fuel and fertiliser. to handle, and does not induce nitrogen deficiency in recipient plants by Leftover foods fruit & vegetable wastes nitrogen stabilization in the compost. and cow dung can be subjected to anaerobic digestion for energy production in a variety • It suppresses disease infestation by of ways.

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Coo ing

Inlet ighting

as tan Manure ertili er obar Soil Soil Out let Scum

Compost tan

Figure 9.14(a): Biogas Figure 9.14(b): Biogas Digester production schematic diagram

Production of Biogas acid, lactic acid), alcohols, ammonia Biogas production is carried out in an airtight (from amino acids), carbon dioxide and cylindrical tank called biogas digester Cow hydrogen. Facultative anaerobes and dung is mixed with equal volume of water and hydrogen producing bacteria Example: made into slurry and fed through the inlet Acetovibrio cellulosolvens, Bacteroid of the biogas unit. The digestion proceeds at cellulosolvens are involved. 37°C with sufficent amount of nitrogen and Methanogenic stage phosphorus. The production of biogas sets In this step, obligate anaerobic methane around 40-50 days, under anaerobic conditions. Production of biogas accomplished in 3 stages namely Hydrolysis, Acetogenesis and Particulate organic matter Methanogenesis (carbohydrate, protein, lipid Steps Hydrolysis

Hydrolytic fermentative stage Amino acid, sugar, alcohol, fatty acid In this step, several microbes secrete different enzymes, which cleave the complex Acidogenesis macromolecules into simpler forms. Organisms that are active in a biogas process Intermediary products (volatile fatty acids, acetate, propionate, ethanol, lactate) during the hydrolysis of polysaccharides include various bacterial groups such Acetogenesis as Bacillus, Clostridium, Cellulomonas.

Acetate H2, CO2 Acetogenic stage Methanogenes (rate limiting step) Through various fermentation reactions, Acetoclastic Hydrogenotrophic the products from hydrolysis are converted mainly into various organic acids (acetic Biogas acid, propionic acid, butyric acid, succinic (mainly methane)

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Infobits

Role of microbes in vermicomposting: Recycling organic wastes through Vermiculture Biotechnology (VBT) is being considered an economically viable solution. Earthworms are regarded as natural bioreactors which proliferate along with other microorganisms and provide required conditions for the biodegradation of wastes. Vermicomposting involves bio oxidation and stabilization of organic material through the interactions between earthworms and microorganisms. Worms like to feed on slowly decomposing organic materials like fruit and vegetable scraps. Worms produce castings that contain beneficial microbes and nutrients, which makes a great soil amendment. Worms are very efficient at breaking down food scraps and can eat over half their body weight in organic matter every day. Vermicasting, also called vermicomposting, is the processing of organic wastes through earthworms . It is a natural, odourless, aerobic process, much different from traditional composting. Earthworms ingest waste, then excrete casts – dark, odourless, nutrient- and organically rich, soil mud granules that make an excellent soil conditioner.

producing bacteria produce Methane gas as sulphate. The methanogenic bacteria include the major end product along with Carbon Methanococcus voltae and Methanobacterium dioxide, Hydrogen and traces of other gases. formicum (Figure 9.14 a, b). Methanogenesis has six major pathways, Small scale biogas unit each converting a different substrate into Methane gas. The six major substrates used The biogas production is carried out in are Carbon dioxide, Formic acid, Acetic an air tight cylindrical tank called biogas acid, Methanol, Methylamine, and Dimethyl digester (Figure 9.15).

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in the water environment. The partly enclosed Deenabandhu model coastal body of water in which river water is mixed It is a biogas production with sea water is called an estuary. Lentic zone model popular in India can be divided into different zones based light which means “Friend of the helpless” penetration and temperature. Eutrophication is an enrichment of water by nutrients, especially Applications nitrogen and phosphorus, which makes the 1. Biogas used as fuel overgrowth of the “algal bloom”. Apart from 2. Used to generate electricity microbes and chemicals, pH, temperature, 3. Biogas is used to run any type of heat dissolved oxygen concentration and salinity are engine in order to generate electrical the physical properties that affect the quality of and mechanical power. biological life. 4. Producing high quality fertilizer. BOD is the amount of dissolved oxygen 5. Reducing water and air pollution. needed by aerobic organisms to breakdown Summary organic material present in a given water sample at certain temperature over a specific period Environmental microbiology is the field of of time. Indicator organisms are frequently science that examines the relationship between used to monitor bacterial contamination of microorganism and their biotic and abiotic water. The most common group of indicator environments. organisms used in water quality monitoring Identification of new microbes and are the coliforms. their products have practical application on Waste water treatment are called sewage protecting the environment as well as human treatment which removes the impurities health. Areomicrobiology is the study of from waste water and sewage. Primary, airborne microorganisms and is one of the secondary, tertiary treatment are employed important modes for the transmission of in the sewage treatment process. Trickling infectious diseases. The air in our atmosphere filter, activated sludge, oxidation ponds is composed of different gaseous molecules. are generally used. The dewatered sludge The air present both interior and exterior used as a fertilizer. Compost is a natural of the environment is called indoor air process in which aerobic and anaerobic and outdoor air. The microorganisms are microorganisms decomposes organic matter discharged out in different forms which are in to valuable manure called as compost. grouped on the basis of their relative size and moisture content. They are aerosols, Indore method and Bangalore method are droplet, droplet nuclei and infectious dust. the different methods used in composting. Hospital – acquired infection are also known Biogas is a type of renewable energy that can as nosocomial infection. Solid and liquid be produced from decomposition of animal impingement, filtration, sedimentation, and plant waste. Hydrolytic fermentative stage, centrifugation, electrostatic, precipitation are Acetogenic stage, Methanogenic stage are the used to enumerate microorganisms in air. three stages involved in the Biogas production. Biogas has many applications. The Aquatic Microbiology is the study of microorgansims and microbial communities

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BIO REMEDIATION

Evalution c. Carbon dioxide d. Neon Multiple choice questions 3. are water droplets containing several types of 1. In 1930, the term microorganisms released in to the air aerobiology was coined from the water sources. a. Droplet nuclei by . b. Infectious dust a. F.C.Meier b. Miquel c. Droplet d. Aerosols c. Carnelly and colleagues 4. Hospital acquired infections are d. None of the above otherwise called as 2. The gas molecules which are more in a. Nosocomial infection atmosphere. b. Gastro intestinal infections a. Nitrogen b. Oxygen

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c. Ocular infection 12. is an open tank d. All the above where algal forms are allowed to grow 5. During summer the upper layer of the a. Trickling filter lake in called b. Oxidation pond a. Hypolimnion b. Epilimnion c. Sludge digester c. Lentic d. None of the above d. None of the above 13. Trickling filter is an example for 6. Is the amount of treatment dissolved oxygen needed by aerobic a. Physical b. Chemical organisms to breakdown organic c. Biological d. None of the materials? above a. BOD b. COD 14. Which one of the following is a good c. DOB d. DOC source for making compost? 7. is called as a. Plastic b. Aluminum Indicator organisms foil a. Escherichia coli c. Vegetable peel d. Polythene b. Staphylococcus aureus 15. Algal boom in pond water is called c. Pseudomonas aeruginosa a. Eutrophication d. None of the above b. Acclimatization 8. In which process of treating sewage,99 c. Algalization percent of all the impurities from the d. Green manuring sewage are removed. 16. The most common toxic algal bloom a. Primary treatment process among the following b. Secondary treatment process a. Euglena b. Microcystis c. Tertiary treatment process c. Paramaecium d. Hydra d. None of the above 17. Organism inhabiting the bottom 9. Primary treatment is a method sediment of aquatic environments a. Physical b. Chemical constitute community c. Biological d. All of the above a. Pelagic b. Benthic 10. Activated sludge process is an example c. Abyssopelagic d. Episammon for treatment 18. Study of flora and fauna of fresh water a. Physical b. Chemical a. Ornithology b. Zoology c. Biological d. Composting c. Paleontology d. Limnology 11. Chlorination is an example for 19. A partly enclosed coastal body of treatment water in which river water is mixed a. Physical b. Chemical with sea water c. Biological a. Lake b. Estuary d. None of the above c. Creek d. Bay 20. Chemical agent used for disinfecting water

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a. Glycols b. Chlorine 4. What is the purpose of bacteriological c. Hydrogen peroxide analysis of water? d. None of the above 5. What is potable water? 21. The main component of natural gas 6. What is sewage? is 7. Define sludge. 8. Explain the sewage treatment a. CO2 b. Carbon monoxide processes. c. O d. Methane 2 9. Is compost a fertilizer? 22. Biogas is a mixture of 10. Describe the process of composting? a. Methane, Nitrogen, Hydrogen 11. What is Biogas? b. Methane, Nitrogen, Oxygen 12. What role can biogas play in supplying c. O2, CO2, N2 our energy needs? d. None of these 13. Draw the light penetration zones of a 23. Which compost method is employed fresh water lake. in the regions with heavy rainfall? 14. What is Eutrophication? a. Heap method 15. Write a note on trickling filter. b. Pit method 16. Discuss the air borne diseases. c. Indore method 17. Write in detail about settle plate d. Banglore method technique. 18. Write in detail about biogas Answer the following production processes. 19. Discuss the Indore method of 1. What are properties favour survival of composting in detail. microorganisms in the atmosphere? 20. Discuss the sludge digestion methods. 2. Define Nosocomial infections 21. Explain the benefits of waste water 3. What is droplet nuclei? treatment?

Student Activity 1. Set up a small scale anaerobic digester for anaerobic digestion using cow dung / fruits & vegetable waste. 2. Instruct the students to bring algal bloom sample from their residential area. 3. Visit nearby sewage treatment plant 4. Set up a mini composter using plastic bin/bottles (Ask students to collect rubbish such as fruit and vegetables wastes from their houses and separate degradable and biodegradable waste and build mini composter). Requirements: soda bottle, soil and organic wastes.

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Figure 10.1: Soil Horizons

10.1.2 Soil Horizons of science dealing with study of soil Each type of soil is characterized by the microorganisms and their activities in soil presence of different horizons which can is known as 'Soil Microbiology'. be seen in a soil profile (Figure 10.1). 10.2 Pioneers of Soil Microbiology The formation of soil horizons depends on climate, living organisms, parent rock Scientists studied the microorganisms material, topography and time; all of from water, air and soil. They recognized which control the weathering of rocks. the role of microorganisms in natural processes. They realized the importance 10.1.3 Physical and Chemical of soil microorganisms in growth and Properties of Soil development of plants. Soil Microbiology emerged as a distinct branch of soil science Physical properties of a soil type depends during first half of the 19th century. on the size of particles, soil texture, soil Sergei N. Winogradsky discovered the temperature and soil pH. autotrophic mode of life among bacteria Chemical properties of soil includes and established the microbiological three main components which provides transformation of Nitrogen and Sulphur. nutrients for plant growth. The three He isolated nitrifying bacteria for the first components are the organic matter, the time and demonstrated the role of these derivatives of parent rock materials and bacteria in nitrification (1890). Further he the clay fraction. demonstrated that free-living Clostridium The fertility of soil depends not only pasteurianum could fix atmospheric on its chemical composition, but also on Nitrogen (1893). He developed the the qualitative and quantitative nature of Winogradsky column (Figure 10.2), microorganisms inhabiting it. The branch a self contained ecosystem for studying the

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Cyanobacteria and algae Air Aerobic zone Nonsulfur Micro- photosynthetic Liquid aerophilic zone bacteria (e.g., Rhodomicrobium) (Less anaerobic) Purple photosynthetic Anaerobic zone bacteria (More anaerobic) (e.g., Chromatium) Green photosynthetic Mud mixed with bacteria (e.g., sulfate and carbonate Chlorobium) salts and cellulose or other organics

Figure 10.2: Winogradsky column

Sulphur cycle. Therefore, he is considered • Organic matter as the 'Father of Soil Microbiology'. • Living organisms M. W. Beijerinck (1888) isolated root One gram of soil contains approximately nodule bacteria in pure culture from one million microorganisms. The soil nodules in legumes and named them as has organic matter, soil solution and soil Bacillus radicola. Thus, he is considered air. All these components are affected by as the 'Father of Microbial ecology'. the activities of microorganisms. Soil is Beijerinck and Winogradsky (1890) a constantly changing medium. The soil developed the enrichment culture solution in agricultural soil has ions like K+, ++ ++ ++ + - technique for isolation of soil organisms, Na , Mg , Ca , Fe , S , NO3, SO4, PO4 and proved independently that transformation others. of nitrogen in nature is largely due to These ions are very essential in culture the activities of various groups of soil media. In a fertile soil, these elements microorganisms (1891). Therefore, in mineral form are supplemented by they are considered as 'Pioneers in Soil organic compound, derived from the Bacteriology'. decomposition of animal and plant residues. Thus the soil is an excellent natural 10.3 Components of Soil medium for growth of microorganisms. The soil is composed of five major 10.4 Soil Microorganisms components Soil contain five major groups of • Mineral matter microorganisms. They are Bacteria, • Water Actinomycetes, Fungi, Algae and Protozoa • Air (Table 10.1).

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Table 10.1: Soil Microorganisms with This is because all kinds of organic refuse example are disposed on the soil Soil Microorganisms Examples Many of the soil bacteria perform useful functions like decomposition of organic Bacteria Agrobacterium matter, conversion of soil constituents into Bacillus useful materials, production of antibiotics Clostridium in the soil and biogeochemical cycling of Pseudomonas elements like Carbon, Nitrogen, Phosphorus, Actinomycetes Actinomyces Iron, Sulphur and Manganese. The bacterial Nocardia population of the soil exceed the population Streptomyces of all other groups of microorganisms in Fungi Aspergillus both number and variety. Fusarium Soil Actinomycetes Alternaria Cladosporum The actinomycetes population is present as many as millions per gram of soil. Soil algae Anabaena The most predominant genera present Oscillatoria in the soil are Nocardia, Streptomyces Nostoc and Micromonospora. Actinomycetes Protozoa Colpoda are capable of degrading many complex Nematodes organic substances and therefore Pleurotricha play an important role in building Heteromita soil fertility. One of the most notable Bacteriophages T4 characteristics of the actinomycetes Bacteriophages is their ability to produce antibiotics. Examples: Streptomycin, neomycin, One teaspoon of erythromycin and tetracycline. productive soil can Soil Fungi contain between 100 million and 1 billion Next to bacterial population in soil, bacteria. These living microorganisms fungi dominates in all kinds of soil. It recycle organic material, promoting soil possess filamentous mycelium composed fertility and supporting plant growth. of individual hyphae. All environmental By practicing conservation tillage, factors which influence the distribution of farmers can maintain biodiversity in bacteria and actinomycetes also influence their soil. the fungal flora of soil. The quality and quantity of organic matter present in the soil have a direct influence on the fungal Soil Bacteria numbers in soil. Fungi are dominant in Among the soil microorganisms, bacteria acidic soils because acidic environment is are most dominant group of organisms. not supportive for the existence of either All kinds of bacteria are found in soil. bacteria or actinomycetes.

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adverse soil conditions. The protozoans Soil microbes create prefer certain species of bacteria for their humus: Humus is nutrition. Protozoa are abundant in the the dark organic upper layer of the soil and their numbers matter in soil. The are directly dependent on bacterial humus is formed when dead plant population. and animal matter are decayed by soil microorganisms. Humus has many HOTS nutrients that improve the fertility of soil, Nitrogen being the most important. “Without fungi even death will be Humus helps soil to retain moisture, incomplete” - Pasteur and encourages the formation of soil Justify the statement. structure. Soil organisms promote plant growth. 10.4.1 Factors Influencing Microbial Population in Soil Soil Algae The major factors that influence the microbial Soil algae are ubiquitous in nature wherever community in soil are moisture and sunlight are available. They are visible to the unaided eye in the form • Moisture of green scum on the surface of soils. • pH Numerically, they are not as many as Fungi, • Temperature Bacteria or Actinomycetes. Some of the • Gases common algae in Indian soil are Chlorella, • Organic and inorganic fertilizer Chalmydomonas, Chlorochytrium, • Organic matter of soil Chlorococcum and Oedogonium. • Types of vegetation and growth stages Blue green algae, or Cyanophyceae, • Ploughing are responsible for Nitrogen fixation. • Season The amount of Nitrogen they fix • Depth of soil depends more on physiological and environmental factors rather than the Infobits organism’s abilities. These factors include intensity of sunlight, concentration of Some soil microbes produce a variety of substances that promote plant inorganic and organic Nitrogen sources growth, including Auxins, Gibberellins and ambient temperature and stability. and antibiotics. Soil Protozoa Soil protozoa are unicellular. They are 10.5 Microbial Interactions characterized by a cyst in their life cycle Microorganisms in soil interact with which can help the species to withstand themselves and lead to beneficial and

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harmful relationships (Flowchart 10.1). Symbiosis (mutualism) Some of the interaction and Mutualism is an example of symbiotic interrelationship have been discussed in relationship in which each organisms this connection in Table 10.2. benefits from the association. One type of mutualistic association is involving the Microbial interactions exchange of nutrients, between two species, a phenomenon called syntrophism. Many Beneficial Harmful microorganisms synthesise vitamins and interactions interactions amino acids in excess of their nutritional requirements. Other have a requirement for one or more of these nutrients. Symbiosis Parasitism Symbiosis is an obligatory relationship Commensalism Amensalism between two populations that benefit Protocooperation Competition both the population. Both populations Flowchart 10.1: Microbial interactions live together for mutual benefit. 10.5.1 Beneficial Interactions A B The beneficial interactions such as symbiosis (mutualism) and commensalism are found to operate among the soil inhabitants. Table 10.2: Types of microbial interaction in soil

Interaction Microorganisms A Microorganisms B

Neutralism No effect No effect

Commensalism + No effect

Amensalism No effect -

Mutualism Synergism ++ Protoco-operation Symbiosis

Competition - -

Parasitism + -

Predation + -

+ = positive effect – = negative effect

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The relationship between algae and producing organisms benefit vitamin and fungi that result in the formation of growth factors requiring organisms. lichen is a classical example of mutualistic intermicrobial relationship (Figure 10.3). 10.5.2 Harmful Microbial Interactions Lichens are composed of primary Harmful microbial interaction is otherwise producer, the phycosymbiont (algae) and described as negative interaction or a consumer the mycosymbiont (fungus) antagonistic interaction. Any inhibitory

ichen morphology effect of an organism created by any means to the other organism is known eproductive unit as harmful interaction or antagonistic lgal cells ungal hyphae interaction and the phenomenon of this activity is called antagonism Ammensalism

lgal layer Ammensalism is the phenonmenon where one microbial species is affected by other species, where as other species is ungal hyphae unaffected by first one. Ammensalism is Figure 10.3: Lichen Morphology accomplished by secretions of inhibitory substances such as antibiotics. Certain Commensalism organisms may be of great practical In a commercial relationship between importance, since they often produce two microbial population, one population antibiotics or other inhibitory substances, is benefited and other population which affect the normal growth of other remains unaffected. Commensalism is an organisms. Antagonistic relationships are unidirectional relationship between two quite common in nature. For example: population. The unaffected population Pseudomonas aeruginosa is antagonistic does not benefit by the action of second towards Aspergillus terreus (Figure 10.4). population. For the receiving population, the benefit provided may be essential. In commensalism, the unaffected population modifies the habitat in such a way that another population is benefited. Benefits A B Figure 10.4: Microbial antagonism Benefits Parasitism For example: A population of facultative This is a relationship in which one of the anaerobes utilizes oxygen and creates a population benefits from the other and habitat suitable for the growth of anaerobes. the host is usually harmed. Parasitism In soil, vitamin and growth factor

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is one of the most complex microbial 10.6 Rhizosphere interactions. The line between parasitism In 1904, L.Hiltner for the first time coined and predation is difficult to define. The the term “rhizosphere” to denote the area parasites feed on the cells, tissues or fluids of intense microbiological activity that of another organisms the host, which is extends several millimeters from the root harmed in this process. system of the growing plants. Harms The region which is adjacent to the A B root system is called rhizophere. The Benefits microbial population on and around root system is considerably higher than the root The parasites depends on the host and free soil or non rhizophere soil. This may lives in intimate physical and metabolic be due the availability of nutrients from contact with the host. All types of plants plants root in the form of root nodules, and animals are susceptible to attack by secretion, lysates, mucigel and sloughed microbial parasites. off cells (Figure 10.5).

hi oplame

hi osphere mm Stele ( ylem↑ phloem↓) oot hair

pidermis

Mucigel (plant Corte bacterial mucilage)

ndodermis

oot cap

Plant mucilage

Sloughed root cap cell

Figure 10.5: Root hair and Rhizosphere

The rhizospere region can be divided Rhizophere Effect into two zones. The rhizophere is a zone of increased • Exorhizosphere microbial community as well as microbial • Endorhizophere activities influenced by the root itself. However the root surface is termed as Greater rhizosphere effect is seen “rhizoplane”. with bacteria (R: S values ranging from

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Pathogenic interactions Mutualistic interactions Increased disease response P P traits pathogen decreased biomass nutrient protection induced systemic content resistance

ndophytic microbiome Plant e udates

Microbiome e udates Potential rhi oplane and endophytic microbiome

Bul soil microbiome

Figure 10.6: Effect of Rhizosphere in plant growth

10 to 20 or sometimes more) than with Infobits actinomycetes or fungi (Figure 10.6). From the agronomic point of view, Bioleaching: the abundance of Nitrogen fixing and Soil microorganisms are very closely Phosphate solublising bacteria in the involved as catalytic agents in rhizosphere of crop plants assumes a many geological processes. These natural significance. include mineral formation, mineral It has been reported that amino acid degradation, sedimentation and requiring bacteria exist in the rhizosphere geochemical cycling. In recent in large numbers than in the root free years, a new discipline of mineral soil. The rhizosphere effect improves the science namely bio-hydrometallurgy physiological conditions of the plant and or microbial mining (mining with ultimately result in higher yield. microbes) is rapidly growing. Broadly speaking, bio-hydrometallurgy deals with the application of biotechnology 10.7 Phyllosphere in mining industry. In fact, The term “Phyllosphere” was coined microorganisms can be successfully by the Dutch Microbiologist Ruinen. used for the extraction of metals The leaf surface has been termed as (Example: copper, zinc, cobalt, lead, Phylloplane and the zone on leaves uranium) from low grade ores. Mining inhabited by the microorganisms as with microbes is both economical and Phyllosphere (Figure 10.7). In forest environmental friendly. vegetation, thick microbial epiphytic

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Figure 10.7: Microscopic appearance of Phyllosphere Bacteria

Infobits

PGPB can promote plant growth. The bacteria include those that are free-living, those that form specific symbiotic relationships with plants (Example: Rhizobia and Frankia), bacterial endophytes that can colonize some or a portion of a plant’s interior tissues, and cyanobacteria (blue-green algae). PGPB may promote plant growth directly usually by either facilitating resource acquisition or modulating plant hormone levels, or indirectly by decreasing the inhibitory effects of various pathogenic agents on plant growth and development, that is, by acting as biocontrol bacteria. It is envisioned that in the not too distant future, plant growth-promoting bacteria (PGPB) will begin to replace the use of chemicals in Agriculture, Horticulture, Silviculture, and environmental cleanup strategies. associations exist on leaves. The Pseudobacterium, Phytomonas are also dominant and useful microorganisms on encountered on the leaf surface. The the leaf surfaces in the forest, vegetation age of plant, its leaf spread, morphology happened to be Nitrogen fixing bacteria and maturity level and the atmospheric like Beijerinckia and Azotobacter. factors greatly influence the phyllosphere Apart from Nitrogen fixing bacteria, microflora. other genera such as Pseudomonas,

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IO OBIO MIC M I IC O O B B IC IO

P C S BI O O IO M OB MIC P

S OBIC MIC OBIO

M IC O O BIC BIO

Figure 10.8: Spermosphere

10.8 Spermosphere There are five major components in the soil, that includes mineral matter, water, The region which is adjacent to the air organic matter and living organisms. seed surface is termed as spermosphere The soil environment is unique in (Figure 10.8). Healthy seeds carry specific several ways. It consists of bacteria, bacterial flora in respect to number and fungi, actinomycetes, algae and protozoa. species. There are several reports in the Several factors influences the moisture, literature on the quantity and quality of pH, temperature, organic and inorganic microorganisms carried by the seeds of matter of the soil. different plants species both externally and internally. When the seed is sown in soil, Microorganisms in soil interact certain interactions between the seed borne themselves and lead to both beneficial microflora and the soil microorganisms and harmful interactions. Beneficial take place under the influence of chemicals interaction includes symbiosis and excluded by the germinating seed. commensalism. Harmful microbial interaction includes parasitism. The Summary region adjacent to the root system is called rhizosphere. Bacteria predominate Soil is the outer most covering of the in rhizophere. Soil and their growth is earth. Soil consists of living and non living influenced by nutritional substances components that contribute its fertility. released from plant tissues.

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The leaf surface has been termed 6. Leaf surface has been termed as as phylloplane and the zone on leaves . inhabited by the microorganisms is a. Rhizosplane b. Spermoplane phyllosphere. The region, which is c. Phylloplane d. All the above adjacent to the seed surface is termed as 7. Spermosphere is spermosphere. a. Leaf and microorganisms Evaluation b. Root and microorganisms Multiple choice questions c. Seed and microorganisms d. All the above 1. Example for soil algae a. Anabaena Answer the following b. Oscillatoria 1. What is soil? c. Nostoc 2. Give examples for the soil bacteria? d. All the above 3. What are the types of microbial 2. Lichens are example for interaction? a. Symbiosis 4. What is harmful microbial interac- b. Parasitism tion? c. Commensalism 5. Define rhizophere. d. All the above 6. Define rhizoplane. 3. The relationship between and that result in the 7. Define phyllosphere. formation of lichen 8. Define spermospere. a. Bacteria and virus 9. Explain parasitism. b. Algae and bacteria 10. Explain commensalism. c. Algae and fungi 11. Give examples for the soil Fungi & d. Virus and fungi Actinomycetes. 4. Harmful interaction is otherwise 12. What are the components of soil? called as 13. Mention the different types of soil a. Mutualism microorganism with help of chart. b. Antagonism 14. Explain – factors influencing c. Commensalism microbial population in soil. d. Symbiosis 15. Explain – microbial interaction. 5. first coined the term 16. Write about symbiosis or mutualism. rhizophere 17. Describe rhizosphere. a. L. Hiltner 18. Explain rhizophere effect. b. Ruinen c. Pasteur d. Koch

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Chapter 11 Agricultural Microbiology

Chapter Outline

11.1 Biogeochemical Cycles 11.2 Biofertilizers 11.3 Biopesticides 11.4 Plant Diseases

The Anabaena azollae-Azolla association is a symbiotic relationship between a blue green alga and a rice plant. Azolla is a small, fast growing floating water fern. In the cavities of leaves of azolla, Anabaena azollae a blue green algae (a cyanobacterium) fixes nitrogen from the air.

Learning Objectives and Nitrogen are microbe mediated and play a very significant role in replenishing After studying this chapter the student the nutrient supply in soil. Certain will be able, microorganisms like bacteria, fungi • To understand the various biogeo and viruses are economically important chemical cycle since they cause plant diseases and are • To know the nitrogen fixation responsible for severe crop losses. The process study that deals with plant diseases is called plant pathology. • To infer about the biofertilizer There are microorganisms capable • To learn the role of biopesticides in of producing plant hormones thereby agriculture enhancing the plant growth. Some micro • To discuss the plant diseases organisms can be used as biopesticides as they have ability to kill insects that Agricultural microbiology is a branch of damage plants. microbiology which deals with the study of microorganisms that are involved in 11.1 Biogeochemical Cycles various agricultural processes taking place Biogeochemical cycling is defined as the in soil, plants and agro industries. movement and conversions of materials Agriculturally important processes like by biochemical activities throughout the Biological nitrogen fixation, nutrient air, water and soil. All living organisms cycling of Carbon, Sulphur, Phosphorus participate in biogeochemical cycling but

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microorganisms play a major role. This is Atmospheric CO2, dissolved carbon because of their high enzymatic activity in oceans and freshwater, organic matter and their ubiquity. are actively cycled carbon reservoirs. Most macro elemental components of Sediments and fossil fuels like coal, living organisms like Carbon, Nitrogen, petroleum and natural gas are slowly Sulphur, Oxygen, Phosphorus and cycled carbon reservoirs. Carbon is Hydrogen are cycled most intensely and cycled between these reservoirs by the other elements like Copper, Chromium, biochemical activities of micro organisms Iron, Zinc, Nickel are cycled less intensely. and other living things (Figure 11.1). The different stages or processes 11.1.1 Carbon Cycle involved in carbon cycle are

Carbon is a macro element present in all 1. Photosynthesis living cells. In microorganisms, they are 2. Decomposition present in all macromolecules like cell wall, cytoplasmic membrane, proteins and 3. Methanogenesis nucleic acids. 1. Photosynthesis

Reservoirs of Carbon: It is a process where atmospheric CO2 is

Reservoirs are the storage places of converted to organic carbon (CH2O)n. nutrients that are present in nature. They This is carried out by higher plants, store nutrients in large amounts for longer photosynthetic bacteria, cyanobacteria periods of time. and algae using radiant energy from

Organic compounds eath in dead organisms

eath eeding

Organic compounds Organic compounds Organic compounds in fossil fuels in green plants in consumers espiration ecay and espiration returns CO to cecompositicn the atmosphere CO released as microbes espiration respire CO in the air and Photosynthesis dissolved in ater removes CO from the particularly oceans Burning environment Figure 11.1: A simplified diagram of Carbon cycle is as follows

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the sun. This can be explained by the equation, Infobits Fistulated cow: CO2 + H2O Æ (CH2O)n + O2 Fistulated cows are very useful in where (CH2O)n represents the organic studying rumen microorganisms and form of carbon (Example: Carbohydrates) ruminant nutrition and are used to which gets incorporated into the treat indigestion in cows. Fistula is photosynthetic organisms. This organic a sampling port that allows access to carbon serves as food for herbivores and the rumen. Rumen is the first and in turn for carnivores. the largest chamber of the stomach of 2. Decomposition cows and other ruminant animals. The organic matter fixed as a result of photosynthesis is eventually degraded by microorganisms to CO2 during processes like respiration and decomposition. When aerobic and anaerobic organisms respire,

CO2 is released into the atmosphere. Much of the CO2 is released when dead organisms decompose in the soil predominantly by the activities of soil microorganisms. is needed for the formation of proteins, Burning of fossil fuels also release CO2 into the atmosphere. amino acids, nucleotides and is present in a number of oxidation states inside the cell. 3. Methanogenesis Nitrogen is cycled between atmosphere,

It is an anaerobic process where CO2 gets organic compounds in living things, soil converted to CH4 (methane) by strict and sediments. anaerobes like methanogens (Example: The processes that are involved in Methanobacterium). Methanogens are Nitrogen cycle are a group of Archaebacteria found in anaerobic environments like swamps, 1. Nitrogen fixation marshes, rumen of ruminants, paddy 2. Nitrification fields and gut of termites. 3. Ammonification 4. Denitrification - + H2 + HCO3 + H Æ CH4 + 3H2O Nitrogen fixation Methane is converted back to carbondioxide Nitrogen is present as N2 (N N) in by a process called Methylotrophy. air (78% N2). The triple bonded state of nitrogen makes it very stable and nitrogen 11.1.2 Nitrogen Cycle in its gaseous state cannot be assimilated The element Nitrogen (N) is a key by plants or animals for their metabolism. constituent in microbial cell. Nitrogen Only few groups of prokaryotes are

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capable of breaking the triple bond and Ammonification use it for building up their proteins and The production of ammonia during aminoacids. The process of reduction of the decomposition of organic nitrogen gaseous nitrogen (N2) to ammonia (NH4) compounds, by micro organisms after is called Nitrogen fixation. This process the death of plants and animals is called is carried out by a group of prokaryotes ammonification (Figure 11.2). Much called diazotrophs. of the ammonia released by aerobic decomposition in soil is taken up rapidly N2+8H Æ 2 NH3 + H2 by plants and micro organisms and is converted to amino acids. Cyanobacteria, Rhizobium and Frankia are some of the examples of diazotrophs that Bacteria like Bacillus, Clostridium, can fix atmospheric nitrogen. The fixed Pseudomonas and fungi like Aspergillus, ammonia gets incorporated into proteins Mucor and Penicillium are few examples and amino acids, thus building up organic of micro organisms that can ammonify. nitrogen. Nitrification The oxidation of ammonia (NH ) to nitrate assimilation 3 NH4 Proteins, amino acids (NO3) is called nitrification. It is carried

itrogen in atmosphere ( )

Plants ssimilation

enitrifying bacteria itrogen fi ing itrates bacteria in root (O) nodules of ecomposers legumes (aerobic and anaerobic bacteria and fungi) itrifying bacteria mmonification itrification

mmonium ( ) itrites ( O )

itrogen fi ing soil bacteria itrifying bacteria

Figure 11.2: Simplified diagram of nitrogen cycle

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out by nitrifying bacteria. It is a two step symbiotic association with other plants or process where ammonia is first converted micro organisms and fix N2. to nitrite (NO2) and then to nitrate (NO3). Organisms capable of BNF are Free living 2NH3 + 3O2 Æ 2HNO2 + 2H2O Aerobic Azotobacter The above given oxidation reaction is Cyanobacteria the first step that produces nitrite. This Anaerobic Clostridium reaction provides energy and is carried out by Nitrosomonas and Nitrosococcus. Symbiotic Rhizobium In the second step, the nitrite is Frankia. oxidized to nitrate BNF by Rhizobium in leguminous plants Leguminous plants belong to the family 2HNO + O Æ HNO 2 2 3 Leguminaceae and bear seeds in pods. This reaction is carried out by Example: Black gram, Green peas, Nitrobacter. Soyabean, Subabul. The bacteria belonging to the genus Rhizobium which can exist in Nitrates are readily assimilated by free living state in soil but can enter into plants but are very water soluble and symbiosis with legume plants and carry rapidly leached from soil. out nitrogen fixation. Denitrification Process of BNF - The reduction of NO3 from soils by It consists of the following steps denitrifying bacteria to gaseous nitrogen 1. Infection of legume roots by Rhizobium is called denitrification. In this process, carried out by bacteria like Pseudomonas, 2. Formation of root nodules Thiobacillus denitrificans, organic 3. Reduction of N N to NH4 in root compounds serve as hydrogen donors and nodules nitrates serve as electron acceptor. 1. Infection of legume roots by Rhizobium Rhizobium recognises and attaches to the NO Æ NO Æ N O Æ N 3 2 2 2 root hairs of legume plant. It invades the Biological Nitrogen Fixation root hairs and secretion of certain nod factors result in root hair curling typically One of the most significant biological called Shepherd’s crook symptom which process taking places on the Earth is leads to the formation of infection thread. biological nitrogen fixation (BNF). This Infection thread is a cellulosic tube like fixation of atmospheric nitrogen carried structure through which Rhizobium moves out by few prokaryotes is cost efficient into the cortex from root hairs. because industrial production of ammonia by Haber’s Bosch process is very expensive. 2. Formation of root nodules Organisms carry out BNF in a free The invaded plant cells are stimulated to living state in soil or they can establish divide repeatedly thus forming a mass

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of tissue on the roots which are called root nodules (Figure 11.3). Root nodules are fleshy light pink colored globose structures seen on the roots. The bacteria inside the root nodules transform into swollen, mishappened forms. These are called bacteroids. The bacteroids are capable of nitrogen fixation (Figure 11.4)

3. Reduction of N N to ammonia in root nodules This biochemical process is catalysed by an enzyme called Nitrogenase present in bacteroids and happens under diminished

O2 levels. The O2 levels in the nodules are controlled by an oxygen binding protein called leghemoglobin. This is a red, iron containing protein which can keep the Figure 11.3: Root nodules in the nodule environment free of oxygen. leguminous plant root Nitrogenase The enzyme nitrogenase is a complex 11.1.3 Phosphorus Cycle enzyme consisting of 2 enzymes, The element phosphorus (P) is an essential dinitrogenase reductase and dinitrogenase. macro element in all living organisms. They Electrons from organic compounds like are found in nucleic acids and phosphate pyruvate are passed on to dinitrogenase esters. It is an essential component of ATP reductase first and then to dinitrogenase and other high energy phosphates and which in turn passes them to N N thus phospholipids. reducing it to NH4. This reduction needs 16 ATP, ferredoxin and cytochromes. Reservoirs of Phosphorus 1. Phosphate rock like apatite, a large - + N2 + 8e + 8H Æ 2NH3 + H2 inert reservoir N N + 4H HN=NH + 2H 2. Marine and aquatic sediments

H2N-NH2 + 2H 2NH3 3. Dissolved phosphates in soils and waters 4. Organic phosphates in dead and living HOTS organisms.

Will nitrogen fixation occur in the Phosphorous transformations mostly presence of air? What will be the fate happen as inter conversion of inorganic to of nitrogenase enzyme in aerobic organic phosphate and insoluble form to condition? soluble phosphates.

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rtical cells t hair

hi obium bacteria

t hairs release a s bstance that attracts hi obium

In ecti n threa

hi obium r li erates an ca ses an in ecti n threa t rm

rtical cells begin t i i e

In ecti n threa gr s int the c rte the r t t ha ir

In ecti n threa releases bacterial cells hich bec me bacter i s bacterial cells membrane isa ears in the r t cells t kinins r m bacteria ca se lant c rtical cells t i i e

t n le t ca

t n le rms r m ra i l i i ing in ecte c rtical cells acter i s er rm s mbi tic nitr gen i ati n

acter i s Figure 11.4: Showing stages of formation of root nodules on legume plant

Infobits Phosphate solubilizations Most of phosphates occur in combination The ability of bacteria to solubilise with Calcium, Iron, Magnesium and phosphates can be tested in the Aluminium (inorganic P) and thus are laboratory by streaking the bacterial insoluble and unavailable to plants and culture in Pikovaskaya agar which micro organisms. Some micro organisms contains tricalcium phosphate. Positive solubilize those insoluble phosphates cultures show clear halo around by producing organic acids. Example: growth. Thiobacillus, Bacillus thus enabling the plants to utilize it.

Phosphate assimilation Plants and micro organisms can readily assimilate soluble forms of inorganic - -2 -3 phosphates like H2PO4 , HPO4 and HPO4 and incorporate them as organic forms of phosphates like ATP, nucleic acids.

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Phosphate mineralisation 3. Sulphur reduction Breakdown of organic phosphates to 4. Organic sulphur compound form soluble inorganic phosphates oxidation or reduction is called mineralisation. Organisms 5. Desulfurylation produce phosphatase enzymes and catalyse mineralisation. The mineralised Sulphide/Sulphur oxidation 0 2- phosphates can be utilized by plants (H2S Æ S Æ SO4 ) (Figure 11.5). It is carried out by prokaryotes under aerobic and anaerobic conditions. Under Phytic acid Inositol Phytase aerobic conditions, H2S is spontaneously oxidized at neutral pH to elemental 11.1.4 Sulphur Cycle sulphur. Elemental sulphur is oxidized to Sulphur is present in sulphur containing suphates by chemolithotrophic bacteria aminoacids. The sulphur cycle involves like Thiobacillus, Beggiatoa. oxidation – reduction reaction between If light is available, H2S can be used as

Sulphate (SO4), Elemental S and H2S and electron donor to carry out photosynthesis hence there is change in the valence states of sulphur from -2 to +6. HOTS The basic steps involved in sulphur cycle are If there is no biogeochemical cycle in 1. Sulphide/ sulphur oxidation the ecosystem, What will happen to 2. Sulphate reduction the earth?

plifting of roc Phosphates in organic eathering compounds of roc Phosphates in roc nimals Plants unoff

etritus Phosphates Phosphates in soil in solution (inorganic)

ecomposition etritivores oc Precipitated (solid) phosphates in soil

Figure 11.5: Phosphorus cycle

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under anoxic conditions by phototrophic is called as assimilatory sulphate sulphur bacteria like Chromatium and reduction. The H2S thus produced is Chlorobium . immediately incorporated into organic compounds. Sulphate reduction 0 When sulphate is present in habitats, Sulphur reduction (S ÆH2S): different groups of microorganisms The dissimilative sulphur reducing are capable of carrying out sulphate bacteria can reduce elemental sulphur reduction. to hydrogen sulphide. Example: Beijerinck described the use of sulphate Desulfuromonas, an obligate anaerobe. 2- (SO4 ) as a terminal electron acceptor Under aerobic conditions, organisms like during anaerobic respiration to form Pseudomonas, Proteus and Salmonella are sulphide (H2S). This process is called also capable of performing this reaction. Dissimilatory Sulphate Reduction(DSR). The anerobic bacteria capable of carrying Organic sulphur compounds out DSR are Desulfovibrio, Desulfococcus, reduction/oxidation Desulfotomaculum (Figure 11.6) This Organic sulphur compounds like dimethyl reaction by sulphate reducers requires sulphide can be used as carbon and energy organic carbon sources like pyruvate or source for many microorganisms. lactate. H2S accumulated in such habitats by the action of sulphate reducers is toxic to Desulfurylation aerobic organisms. It is a process where organic sulphur compounds are used up by microorganisms The reduction of sulphate to H S, for 2 for energy to produce H S. building up aminoacids and proteins 2

er bic c n iti ns lemental Be iatoa sl r SO ci e siti n hiobacillus

S Be iatoa SO hiobacillus Micr bial i ati n rning ssil els e cti n b esul o SO ibrio

ssimilati n S b lants an esul uromonas bacteria S slhrl S gr s ec m siti n r teins b micr bes r le an green issimilat r h t tr hic bacteria

S r le an green h t tr hic bacteria naer bic c n iti ns m stl s il an se iments lemental s l r

Figure 11.6: Sulphur cycle

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11.2 Biofertilizers Based on the nutrients that they provide, biofertilizers are of the following types In India, the availability and affordability of fossil fuel based chemical fertilizers Nitrogenous biofertilizers- at the farm level have been ensured • Rhizobium, only through imports and subsidies. • Azotobacter, Indiscriminate and imbalanced use of • Azospirillum chemical fertilizers, especially urea, along with chemical pesticides and unavailability • Frankia of organic manures has led to considerable Phosphate solubilisers reduction in soil health. Biofertilizers can • Bacillus act as a renewable supplement to chemical • VAM fertilizers and organic manures. They have the capacity to produce natural resistance 11.2.1 Rhizobium in plants against pests and soil borne diseases, adding fertility to soil. Rhizobium – legume symbiosis is a well studied plant microbe interaction and Nitrogen fixation by leguminous and Rhizobium is the most extensively used other crops is reported to be 44 million metric nitrogenous biofertilizer in India. tons per annum. The appropriate strain of Rhizobium can increase the crop yield up to Rhizobium is a gram negative, non-spore 10-35%. Also, residual N is beneficial for the forming aerobic bacillus inhabiting the next crops grown in the same field. soil in a free living state. The colonies of Rhizobium on YEMA (Yeast Extract It has been estimated that 40-250 kg Mannitol Agar) plate are gummy, pale white N / hectare(ha) / year is fixed by different in colour (Figure 11.7). They can establish legume crops by the microbial activities of symbiotic relationship with leguminous Rhizobium. plants and fix atmospheric nitrogen thereby Definition greatly improving soil fertility. Biofertilizers are preparations containing beneficial micro organisms like N2 fixers,

PO4 solubilizers in a viable static state intended for seed or soil application and designed to improve soil fertility.

Advantages 1. They reduce the need for chemical fertilizers. 2. They provide the plant with certain vitamins, plant growth promoting substances and increase the vigour of the plant. Figure 11.7: Pale pink mucoid colonies of Rhizobium on Yeast Extract Mannitol 3. It is cheap and cost effective. Agar plate

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Mass production of Rhizobium Method of application of Rhizobium to The flowchart explaining the mass plants production of Rhizobium biofertilizer is Carrier based Rhizobium inoculants are given below mixed with water to form slurry to which the seeds of plants are added (Figure 11.8). The coated seeds are dried in shade and Efficient N fixing strain of Rhizobium 2 used for sowing. are selected and initially grown in YEM broth for 24 hours. 11.2.2 Phosphate Solubilizers Several soil bacteria like Pseudomonas and Bacillus possess the ability to convert Inoculum level is increased from insoluble mineral phosphates into soluble 250 ml to 1000 ml in large flasks form by secreting organic acids thereby making it available to plants. For mass cultivation and inoculant Cultures are then grown in large preparation, the cultures are grown in fermentor for large scale production Pikovaskaya broth for 7-18 days and mixed in suitable carrier like peat or lignite. After curing for a week, the Broth is blended with powdered carrier inoculants are packed and made ready material (peat or lignite) for use in a similar manner as Rhizobium inoculants.

Packing in polythene bags 200g per bag 11.2. 3 VAM Mycorrhiza means fungus root. It describes the symbiotic association Curing at 25°C between plant and fungus. Vesicular Arbuscular Mycorrhiza (VAM) is an endomycorrhiza which is used as a fungal Dispatch to farmers biofertilizer. They mobilize the soluble

ppressorium pidermis at entry point ypodermis Intracellular Intercellular hyphae Corte hypha in air channel

rbuscules esicle

Figure 11.8: Showing the colonization of VAM fungi in root cells of plants

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phosphates in the root zone of plants and satisfy the phosphorus nutrition of plants.

Morphology VAM is an example of endomycorrhiza meaning, the storage organelles of phosphates like vesicles and arbuscles are seen intracellularly. Vesicle is a globose structure and arbuscle is a tree like branching structure present in the root cortical cells Figure 11.10: Microscopic view of (Figure 11.9). VAM fungi are naturally most Anabaena azollae prevalent in angiosperms. gymnosperms, pteridophytes and bryophytes. fixation and photosynthesis. Most of the filamentous forms have specialized large, thick walled cells called heterocysts which are sites of nitrogen fixation. Example: Nostoc, Anabaena is examples for filamentous BGA. Gleocapsa is an example of unicellular BGA. Some of the filamentous forms do not possess heterocysts but still fix atmospheric nitrogen. Since they need standing water for their growth, BGA can effectively colonize paddy fields and enrich the soil Figure 11.9: The fresh water fern Azolla. with nitrogen.

Mass production Mass cultivation of BGA Root based inoculum is used for preparing Applying BGA to paddy fields can reduce VAM biofertilizer (Figure 11.10). The the amount of chemical nitrogenous selected spores of VAM fungi are allowed fertilizer applied for the growth of paddy to infect plants like onion, sorghum and crop. Therefore cultivation of BGA in large other grasses. After 3-4 months, the roots quantities is necessary. Mass cultivation of along with the soil are macerated or BGA has the following steps. pelleted with an inert material and packed 1. Isolation of BGA in polythene pouches which can be used 2. Mass cultivation of BGA as biofertilizer. Isolation of BGA 11.2.4 Cyanobacteria / Blue green BGA can be isolated from soil or paddy Algae fields. Appropriate dilutions from serially Blue green algae are single celled or diluted algal sample are inoculated in filamentous prokaryote capable of nitrogen the liquid flasks containing algal media

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like BG-11 or Pringsheim’s media. After Anabaena azollae (Figure 11.10). The fern several weeks of incubation at 28°C, and the cyanobacteria exhibit symbiotic the individual colonies are picked up, relationship in which Anabaena provides identified and stored. This can be used as the fern with fixed nitrogen and fern starter culture for mass cultivation. Mass provides niche for the cyanobacteria culture can be done in 2 ways. free from competition from other microorganisms. Mass cultivation of BGA Azolla can be used as a nitrogenous 1. Open air shallow culture: biofertilizer for paddy crop. When applied into the paddy fields, Azolla provides Pits are prepared (6΄ × 3΄ × 9΄) nitrogen nutrition to standing rice crop and can reduce the need for synthetic fertilizers. Filled with soil and 200g of superphosphate and water is added Infobits

Mycorrhiza and orchid germination

Starter culture is sprinkled over water In the early stages of their life cycle, all terrestrial orchids are non photosynthetic, totally lacking 1 week chlorophyll and relying on carbon(C) acquired from a fungal symbiont (Mycorrhiza) for growth until the production of the first green leaves Algal scum is formed on the surface above the ground, a nutritional strategy termed mycoheterotrophy. Around 200 species of orchids Water is allowed to dry and the dried remain achlorophyllous throughout up algal flakes are collected and stored their lifetimes. Species such as in poly bags Galeola, Gastrodia, Corallorhiza, Rhizanthella and many others The dried algal flakes around continue to gain carbon from 10kg/ha can be applied in paddy fields mycorrhizal fungi. after transplantation.

11.2.5 Azolla Azolla is a floating freshwater fern. The plant has a branched stem, deeply bilobed leaves which are arranged alternately on the stem and each leaf has a dorsal and ventral lobe (Figure 11.9). The dorsal lobe houses the cyanobacterial symbiont

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Mass multiplication of Azolla detrimental to ecosystem if the usage is prolonged and pests may develop resistance Small plots (50-100 sq.m) or concrete to the pesticides. tanks with standing water are The term biopesticides refers to inoculated with Azolla compounds that are used to manage agricultural pests by means of specific biological effects. It refers to products containing biocontrol agents like natural pH is adjusted to 8.0 with lime. substances such as plants, certain minerals, animals, micro organisms including their genes or metabolites. They are an important part of Integrated Pest Management (IPM) Superphosphate is applied as a nutrient strategy in controlling the pest. and carbofuran is applied to deter insects Advantages They are less toxic to humans and After 20 days, Azolla biomass can be environment and they do not leave harvested and used as biofertilizer for harmful residues. rice plantings They affect only the target pest. They cause long term suppression of Method of application of Azolla in rice pest populations since they persist in the fields environment. Azolla is grown on the flooded rice fields Microbial biopesticides are of three kinds prior to planting for 2-3weeks. Then water 1. Bacterial biopesticide is drained and Azolla is incorporated into 2. Fungal biopesticide the soil followed by rice transplantation within a week’s time. 3. Viral biopesticide 11.3.1 Bacterial Biopesticide HOTS Bacteria like Bacillus thuringiensis, Bacillus papillae and Bacillus lentimorbus Why biofertilizer are preferred to have the potential to kill certain insect chemical fertilizer? pests and are entomopathogenic. Bacillus thuringiensis 11.3 Biopesticides It is a gram positive, spore forming, rod Pests are insects that damage crop plants shaped soil bacterium. During sporulation, and stored products. They feed on leaves the bacterium produces insecticidal and roots or suck the sap of the plants proteins as parasporal crystals. These are causing severe crop losses. Chemical called delta endotoxin also called as Cry pesticides sprayed on plants can be proteins. Cry proteins are specifically

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toxic to insects belonging to Lepidoptera, Various species of Bt are able to work Coleoptera and other few insect orders. against cotton boll worm, cabbage worm Mode of action of Bt and gypsy moths. The Bt cells sprayed on the leaves have to be ingested by the larval forms of the Infobits insects in order to exert its action. This is Photograph of a cotton plant showing because the Bt toxin gets activated in the opened and unopened bolls. BT insect gut at a specific pH. cotton is a genetically modified cotton Process plant (GM crop) which has the gene for the crystal toxin integrated in its Insect ingests the parasporal crystals genome. Crystal toxin is expressed containing Cry protein in plant parts which reduce the need for spraying pesticides. BT cotton is the only GM crop approved for The crystals dissolve in the alkaline commercial cultivation in India. environment of the insect gut 11.3.2 Fungal Biopesticides These entomopathogenic fungi attack The solubilised inactive delta endotoxin are insects and cause diseases in insect body cleaved rendering them active which lead to insect death. Two prominent fungi used as mycopesticide are • Beauveria bassiana which causes white The activated toxin binds to specific muscardine disease receptor of the midgut epithelium • Metarhizium anisopliae which causes green muscardine disease

This leads to the insertion of toxin thereby Mode of action of Beauveria bassiana developing pores in the epithelium Beauveria bassiana, a filamentous fungus belongs to class Deuteromycetes also called imperfect fungi. It can be successfully used Cell lysis and disruption of epithelium releases against Colarado potato beetle, (Figure 11.11) the cell contents leading to insect death. Codling moth and American boll worm. This fungus invades the haemocoel of Symptoms insects through spores. Once the spores attach to the cuticle, it germinates and • Larvae stops feeding the hyphae penetrates the insect cuticle • Larvae becomes sluggish and static (cuticle is the outer membrane of insects) • Water oozes out from the body Penetration is aided by formation of • Larvae dies and falls off the leaf appresorium and penetration peg. The fungi

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secrete chitinases, lipases and proteases genus Baculoviruses contains 3 subgroups. which can dissolve the cuticle. The hyphae • Nuclear Polyhedrosis viruses (NPVs) enter the haemolymph and proliferate • Granulosis viruses (GVs) and colonise the entire insect and release blastospores. Insect death occurs due to • Non occluded viruses nutrient depletion of the haemolymph or by Mode of action of NPV toxaemia by secretion of toxic metabolites. The virus enters the insect body via ingestion by insects and infects the midgut cells by membrane fusion. The NPV uncoat within the nucleus of cells and pass through the intestinal epithelium (Figure 11.12) and establish a systemic infection of the haemocoel.

Figure 11.11: Picture of insect infected Symptoms with Beauveria bassiana Discoloration (larvae turns brown or yellow) Biopesticides • Decomposition or softening of larvae registered in India: • Lethargy 1. Bacillus thuringien- • Infected larvae hang upside down twigs sis var. israelensis • Larvae become swollen with fluid 2. Bacillus thuringiensis var. kurstaki containing virus and eventually die 3. Bacillus thuringiensis var. galleriae turning black in color. 4. Bacillus sphaericus Mass production of NPV 5. Trichoderma viridae NPV are mass produced in laboratory 6. Trichoderma harzianum using suitable larval hosts. The fifth stage 7. Pseudomonas fluorescens larvae are fed with food infected with

11.3.3 Viral Biopesticides Viral insecticides are pathogens that attack insects and other arthropods. Viral pesticides are used to control Lepidopteran larvae like Helicoverpa, Spodoptera sp on Cotton, Corn, Sorghum, tomatoes. Baculoviruses are the commonly used viral biopesticide. They are extremely small and Figure 11.12: Picture of larvae are composed of double stranded DNA. The infected with NPV

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NPV. After 4-5 days, the dead larvae are Rusts collected and macerated. The liquid is It is a plant disease caused by the rust centrifuged and the pellet containing the fungus of the class Uredinomycetes. viruses is suspended in sterile distilled Example: Puccinia sp. The leaves have water. This viral suspension can be used characteristic spots or pustules which are for spraying in the fields. rust colored, yellow or brown that bear spores of the infecting fungi. 11.4 Plant Diseases Smuts The study of the nature, causative agent, This plant disease is caused by a fungus development and control of plant diseases of the order Ustilaginales. The plant parts is called plant pathology. The study of contain black, powdery masses of spores plant diseases is important because man that appear as sooty smudges. is directly and indirectly dependent upon plants for his survival and plants are the Wilts source of food, fibre and drugs. It is due to the deficiency of water in the foliage, which results in the death of Impact of plant diseases on human shoots and large branches. Several fungi welfare like Fusarium, Verticillium cause wilt by History proves us that plant diseases plugging up the xylem and phloem vessels had caused severe famine in the early of the plant. centuries as a result of which millions of people died and was forced to migrate Downy mildew out of their country. Plantations were This disease is characterised by the wiped out and agro industries failed and formation of downy growth (covered shortage of food resulted due to microbial with soft, fine hairs) on the undersurface attack on plants. of the leaves with a corresponding Example: In 1840, late blight disease of yellowish patch on the top of the leaves. potato has caused severe famine in Ireland The causative fungus belongs to the order In 1942, Bengal famine in India was Perenosporales due to Helminthosporium disease of rice The other fungal diseases of plants caused by Helminthosporium oryzae. include Powdery mildews, Rots, Damping The various diseases caused on plants off, Leaf spots (Figure 11.13) and Blights. by micro organisms can be broadly 11.4.2 Bacterial Diseases grouped into the following kinds. The common bacterial diseases of plants 11.4.1 Fungal Diseases are bacterial leaf spot, crown gall and fire blight. Most of the diseases of plants are caused by fungi. They exhaust all the nutrients from Bacterial leaf spot the plants and exhibit a wide variety of Plants that are infected with bacterial symptoms on leaves, stem and inflorescence. leaf spot will develop dark-colored, The common symptoms are given below. water-soaked spots that are accompanied by

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Rust Smut of corn Verticillium wilt

Downy mildew on upper and lower surface of grape leaf Figure 11.13: Various fungal diseases in plants encasing yellowing halos. Continuous rain Crown gall and moisture will cause the coalescence Crown gall is a root and stem disease that of the spots. Severely infected leaves will is most commonly found on woody plants. defoliate prematurely. Bacterial leaf spot is Infected plants will develop smooth, light- a common nuisance of citrus (Figure 11.14) colored galls on its roots and stems. These and stone fruit trees and vegetables, as well formations inhibit the plant’s ability to as other indoor and outdoor foliage plants. transport nutrients and water throughout

Figure 11.14: Showing citrus canker lesions caused by the bacteria Xanthomonas citri

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Figure 11.15: Photograph of a tree Figure 11.16: Fire blight of apple caused showing galls caused by Agrobacterium by Erwinia amylovora the plant. This results in loss of vigor diseases are very host specific, and others of plant which is also accompanied by are general, over many hosts. Symptoms growth stunt and dieback of branch and include stunting, mosaic, ring spots, color twigs (Figure 11.15) Crown gall disease breaks, and distortion. is a soil-borne bacterial disease caused by Common plant viruses are tobacco Agrobacterium sp. mosaic virus (Figure 11.17) Cauliflower mosaic virus, Impatiens Necrotic Spot Virus Fire blight (INSV), tomato spotted wilt virus, ajuga Fire blight is a destructive bacterial disease viruses, and cucumber mosaic virus that is especially threatening to rosebushes and pome fruit trees like apple and pear. Trees and plants that are infected with fire blight will display tan-colored, bacterial ooze near the points of infection (Figure 11.16) The infected areas become necrotic, turn black, wilt and become deformed.

11.4.3 Viral Diseases Viruses are obligate parasites and require a wound to gain entrance to a plant cell. In nature, they depend primarily on biological agents such as nematodes, insects and man for their dissemination. Once duplication starts, the virus is translocated from cell to cell through the plasmodesmata and to distant plant parts by the phloem. Some viral Figure 11.17: Photograph showing tobacco leaves affected with TMV

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Late blight of potato two flagella) with one tinsel flagellum Causative organism: Phytophthora infestans directed anteriorly and one whiplash flagellum directed posteriorly. After Late blight is the disease that swimming on the surface of the host plant triggered the Irish potato famine of the surface, zoospores encyst and infect the 1840s which resulted in the death and plant. During sexual reproduction, mating starvation of 1 million people. It also between opposite mating types happen. was the first plant disease for which a A nucleus from the antheridium (Male) microorganism was proved to be the enters the oogonium (female). Following causal agent, leading to the birth of plant karyogamy (the fusion of two nuclei), a pathology as a science (Figure 11.18) thick-walled, diploid oospore is formed. Phytophthora infestans is a member From oospore, sporangium develops and of the oomycetes, a group of organisms the life cycle (Figure 11.19) continues. sometimes referred to as the “water molds”. During asexual reproduction, Lesions on stems and leaves Phytophthora infestans produces sporangia Late blight of potato is identified by black/ on sporangiophores. In cool, wet brown lesions on leaves and stems that may conditions, zoospores will form and emerge be small at first and appear water soaked from the sporangia after about two hours. or have chlorotic borders, but soon In warmer conditions, sporangia may expand rapidly and become necrotic. function as a single spore and germinate In humid conditions, Phytophthora directly. Zoospores are biflagellate (have infestans produces sporangia and

Symptoms

Figure 11.18: Photograph showing blight lesions on potato leaves, stem and tuber.

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S rangi m sres

S rangi m S r lati n rm ng lant in s ring erm t be

Os re

S r lati n n lea

Ognim ntheri i m

S rangi m Se al re r cti n I cc rs nl hen b th mating t es resent

In ecte lant his is a sim li ie isease c cle r late blight tat Figure 11.19: Disease cycle for late blight of potato sporangiophores on the surface of infected can occur from 3-26°C, but the optimum tissue. This sporulation results in a visible range is 18-22°C. Sporangia germinate white growth at the leading edge of lesions directly via a germ tube at 21-26°C. on abaxial (lower) surfaces of leaves. As many lesions accumulate, the entire plant Control can be destroyed in only a few days after Fungicide applications are an important the first lesions are observed means of late blight management, Lesions on tubers particularly in humid areas. They must be Tuber infection symptoms are a darker applied before plants are exposed to spores. brown sometimes purplish area on Copper hydroxide (2g/lit), chlorothalonil the tuber surface. The internal rot is a (2g/lit) and mancozeb (2.5g/lit) fungicides reddish brown granular rot which can are the standard protectants used for remain close to the surface or progress to control. they are usually applied every the centre of the tuber. Rot development seven to ten days for best protection. is irregular and sometimes threadlike through the tuber flesh. Late blight causes Summary skin damage which allows opportunistic Carbondioxide fixation and Biological bacteria to invade and cause soft rotting. Nitrogen Fixation are the most significant biological processes taking place on planet Epidemiology Earth. Methanogenesis is an anaerobic Temperature and moisture are the most process converting CO2 to CH 4. It is carried out by methanogens like Methanobacterium important environmental factors affecting sp. Phosphorous transformations mostly late blight development. Sporangia are formed happen as inter conversion of inorganic to on the lower leaf surfaces and infected stems organic phosphate and insoluble form to when relative humidity is < 90%. Sporulation

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soluble phosphates.Purple and green sulphur Green algae are prokaryotes that can perform bacteria store sulphur as granules as a result both photosynthesis and nitrogen fixation. of which they appear yellow in colour. Biopesticides refer to compounds that Biofertilizers are preparations are used to manage agricultural pests by containing beneficial micro organisms like means of specific biological effects. Bacillus N2 fixers, PO4 solubilizers in a viable static thuringiensis produces crystal toxin which state intended for seed or soil application is detrimental to insects. Fungal and viral and designed to improve soil fertility. biopesticides establish systemic infection Azolla and Anabaena share symbiotic in the body of insects to kill them. Smuts, relationship in which Anabaena provides rusts, downy and powdery mildews are fixed atmospheric nitrogen to Azolla. some of the important plant diseases Vesicles and arbuscles in VAM fungi are caused by fungi. the storage organelles of polyphosphates.Blue

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Evaluation 7. The other name for Blue Green Algae is a. Green algae Multiple choice questions b. Brown algae 1. The conversion of c. Cyanobacteria atmospheric nitrogen to ammonia by d. Blue algae prokaryotes is called 8. The selective media for Rhizobium a. Biological Nitrogen Fixation YEMA contains the sugar b. Nitrification a. Maltose c. Ammonification b. Mannitol d. Denitrification c. Glucose 2. The oxidation of sulphide is carried d. Lactose out by 9. Solubilisation of inorganic phosphates a. Thiobacillus by Bacillus is brought about by the b. Purple bacteria a. Production of enzymes c. Beggiatoa b. Production of organic acids d. Both a and b c. Production of alkali 3. Phosphate solubilisation by bacteria is mediated by the production of d. Mineralisation a. Organic acids 10. The mass cultivation of VAM is b. Phosphatases preferably done in c. Phosphoric acid a. Sorghum roots d. Phytases b. Rice roots

4. The process of production of CH4 c. Potato roots from CO2 is called d. Cotton roots a. CO2 fixation 11. is an example of b. Methylotrophy entomopathogenic fungi c. Methanogenesis a. Verticillium d. Photosynthesis b. Beauveria bassiana 5. The reduction of sulphate for building c. Metarhizium anisopliae up aminoacids and proteins is called d. All of the above a. Desulfurylation b. Assimilatory sulphate reduction 12. The toxic effect of Bacillus thuringiensis is due to c. Dissimilatory sulphate reduction a. Cry protein d. Sulphur reduction 6. An example of nitrogenous b. Delta endotoxin biofertilizer c. Parasporal crystals a. Bacillus d. All of the above b. Pseudomonas c. Rhizobium d. VAM

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13. The action by mycopesticide depends 13. Explain the mass production of on the phosphate solubilisers. a. Ingestion of fungi by insects 14. What is Azolla-Anabaena symbiosis? b. Penetration of cuticle by fungi 15. What is crystal toxin? Write short c. Ingestion of leaves infected with notes on BT cotton. fungi 16. Explain the process of root nodule d. Ingestion of spores by fungi. formation with appropriate diagram. 14. Crown gall is caused by Agrobacterium 17. Give in detail the reactions involved in on phosphorus/carbon/sulphur/nitrogen a. Monocots cycle. b. Dicots only 18. Explain the mass production of VAM. c. Monocots and dicots 19. Give detailed account on Bacillus d. Ferns thuringiensis. 15. Smut is a disease caused by a. Puccinia sp Student Activity b. Ustilago sp • Sow two groundnut seeds in a c. Verticillium plastic cup/earthern pot. After d. None of the above one month, pull out the plant and observe the root system for Answer the following nodules. 1. What is the role of purple and green • Collect pictures of all organisms bacteria in sulphur cycle? involved in sulphur cycle and 2. What is nitrogenase? Give its function. prepare a collage showing its role. 3. Define biofertilizers. • Prepare a chart work showing the 4. What is VAM? biogeochemical cycles of carbon, 5. Define biopesticides. nitrogen and phosphorus 6. What is a smut? • Collect pictures of various diseases 7. What is NPV? of plants and prepare a chart. 8. List out Bacterial diseases of plants. • Prepare a model on the mode of action of BT. 9. What is the end result of decomposition of organic matter in carbon • Collect diseased parts of plants cycle? Give the role of microorganisms and identify the symptoms of the with examples. disease. 10. What is the function of leghemo-globin? 11. Give the method of application of Late blight of potato Azolla in paddy fields. https://youtu.be/2Y77KEYuw_g 12. Give the salient features of https://www.youtube.com/watch?v=p9Koqk Rhizobium.

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Chapter 12 Medical Microbiology Chapter Outline

12.1 Microbial Infections of the Human Body 12.2 Skin and Wound Infections 12.3 Respiratory Tract Infections 12.4 Gastrointestinal Tract Infections 12.5 Ocular Infections 12.6 Urinary Tract Infections

12.7 Reproductive Tract Infections Medical Microbiology or Clinical Microbiology plays 12.8 Infections of the an important role by providing the necessary diagnostic testing, means of epidemiological detection, and future Nervous System innovation required in an era of emerging and reemerging 12.9 Systemic infectious diseases. Infections

Learning Objectives 12.1 Microbial Infections of the Human Body After studying this chapter the student Medical microbiology is the branch will be able, of microbiology which deals with • To describe the importance of prevention, diagnosis and treatment of medical microbiology. infectious diseases. There are four kinds • To understand the types and sources of microorganisms that cause infectious of infections. diseases. They are bacteria, fungi, parasites and viruses. Any disease that spreads • To know the types of infectious from one host to another, either directly diseases and virulence factors of the or indirectly is said to be a communicable pathogen. disease. Chicken pox, measles, genital • To tell the etiological agents of skin herpes, typhoid fever and tuberculosis are wound respiratory, gastro intestinal, examples of such diseases, that are easily ocular, urinary, reproductive, ner- spread from one person to another. vous system and systemic infections. A non communicable disease does • To know the causative agents of not spread from one host to another. varoius human diseases and their For example, Clostridium tetani, a soil portal of entry.

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inhabitant, produces Tetanus when it is some of the virus producing respiratory introduced into a wound or an abrasion. infections. Tetanus is thus an infectious disease, but c. Ingestion not communicable. Intestinal infections are generally Infectious disease occurs when the acquired by the ingestion of food or infecting microorganism causes damage drinks contaminated by pathogens. to the host. The term infection refers to Infection transmitted by ingestion may the establishment of the microorganisms be waterborne (cholera), food borne in the tissues resulting in injury or (typhoid) or fecal-oral route (dysentery). harmful effect to the host. Infection is a d. Inoculation pathological condition due to the growth of microorganisms in a host. To initiate an Pathogens, in some instances, may be infection, a pathogenic microbe enters the inoculated directly into the tissues of the tissues of the body by a characterization host. Tetanus spores implanted in the route, the portal of entry. depth of wounds, rabies virus deposited subcutaneously by dog bites, inoculation 12.1.1 Routes of Infections through unsterile syringes and surgical equipments are examples that enter There are various ways in which through direct inoculation. microorganisms enters into the host are explained below. e. Congenital Some pathogens are able to cross the a. Contact placental barrier and infect the fetus in Infection may be acquired by contact uterus. Bacteria like Treponema pallidum, which may be direct or indirect. Sexually viruses like Rubella, Cytomegalovirus transmitted diseases such as syphilis parasite like Toxoplasma gondi are some of and gonorrhea spread by direct contact. the organisms that enter through placenta Indirect contact may be through the and cause disease in the newborn. agency of inanimate objects such as clothing, pencils or toys which may be 12.1.2 Types of Infections contaminated by a pathogen from one person to another. Pencils shared by Infections may be classified in various school children may act as fomites in ways. Initial infection with a parasite the transmission of diphtheria, and face in a host is called a primary infection. towels in trachoma. Subsequent infections by the same parasite in the host are termed reinfections. When b. Inhalation a new parasite sets up an infection in Respiratory infections such as influenza a host whose resistance is lowered by and tuberculosis are transmitted by a preexisting infectious disease, this is inhalation of the pathogen in droplet and termed secondary infection. droplet nuclei that are shed by the patients When in a patient already suffering during sneezing, speaking or coughing. Common cold virus, Adenovirus is from a disease, a new infection is setup

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from another host or other external 1. When the skin is breached normal sources it is termed cross infection. Cross flora enters the tissues. infections occurring in hospitals are 2. When the urethral organisms ascend, called nosocomial infections. Iatrogenic they cause urinary tract infection infection refers to physician induced 3. When a patient is treated with infections resulting from investigative, antibiotics, normal flora is eliminated therapeutic or other procedures. and replaced by potential pathogens Depending on whether the source 4. When the intestine is perforated, of infection is from the host’s own body normal flora enter the previously or from external sources, infections are sterile body parts classified as endogenous or exogenous, 5. Similarly when the pH of the vagina respectively. increases potential pathogens occupy Endogenous infection the space. Endogenous infections are acquired from However normal flora helps host against the host himself from the normal flora of pathogen and benefits the host in many the body. ways Microorganisms are present in certain • Normal flora of skin produces fatty areas of the body in all human beings. They acids which inhibit other species are called normal flora. The common areas • Intestinal bacteria secrete antibacterial are Nose, Mouth, Teeth, Throat, Intestine, substances (bacteriocins, colicins) and Urethra, Vagina and Skin (Figure 12.1). many metabolic products that prevent other species to survive.

acterial l ra in a n rmal ers n in the c mm nit

er res irat r tract Staphylococcus sp. Streptococcus sp. Streptococcus pneumoniae Viridans streptococcus Haemophilus sp. naerobes Skin Staphylococcus sp. Coryne orm bacteria or iptheroids Propionibacterium sp.

astr intestinal tract naerobes Enterococcus sp. Enterobacteriaceae Escherichia coli Klebsiella sp. Streptococcus sp. Lactobacillus sp.

enital tract Lactobacillus sp. Streptococcus sp. Streptococcus a alactiae Figure 12.1: Microorganisms present as normal flora (There are many organisms in a site. Only few are listed)

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• Because of their large numbers other Transmission may be mechanical or species do not have space in the biological. Mechanical transmission is the intestine passive transport of the pathogens on the • Acidic environment created by vaginal insects feet or other body parts. Example: Lactobacilli suppresses growth of other Houseflies can transfer the pathogens of bacteria. Typhoid fever and Bacillary dysentery

HOTS Infobits The story of typhoid Mary How do normal flora help host against The classic example of role of carriers pathogenic microorganisms? in disease transmission is the story of Mary Mallon. Exogenous sources of infections Mary Mallon was an Irish Human beings: The commonest source immigrant who worked as a cook of infections in human are from other in New York in the early twentieth human beings. The parasite may originate century. Over seven years, from from a patient or a carrier. A patient is 1900 to 1907, Mallon worked for a person who harbours the pathogenic number of different households. microorganism and suffers from ill effect Unknowingly spreading illness to because of it. A healthy carrier is the the people who lived in each one. one who harbours the pathogens but has Later George Soper, tracked Mallon never suffered from the disease caused by linked 22 cases of typhoid fever the pathogen. A convalescent carrier is through her. He discovered that one who has recovered from the disease Mallon was a carrier for typhoid but and continues to harbor the pathogen in was immune to it herself. Although his body (Figure 12.2). active carriers had been recognized before, this was the first time that Animals: Many pathogens are able to an asymptomatic carrier of infected infect both human beings and animals. had been identified. Epidemiologists Infectious disease transmitted from were able to trace 51 cases of typhoid animals to human beings are called fever and three deaths directly to zoonoses. Zoonotic diseases may be Mallon, who is remembered as bacterial (Example: plague from rats) or “Typhoid Mary”. She was forced to viral (Example: rabies from dogs). prison and then released under the Insects: Blood sucking insects may transmit conditions that she could no longer pathogens to human beings. The diseases so be a cook. She assumed a false name caused are called arthopod borne diseases. and began cooking again and of Insects such as mosquitoes, ticks, mites, course, infecting numerous people. flies, fleas and lice that transmit infections She was again prisoned where she are called vectors. died 26 years later of pneumonia.

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Figure 12.2: The steps involved when a microbe causes disease in a host

(shigellosis) from feces of infected people Localised infections: An infection to food. Such vectors are called mechanical that is restricted to a specific location or vectors. region within the body of the host is called Biological transmission is an active localised infection. process and is more complex. The pathogens Generalised infections: An infection multiply in the body of the vectors often that has spread to several regions or areas undergoing part of a developmental cycle in the body of the host. This involves in it. Such vectors are termed biological the spread of the infecting agent from vectors. Example: Aedes aegypti mosquito the site of entry through tissue spaces or transmitting dengue, Anopheles mosquito channels, along the lymphatics or through transmitting malaria. the bloodstream.

Soil: Some pathogens are able to survive Circulation of bacteria in the blood is in the soil for very long periods. Spores known as Bacteremia. Septicemia is the of tetatus bacilli may remain viable in the condition where bacteria circulate and soil for several decades and serve as the multiply in the blood, form toxic products source of infection. and cause high fever. Pyemia is a condition where pyogenic bacteria produce Water: Water may act as the source of septicemia with multiple abscesses in the infection due to contamination with internal organs such as the spleen, liver pathogenic microorganisms. Example: and kidney. Cholera causing Vibrio cholerae. Occurrence of a disease Food: Contaminated food materials may To understand the full scope of a disease, act as source of infection. The presence we should know about its occurrence. of pathogens in food may be due to Epidemiology involves in the study of the external contamination. Example: Food frequency and distribution of disease and contaminated by Staphylococcus. other health related factors in defined populations. The incidence of a disease 12.1.3 Types of Infectious Diseases is the number of people in a population Infectious diseases may be localised or who develop a disease during a particular generalised. time period. The prevalence of a disease

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is the total number of existing cases with respect to the entire population. Facts about Fever: Depending on the spread of infectious Fever is as more healthful than harmful. disease in the community, they may be An experiment with vertebrates shows classified into different types. that fever increases the rate of antibody synthesis. Increased temperatures • Endemic diseases are those which are stimulate the activities constantly present in a particular area. of T cells and increase Typhoid fever is endemic in most parts the effectiveness of of India. interferon. Fever • Epidemic disease is one that spreads appears to enhance rapidly, involving many persons in phagocytosis. Fever almost never an area at the same time. Example: occurs as a single response; it is usually Epidemic of Dengue in 2017. accompanied by chills. The explanation • A pandemic is an epidemic that spreads lies in the natural physiological through many areas of the world interaction between the thermostat in involving very large numbers of persons the hypothalamus and the temperature within a short period. Example: H1N1 of the blood. For example: If the Influenza outbreak in 2009. Ebola thermostat has been set (by pyrogen) at outbreak in 2014-2016 in West Africa 102°F but the blood temperature is 99°F, was the largest in history and first ever the muscles are stimulated to contract epidemic, affecting multiple countries. involuntary (shivering) as a means of • If a particular disease occurs only producing heat. In addition, the vessels occasionally, it is called a sporadic in the skin constrict, creating a sensation disease. The most commonly of cold and the piloerector muscles in occurring sporadic diseases in India the skin develops ‘goose bumps’. are Diphtheria and Hepatitis A and E. Severity or duration of a disease • A latent disease is one in which the causative agent remains inactive for Another useful way of defining the scope a time but then becomes active to of a disease is in terms of its severity or produce symptoms of the disease. duration. • An acute disease is one that develops 12.1.4 Interaction between Microbes rapidly but lasts for a short time. and Host • A chronic disease develops more Pathogen is a microorganism which causes slowly, and the body’s reactions may disease. be less severe, but the disease is likely Pathogenecity is the ability of a to be continual or recurrent for long pathogen to produce disease. periods. Virulence is the degree of pathogenecity • A disease that is intermediate between of a microorganism. Virulence is not acute and chronic is described as a generally attributable to a single property subacute disease.

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but depends on several parameters related to intestine, Haemotoxin lyses red blood the organism, the host and their interaction. cells, and Nephrotoxins damages the Microorganisms first enter the body, kidneys. survive, multiply and elaborate many A toxin molecule secreted by a living factors and produce the disease. bacterial cell into the infected tissue is an Adhesion: The initial event in the exotoxin. A toxin that is not actively secreted pathogenesis of many infections is the but is shed from the outer membrane is an attachment of the bacteria to body endotoxin. the difference between exotoxin surfaces. Adhesions may occur as and endotoxin were given in Table 12.1. organized structures, such as fimbriae and Production of enzymes pili. Adhesions serve as virulence factors. Some enzymes like proteases, DNAases Capsule: It is an envelope or slime and phospholipases are produced and they layer surrounding the cell wall of certain help in destruction of the cell structure microorganisms. Capsule plays important and to hydrolyse host tissues. roles in immune evasion as it inhibit’s phagocytosis, as well as protecting the Antigenic variation bacteria while outside the host. Microorganisms evade the host immune responses by changing their surface Toxins: Toxins are specific chemical antigens. Antigenic drift and antigenic products of microbes, plants and some shift are common in influenza viruses. animals that are poisonous to other The distinction between the commensal organisms. Toxigenicty is the power to and the organism associated with disease. produce toxins. A toxin is named according to specific 12.1.5 Diagnostic Cycle target of action: Neurotoxin acts on the Specific diagnosis is important for better nervous system. Enterotoxin acts on the patient care, use of appropriate antibiotics

Table 12.1: Differences between endotoxin and exotoxin Exotoxins Endotoxins Heat labile proteins, secreted by certain Heat stable polysaccharide proteins, lipid species of bacteria and diffuse readily into complex which form an integral part of the the surrounding medium cellwall of Gram negative bacteria Proteins with a strong specificity to a target A Lipopolysaccharide (LPS), which is part cell and extremely powerful sometimes of the outermembrane of gram negative deadly cell walls Highly immunogenic Less immunogenic Toxoids can be made by treating toxins Toxoids cannot be made with formalin Produced mainly by Gram positive bacteria Produced by Gram negative bacteria but also by some Gram negative bacteria

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and to initiate appropriate preventive believed to be present in the clinical sample. measures. The diagnostic cycle begins Quality of patient specimens and their when the clinician takes a microbiological transport to the laboratory is important. sample and ends when a clinician receives the laboratory report and uses Infections and samples used the information to manage the condition Respiratory tract infections: Nasal and (Figure 12.3). bronchial washings, throat and nasal swabs, sputum. The steps in diagnostic cycle are Eye infections: Conjunctival swab or 1. Clinical request and provision of scraping. clinical information. Wound infections: Pus, skin scraping, 2. Collection and transport of appropriate wound swap. specimens. Gastrointestinal infections: Stool, rec- 3. Laboratory analysis. tal swabs. 4. Interpretation of microbiology report Genital infections: Vesicle fluid or and use of the information. swab. Specimen Collection and transport: Urinary tract infections: Urine. It is important to collect the specimen appropriately and protect it from Blood borne infections: Blood. contamination. Transport media are used Nervous system infections: Cerebro- that are compatible with the organism spinalfluid (CSF).

Figure 12.3: The steps in diagnostic cycle

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Figure 12.4: Different approaches of diagnosis

Laboratory diagnosis of infectious agents can enter through skin breaks that are Direct diagnosis: It is the not readily apparent, and the larval demonstration of the presence of an forms of a few parasites can penetrate infectious agent, antigen or nucleic acids the intact skin. The skin has up to seven Indirect diagnosis: It is the layers (Figure 12.5) of ectodermal tissue demonstration of presence of antibodies and guards the underlying tissues viz; to a particular infectious agent, cytopathic muscles, bones, ligaments and internal effects, haemagglutination, inclusion organs. Nearly all human skin is covered bodies and neutralization. with hair follicles. Because it interfaces with the environment, skin plays an The different approaches for diagnosis important role in protecting the body or identification of infectious agents are against pathogens and excessive water shown in Figure 12.4. loss. Its other functions are insulation, temperature regulation, sensation, 12.2 Skin and Wound Infections synthesis of vitamin D, and the The skin, which covers and protects the protection of vitamin B folates. Severely body, is the body’s first line of defense damaged skin will try to heal by forming against pathogens. As a physical barrier, scar tissue. This is often discolored and it is almost impossible for the pathogens depigmented. to penetrate it. However, microorganisms

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n ections ac uired Kni e Needle nsect throu h s in cut stic animal horn n ury bite pric

ct s ri er s Strat m c rne m i ermis glan Strat m l ci m Strat m granlsm Strat m actile mcsm c r scle Strata m germinati m ct ermis s ri er s glan

Sriers glan ise tiss e rter Sbctis h ermis er e acinian c r scle M scle

Figure 12.5: Structure of skin

12.2.1 Structure of Skin and cushions the body from stress and Skin is composed of three primary strain. The dermis is tightly connected to layers: the epidermis, the dermis and the the epidermis by a basement membrane. hypodermis. It also harbors many nerve endings that provide the sense of touch and heat. It Epidermis contains the hair follicles, sweat glands, It forms the water proof, protective wrap sebaceous glands, apocrine glands, over the body’s surface. It also serves as a lymphatic vessels and blood vessels. barrier to infection. It is made up of stratified The blood vessels in the dermis provide squamous epithelium with an underlying nourishment and waste removal from basal lamina. The outermost layer of the its own cells as well as from the Stratum epidermis, the stratum corneum, consists basale of the epidermis. of dead cells that contain a waterproofing Hypodermis protein called keratin. The epidermis contains no blood vessels and cells in the Subcutaneous tissue (also hypodermis and deepest layers are nourished exclusively subcutis) is not part of the skin, and lies by diffused oxygen from the surrounding below the dermis of the cutis. Its purpose air. The main types of cells present in is to attach the skin to underlying bone epidermis are Merkel cells, keratinocytes and muscle as well as supplying it with with melanocytes and Langerhan cells. blood vessels and nerves. It consists of loose connective tissue, adipose Dermis tissue and elastin. The main cells are The dermis is the layer of skin beneath the fibroblasts, macrophages and adipocytes epidermis that consists of epithelial tissue (subcutaneous tissue contains 50% of body

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fat). Fat serves as padding and insulation anaerobic and inhabit hair follicles. for the body. These bacteria produce propionic acid, The hair follicles, sweat gland which helps maintain the low pH of skin, ducts, and oil gland ducts in the dermis generally between 3 and 5 provide passageways through which the microorganisms can enter the skin and 12.2.3 Wound Infection penetrate deeper tissues. Perspiration Wound can be defined as any interruption provides moisture and some nutrients for of continuity of external or internal microbial growth. However, it contains surfaces caused by violence salt, which inhibits many microorganisms; Wounds may occur following: surgery, the enzyme lysozyme, which is capable of trauma or injections breaking down the cell walls of certain Wound infections may occur mainly bacteria and antimicrobial peptides. after surgical procedures Sebum, secreted by oil glands, is a mixture of lipids (unsaturated fatty acids), proteins, Wound sepsis is the result of cross and salts that prevents skin and hair from infection from human sources and from drying out. Although the fatty acids other outside sources. inhibit the growth of certain pathogens, Bacteria associated with wound sebum, like perspiration, is also nutritive infections for many microorganisms Many bacteria are associated with wound infection (Figure 12.6). The normal 12.2.2 Normal Microbiota of the Skin flora may also cause infection. The most The skin’s normal microbiota contains common normal flora of the skin are: relatively large numbers of Gram positive Staphylococci, and various Streptococci, bacteria, such as Staphylococci and Sarcina sp, anaerobic Diphtheroids, Gram Micrococci. Bacteria in the skin tends to negative rods and others. be grouped into small clumps. Vigorous In ecte area washing can reduce their numbers but s will not eliminate them. Microorganisms hite l celis remaining in hair follicles and sweat Skin glands after washing will soon reestablish the normal populations. Areas of the body with high moisture, such as armpits and between the legs, have higher populations acteria of microorganisms. They metabolize Figure 12.6: Bacterial infection secretions from the sweat glands and are on the skin the main contributors to body odour. Also part of the skin’s normal microbiota Factors determining the ecology of the are Gram positive pleomorphic rods called skin bacteria diphtheroids. Some diphtheroids, such The main factors that determine the as Propionibacterium acnes, are typically ecology of skin bacteria:

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• Climate: Temperature and humidity • Extremes of age: Very old and very • The effect of free fatty acids young people are susceptible to infection. • Maintenance of the flora by products of skin secretions and other bacterial • Diabetes mellitus: Hormonal imbal- inhibitors. ance increases susceptibility. • Steroid therapy: Immune responses Defence against infection are affected. • Intact skin: Normal uninterrupted skin • Obesity: Increases susceptibility. provides protection against invasion by bacteria. • Malnutrition: General health status affected. • Lysozyme in sweat: The enzyme lysozyme provides protection against • Immunocompromised individual: Gram positive bacteria by lysing the Immune system will not function cell wall. properly. • IgA antibodies in the sweat and • Presence of remote infection at the secretions provide first line of defense time of surgery. against infection. b) Exogenous Factors • Inhibitors like unsaturated fatty acids • Use of unsterile instruments: provide protection against bacteria. They carry pathogens. • Bacteriocins produced by the normal • Surgeons hands / from health workers: flora prevent the establishment of May carry pathogens. other bacteria. • Air / Hospital environments: Unclean Factors responsible for wound environment harbur pathogens. infections c) Endogenous Factors a) Host factors • Wound contamination from the The following factors help the organisms patient source: From the normal flora. to survive and produce the infections: • Wound penetrating through structures containing normal flora. HOTS • Surgical procedures involving mucous membranes harbouring normal flora. 1. What are the possible infecting agent you could pick up when you • Patients carrying pathogens in their are injured while playing on the nose, throat, axilla. ground? List them and name the Etiological agents diseases that they could cause. Etiological agents like Pseudomonas 2. What are the possible infectious aeruginosa, Staphylococcus aureus, agent that can infect you when you Proteus, member of enterobacteriaceae are injured by a rusted nail? anaerobic organisms, anaerobic cocci and bacteroides cause infections.

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Post operative infections Gasgangrene organisms like Clostridium perfringens, Staphylococcus aureus and Clostridium tetani may cause post operative infections. Route of entry Wounds may occur following surgery, trauma or injections. Wound infections Figure 12.7: Cellulitis may occur mainly after surgical procedures. Wound sepsis is the result of As soon as the organisms enter the cross infection from human sources and skin they multiply and produce from other outside sources. Infections of various toxins that kill the cells and skin are listed in Table 12.2. produce cellulitis. Further damage leads to necrosis and ulcer formation Mechanisms of damage (Figure 12.8). 1. Organisms enter through the skin, multiply there and produce the disease 2. Organisms multiply in the skin and in the skin. produce disease in internal organs. For For example, impetigo, abscess and example some Group A Streptococci cellulitis (Figure 12.7) are caused multiply in the skin and produce disease by Staphylococcus aureus and known as Acute Glomerulonephritis Streptococcus pyogenes.

Table 12.2: Bacterial Infections of the skin Disease Pathogen Signs and Symptoms Transmission Cellulitis Streptococcus Localised inflammation Through cut or abrasion pyogenes of dermis and hypodermis; skin red, warm, and painful to the touch Erysipelas Streptococcus Inflamed, swollen patch Through cut or abrasion pyogenes of skin, often on face; may be suppurative Impetigo Staphylococcus Vesicles, pustules, Highly contagious, aureus, and sometimes bullae especially via contact Streptococcus around nose and mouth pyogenes Wound infections Pseudomonas Formation of biofilm in Exposure of wound aeruginosa, others or on wound to microbes in environment; poor wound hygiene

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causing damage to the kidneys. Some airborne pathogens. Coarse hairs in the times Corynebacterium diphtheriae nose, filter large dust particles from the air. may multiply in the skin and affect the The nose is lined with a mucous membrane heart due to the toxin that contains numerous mucous secreting cells and cilia. The upper portion of the throat also contains a ciliated mucous membrane. The mucous moistens inhaled air and traps dust and microorganisms. The cilia help to remove these particles by moving them towards the mouth for elimination.

12.3.1 Structure of Respiratory Tract The structure of respiratory tract is divided into two main parts viz: upper respiratory tract (URT) and lower respiratory tract (LRT). Upper respiratory tract includes Figure 12.8: Ulcer formation mouth, nose, nasal cavity, sinuses, throat or pharynx, epiglottis and larynx. 3. Sometimes organism may multiply in the skin and produce the toxin which Lower respiratory tract includes affect the Central Nervous System trachea, bronchi, bronchioles, lungs and (CNS) and the effects seen. In the alveoli (Figure 12.9). case of Clostridium tetani infection, convulsions and paralysis occur due to the production of a powerful toxin.

12.3 Respiratory Tract Infections With every breath, we inhale several microorganisms and therefore the respiratory system is a major portal of entry for pathogens. In fact, respiratory system infections are the most common type of infections and among the most damaging. Some pathogens that enter via respiratory route can infect other parts of the body, such as skin incase of measles, mumps and rubella. The upper respiratory system has Figure 12.9: Structure of human several anatomical defenses against respiratory tract

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12.3.2 Normal Defenses against Infections iii. Resident macrophages kill the 1. Arrangement of nose: There is no organisms direct entry of air into (LRT). b. Specific manner 2. Broncho constriction helps to trap the Secretory IgA antibody: Forms first microorganisms. line of defense 3. Cough reflex expels the microbes out Respiratory tract infection are divided side. into upper respiratory tract (URT) tract 4. Mucociliary blanket traps the infection and lower respiratory tract (LRT) organisms. infection. Infection of the respiratory tract are listed in the Table 12.3. 5. Mucosal factors: Kill the organisms by a. Non specific way URT: Infections are Sinusitis, Pharyn- i. Lysozyme: Cell wall of Gram gitis Laryngitis and Epiglotitis positive organism are lysed LRT: Infections are Trachiitis, Tracheo ii. Influenza virus inhibitors do bronchitis, Bronchitis, Alveolitis and not allow the virus to multiply Pneumonia (Figure 12.10).

Table 12.3: Microbial diseases of the respiratory system Upper respiratory system Diseases Pathogen Symptoms Bacterial diseases Epigottitis Haemophilus influenzae Inflammation of the epiglottis Streptococcal Streptococci, especially Inflamed mucous membranes of the pharyngitis (strep Streptococcus pyogenes throat; throat) Diphtheria Corynebacterium Bacterial exotoxin interferes with diphtheriae protein synthesis; damages heart, kidney, and other organs; membrane forms in throat; cutaneous form also occurs; Otitis media Several agents, especially Accumulations of pus in middle ear Staphylococcus aureus, build up painful pressure on eardrum Streptococcus pneumonia and Haemophilus influenza Viral diseases Common cold Rhino virus Familiar symptoms of coughing, sneezing, running nose. (Continued)

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Table 12.3: Microbial diseases of the respiratory system (Continued) Lower respiratory system Bacterial diseases Pertussis (whooping Bordetella pertussis Cilia in upper respiratory tract cough) inactivated, mucus accumulates, spasms of intense coughing to clear mucus; Tuberculosis Mycobacterium Tubercle bacilli entering lungs tuberculosis survive phagocytosis, reproduce in macrophages; tubercles formed to isolate pathogen; defenses eventually fail, and infection becomes systemic; Viral diseases Respiratory syncytial Respiratory syncytial A serious respiratory disease of virus (RSV) virus infants; Fungal diseases Blastomycosis Blastomyces dermatitidis Abscesses; extensive tissue damage; Bacterial pneumonia Pneumococcal Streptococcus pneumonia Infected alveoli of lung fill with pneumonia fluids; interferes with oxygen uptake Haemophilus Haemophilus influenzae Symptoms resemble pneumococcal influenzae pneumonia pneumonia

rmal in i e trachea r nchi le Lng r nch s ir sacs al e li

Fli in air sacs

ne m nia Figure 12.10: Diseased person with Pneumonia

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12.4 Gastrointestinal Tract Infections Difference between infection and intoxication Human systems function by the energy produced from the digested food Microbial diseases of digestive system molecules. The food is swallowed through are typically transmitted by a fecal oral mouth and digested in the gastro intestinal route. Most such diseases result from the tract. The food we consumed should be ingestion of food or water contaminated free of contaminations. The contaminated with pathogenic microorganisms or their food causes gastrointestinal infections. toxins. These pathogens usually enter the food or water supply after being shed in Through contaminated food and water the feces of people or animals infected the pathogens are ingested and they enter with them. the GIT. In the small intestine they initiate an infection. Many times the pathogens • Each milliliter that cause intestinal infections multiply of saliva can in the GIT and produce their pathogenic contain millions effect in the intestine itself. Example: of bacteria Shigellosis, Cholera. • Stomach/small intestine has very The gastrointestinal tract (GIT) or few microorganisms because of alimentary canal includes the mouth, hydrochloric acid present in the pharynx, throat, oesophagus (food Tube stomach. lead to the stomach), stomach, small and • Large intestine harbours microbial large intestine. It also includes accessory population exceeding 100 billion structures salivary glands, liver, gall of bacteria per gram of feces (40% bladder and pancreas lying outside the fecal masses contain microbial cell GIT. Secretions of these organs enhance the material) digestion of food molecules (Figure 12.11). • Large intestine microbial population astr intestinal tract mainly contain anaerobes and

Sali ar Mth facultative anaerobes. glan s After ingestion of pathogenic microorganisms, localization and Li er multiplication of organisms takes place in the en m St mach GIT and is called infection. Microorganisms ancreas may penetrate into intestinal mucosa and all bla er grow there or they may penetrate to other organs. Gastroenteritis is usually classified Small Large intestine intestine as either infection or intoxication. Food ect m borne diseases can arise from either ns infection or intoxication. In both cases, Figure 12.11: The structure of bacterial toxins are typically responsible for Gastro intestinal tract producing disease signs and symptoms. In a food infection the microbial agent ingested

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colonise in the gut and then produces toxins 12.4.2 Normal Defences against GI that damage host cells. Tract Infections In case of food intoxication the toxins High acidic nature of stomach acts produced by bacteria in the food are as natural defensive mechanism. It ingested. Infection and intoxication differ eliminates potentially pathogenic in their onset of symptoms. Infections are microorganisms. Many bacteria like characterized by a delay in the appearance Eschericha coli and Salmonella survive of gastrointestinal disturbance until the in the acidic environment of stomach pathogen increases in number or affects for an hour. Nitric oxide produced in the invaded tissue. Infection is correlated stomach by oxidation of ingested nitrates with onset of fever, one of the basic body’s combines with the stomach acids and general responses to an infective organism. kills the bacteria in less than an hour. In case of intoxication, the symptoms are Lysozyme of saliva has antimicrobial characterized by sudden appearance of properties. Small intestine contains gastrointestinal disturbances like cramping, important antimicrobial defences like nausea, vomiting or diarrhoea. specialized granule filled cells called paneth cells. These cells are able to 12.4.1 Microbial Flora of phagocytose bacteria and also produce Gastrointestinal Tract antibacterial proteins called defensins. The stomach and gastrointestinal tract are Globet cells secrete a gel forming not sterile and are colonized by the organisms mucin (major component of mucus), that perform functions beneficial to the reduce the pathogens reaching the host, including the manufacture of essential deeper tissues. Peyer’s patches of ileum vitamins. Escherichia coli found in the stimulates the host to secrete IgA intestine help the body to produce vitamin K antibodies (Figure 12.12). and Bifodobacteria can synthesizes vitamins such as vitamin B12, folate, and riboflavin. HOTS Humans cannot produce these vitamins. The normal flora changes according to the What is likely to happen to a child who diet, age, cultural conditions and the use of drinks contaminated water? antibiotics (Table 12.4).

Table 12.4: Normal flora of human gastrointestinal tract At human birth Stomach and intestine are sterile Breast fed babies Lactobacillus bifidus Bottled milk fed babies Enteric bacteria, Lactobacillus bifidus, Enterococci, Clostridium sp Small intestine Lactobacilli, Enterococcus faecalis, Escherichia coli Large intestine Anaerobic bacteria, Streptococci, Bacteroides, Bifidobacteriumbifidum

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12.4.3 Terms used in GIT Infections Fl li i s Gastroenteritis: Inflammation of lining of Sali a Ls me stomach and intestine. It is a syndrome rmal bacterial l ra characterized by nausea, vomiting, diarrhea, abdominal discomfort. Fl li i s eristalsis Diarrhea: Condition in which feces, are discharged from the bowels frequently and in a liquid form. ci Dysentery: Inflammatory disorder of the GIT associated with pus and blood in

Fl g t c ntents feces. eristalsis Mcs ile Gastritis: Inflammation of the stomach Secret r lg Lmhi tiss e rmal l ra lining that results in swelling. eristalsis e er s atches She ing an rere licati n She ing an re lacement Enteritis: Inflammation of the e itheli m e itheli m Mcs rmal l ra intestinal mucosa Figure 12.12: Natural defence Colitis: Inflammation of the colon mechanism in the Gastrointestinal Tract Hepatitis: Inflammation of the liver Enterocolitis: Inflamation involving the Stomach is acidic mucosa of both large and small intestine. because of the presence of hydrochlo- Peritonitis: Inflammation of ric acid. So in this peritoneum (it is the serous membrane acidic condition organisms gen- that forms the lining of the abdominal erally not survive except one bac- cavity). Infections of digestive system are terium Helicobacter pylori. This listed in Table 12.5. bacterium is the leading cause of stomach ulcers. This bacterium has Botulism is a special maximum evidence of correlation case of intoxication with the development of stomach because, the ingestion and intestinal cancer. of the preformed St mach toxin affects the nervous system rather than GIT. Infant Botulism is the infectious form of Botulism which results when spores of Clostridium botulinum H. pylori swallowed colonise in the intestine. Botulism spores can be found in honey.

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Table 12.5: Diseases of the digestive system Infection Pathogen Symptoms Bacterial Diseases Staphylococcal food Staphylococcus aureus Nausea, vomiting, and diarrhea poisoning Shigellosis (bacillary Shigella sp- Tissue damage and dysentery dysentery) Salmonellosis salmonella enterica Nausea and diarrhea Typhoid fever Salmonella typhi High fever, significant mortality Cholera Vibrio Cholerae Diarrhea with large water loss Yersinia gastroenteritis Yersinia enterocolitica Abdominal pain and diarrhea, usually mild; may be confused with appendicitis Viral Diseases Mumps Mumps virus Painful swelling of parotid glands Paramyxoviridae Viral gastroenteritis Rotavirus Vomiting, diarrhea for 1 week Fungal Diseases Ergot poisoning Claviceps purpurea Restricted blood flow to limbs; hallucinogenic Aflatoxin poisoning Aspergillus flavus Liver cirrhosis; liver cancer

12.5 Ocular Infections susceptible because it is covered with eyelid that provides warm, moist and enclosed A number of microorganisms cause environment in which contaminating infection when introduced into the organisms can quickly establish a focus mucosa of the eye. In general, bacterial of infection. However, eyelid and tears eye infections can lead to inflammation, protect the external surfaces of the eye, irritation, and discharge, but they vary in both mechanically and biologically severity. Some are typically short-lived, and (Figure 12.13). others are chronic and lead to permanent eye damage. Prevention requires limiting Factors that Protect the External the exposure to contagious pathogens. Surfaces of the Eye When infections do occur, prompt treatment with antibiotics can often limit 1. Eyelid gives mechanical protection to or prevent permanent damage. the surfaces 2. Tears (a) make the surfaces The external surfaces of the eye viz. the moist and prevent drying. (b) contains conjunctiva and cornea are susceptible to lysozyme- an enzyme that lyses the cell wall infection. These are exposed to external of Gram positive bacteria. (c) contains IgA world and are easily accessible to infective antibodies that provide first line defense agents. Particularly the conjunctiva is against viruses.

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Figure 12.13: Structure and parts of eye

Infection of Eyelid can be caused by many different kinds of Most common cause of eyelid infection is viruses and bacteria. Staphylococcus aureus. Trachoma Infection involves lid margins and Trachoma, or granular conjunctivitis, is a cause blepharitis. common cause of preventable blindness that When the eyelid glands or follicles is rare in the United States but widespread are affected stye (sticky eye) is seen in developing countries, especially in Africa (Figure 12.14). and Asia. The condition is caused by the Infected eyelid and conjunctiva same species that causes neonatal inclusion

yelid margin conjunctivitis in infants, Chlamydia Infected eyelid trachomatis. Chlamydia trachomatis can be transmitted easily through fomites such Pus accumulation as contaminated towels, bed linens, and clothing and also by direct contact with infected individuals. Chlamydia trachomatis Figure 12.14: Infected eyelid and can also spread by flies that transfer infected conjunctiva mucous containing Chlamydia trachomatis Conjunctivitis (inflammation of from one human to another. Infections of conjunctiva) Conjunctivitis or pink eye eye are listed in Table 12.6.

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Table 12.6: Bacterial infections of the eye Disease Pathogen Signs and Symptoms Transmission Acute bacterial Haemophilus Inflammation of Exposure to conjunctivitis influenza conjunctiva with secretions from purulent discharge infected individuals Bacterial keratitis Staphylococcus Redness and irritation Exposure to epidermidis, of eye, blurred vision, pathogens on Pseudomonas sensitivity to light; contaminated aeruginosa progressive corneal contact lenses scarring, which can lead to blindness Neonatal Chlamydia Inflammation of Neonate exposed to conjunctivitis trachomatis, conjunctiva, purulent pathogens in birth Neisseria discharge, scarring canal of mother gonorrhoeae and perforation of with chlamydia or cornea; may lead to gonorrhea blindness

12.6 Urinary Tract Infections as parasites, protozoa and fungi also occured. Microorganisms invloved in UTI The urinary system is composed of organs are listed in Table 12.7. that regulate the chemical composition and the volume of the blood excrete mostly nitrogenous wastes products and Kidney Pyelonephritis water. The urinary system consists of (Kidney infection) two kidneys, two ureters, a single urinary bladder and a single urethra. Wastes are Ureter Ureteritis removed from the blood as it circulates (Ureter infection) through the kidneys (Figure 12.15). Infections of the kidney, ureter and bladder constitute Urinary Tract Infections Cystitis (UTI). When infection occur in the kidney Bladder (Bladder infection) and ureter it is called upper urinary Urethra Urethritis

LOWER TRACT UPPER TRACT (Urethra infection) tract infections and bladder downwards is called lower urinary tract infections. Figure 12.15: Structure of lower and Urinary tract infection is common in upper urinary tract infection females than males. The urinary system normally contains few microbes but it is 12.6.1 Predisposing Factors for UTI subjected to opportunistic infections that can be quite troublesome. Almost all such Urinary tract infection is common in infections are caused by bacteria although females than in males. The urethra in occasional infections by pathogens such females are shorter and wider and is

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Table 12.7: Microorganisms involved in UTI Microorganisms Examples Bacteria (most common) Escherichi coli, Klebsiella, Enterobacter, Proteus Viruses Adenovirus, Mumps Fungi Trichomonas vaginalis, Schistosoma haematobium Parasites Candida less effective in preventing the bacteria Pathogenesis of cystitis in woman entering the bladder. Sexual intercourse Bladder infections can result from the is a predisposing factor in females. High downward migration of organisms from incidence is seen in pregnant women due an infected kidney. But majority arise by to hormonal changes and impairment ascent of pathogens from the rectum and of urine flow due to pressure on urinary vagina to the urethra meatus and bladder, tract. leading to cystitis. If left untreated, the infection can further ascend to involve the Infobits kidneys (pyelonephritis) (Figure 12.16). Many of the bacteria which cause The rectum and vagina function as UTI’s have developed resistance to the reservoir of bacteria for sporadic antibiotics. Probiotics are a great infections defence in such cases. Research has In men, the longer urethra is believed turned to probiotic (Lactobacillus) to protect against ascending infections. strain that have demonstrated the When Escherichia.coli (and other best result. This particular probiotic Gram Negative rods) causes UTI, usually bacteria also stimulates immune the number of organisms in freshly passed function, lowers acidity levels in the urine is more than 100,000 organisms/ml. urinary tract, and discourages the This is called “significant bacteriuria”. growth of UTI causing organisms. Counts less than this is associated with contaminants from urethra or externalia. 12.6.2 Urinary Tract Infection caused Infection of urinary tract are listed in by Escherichia coli Table 12.8. Escherichia coli is the predominant cause Obesity increases of UTI. the risk of UTI’s It is a normal flora of the gut and can in men. A 2013 cause extra intestinal infections (UTI, study examineed Wound infection.) UTI (it can also be how obesity affected the chance of involved in other infections like wound developing UTI and it was found that infection peritonitis) UTI is common in obese men were twice more likely to (a) married women (b) elderly men with develop the UTI than obese women. prostate enlargement.

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Stages of a urinary tract infection

Acute kidney Bacteria continues to cascade 5 injury on to the kindneys, leading to acute kidney injury. Kidneys

Infection of the renal parenchyma 4 Pyelonephritis causes an inflammatory response called pyelonephritis.

Bacteria ascends towards the Ureters 3 Ascension kidneys via the ureters.

Pathogen penetrates bladder Uroepithelium Bladder and bacteria replicates, poten. 2 penetration tially forming biofilms.

Urethra Pathogen colonizes the urethra and ascends towards Colonization 1 the bladder.

Figure 12.16: Various stages of a urinary tract infection

Table 12.8: Microbial Diseases of the Urinary System Disease Pathogen Symptoms Bacterial Diseases of the Escherichia coli, Difficulty or pain in Urinary system Staphylococcus urination Cystitis (Urinary bladder saprophyticus infection) Pyelonephritis (Kidney Primarily Escherichia coli Fever; back or flank pain infection) Leptospirosis (Kidney Leptospira interrogans Headaches, muscular aches, infection) fever; kidney failure a possible complication

12.7 Reproductive Tract Infections tract or introduced from the outside during sexual contact or medical Reproductive tract procedures. It occur both in men and infections are caused women. Based on mode of infection by organisms normally reproductive tract infections are present in the classified into three types: reproductive or genital

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1. Sexually Transmitted Disease reproductive infections spread through non It is caused through means of sexual sexual routes are usually more common. contact. Examples: Chlamydia, Gonnorhea, Chancroid, and Acquired 12.7.1 Mode of Transmission Immuno Deficiency Syndrome (AIDS). Reproductive tract infections are caused by pathogenic bacteria, parasite, virus. It 2. Endogenous Infections is mainly caused by pathogens entering These are caused by the overgrowth of into the body through the mucous mem- organisms normally present in the genital branes during unprotected vaginal, oral, tract of healthy women. Example: Bacterial anal intercourse with an infected part- Vaginosis or Vulvo Vaginal Candidiasis. ner. In developing countries bacterial 3. Iatrogenic Infections infections like Gonorrhoea, Chlamydia, Syphilis, Bacterial Vaginosis, Lympho- These infections are associated with granuloma Venereum, Trichomoniasis, improperly performed medical procedures Chancroid, and viral infections caused such as unsafe abortion or poor delivery by Human Papilloma Virus, Hepatitis B practices. The endogenous organisms Virus, Herpes Simplex Virus, Human Im- in the vagina or sexually transmitted munodeficiency Virus are very common. organisms in the cervix may be transferred during a transcervical procedure into the 12.7.2 Normal Flora of Reproductive upper reproductive tract and cause serious Tract infections of the uterus, fallopian tubes, and other pelvic organs. Mycobacterium smegmatis, a harmless In men reproductive tract infections commensal found in the smegma of transmitted by sexual contact are much the genitalia of both men and women. more common than by endogenous or (Figure 12.17 & 12.18). In nomal iatrogenic reproductive infections. In women men aerobic and anaerobic bacteria,

Fimbriae Ovary Uterine Cavity

Uterus Fallopian tube Internal Os

Endocervical canal Cervix

Endocervix External Os

Ectocervix Vagina

Figure 12.17: Female reproductive system

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Figure 12.18: Male reproductive system lactobacilli, alpha haemolytic Streptococci, 12.7.3 Pathogenesis Chalmydia trachomatis and Ureaplasma After the entry of pathogenic organisms, urealyticum may also be present. with sufficient incubation time, symptoms The adult female genital tract has a are clearly manifested in the affected very complex microflora. The character individual. The most common symptoms of the population changes with the include unusual vaginal discharge, penile variation of the menstrual cycle. Mostly discharge, pelvic pain, itching, abnormal the predominant bacteria are acid tolerant or heavy vaginal bleeding, rashes, warts, Lactobacilli. Glycogen is accumulated in lesions, burning or pain during urination. the vaginal wall due to ovarian hormonal However most of the infections are activity. The breakdown of glycogen asymptomatic, which act as a effective by the lactic acid bacteria (Doderlien’s control of reproductive tract infections. bacillus) leads to the formation of acidic Diseases of reproductive system are listed pH (4.4-4.5). This acidic nature prevents in Table 12.9. the vagina from bacterial vaginosis and yeast infections. However before Infobits puberty and after menopause there is Tamilnadu has AIDS testing centres no glycogen formation. The normal at all district head quarters with more flora during this period contain normal than 55 Anti Retroviral Therapy(ART) skin microorganisms. The vaginal pH centres and 750 (ICTC)-Integrated is mild alkaline. The normal vaginal (voluntary) and confidential flora often includes Listeria, anaerobic counselling and testing centres under Streptococci, Mycoplasma, Gardnerella the national AIDS control programme vaginalis, Neisseria, Spirochetes, Candida, at district level government hospitals Staphylococcus epidermidis. and medical colleges across the state.

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Table 12.9: Microbial diseases of the reproductive system

Disease Pathogen Symptoms Bacterial Diseases Gonorrhea Neisseria Painful urination, discharge of pus in gonorrhoeae males, abnormal vaginal discharge in females Nongonococcal Chlamydia Painful urination and watery discharge, urethritis (NGU) trachomatis or other Chronic abdominal pain in females bacteria, including Mycoplasma hominis and Urea plasma urealyticum Syphilis Treponema pallidum Initial sore at site of infection, later skin rashes and mild fever; final stages may be severe lesions, damage to cardiovascular and nervous systems. Lymphogranuloma Chlamydia Swelling in lymph nodes in groin venereum (LGV) trachomatis Viral Diseases Genital Herpes Herpes simplex virus Painful vesicles in genital area type 2; HSV type 1 Genital warts Human papilloma Warts in genital area viruses AIDS Human loss of appetite, weight loss, Immunodeficiency persistent cough, attack on T cells virus (HIV) (immunocompromise), easily prone to fungal and other bacterial pathogens as secondary opportunistic infections. Fungal Diseases Candidiasis Candida albicans Severe vaginal itching, yeasty odor, yellow discharge Protozoan Diseases Trichomoniasis Trichomonas Vaginal itching, greenish yellow discharge vaginalis

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12.8 Infections of the Nervous System System (CNS) and Peripheral Nervous System (PNS). The Central Nervous Some of the most devastating infectious System (CNS) consists of brain and diseases are those that affect the nervous spinal cord. It controls most functions system, especially the brain and the spinal of the body and mind. The peripheral cord. Damage to these areas can lead to nervous system (PNS) consists of all deafness, blindness, learning disabilities, the nerves that branch off from the paralysis and death. Microbial infections of brain and spinal cord. These peripheral CNS are infrequent but often have serious nerves are the lines of communication consequences. In pre antibiotic times, they between the CNS, the various parts of were almost always fatal. An infection of the body and the external environment CNS can be life threatening condition, (Figure 12.19). especially for children with weakened immune system. These infections need Brain and spinal cord are covered quick diagnosis and immediate treatment by three layers of membranes called by an infectious disease specialist. Bacteria, meninges. These layers are the outermost Fungi and viruses are the most common dura mater, the middle arachnoid mater, causes of CNS infections. and the innermost pia mater. Between the pia mater and arachnoid membranes is a 12.8.1 Structure of Nervous System space called the subarachnoid space, in which there is cerebrospinal fluid (CSF) The human nervous system is organized circulating. into two divisions: The Central Nervous

Scal Sk ll b ne ra mater en s bl rachn i mater S barachn i s ace c ntains en s cerebr s inal bl li ia mater rain cerebr m

erebr s inal l i ia mater rachn i Meninges mater entricles ra mater S barachn i s ace brain

Figure 12.19: Structure of central nervous system

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12.8.2 Barriers of CNS and are therefore more selective in passing Dyes such as Trypan blue injected into materials (Figure 12.20). The blood brain the systemic circulation stain virtually barrier (Figure 12.21) is due to the cellular all tissues, with the exception of the configuration of cerebral capillaries, the brain and spinal cord. This blood brain choroid plexus and arachnoid cells. It barrier excludes most macromolecules, acts as a natural barrier that prevents microorganisms, immunocompetent cells the invasion of microorganisms into and antibodies. Even pathogens that are the brain. If this is breached organisms circulating in the bloodstream usually enter the brain. The blood CSF barrier cannot enter the brain and spinal cord (Figure 12.22) (also calle brain CSF because of blood brain barrier. Certain barrier) consists of endothelium with capillaries permit some substances to fenestrations, and tightly joined choroid pass from the blood into the brain but plexus epithelial cells. It acts as a natural restricts others. These capillaries are less barrier that prevents the invasion of permeable than others within the body microorganisms into the meninges.

Capillary (general) Capillary (brain) cell Astrocyte forming capillary wall Tight junction Pore (no pores) Water-lined Substance Substance passage pore in blood in blood Carrier- mediated Lipid-soluble Lipid-soluble transport substances substances

Figure 12.20: Capillaries of brain

Drugs cannot cross the blood brain barrier unless they are lipid soluble. Glucose and many amino acids are not lipid soluble, but they can cross the barrier through special transport systems. The lipid soluble antibiotic Chloramphenicol enters the brain readily. Penicillin is only slightly lipid soluble, but, if it is taken in very large doses, enough may cross the barrier to be effective. Inflammations of the brain tend to alter the blood brain barrier in such a way as to allow antibiotics to cross that would not be able to cross if there were no infection. Antibodies found in the normal CNS are derived from the serum and are present at low levels compared to serum levels. There are a few phagocytic cells and complement is also largely excluded. CSF is especially vulnerable because it lacks many of the defenses found in the blood, such as phagocytic cells. It is not easy for the microorganisms to enter CNS but it hampers their clearance once it is penetrated.

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Tight junction Blood Endothelium

Basement membrane Astrocyte

Brain

Figure 12.21: Blood brain barrier

Fenestration Blood Endothelium

Basement membrane Chorid cells

Tight junction Cerebrospinal fluid

Figure 12.22: Blood CSF barrier

12.8.3 Routes through which infection caused by a variety of microorganisms enter nervous system different agents. • Skull or bone fractures • Encephalitis is defined as inflammation • Medical procedures of the brain. Unlike an abscess, which is a localised area of bacterial or fungal • Peripheral nerves growth, Encephalitis is usually due to • Blood or lymph viruses that produce more widespread intracellular infections. 12.8.4 Clinical Manifestations of • Brain abcess is a focus of purulent Nervous System Infections infection and is usually due to bacteria. Some of the symptoms of nervous System Brain abscesses develop from either a infections are headache, fever, stiff neck, contiguous focus of infection (such as focal signs, seizures, confusion, weakness, the ears, the sinuses, or the teeth) or hallucinations, stupor, coma, abnormal hematogenous spread from a distant behavior and sleep disorder focus (such as the lungs or heart, particularly with chronic purulent 12.8.5 Infections of Nervous System pulmonary disease, subacute bacterial • Meningitis is an inflammation of endocarditis, or cyanotic congenital the meninges (membrane covering heart disease). In many cases the the brain). Meningitis is a diffuse source is undetectable.

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Etiological agents of Meningitis such as flu and typhoid affect the entire This can be caused by a wide range of body. Bacteria can enter the circulatory and microorganisms and can be classified as lymphatic systems through acute infections pyogenic and non pyogenic meningitis. or breaches of the skin barrier or mucosa. In pyogenic meningitis infiltration of Breaches may occur through fairly common pus cells (neutrophils) will be seen. In occurrences, such as insect bites or small Non pyogenic or aseptic meningitis infiltration of lymphocytes may be seen. wounds. Even the act of tooth brushing, Diseases of nervous system are listed in which can cause small ruptures in the gums Table 12.10. may introduce bacteria in to the circulatory system. In most cases, the Bacteremia 12.9 Systemic Infections result from such common exposure is transient and remains below the threshold An infection that is in the bloodstream is of detection. In severe cases, bacteremia called a systemic infection. Systemic diseases can lead to septicemia with dangerous

Table 12.10: Microbial diseases of the Nervous system Diseases Pathogen Portal of Entry Method of Transmission Bacterial Diseases Haemophilus Haemophilus. Respiratory Endogenous infection; influenzae meningitis Influenzae tract aerosols Meningococcal Neisseria Respiratory Aerosols meningitis meningitidis tract Pneumococcal Streptococcus Respiratory Aerosols meningitis pneumoniae tract Tetanus Clostridium tetani Skin Puncture wound Botulism Clostridium Mouth Food borne intoxication botulinum Viral Diseases Poliomyelitis Poliovirus Mouth Ingesting contaminated water (fecal oral route) Rabies Lyssavirus, includ- Skin Animal bite ing rabies virus Fungal Diseases Cryptococcosis Cryptococcus Respiratory Inhaling soil contaminate neoformans route with spores Protozoan Diseases African Trypanosoma Skin Tsetse fly trypanosomiasis brucei Rhodesiense, Trypanosoma brucei gambiense

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complication such as Toxemia sepsis and the common cause of gastroenteritis. The Septic shock. In these situations, it is often gut flora and natural defence mechanism the immune response to the infection that by defensins bacteriocins, globet cells, IgA result in the clinical signs and symptoms antibodies protect the individual against rather than microbes themselves. pathogenic infection. Diarrhea, dysentery, vomitting are the common symptoms of GIT. Summary Oral rehydration therapy, proper hygiene to be In the branch of medical microbiology we manifested to reduce the risk of gastroenteritis. discussed about prevention, diagnosis and The external structure and parts of the treatment of infectious diseases. Infections eye are easily susceptible to infections. The are acquired through contact, inhalation, eyelids, tears, lysozyme, IgA are the natural ingestion, inoculation and congenital. Sources defence against infections. Conjunctivitis of infections are endogenous and exogenous and Trachoma are the common eye diseases. in origin. Normal flora are organisms present Proper diagnosis and treatment should be in certain areas of the body. Infectious suggested. diseases may be generalised or localised. Uninary tract infections are more Based on the occurrence of infectious diseases common in females than in males. There are the infection may be epidemic, endemic, or many predisposing factors making female sporadic. There are various virulence factors prone to the infections. The predominant which are responsibility for the pathogenicity. causative agents in urinary tract infection is Skin is the first line of defence against Escherichia.coli. The number of organisms pathogen. Normal uninterrupted skin in freshly passed urine is more than 100,000 provides protection against “invasion by organisms/ml. It is called significant bacteria”. Many exogenous and endogenous bacteriuria. factors are responsible for wound infections. The infections spread through The mechanism of damage may be in the reproductive tract by direct contact skin or some cases its spreads to the internal is called sexually transmitted disease. organs and CNS system. Mostly these infections are asymptomatic Respiratory system of both lower and in women. upper is the major path for entry of pathogens. Nervous system infection affect The infections of upper respiratory tract are brain and spinal cord. They are of two sinusitis, pharyngitis, laryngitis and epiglottitis. types meningitis and encephalatis. An The infection of lower respiratory tract are infection that is in the blood stream trachitis, tracheobronchitis, bronchitis, and is called systemic infections. Systemic alveolitis. diseases like flu and typhoid affect the Gastrointestinal tract infections are entire body. infections of the digestive system. The food borne infection and food intoxication are

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Modes of Infection

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ICT CORNER Respiratory Tract Infections

Know the myths of cold

STEPS: • Use the link or scan the QR code given below. “Cells Alive” home page will open. You can select any topic you wish. For example click “understanding colds” • “Understanding Colds” page will open. You can go through anatomy of the nose, CAT scan view etc.. • At the top left of the page click on “Menu” and select “Treatments” and analyze. • Also select “Special features” and go through the topic. Also you can select how penicillin kills bacteria in the “Cells Alive” page, and know the action of penicillin against bacteria.

Step1 Step2 Step3 Step4

URL: https://www.cellsalive.com/toc_micro.htm

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Evaluation c. Stratum mucosum Multiple choice questions d. All the above* 7. Adipose tissue is present in which 1. Syphilis is layer of the skin disease a. Epidermis a. Sexually transmitted disease b. Dermis b. Respiratory tract disease c. Hypodermis * c. Gastro tract disease d. All the above d. Urinary tract disease 8. Which are all the cells present in the 2. is the person epidermis of the skin? who harbours the pathogenic a. Merkel cells microorganisms and suffers from till effect because of it? b. Keratinocytes a. Carrier c. Melanocytes b. Healthy carrier d. All the above * c. Patient 9. Sense of heat and touch is provided d. All the above by the nerves present in which of the following? 3. Circulation of bacteria in the blood is a. Epidermis known as a. Septicimia b. Dermis* b. Pyemia c. Muscle c. Bacterimia d. Adipose tissue 10. Keratinocytes are present in Dermis d. None of the above (1). 4. Stratum corneum is seen in which of Epidermis does not contain blood the following? vessels (2). Which of the above a. Dermis statements is/are true? b. Subcutaneous tissue a. 1 only c. Muscle b. 2 only* d. Epidermis* c. Both 1&2 5. Which of the following is the structure d. Neither 1 nor 2 present in epidermis? 11. From the skull down to the brain, a. Stratum mucosum select the arrangement of layers of b. Stratumlucidum* meninges from the following: c. Stratum granulosum a. Dura mater/Arachnoid mater/Pia d. Stratum germinativum mater * 6. Dermis contains which of the following? b. Arachnoid mater/Dura mater/Pia a. Stratum germinativum mater b. Stratum granulosum

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c. Pia mater/Arachnoid mater/Dura c. Secretion mater d. None of these d. Dura mater/Pia mater/Arachnoid 17. nature of stomach act as mater a natural defense mechanism. 12. Cerebrospinal fluid (CSF) is present a. Acidic in which of the following? b. Neutral a. Perivascular spaces c. Alkaline b. Sub arachnoid space * d. None of the above c. Between skull and dura mater 18. Traveller”s diarrhea is caused by d. Sub dural space a. Escherichia coli 13. Which of the following organisms, b. Staphylococcus aureus notincluded in causing pyogenic c. Vibrio cholerae meningitis? d. All the above a. Staphylococcus aureus 19. is the predominant cause b. Streptococcus pyogenes of UTI? c. Neisseria meningitidis a. Staphylocous aureus d. Mycobacterium tuberculosis b. Escherichia coli e. Leptospira (ser.var) c. Salmonella icterohaemorrhagiae d. Streptococcus pyogenes 14. Which of the following organisms, 20. fungi involved in notincluded in causing aseptic urinary tract infection? meningitis? a. Klebsiella a. Staphylococcus aureus b. Candida sp b. Streptococcus pyogenes c. Penicillium c. Neisseria meningitidis d. Escherichia coli d. Mycobacterium tuberculosis 21. During the breakdown of glycogen 15. antibody gives first line by lactobacilli in the vagina, makes defense against respiratory tract vaginal pH as . infections. a. Acidic a. IgM b. Neutral b. IgA c. Alkaline c. IgD d. None of the above d. IgE 22. In typhoid, organisms are acquired 16. The nose is lined with through which of the following membrane. routes? a. Mucous a. Oral* b. Epithelial b. Respiratory

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c. Skin 12. List out the normal defense against d. Blood transfusion Respiratory tract infections? 23. Which of the following is the causative 13. Describe microbial disease of upper agent of typhoid? respiratory tract infection? a. Salmonella enteritidis 14. Define gastroenteritis and enteroco- b. Salmonella typhimurium litis? c. Salmonella typhi* 15. State the difference between dysentery d. All the above and diarrhoea? 16. What is the difference between food borne infection and intoxication? Answer the following 17. Give the normal flora of the 1. Define congenital infection? gastrointestinal tract of humans? 2. What is meant by nosocomial infec- 18. Explain the various pathogenic tion? bacteria involved in gastroenteritis? 3. Define the term bacteremia, septice- 19. What is called significant bacteriuria? mia pyremia? 20. Explain the predisposing factors for 4. Explain mode of transfer of infection? urinary tract infection? 5. Define a wound. 21. List out the organisms causing urinary 6. What are the causes of wound? tract infections? 7. Name two types of CNS infections. 22. Define iatrogenic infection. 8. Give the names of the etiologic agents 23. Explain the role of lactobacilli in the of wound infection. prevention of bacterial vaginosis. 9. State the defenses of skin organisms 24. Give detailed study of various against the bacterial invasion. bacterial, fungal and viral infectious 10. Describe the factors responsible for diseases of reproductive tract wound infections. infection. 11. List out the pathogen that causes 25. Give the list of agents causing typhoid Otitis media? and paratyphoid.

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Student Activity (1) 1. Get information from your parents/neighbor about types of diseases one gets due to contamination. Example: If you drink contaminated water, you get diarrhoea.

No After certain activity Getting a disease Preventive method to advocate 1 Contaminate Diarrhoea Don’t drink or Boil, cool drinking water and drink 2 3 4 5

2. Give a list of organisms present as normal flora of the skin (include other than that is given in the text book).

3. Prepare model of respiratory tract with innovations. Prepare a list of URT infections with the etiologic agents and prevention. Observe a chronic smoker. He coughs very often. List out the reasons for his cough. collect information from nearby neighbors kids (10). How many of them are immunized DTP vaccinated? Where corporation No Kid’s name DOB Immunized on or pvt 1 2 3

4. Student is asked to prepare a model of GIT with their innovations. See for example: What all the organisms that can be transmitted through the fly contaminated food. Give a list. 5. (1) Write an assignment on Madras eye (conjunctivitis due to viruses) (2) Write Dos and Don’s when a dust particle comes into your eye. 6. 1) Draw the structure of urinary tract in a chart board using your innovation. Label the parts (make a poster presentation material with flow of urine from kidney to urethra). 7. Prepare a chart showing all sexually transmitted diseases. Collect the disease photographs from the net. 8. Write a chart showing differences between pyogenic and aseptic meningitis.

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Chapter 13 Immunology

Chapter Outline

13.1 Historical Background 13.2 Organs of the Immune System 13.3 Cells of Immune System 13.4 Immunity 13.5 Antigens 13.6 Antibodies 13.7 Antigen– Antibody Reactions Non specific immunity or Innate immunity has four types of defense barriers namely anatomical barriers, chemical barriers, phagocytic barriers and inflammatory barriers.

Learning Objectives • To understand the properties of antigen. After studying this chapter the student • To describe the basic structure will be able, and function of immunoglobulin • To gain knowledge on the history of (antibodies). immunology and Know the Nobel • To explain the mechanism of prize winners in immunology. antigen-antibody interactions • To know the structure and functions and their applications in clinical of primary and secondary lymphoid laboratory. organs. • To know the cells of immune system and understand the role of 13.1 Historical Background granulocytes, mast cells, macrophages, Immunology is the study of immunity to dendritic cells and lymphocytes. diseases. Immunology began as a branch of • To define immunity and Microbiology. Its origin is usually attributed Differentiate between innate to Edward Jenner who introduced immunity and acquired immunity. variolation in 1796.

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The success of vaccination enabled the extension of Jenner’s strategy of the World Health Organization to vaccination to other diseases. announce in 1979 that small pox had Pasteur used attenuated culture and been eradicated. Late in 19th century called it vaccine (Latin vacca, cow) in Robert Koch proved that infectious honour of Edward Jenner. Table 13.1 and diseases are caused by microorganisms. Figure 13.1 list and shows the scientist who The discoveries of Koch stimulated contributed to the field of immunology.

Table 13.1: Scientists and their contributions to immunology Year Name of the Contributions to immunology Scientists 1796 Edward Jenner Discovered that cowpox or vaccinia, induced protection against human small pox Discovery of humoral and cellular immunity 1890 Von Behring and Gave the first insights into the Kitasto (von Behring mechanism of immunity earned the Nobel Prize in medicine in 1901) 1930’s Kabat Showed that gamma - globulin (now immunoglobulin) a fraction of serum exhibited the active component of immunity 1883 Elie Metchnikoff He observed that certain white blood cells, which he termed phagocytes, were able to ingest microorganisms and other foreign material

1903 Sir Almoth Wright Reported that antibodies could aid in the process of phagocytosis. Wright called these antibodies ‘opsonins’

1996 Claman, Chaperon and Discovered the presence and Triplett cooperation of B cells and T cells (Continued)

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Year Name of the Contributions to immunology Scientists Specificity of immune response Around 1900 Jules Bordet Demonstrated that nonpathogenic substances, such as red blood cells from other species, could also serve as antigens Karl Landsteiner Showed that injecting an animal with almost any non-self, organic chemical could induce production of antibodies that would bind specifically to the chemical Molecular immunology 1959 Porter Separated fragments of immunoglobulin Edelman Heavy and light chains of antibodies were separated by him 1965 Putnam, Hirschmann Discovered constant and variable and Craig regions of immunoglobulin 1979 Kung et al. Described the first monoclonal antibody identifying a T cell subset 1982-83 Allison et al and Isolated T cell receptor Haskins et al. Immunogenetics and Genetic Engineering 1936 Gorer Discovered the major histocompatibility antigens 1968 McDevitt and Tyan Showed that immune response genes were linked to the genes of the major histocompatibility complex (MHC) 1974 Doherty and Reported that recognition of antigen Zinkernagel by T cells was restricted by MHC molecules 1978 Tonegawa et al. Demonstration of immunoglobulin gene rearrangement

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Infobits

Nobel Prizes for immunologic research

Year Recipient Country Research 1908 Elie Metchnikoff Russia Role of phagocytosis (Metchnikoff) and Paul Ehrlich Germany antitoxins (Ehrlich) in immunity 1913 Charles Richet France Anaphylaxis 1919 Jules Bordet Belgium Complement-mediated bacteriolysis 1930 Karl Landsteiner United States Discovery of human blood groups 1972 Rodney R. Porter Great Britain Chemical structure of antibodies Gerald M. United States Edelman 1977 Rosalyn R. Yalow United States Development of radioimmunoassay 1980 George Snell United States Major histocompatibility complex Jean Dausset France Baruj Benacerraf United States 1984 Cesar Milstein Britain Technological advances in the development George E. Kohler Germany of monoclonal antibodies 1991 E. Donnall United States Transplantation immunology Thomas United States Joseph Murray 2002 Sydney Brenner South Africa Genetic regulation of organ development H. Robert Horvitz United States and cell death (apoptosis) J. E. Sulston Great Britain 2008 Harald zurHausen Germany Role of HPV in causing cervical cancer Françoise Barré- France (Hausen) and the discovery of HIV Sinoussi Luc France (Barré-Sinoussi and Montagnier) Montagnier 2011 Jules Hoff man France Discovery of activating principles of innate Bruce Beutler United States immunity (Hoff man and Beutler) and role Ralph Steinman United States of dendritic cells in adaptive immunity (Steinman)

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Elie Metchnikoff karl Landsteiner Emil von Behring

Paul Ehrlich Robert Koch Niels K.Jerne

Jules J.B.V.Bordet Max Theiler Rodney R.Porter

Figure 13.1: Notable Scientists who contributed to the development of Immunology

13.2 Organs of the Immune System of B cells. The secondary lymphoid organs serve as sites where lymphocytes interact The immune system consists of with antigen and undergo proliferation structurally varied organs that are and differentiation into antigen specific distributed throughout the body. Based on effector cells. The spleen, lymph nodes function, the organs can be divided into and mucosal associated lymphoid tissues primary and secondary lymphoid organs (MALT) are secondary lymphoid organs. (Figure 13.2). The primary lymphoid These are discussed in more detail below. organs are responsible for providing the appropriate microenvironments for the 13.2.1 Primary Lymphoid Organs development and maturation of antigen sensitive B and T cells. The thymus is the a. Thymus primary lymphoid organ for development The thymus is a highly organized of T cells and the bone marrow is the lymphoid organ located above the heart. primary lymphoid organ for development The thymus consists of two lobes. Each

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asle

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Figure 13.5: Structure of Spleen arterioles entering the spleen, forming populated largely by T lymphocytes areas of white pulp. The inner region of and also interdigitating dendritic cells white pulp is divided into a Periarteriolar thought to have migrated from tissues to Lymphoid Sheath (PALS) containing the node. These interdigitating dentritic mainly T cells. The spleen filters the blood cells express high levels of class II MHC and traps blood borne microorganisms molecules, which are necessary for and antigens. Once trapped by splenic presenting antigen to T helper (TH) cells. macrophages or dendritic cells, the Lymph nodes taken from neonatally pathogen is phagocytosed, killed and thymectomized (removal of thymus from digested. new born animal) mice have unusually few cells in the paracortical region; b. Lymph nodes the paracortex is therefore sometimes The lymph nodes are encapsulated referred to as a thymus dependent round structures located at the junction area in contrast to the cortex, which of major lymphatic vessels. Lymph is a thymus independent area. The node is morphologically divided into inner most layer of a lymph node, the three regions: the cortex, the paracortex medulla, is more sparsely populated with and the medulla (Figure 13.6). The lymphoid lineage cells; of those present outer most layer, the cortex contains many are plasma cells actively secreting lymphocytes (mostly B cells), antibody molecules. Lymph nodes are macrophages and follicular dendritic specialized to trap antigen from regional cells arranged in primary follicles. After tissue spaces. As antigen is carried into a antigenic challenge, the primary follicles lymph node by the lymph, it is trapped, enlarge into secondary follicles, each processed and presented together with containing a germinal centre. Beneath class II MHC molecules by interdigitating the cortex is the paracortex which is dendritic cells in the paracortex, resulting

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Figure 13.6: Structure of Lymph node in the activation of TH cells. Activated c. MALT and SALT TH cells release cytokines needed for The specialized lymphoid tissue in mucus B cell activation. Thus lymph nodes membranes is called mucosal associated represent one environment where B lymphoid tissue (MALT). There are several cells differentiate into memory cells and types of MALT. The system most studied is antibody – secreting plasma cells. the gut associated lymphoid tissue (GALT). Lymph draining the extra cellular GALT include the tonsils, adenoids, and spaces of the body carries antigen from appendix and specialized structures called the tissues to the lymph node through the peyer’s patches (Figure 13.7) in the small afferent lymphatics. Lymph leaves by the intestine, which collect antigen from the efferent lymphatic in the medulla. Naive epithelial surfaces of the gastrointestinal lymphocytes (mature lymphocytes not tract. In peyer’s patches, the antigen is yet exposed to an antigen) enter the node collected by specialized epithelial cells called from the blood stream through specialized M cells (Figure 13.8). The lymphocytes post capillary venules and leave with the form a follicle consisting of a large central lymph through the efferent lymphatic. dome of B lymphocytes surrounded by

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13.3 Cells of the Immune System gives rise to B lymphocytes, T lymphocytes and natural killer (NK) cells. All blood cells arise from a type of cell called the hematopoietic stem 13.3.2 Types of Leukocytes cell(HSC). Stem cells are cells that can differentiate into other cell types. They The cells responsible for both innate are self renewing and they maintain their immunity and acquired immunity are population level by cell division. This the leukocytes (Greek leukos, white chapter describes the formation of blood and kytos cell). The average adult has cells and the properties of the various cells approximately 7400 leukocytes (white of the immune system. blood cells) per cubic millimeter of blood (Table 13.2). The average value 13.3.1 Hematopoiesis shifts substantially during an immune response. In defending the host against Hematopoiesis is the formation and pathogenic microorganisms, leukocytes development of blood cells of all types. cooperate with each other first to In humans, hematopoiesis begins in the recognize the pathogen as an invader and yolk sac in the first weeks of embryonic then to destroy it. The different types of development. In the third month of leukocytes are now briefly described. gestation, the stem cells migrate from the yolk sac to the fetal liver and then to a. Granulocytes the spleen. Hematopoiesis continues in Granulocytes have irregularly shaped these two organs from the third to the nuclei with two or five lobes. Their seventh month of gestation. As gestation cytoplasm has granules that contain reactive continues, the site of hematopoiesis substances that kill microorganisms and gradually shifts to the bone marrow such enhance inflammation. Three types of that it becomes the principle site at the granulocytes exist: basophils, eosinophils, time of birth. and neutrophils. Because of the many lobed (3-5) nuclei, neutrophils are also As hematopoietic stem cells can give called polymorphonuclear neutrophils rise to all of the different types of blood or PMNS (Figure 13.11). cells, they are often known as pluripotent stem cells. The different types of blood i) Basophils cells and their lineage relationships are Basophils are found in blood. Basophils summarized in Figure 13.10. We shall be have irregularly shaped nuclei with two concerned here only with the cells derived lobes and granules that stain bluish from the myeloid progenitor and the black with basic dyes. Basophils are non common lymphoid progenitor. phagocytic cells that release specific The myeloid progenitor gives rise to compounds from their cytoplasmic erythrocytes, neutrophils, eosinophils, granules which include histamine, basophils, monocytes, mast cells and prostaglandins, serotonin, and platelets. The common lymphoid progenitor leukotriens. Because these compounds

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r thr c te r thr i r genit r Figure 13.10: Hematopoiesis affect vascular permeability they are Table 13.2: Normal Adult Blood Count termed vasoactive mediators. Vasoactive mediators play a major role in certain Cell type Cells/ % allergic responses such as eczema, hay mm3 WBC fever and asthma. Red blood cells 50,00,000 – Platelets 2,50,000 – HOTS Leukocytes 7,400 100 Neutrophil 4320 60 Heard about stem cell treatment! Lymphocytes 2160 30 Why do we need stem cells bank? Monocytes 430 6 Eosinophils 215 3 Basophils 70 1

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ii) Eosinophils migrate to the site of tissue damage and Eosinophils have a two lobed nucleus infection, where they become the principle connected by a slender thread of chromatin phagocytic and microbicidal cells. and granules that stain with acidic dyes. Unlike basophils, eosinophils, migrate b. Mast cells from the blood stream into tissue spaces, Mast cells are bone marrow derived especially mucous membranes. They are cells that differentiate in the blood and important in the defense against protozoan connective tissue. Although they contain and helminth parasites, mainly by releasing granules with histamine and other cationic peptides and reactive oxygen pharmacologically active substances intermediates, into the extracellular fluid. similar to those in basophils, they arise These molecules damage the parasite from a different cellular lineage. Mast plasma membrane, killing it. Eosinophils cells, along with basophils, are important also play a role in allergic reactions. in the development of allergies and hypersensitivities. M ltil be cle s ran les c. Monocytes and Macrophages Monocytes are mononuclear leukocytes. hagsme They are produced in the bone marrow etrhil and enter the blood, circulate for about eight hours, enlarge, migrate to the tissues and mature into macrophages or dendritic lcgen cells (Figure 13.12 a). Macrophages are derived from ran le monocytes and are classified as as hil mononuclear phagocytic leukocytes. However, they are larger than monocytes, contain more organelles that are ran le critical for phagocytosis and have a plasma membrane with microvilli. Macrophages have receptors to recognize sin hil common components of pathogens. Figure 13.11: Structure of granulocytes These receptors include mannose and fructose receptors and a special class of iii) Neutrophils molecules called toll like receptors. Toll Neutrophils have three to five lobed like receptors bind lipopolysaccharide nucleus. Like macrophages, neutrophils (LPS), peptidoglycan, fungal cell wall have receptors for antibodies and component called zymosan, viral nucleic complement proteins and are highly acids and foreign DNA. These microbial phagocytic. However, unlike macrophages, molecules are examples of pathogen neutrophils do not reside in healthy tissue associated molecular patterns (PAMPs) but circulate in blood so they can rapidly (Figure 13.12 c).

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Lssme

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bc Figure 13.12: (a) Structure of Monocytes (b) Phagocytosis by a Macrophage (c) Dendritic Cell

PAMPs enable macrophages to • Alveolar macrophages in the lung distinguish between potentially harmful • Histiocytes in connective tissue microbes and other host molecules. • Kupffer cells in the liver After the pathogen is recognized, the macrophages’, pattern recognition • Mesangial cells in the kidney receptors (Example: Toll like receptors) • Microglial cells in the brain bind the pathogen and phagocytose it. • Osteoclasts in bone Macrophages also have receptors for d. Dendritic cells antibodies and complement proteins. Both antibody and complement proteins Dendritic cells are not a single cell type. can coat microorganisms and enhance They are a heterogeneous group of cells so their phagocytosis. This enhancement named because of their Dendron (neuron) is termed opsonization. Macrophages like appendages (Figure 13.12d). They spread throughout the body and take up arise from various hematopoietic cell residence in specific tissues. Macrophages lineages. Most dendritic cells are tissue serve different functions in different bound, where they play an important role tissues and are named according to their in bridging innate immunity and acquired tissue location. immunity.

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Dendritic cells can be classified by their cells, and NK (natural killer) cells. Clusters location: of differentiation are group of monoclonal • Langerhans cells found in the skin antibodies that identify the same cell and mucus membranes surface molecule. The cell surface molecule is designated CD (cluster of differentiation • Interstitial dendritic cells which followed by a number (CD1, CD2). populate most organs (heart, lungs, liver, kidney, gastrointestinal tract) i) B Lymphocytes • Interdigitating cells present in T cell B lymphocytes mature within the bone areas of secondary lymphoid tissue marrow. When they leave bone marrow, and the thymic medulla. each expresses a unique antigen binding receptor on its membrane. The B cell • Circulating dendritic cells in the receptor is a membrane bound antibody blood and lymph. molecule (Figure 13.13a). When a naive All the above dendritic cells express high B cell, first encounters the antigen that levels of both class II MHC molecules. matches its membrane bound antibody, They are more potent antigen presenting the binding of the antigen to the antibody cells than macrophages and B cells. causes the cell to divide rapidly. Its Another type of dendritic cell, called the progeny differentiate into memory B cells Follicular dendritic cell has a different and effector B cells called plasma cells origin and function from antigen Memory B cells have a longer life span presenting dendritic cells described above. than native cells. They express the same Follicular dendritic cells do not express membrane bound antibody as their parent class II MHC molecules and therefore do naive B cell. Plasma cells do not express not function as antigen presenting cells. membrane bound antibody. Plasma cells Follicular dendritic cells express high secrete large quantities of antibodies. level of membrane receptors for antibody Secreted antibodies are the major effector and complement. Binding of circulating molecules of humoral immunity. antibody-antigen complexes by these receptors facilitates B cell activation in ii) T Lymphocytes lymph nodes. T lymphocytes also arise in the bone marrow. T cells then migrate to the Dendritic cells are similar to macrophages thymus to mature. During its maturation in their ability to recognize specific PAMPs within thymus, the T cells express a on microorganisms. They also posses unique antigen binding molecule called pattern recognition receptors (PRRs) to the T cell receptor (Figure 13.13b) on bind and phagocytose the pathogen. its membrane. Unlike membrane bound e. Lymphocytes antibodies on B cells, which can recognize Lymphocytes are the major cells of the antigen alone, T cell receptor can specific immunity. Lymphocytes can be recognize only antigen that is bound to divided into three populations: T cells, B MHC molecules. There are two major

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B cell Receptor T cell Receptor BC C recognition ecognition

ight chain C C eavy chain αβ ε δ γ ε lgβ lgα

IM IMa ζ ζ Signaling Signaling Figure 13.13: (a) B cell receptor. (b) T cell receptor types of MHC molecules. Class I MHC There are two subpopulations of T cells: molecules are expressed by all nucleated T helper (TH) and T cytotoxic (TC) cells. cells. Class II MHC molecules are Although a third type of T cells called a T expressed only by antigen presenting suppressor (TS) cell, has been postulated, cells. When a naive T cell encounters recent evidence suggests that it may not be antigen combined with an MHC distinct from the TH and TC subpopulations. molecule on a cell the T cell proliferates T cells displaying CD4 function as TH cells and differentiates into memory T cell and whereas; those displaying CD8 function as various effector T cells. TC cells (Figure 13.14).

(a) B cell (b) cell (c) C cell C C C C

ntigen binding receptor (antibody) Figure 13.14: Distinctive membrane molecules on lymphocytes

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After a T cell recognizes and interacts ntib ies arget cell in ecte H ith ir s with an antigen-MHC class II molecule ir s antigenn complex, the cell is activated. It becomes an effector cell that secretes cytokines. The secreted cytokines activate B cells, a ntibntib rece t r TC cells, macrophages and various other arget cell in ecte ith ir s n c ate cells that participate in the immune ith antib response.

Under the influence of TH derived cellcell cytokines, a TC cell that recognizes an antigen-MHC class I molecule complex b proliferates and differentiates into a arget cell l sis cytotoxic T lymphocyte (CTL). Cells that display foreign antigen complexed with a class I MHC molecule are called altered self cells. CTL destroy virus infected cells cellcell and tumor cells. c iii) Natural killer (NK) Cells (Null cells) Figure 13.15: Antibody-Dependent Cell-Mediated Cytotoxicity NK cells are a small population of large, non phagocytic granular lymphocytes that play an important role in innate Stem Cell: A cell from immunity. The major NK cell function is which differentiated to destroy cancer cells and cells infected cells are derived. with microorganisms. They recognize Stem cells are their targets in one of two ways. They can classified as totipotent, pluripotent, bind to antibodies that coat infected or multipotent, or unipotent depending cancer cells. Thus the antibody bridges on the range of cell types that they the two cell types. This process is called can generate. antibody dependent cell mediated Necrosis: Morphologic changes that cytotoxicity (ADCC) (Figure 13.15) accompany death of individual cells or The second way that NK cells recognize groups of cells and that release large infected cells and cancer cells relies on the amounts of intracellular components to presence of specialized proteins on the the environment, leading to disruption surface of all nucleated host cells known and atrophy of tissue. as class II MHC molecules. If a hosts cell loses this MHC protein, as when some viruses or cancers overtake the cell, the 13.4 Immunity NK cells kill it by releasing pore forming To establish an infection, an invading proteins and cytotoxic enzymes called microorganism must first overcome many granzymes (Figure 13.16). surface barriers, such as skin, degradative

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atural iller cell

ctivatinging InhibitingIn receptor receptor bi uitous M C class I molecule molecule Perforin and gran ymes bnormal cell lac ing ormal M C class I cell molecule o attac ill

(a) (b) Figure 13.16: The system used by natural killer cells to recognize normal cells and abnormal cells that lack the Major Histocompatibility Complex Class I surface molecule enzymes and mucus. These surface barriers material. They are non-specific immune have either direct antimicrobial activity or response or innate immunity or natural inhibit attachment of the microorganism immunity and specific immune response to the host. Any microorganism that or acquired immunity or adaptive penetrates these barriers encounters two immunity. levels of resistance: nonspecific resistance I. Innate immunity mechanisms and the specific immune Innate immunity refers to those general response. defence mechanisms that are inherited as part of the innate structure and function 13.4.1 Types of Immunity of each animal (such as skin, mucus and The term immunity (Latin immunis, lysozyme). Innate immunity is the first free of burden) refers to the general line of defence against any microorganism ability of a host to resist infection or or foreign material encountered by the disease. There are two interdependent vertebrate host. Innate immunity defends components of the immune response to against foreign invaders equally and lacks invading microorganisms and foreign immunological memory.

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II. Acquired immunity 13.4.2 Mechanisms of Innate Immunity Acquired immunity refers to the type A potential microbial pathogen invading a of specific immunity that develops after human host immediately confronts a vast exposure to a suitable antigen. The array of nonspecific defence mechanisms. effectiveness of acquired immunity increases Many direct factors (nutrition, physiology, on repeated exposure to foreign agents such fever, age, genetics) and equally as many as viruses, bacteria or toxins. So acquired indirect factors (personal hygiene, immunity has memory. The innate immunity socioeconomic status, living conditions) and acquired immunity work together to influence all host microbe relationships. eliminate pathogenic microorganisms and In addition to these direct and indirect other foreign agents. Although innate systems factors, a vertebrate host has the following predominate immediately upon initial four non specific defence mechanisms. exposure to foreign substances, multiple A. Physical barriers bridges occur between innate and acquired immune system components (Figure 13.17). B. Chemical mediators

Innate immunity

Physical barriers Cells Chemical barriers

Skin, mucous membranes pH, lipids, enzymes

Pattern recognition molecules PMN’s monocytes, macrophages, eosinophils, NK cells

Cytokines Cytokines Antibodies Cytokines

B cells T cells

Ag Specific receptors

Acquired immunology Figure 13.17: The interrelationship between innate and acquired immunity

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C. Phagocytosis a minute or 10,000 each. Microbes larger than D. Inflammation 10μm are trapped by hairs and cilia lining the nasal cavity. The cilia in the nasal cavity beat A. Physical barriers toward the pharynx, so that mucus with its i) Skin trapped microorganisms is moved toward Intact skin contributes greatly to host the mouth and expelled. Microbes smaller resistance. It forms a very effective than 10μm pass through the nasal cavity mechanical barrier to microbial invasion. and are trapped by the mucociliary blanket Its outer layer consists of thick, closely and the trapped microbes are transported packed cells called keratinocytes, The by ciliary action that moves them away from skin is slightly acidic (around pH 5-6) lungs. Coughing and sneezing reflexes clear due to skin oil, secretion from sweat the respiratory system of microorganisms glands and organic acids produced by by expelling air forcefully from the lungs commensal Staphylococci. It also contains through the mouth and nose, respectively. a high concentration of sodium chloride Salivation also washes microorganisms and is subject to periodic drying. from the mouth and nasopharyngeal areas into the stomach. ii) Mucous membranes The mucous membranes of the eye iv) Gastrointestinal tract (conjunctiva), the respiratory, digestive and Most microorganisms that reach the urogenital systems withstand microbial stomach are killed by gastric juice. invasion. The intact stratified squamous (pH 2-3). However, organisms embedded epithelium and mucus secretions form a in food particles are protected from protective covering that resists penetration gastric juice and reach the small and traps many microorganisms. Many intestine. There microorganisms are mucosal surfaces are bathed in specific damaged by various pancreatic enzymes, antimicrobial secretions. One antibacterial bile, enzymes in intestinal secretions and substance in these secretions is lysozyme, an GALT system. Normal microbiota of the enzyme that lyses bacteria. Mucous secretions large intestine is important in preventing possess the iron binding protein, lactoferrin. the establishment of pathogens. The Lactoferrin sequesters iron from the plasma mucous membranes of the intestinal reducing the amount of iron available to tract contain paneth cells. These cells invading microbial pathogens and prevents produce lysozyme and cryptins (toxic for their ability to multiply. Mucous membranes bacteria). produce lactoperoxidase, an enzyme that v) Genitourinary tract catalyzes the production of superoxide Under normal circumstances, the kidneys, radicals, reactive oxygen intermediate that is ureters and urinary bladder of mammals toxic to many microorganisms. are sterile. Urine within the urinary bladder iii) Respiratory system is also sterile. In addition to removing The mammalian respiratory system has microbes by flushing action, urine kills strong defense mechanisms. The average some bacteria due to its low pH and the person inhales at least eight microorganisms presence of urea and other metabolic end

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products (uric acid, hippuric acid, indican, i) Cationic peptides fatty acids, mucin, and enzymes). The Cationic peptides are found in humans. acidic environment (pH 3-5) of the vagina There are three generic classes of cationic is unfavorable to most microbes. peptides that have the ability to damage vi) Eye bacterial plasma membrane.

The conjunctiva is specialized mucus Classes of Cells that produce secreting epithelial membrane that lines the Cationic Peptides interior surface of each eyelid and the exposed Cathelicidins neutrophils, surface of the eye ball. It is kept moist by the respiratory cells and continuous flushing action of tears. Tears alveolar macrophages contain large amounts of lysozyme, lactoferrin, Defensins primary granules of and antibody and thus provide chemical as neutrophils, intestinal well as physical protection (Figure 13.18). paneth cells and in in- B. Chemical mediators testinal and respiratory • Antimicrobial peptides epithelial cells Histatin Found in human They are low molecular weight proteins saliva. It has that exhibit broad spectrum antimicrobial antifungal activity. activity toward bacteria.

ntimicrobial factors in saliva (lyso ymes yso yme in pero idase lactoferrin tears and other myelopero idase) secretions emoval of particles by rapid passage Commensals of air over turbinate Mucus cilia bones hairs

Sin physical barrier fatty acids cid commensals

apid p Commensals change Paneth s cells Peristalsis

p and lushing of commensals urinary tract of vagina

Figure 13.18: Physical Barriers

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ii) Bacteriocins to eliminate pathogens. Complements Bacteriocins are produced by gram are soluble proteins and glycoproteins negative and gram positive bacteria. mostly produced by hepatocytes. More For example, Escherichia coli synthesize than 20 types of complements are present bacteriocins called colicins. Colicins in serum found circulating normally in causes cell lysis. human body in inactive forms (called as zymogens or proenzymes). Complement • Cytokines activation is triggered by an antibody Cytokines are proteins made by cells when it is bound to the antigen. It can that affect the behavior of other cells. also be triggered by some components of When released from mononuclear innate immunity. Thus the complement phagocytes, they are called monokines. system works in both innate and acquired When released from T lymphocytes immunity. they are called lymphokines. When Complement activation and cell lysis released from leukocytes they are called interleukins. Cytokines are required for The complement activation occurs via regulation of both the nonspecific and three pathways which are: specific immune responses. Interferons 1. Classical pathway (IFNS) are a group of cytokines produced 2. Alternative pathway by virus infected cells. Several classes 3. Lectin pathway (or mannose binding of interferons are recognized. IFN γ is lectin pathway) synthesized by virus infected leukocytes, Classical pathway, activated by antigen stimulated T cells and natural antigen-antibody reaction, Alternative killer cells. IFN α / β is derived from pathway, activated on microbial cell virus infected fibroblasts. Interferons surfaces, and Mannose binding Lectin prevent viral replication and assembly, pathway, activated by a plasma lectin that thereby limiting viral infection. binds to mannose residues on microbes Another group of noteworthy cytokines (Figure 13.19). are endogenous pyrogens which elicit Functions of complements fever in the host. Examples of endogenous Some major functions of complements pyrogens include interleukin – 1, are: Interleukin – 6 and tissue necrosis factor. All are produced by host macrophages in • Opsonization and phagocytosis response to pathogens. • Cell lysis • Complement system • Chemotaxis The complement system is a part of the • Activation of mast cells and immune system, consists of a series of basophils and enhancement of proteins that interact with one another inflammation in a highly regulated manner, in order • Production of antibodies

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Classical path ay MB path ay lternative path ay

C Microbe ntibody Microbe MB PS ntigen ntibody Microbe Comple MB Cb Comple efensin Microbe

C ctivates M SP

C C r.C s lternative C convertase

C Cb C Ca Ca Ca M SP O C r.C s Cb Membrane Ca C C convertase ttac Convertase Comple C

Figure 13.19: Complement pathways

• Immune clearance and inflamma- neutrophils and tissue tion by attracting macrophages macrophages. Phagocytosis may and neutrophils. be enhanced by a variety of factors C. Phagocytosis collectively referred to as opsonins which consist of antibodies and i. Phagocytosis is the ingestion various serum components of by phagocytic cells of invading complement. foreign particles such as bacteria. After ingestion, the foreign particle ii. Phagocytic cells use two basic is entrapped in a phagocytic mechanisms for the recognition of vacuole (phagosome), which microorganisms. Opsonin dependent fuses with lysosomes forming the and opsonin independent phagolysosome. The lysosomes iii. Phagocytes use pathogen release their powerful lytic recognition receptors to detect enzymes which digest the particle. pathogen associated molecular (Figure 13.20). Phagocytosis is patterns on microorganisms. conducted by blood monocytes, Toll like receptors are a distinct

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acteri m bec mes attache t membrane e aginati ns calle se ia

acteri m is ingeste rming hag s me

hag s me ses ith lssme

acteri m is kille an then igeste b l s s mal en mes

igesti n r cts are release r m cell ab Figure 13.20: (a) Scanning electron micrograph of alveolar macrophage phagocytosis of E. coli bacteria on the outer surface of a blood vessel in the lung pleural cavity. (b) Steps in the phagocytosis of a bacterium.

class of pathogen recognition of the capillary network. The engorged receptors. capillaries are responsible for tissue redness (erythema) and an increase in D. Inflammation temperature. Tissue damage caused by a wound or by 2. An increase in capillary permeability an invading pathogenic microorganism facilitates an influx of fluid and cells induces a complex sequence of events from the engorged capillaries into collectively known as inflammatory the tissue. The fluid that accumulates response. Inflammation can either be ( exudate) has much higher protein acute or chronic. The gross features content. Accumulation of exudate were described over 2000 years ago and contributes to tissue swelling are still known as the cardinal signs of (edema) inflammation: redness (rubor), warmth (calor), pain (dolor), swelling (tumor), and 3. Influx of phagocytes from the loss of function (functiolaesa) capillaries into the tissues is facilitated by increased capillary permeability. As The cardinal signs of inflammation phagocytic cells accumulate at the site reflect the three major events of an and begin to phagocytoses bacteria, inflammatory response. they release lytic enzymes, which 1. Vasodilation (an increase in the can damage nearby healthy cells. The diameter of blood vessels) of nearby accumulation of dead cells, digested capillaries occurs as the vessels that material and fluid forms substances carry blood away from the affected area called pus. constrict. This results in engorgement

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namely (1) Memory (2) Specificity Infobits (3) diversity and (4) discrimination between self and non self. Reactive Nitrogen Species: Highly cytotoxic antimicrobial compounds 1) Memory formed by the combination of nitric We rarely suffer twice from diseases such as oxide and superoxide anion within measles, mumps, chicken pox, whooping phagocytes such as neutrophils and cough and so on. The first contact with an macrophages. infectious organism clearly imprints some Reactive Oxygen Species (ROS): memory so that the body is effectively Highly reactive compounds such as prepared to repel any later invasion by superoxide anion O2, hydroxyl radicals that organism. (OH)(OH–), hydrogen peroxide By following the production of (H O ), and hypochlorous acid 2 2 antibody on the first and second contact (HClO) that are formed from oxygen with antigen, we can know the basis for the under many conditions in cells and development of immunity. For example, tissues, including microbe-activated when we inject a bacterial product such as innate responses of phagocytic cells; staphylococcal toxoid into a rabbit, several have anti-microbial activity. days elapse before antibodies can be detected in the blood. These reach a peak 13.4.3 Acquired Immunity and then fall. If we now allow the animal to rest and then give a second injection Lower animal forms possess so called innate of staphylococcal toxoid, the cause of or non-specific immune mechanisms such events is dramatically altered. Within two as phagocytosis of bacteria by specialized to three days the antibody level in the cells. Higher animals have evolved an blood raises steeply to reach much higher adaptive or acquired immune response. values than were observed in the primary This acquired immune response provides response. This secondary response is a flexible, specific and more effective characterized by a more rapid and more reaction to different infections. abundant production of antibody. This • Definition of Acquired (Adaptive) explosive production of antibodies is due Immunity to the tuning up of the antibody forming Acquired (adaptive)immunity refers to system to provide a population of memory the type of specific immunity that a host cells after first exposure to antigen. develops after exposure to a suitable The principle of memory is involved in antigen. vaccination. • Important features of acquired 2) Specificity immunity The establishment of immunity by one This is the immunity one develops organism does not provide protection throughout life time. Adaptive or acquired against another unrelated organism. After immunity has four important features an attack of measles we are immune to

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I IMM I I IMM I ra i res nes sl res nes

Macr hage rimar hite cell bl cell

at ral killer cell ntib ies cell

en ritic cell etrhil cell at ral killer cell

sin hil cell cell as hil

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further infection but are susceptible to B cells and antibody secreting plasma polio or mumps viruses. Thus the body cells. A single plasma cell can secrete can differentiate specifically between the more than 2000 molecules of antibody two organisms. per second. Circulating antibodies bind to microorganisms, toxins and extracellular 3) Diversity viruses, neutralizing them or tagging them The immune system is able to generate for destruction by phagocytes and other an enormous diversity of molecules such mechanisms. as cellular receptors and soluble proteins, The cellular (cell mediated) immunity including antibodies that recognize consists of the T cells. Each T cell trillions of different foreign substances. expresses antigen receptors called T cell 4) Discrimination between self and receptors (TCRS). Unlike membrane nonself bound antibody on B cells, which can The specific immune system almost recognize antigen alone, T cell receptors responds selectively to non self and can recognize only antigen that is bound produces specific responses against the to MHC molecules. There are two major stimulus. This is possible because host types of MHC molecules. Class I MHC cells express a unique protein on their molecules are expressed by all nucleated surface, making them as residents of that cells. Class II MHC molecules are expressed host or as self. Thus the introduction of only by antigen presenting cells such as materials lacking that unique self marker dendritic cells, macrophages and B cells. results in their attack by the host. When a naive T cell encounters antigen combined with an MHC molecule on a cell, 13.4.4 Humoral and Cellular Immunity the T cell proliferates and differentiates into memory T cells and various effector Two branches or arms of specific immunity T cells (helper T cells, cytotoxic T cells and are recognized: humoral (antibody regulatory T cells). Specific kinds of T cells mediated) immunity and cellular (cell directly attack target cells infected with mediated) immunity (Figure 13.21). viruses or parasites, transplanted cells or Humoral (antibody mediated) immunity organs and cancer cells. T cells can induce The antigen specific arm of the humoral target cell suicide (apoptosis), lyse targets immunity consists of the B cells. Each B cell cells, or release chemicals (cytokines) expresses a unique antigen binding receptor that enhance specific immunity and non on its membrane. The B cell receptor (BCR) specific defences such as phagocytosis is membrane bound antibody molecule. and inflammation. When a naive B cell first encounters the antigen that matches its membrane bound 13.4.5 Types of Specific Immunity antibody, the binding of the antigen to the Specific immunity can be acquired by antibody causes the cell to divide rapidly. natural means actively through infection Its progeny differentiate into memory or passively through receipt of preformed

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ntigens

F reign ir ses acteria arasites F ngi r teins

Intemali e antigen igeste b cell

ltere sel cell resents antigen

lass II lass I M M cell cell cell rece t rs rec gni e antigen b n t M m lec les

cti ate cell cti ate Ls rec gni e an kill in ing antigen M altere sel cells acti ates cells

t t ic l m h c te L

cti ate cell secretes ell me iate res nse c t kines that c ntrib te t acti ati n cells cells m ral res nse an ther cells

ntigen

cell cells interact ith antigen b secreting ntib bin s antigen an i erentiate int lasma cells an acilitates its clearance antib secreting lasma cells r m the b

Figure 13.21: Overview of the humoral and cell-mediated branches of the immune system. In the humoral response, B cells interact with antigen and then differentiate into antibody-secreting plasma cells. The secreted antibody binds to the antigen and facilitates its clearance from the body. In the cell-mediated response, various subpopulations of T cells recognize antigen presented on self-cells. TH cells respond to antigen by producing cytokines. TC cells respond to antigen by developing into cytotoxic T lymphocytes (CTLs), which mediate killing of altered self-cells (Example: virus-infected cells).

antibodies as through colostrum. Specific Passive Immuno- immunity can be acquired by artificial therapy: Treatment of means actively through immunization or an infectious disease passively through receipt of preformed by administration antibodies as with antisera. of previously generated antibodies specific for the infectious pathogen.

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13.5 Antigens system that the antigen encounters. The factors that influence immunogenicity can Substances capable of inducing a specific be divided under two categories. immune response are called antigens. The molecular properties of antigens and the 1. Contribution of the immunogen to way in which these properties ultimately immunogenicity contribute to immune activation are central 2. Contribution of the biological system to to our understanding of the immune system. immunogenicity

13.5.1 Immunogenicity Versus 1. Contribution of the immunogen to Antigenicity immunogenicity Two properties are exhibited by Immunogenicity is determined in part, antigens; they are immunogenicity and by the following four properties of the antigenicity. Immunogenicity is the immunogen. ability of an antigen to induce a humoral A. Foreignness and / or cell mediated immune response. The immune system normally discriminates B cells + antigen effector B cells (Plasma between self and non self, so that only cells) + memory B cells molecules that are foreign to the host are

T cells + antigen effector T cells (TC, TH immunogenic. For example, albumin isolated cells) + memory T cells from the serum of a rabbit and injected back Although a substance that induces a into the same or another rabbit will not specific immune response is usually called induce an immune response but the same an antigen, it is more appropriately called protein when injected into other vertebrate an immunogen. Antigenicity is the ability species (rat) will induce an immune response. of an antigen to combine specifically with B. Molecular size the final products of the above responses. There is a correlation between the size of a (antibodies and/or cell surface receptors). macromolecule and its immunogenicity. The All immunogens are antigens but all best immunogens tend to have molecular antigens are not immunogens. Some small mass approaching 100,000 daltons (Da). molecules called haptens are antigenic but Generally, substances with a molecular incapable, by themselves, of inducing a mass less than 5000-10000 Da are poor specific immune response. In other words immunogens; however a few substances haptens lack immunogenicity. Examples with a molecular mass less than 1000 Da of haptens are dinitrophenol, penicillin have proven to be immunogenic. and m-amino benzene sulphonate. C. Chemical composition and complexity Proteins are the most potent immunogens 13.5.2 Factors that Influence with polysaccharides ranking second. In Immunogenicity contrast, lipids and nucleic acids of an Immunogenicity is not an intrinsic property infectious agent generally do not serve as of an antigen but rather depends on a number immunogens unless they are complexed of properties of the particular biological with proteins or polysaccharides (examples-

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C. Adjuvants (Figure 13.23 & 13.24). Most T cells The response an immunogen is often recognize only peptides combined with enhanced if it is administered as a mixture MHC molecules on the surface of antigen with adjuvants. Adjuvants are substances presenting cells and altered self cells. that enhance the immunogenicity of () an antigen. Adjuvants function in one COO or more of the following ways. (1) by eme prolonging retention of the immunogen (2) by increasing the effective size of the immunogen or (3) by stimulating the local influx of macrophages and/ or other immune cell types to the injection site and promoting their subsequent activities. Example: Freund’s incomplete antigen, Freund’s complete antigen, ( ) Mycobacterium tuberculosis, Aluminum potassium sulphate (alum) and Bacterial lipopolysaccharide (LPS).

13.5.3 Epitopes Figure 13.23: Diagram of sperm whale myoglobin showing locations of five Immune cells do sequential B-cell epitopes not interact with or recognize an entire immunogen molecule ntigen resenting cell instead; lymphocytes recognize discrete sites on the macromolecule called epitopes or antigenic determinants. Epitopes are lass II M the immunologically active regions of an immunogen that bind to antigen specific membrane receptors on lymphocytes or etie to secreted antibodies. Antigenic epitopes may consist of a single epitope or have varying number of the same epitope on the same molecule (Example: polysaccharides). The size of a single epitope may be 4 or ell 5 aminoacid or monosaccharide residues. The recognition of antigens by T cells and Figure 13.24: Schematic diagram of the B cells is fundamentally different B cells ternary complex formed between a T-cell recognize soluble antigen when it binds receptor (TCR) on a TH cell, an antigen to their membrane-bound antibody. and a class II MHC molecule

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13.5.4 Haptens and the Study of the early 1940s. They demonstrated that Antigenicity the gamma globulin fraction of serum The pioneering work of Karl Landsteiner proteins that migrated most slowly in in the 1920s and 1930s created a simple, electrophoresis contained most of the chemically defined system for studying serum antibodies. This section deals with the binding of an individual antibody to the structural and biological properties of a unique epitope on a complex protein antibodies (immunoglobulins). antigen. Landsteiner employed various Definition of antibodies haptens(small organic molecules that Antibodies are glycoproteins present in are antigenic but not immunogenic). serum gamma globulins produced by Chemical coupling of a hapten to a B-lymphocytes (B cells) or Plasma cells in large protein called a carrier, yields an response to exposure to antigen. Antibodies immunogenic hapten-carrier conjugate. are also known as immunoglobulins. 13.5.5 Cross-Reactivity They react specially with that antigen in When two antigens possess structurally vivo or in vitro and are hence a part of the similar antigenic determinants, the adaptive immune response specifically, antibodies obtained to one of these humoral immunity. antigens tend to react with the other antigen. These reactions are called cross 13.6.1 Structure of an Immunoglobulin reactions. Molecule 1. Basic unit Infobits The basic structural unit (monomer) of an immunoglobulin molecule consists of Penicillin Allergy: New antigens are four polypeptide chains linked covalently produced by altering epitopes. This can by disulfide bonds (Figure 13.25). The be done by conjugating haptens to the four-chain structure is composed of molecule. A classic example in human two identical light (L) and two identical medicine is the allergic response of heavy (H) polypeptide chains. Every some persons to penicillin. A derivative immunoglobulin can be represented by of penicillin, penicilloic acid acting as the general formula (H L ) . a hapten, can couple with body protein 2 2 n and elicit an immune response that a) Light chains can be harmful, even life threatening, Light Chains have a molecular weight of thus excluding this antibiotic from use approximately 25000 Da and are composed in certain individuals. of about 220 amino acids. Light chains are common to all immunoglobulin classes and are of two types – kappa (κ) or lambda 13.6 Antibodies (λ) - based on their structural differences. The first real chemical information A given immunoglobulin molecule may regarding the structure of antibodies contain either identical κ or λ chains but was provided by Tiselius and Kabat in never both.

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eavy chain ight chain μγαδ ∈ or κ or γ

S S

S S S S Cl inge S S Cl S S ntigen binding C SS S SS C S SS SS S COO S SS COO CC Biological C activity CO SS CO SS C SS

COO COO Figure 13.25: Structure of Immunoglobulin b) Heavy chains i. Four known subclasses of the γ chain Heavy chains have a molecular weight of exist – γ1, γ2, γ3 and γ4 - which yield approximately twice that of light chains IgG1, IgG2, IgG3 and IgG4. (57000-70000 Da) and twice the number of ii. Two subclasses of the α chain are amino acids (about 440). Five antigenically known – α1 and α2 - which yield distinct isotypes of heavy chains are IgA1 and IgA2. recognized-gamma (γ), alpha (α), mu iii. Two subclasses of the μ chain are (μ), delta (δ) and epsilon (ε) – based known – μ1 and μ2 - which yield on structural differences in the carboxy IgM1 and IgM2. terminal portion of heavy chains. The heavy chains isotypes form the basis of five iv. No subclasses of the δ and ε (IgD classes of immunoglobulin molecules – IgG and IgE) are known. (contains γ chain), IgA (contains α chain), 2. Disulfide bonds IgM (contains μ chain), IgD (contains δ chain) and IgE (contains ε chain). Five Disulfide bonds hold together the heavy chain classes of immunoglobulin four polypeptide chains in normal can be easily remembered as GAMDE. immunoglobulin molecules and are of Heavy chain classes are again subdivided two types namely interchain bonds and into subclasses of molecules. intrachain bonds.

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a. Inter chain bonds occur between in the constant region (CH1, CH2, CH3, heavy chains (H-H), heavy and light and CH4). Each light chain consists of two chains (H-L) and light chains (L-L). domains – one in the variable region (VL) H-H bonds occur primarily in the and one in the constant region (CL). hinge region and can vary in number 5. Fragments. from 1-15 depending on the class and subclass of the immunoglobulin Proteolytic (peptide bond -splitting) molecules. enzymes such as papain and pepsin are used to degrade immunoglobulin b. Intra chain bonds are stronger than molecules into definable fragments to interchain bonds and occur within the facilitate study of their structure. individual chain type, with the number of bonds varying depending on the i. Treatment of the monomeric type (light chains have two, human basic unit with the enzyme papain γ, α and δ heavy chains have four and splits it into two Fab fragments human μ and ε heavy chains have (Fragment-antigen binding) five). The distribution of intrachain and one Fc fragment. These Fab disulfide bonds forms the basis for fragments can bind but cannot division of each immunoglobulin into precipitate the antigen; therefore, domains. they are monovalent, possessing only one combining site each. 3. Regions ii. Treatment of the immunoglobulin Each heavy and light chain consists of molecule with pepsin results two segments, the variable region and in digestion of most of the Fc the constant region. The variable (V) fragment, leaving one large region shows a wide variation in amino fragment that consists of two Fab acid sequence in the amino terminal fragments joined by covalent bonds, portion of the molecule. The areas of termed the F(ab’)2 fragment. The high variability in the variable region F(ab’)2 fragments has two antigen of heavy (VH) and light (VL) chains combining sites. Therefore it is are called hypervariable regions or bivalent, possessing the ability to complementarity determining regions bind and precipitate an antigen (CDRs). Hypervariable regions are most (Figure 13.26). intimately involved in formation of the antigen binding site. 6. Hinge region Hinge region is the portion of heavy chain 4. Domains between the CH1 and CH2 domains. It is Each immunoglobulin chain consists highly flexible and allows for movement of a series of globular regions enclosed of the Fab arms in relation to each other. by disulphide bonds. Each heavy chain The S values (sedimentation coefficient consists of four or five domains - one in that is expressed in Svedberg units(s)) of the variable region (VH ) and three or four immunoglobulins range from 7S- 19S.

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is l i e L chain bns

ss ss ss ss Fab chain e sin igesti n

ss ss ss ss

Fc ragments a ain igesti n Merca t ethan l re cti n Fab Fab

ss ss ss ss SS S S S S L chains Fc S S

chains Figure 13.26: Prototype structure of IgG, showing chain structure and interchain disulfide bonds

13.6.2 Immunoglobulin Function HOTS There are three major effector functions that enable antibodies to remove antigens Which antibody protects the new born and kill pathogens. Opsonization promotes for few months against infections? antigen phagocytosis by macrophages and neutrophils. Complement activation by IgM and IgG can activate a pathway that 13.6.3 Properties and Activities of leads to the generation of a collection of Immunoglobulin Classes proteins that can perforate cell membranes. Each immunoglobulin class differs in Antibody-dependent cell-mediated its general properties, distribution in cytotoxicity (ADCC) can cause NK cell the body and interaction with other mediated death of target cells when antibody components of the host defensive bound to the target cells associates with Fc systems. receptors of natural killer (NK) cells.

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i) IgG mucosal associated lymphoid 1. IgG is the major immunoglobulin tissue (MALT). It is also found in in human serum, accounting for saliva, tears, and breast milk. 80% of the immunoglobulin pool. 2. It consists of two monomeric 2. It is present in blood plasma and tissue units plus J chain and secretory fluids. It has a monomeric structure. component (Figure 13.29). 3. IgG class acts against bacteria and 3. The dominant subclass of sIgA viruses by opsonizing the invaders is sIgA2 which is unique for its and neutralizing toxins and viruses. absence of a covalent bond between the light and heavy chains. In this 4. IgG molecules are capable of fixing subclass, light chains are linked by complement, except for IgG4. disulphide bonds. 5. It is the major antibody in the 4. It has a half life of 5-6 days. It is secondary immune response and it responsible for local immunity. has half life of 23 days. 5. The sIgA molecules protect 6. IgG is the only immunoglobulin mucosal surfaces by reacting molecule able to cross the placenta with the surface of potential and provides natural immunity in pathogens and interfering with utero and to the neonate at birth. their adherence and colonization. ii) IgA It also plays a role in the alternative It is present in the serum and in various complement pathway. bodily secretions and thus takes two forms – iii) IgM serum IgA and secretory IgA (sIgA) 1. IgM accounts for about 5-10% of A) Serum IgA the serum immunoglobulin pool. 1. It accounts for about 12% of serum 2. It has a pentameric structure immunoglobulin. consisting of five monomeric units 2. In humans, over 80% of serum IgA linked by J chain and disulphide exists in a monomeric form and the bonds at the Fc fragment remaining existing as polymers in the (Figure 13.27). form of dimers, trimers or tetramers. 3. It is the predominant antibody in In polymeric IgA, the monomeric the primary immune response to units are linked by disulphide bonds most antigens and the predominant and joining (J) chain. antibody produced by the fetus. 3. Serum IgA fixes complement via 4. It is the first immunoglobulin the alternative pathway. It has a made during B cell maturation half life of 5 days. and individual IgM monomers are B) Secretory IgA expressed on B cells, serving as the 1. Secretory IgA is the antibody component of the B cell primary immunoglobulin of receptor (BCR).

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Secretory component eavy chain

ight chain

Chain

lgM Secretory lg Figure 13.27: Structural models of IgM and secretory IgA. IgM has a pentameric structure linked by J chain at the Fc fragment. Secretory IgA has a dimeric structure plus J chain plus secretory component and is shown in the dominant IgA2 subclass, which is unique for its absence of a convalent bond between the light and heavy chains. Light chains are linked by disulfide bonds.

5. IgM tends to remain in the B cell receptor complex. Therefore bloodstream, where it agglutinates their function is to signal the B cell (clumps) bacteria, activates to start antibody production upon complement by the classical initial antigen binding. pathway and enhances the ingestion 5. It has a half life of 2-3 days. of pathogens by phagocytic cells. v) IgE 6. It has a half life of approximately 5 days. 1. IgE accounts for only 0.004% of serum immunoglobulin. It iv) IgD has a monomeric structure. It 1. IgD accounts for about less than is also called reagin or reaginic 1% of the total immunoglobulin antibody. pool. 2. The skin sensitizing and 2. One unique structural feature is anaphylactic antibodies belong to the presence of only a single H-H this class. inter chain bond along with two 3. The Fc portion of IgE can bind to H-L interchain bonds. Fc receptors specific for IgE that 3. It has a monomeric structure are found on mast cells, eosinophils similar to that of IgG. and basophils. Thus these cells can 4. IgD antibodies are abundant in become coated with IgE molecules. combination with IgM on the surface When two cell- bound IgE molecules of B cells and thus are part of the are cross linked by binding to the

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Infobits i) Isotype Isotypic determinants are constant region determinants that collectively define each heavy chain class and subclass and each light chain type and subtype within a species. Each isotype is encoded by a separate constant region gene and all members of a species carry the same constant region genes. Within a species, each normal individual will express all isotypes in the serum. Different species inherit different constant region genes and therefore express different isotypes. Therefore, when an antibody from one species is injected into another species, the isotypic determinant will be recognized as foreign, inducing an antibody response to the isotypic determinants on the foreign antibody. ii) Allotype Although all members of a species inherit the same set of isotype genes, multiple alleles exist for some of the genes. These alleles encode subtle aminoacid differences, called allotypicdeterminnatsthat occur in some. The unique amino acid sequence of the VH and VL domains of a given antibody can function not only as an antigenic binding site but also as a set of antigenic determinants. Therefore, the idiotypic determinants are generated by the conformation of the heavy and light chain variable regions. Each individual determinant is called an idiotope and the sum of the individual idiotopes is the idiotype. Anti-idiotype antibody is produced by injecting antibodies that have minimal variation in their isotypes and allotypes, so that the idiotypic difference can be recognized.

Polyclonal Antibody: A mixture of antibodies produced by a variety of B-cell clones that have recognized the same antigen. Although all of the antibodies react with the immunizing antigen, they differ from each other in amino acid sequence. Breast Milk: Breast milk is uniquely suited to the human infant’s nutritional needs and is a live substance with unparalleled immunological and anti-inflammatory properties that protect against a host of illnesses and diseases for both mothers and children. All five classes of immunoglobulins have been found in human milk, but by far the most abundant type is IgA, specifically the form known as secretory IgA. Antitetanus Serum: Antitetanus serum, also known as tetanus immune globulin (TIG) is made up of antibodies against the tetanus toxin. It is used to prevent tetanus in those who have a wound that is at high risk and have not been fully vaccinated with tetanus toxoid. It is also used to treat tetanus along with antibiotics and muscle relaxants. It is given by injection into a muscle.

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same antigen, the cells degranulate. 13.7.1 Three stages of Antigen – This degranulation releases Antibody Reactions histamine and other mediators of a) Primary stage inflammation. The reactions between antigen and 4. IgE also stimulates production of antibody occur in three stages. The an excessive number of eosinophils primary stage is the initial interaction in the blood (eosinophilia) and between the two without any visible increased rate of movement effects. This reaction is rapid and obeys of the intestinal contents (gut the general laws of physical chemistry hypermotility) which aid in the and thermodynamics. The reaction is elimination of helminthic parasites. reversible. The combination between IgE has a half life of 2-3 days. antigen and antibody is effected by the weaker intermolecular forces such as 13.6.4 Antigenic Determinants on electrostatic forces, hydrogen bonds, Van Immunoglobulins der Waals forces and hydrophobic forces. Since antibodies are glycoproteins, they can The primary reaction can be detected themselves function as potent immunogens by estimating free and bound antigen to induce an antibody response. Such anti-Ig or antibody separately in the reaction antibodies are powerful tools for the study mixture by a number of physical and of B cell development and humoral immune chemical methods including the use of response. The antigenic determinants or markers such as radioactive isotopes, epitopes, on immunoglobulin molecules fluorescent dyes or enzymes. fall into three major categories: isotypic, b) Secondary stage allotypic and idiotypicdeteminants, which are located in characteristic portions of the The primary stage is followed by the molecule. secondary stage leading to demonstrable events such as precipitation, agglutination, 13.7 Antigen – Antibody Reactions lysis of cells, killing of live antigens, neutralization of motile organisms, Antigen and antibody combine with each complement fixation and enhancement of other specifically and in an observable phagocytosis. manner. The exquisite specificity of antigen-antibody interactions has c) Tertiary stage led to the development of a variety of Some antigen-antibody reactions immunological assays. These assays can occurring in vivo initiate chain reactions be used to detect the presence of either that lead to neutralization or destruction antibody or antigen. These assays are also of injurious antigens or to tissue damage. helpful in diagnosing diseases, monitoring These are the tertiary reactions and epidemiological surveys and identifying include humoral immunity against molecules of biological or medical interest. infectious diseases as well as clinical Antigen-antibody reactions in vitro are allergy and other immunological known as serological reactions. diseases.

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13.7.2 General Features of Antigen – 13.7.3 Measurement of Antigen and Antibody Reactions Antibody Antigen-antibody reactions have the Many methods are available for the following general characteristics: measurement of antigens and antibodies 1. The antigen-antibody reaction is participating in the primary, secondary and specific. An antigen combines only tertiary reactions. Measurement may be in with its homologous antibody and terms of mass (Example: mg Nitrogen) or vice versa. However, the specificity more commonly as units or titre. The antibody is not absolute and cross reactions titre of a serum is the highest dilution of the may occur due to antigenic serum which gives an observable reaction similarity or relatedness. with the antigen in the particular test. The titre of a serum is influenced by the nature 2. An entire molecule reacts and not and quantity of the antigen and the type and fragments. conditions of the test. Antigens may also be 3. There is no denaturation of the titrated against sera. antigen or the antibody during the Two important parameters of reaction. serological tests are sensitivity and 4. The combination occurs at the specificity. Sensitivity refers to the ability surface. of the test to detect even very minute 5. The combination is firm but quantities of antigen and antibody. reversible. The firmness of the union When a test is highly sensitive, false is influenced by the affinity and negative results will be absent or minimal. avidity of the reaction. Affinity is the Specificity refers to the ability of the test strength of binding of one molecule to detect reactions between homologus to another at a single site, such as antigens and antibodies only. When a test the binding of a monovalent Fab is highly specific, false positive results fragment of antibody to a monovalent will be absent or minimal. Some tests are antigen. Avidity is the sum total of the qualitative and others are quantitative. strength of binding of two molecules The various tests used for detection of to one another at multiple sites. antigen and antibodies are given below: 6. Both antigen and antibody 1. Precipitation tests participate in the formation of agglutinates or precipitates. 2. Agglutintion tests 7. Antigens and antibodies can 3. Complement Fixation test combine in varying proportions, 4. Immunofluorescence unlike chemicals with fixed valence. 5. Radio immuno assay Both antigens and antibodies 6. Enzyme linked immuno sorbent assay are multivalent. Antibodies are 7. Western Blotting technique bivalent. Antigens may have valencies up to hundreds. 8. Neutralization test

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In this section Agglutination and Precipitation reaction in gels Precipitation reactions will be described There are several advantages in in detail. allowing precipitation to occur in a gel rather than in a liquid medium. The 1. Precipitation reactions reaction is visible as a distinct band of When a soluble antigen combines with its precipitation, which is stable and can antibody in the presence of electrolytes be stained for preservation, if necessary. (NaCl) at a suitable temperature and pH, Imunodiffusion is usually performed in the antigen-antibody complex, forms an 1% agarose gel. Different modifications insoluble (visible) precipitate and this of the test are available. reaction is called precipitation. When instead of sedimenting, the precipitate • Single Diffusion in One Dimension remains suspended as floccules, the (Oudin Procedure) reaction is known as flocculation. • Double Diffusion in One Dimensions • Applications of precipitation reactions (Oakley-Fulthorpe Procedure) The following types of precipitation tests • Single Diffusion in Two Dimensions are in common use: (Mancini Procedure) a) Ring test • Double Diffusion in Two Dimensions This test consists of layering the antigen (Ouchterlony Procedure) solution over a column of antiserum in a Immunoelectrophoresis narrow tube. A visible precipitate forms at Immunoelectrophoresis was devised the junction of the two liquids. Examples by Grabar and Williams (1953). This of ring precipitation test are the C- reactive method consists of two steps. The first protein test, Ascoli’s thermoprecipitin step is agarose electrophoresis of the and the grouping of streptococci by the antigen. Rectangular trough is then Lancefield technique. cut into the agarose gel parallel to the b) Slide test direction of the electric field and is filled When a drop of antigen and a drop of with the antiserum. By diffusion, lines antiserum are placed on a slide and mixed by of precipitation develop with each of the shaking, floccules appear. The VDRL test for separated compounds (Figure 13.28). syphilis is an example of slide flocculation. This method is used to detect normal and c) Tube test abnormal serum proteins. A quantitative tube flocculation test is 1. Semisolid agar layered on the glass used for the standardization of toxins slide. A well for antigen and a trough and toxoids. Serial dilution of the toxin / for antiserum cut out of agar. toxoid is added to the tube containing a 2. Antigen well filled with human serum. 3. Serum separated by electrophoresis. fixed quantity of the antitoxin. The toxin 4. Antiserum trough filled with antiserum or toxoid that flocculates optimally with to whole human serum. one unit of the antitoxin is defined as the 5. Serum and antiserum allowed to Lf (Lethal Flocculation) dose. diffuse into agar.

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Figure 13.28: Immunoelectrophoresis

6. Precipitin lines form for individual directly by antibody. An example is the serum proteins agglutination of group A erythrocytes by • Counterimmunoelectrophoresis anti-A sera. • Rocket Electrophoresis Indirect (Passive) agglutination test 2. Agglutination reactions Passive agglutination refers to agglutination of antigen coated cells or inert When a particulate antigen is mixed with particles (bentonite or latex particles) its antibody in the presence of electrolytes which are passive carriers of soluble an- at a suitable temperature and pH, the tigens. An example is the latex agglutina- particles are clumped or agglutinated, and tion for detection of rheumatoid factor. the reaction is called agglutination. When instead of the antigen, the antibody Agglutination is more sensitive than is adsorbed to carrier particles in test for precipitation for detection of antibodies. estimation of antigen, this technique is Agglutination occurs optimally when known as reverse passive agglutination. antigens and antibodies react in Hemagglutination inhibition method equivalent proportions. Incomplete or monovalent antibodies (having only one The inhibition of agglutination of antigen combining site) do not cause antigen-coated red blood cells by agglutination, though they combine with homologous antigen is a highly sensitive the antigen. They may act as blocking and specific method for detecting small antibodies inhibiting agglutination by the quantities of soluble antigen in blood or complete antibody added subsequently. other tissue fluids. The principle of this method is that antibody preincubated Direct agglutination test with soluble homologous antigen will be In the direct technique, a cell or insoluble inactivated when incubated with antigen particulate antigen is agglutinated coated red blood cells.

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This method is used in the detection of test tubes, the agglutination titre of the HBs Ag in hepatitis and in the detection serum can be estimated. Widal test done of factor VIII antigen in hemophilia. for typhoid and Weil Felix test done for Hemagglutination inhibition is also rickettsial infections are examples of used to detect antibodies against certain Tube agglutination. viruses (Arbovirus, Influenza, Measles Latex agglutination test and Rubella). These viruses are able to Here latex particles are used as passive agglutinate red blood cells because they carriers for adsorbed soluble antigens. possess hemagglutinins on their outer The most widespread application of latex surfaces. agglutination has been in the detection of rheumatoid factor. In rheumatoid • Applications of agglutination arthritis, the patient’s produces reactions rheumatoid factor. Rheumatoid factor a) Slide agglutination is a pentameric IgM antibody directed When a drop of the appropriate antiserum against IgG. The test consists of coating is added to a smooth uniform suspension latex particles with IgG and reacting them of a particulate antigen in a drop of saline with the patient serum. Agglutination on a slide, agglutination takes place. A indicates a positive test. Latex positive result is indicated by the clumping agglutination tests are also employed in together of the particles and the cleaning the clinical laboratory for detection of of the drop. Mixing the antigen and the HBs Ag, ASO (Antistreptolysin O) and antiserum by gently rocking the slide CRP (Carbohydrate Reactive Protein) facilitates the reaction. Coombs test (antiglobulin test) It is essential to have on the same slide a This test was devised by Coombs, control consisting of the antigen suspension Mourant and Race (1945) for the in saline, without the antiserum, to ensure detection of anti-Rh antibodies that do that the antigen is not autoagglutinable. not agglutinate Rh-positive red blood Agglutination is visible to the naked eye cells in saline. When sera containing but may sometimes require confirmation incomplete anti-Rh antibodies are mixed under the microscope. Slide agglutination with Rh- positive red blood cells, the is a routine test for the identification antibody globulin coats the surface of of many bacterial isolates from clinical the red blood cells, though they are not specimens. It is also the method used for agglutinated. When such red blood cells blood grouping and cross matching. coated with antibody globulin are washed b) Tube agglutination free of all unattached protein and treated This is a standard quantitative method with a rabbit antiserum against human for measurement of antibodies. When gammaglobulin (antiglobulin or Coombs a fixed volume of a particulate antigen serum), the cells are agglutinated. This suspension is added to an equal volume is the principle of the Coombs test of serial dilution of an antiserum in (Figure 13.29).

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Figure 13.29: Antiglobulin (Coombs) test Rh positive erythrocytes (1) are mixed with incomplete antibody (2). The antibody coats the cells (3) but, being incomplete, cannot produce agglutination. On addition of antiglobulin serum (4) which is complete antibody to immunoglobulin, agglutination takes place.

The Coombs test may be of the direct 2. The evaluation of hemolytic disease or the indirect type. of the newborn. Applications of coombs test 3. The diagnosis of autoimmune 1. Erythrocyte typing in blood banks. hemolytic anemia.

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Summary than that of neutrophils. They play a role in the defense against parasitic organisms. Immunology began as a study of Macrophages and neutrophils are the the response of the whole animal to accessory cells of the immune system infection. Over the years, it has become that phagocytose and degrade antigens. progressively more basic, passing through Dendritic cells are antigen presenting cells. phases of emphasis on serology, cellular They play an important role in T cell immunology, molecular immunology and H activation by processing and presenting immunogenetics. antigen bound to class II MHC molecules. The thymus and bone marrow are the Lymphocytes can be subdivided into B primary lymphoid organs. The primary lymphocytes, T lymphocytes and null cells lymphoid organs provide sites for the (NK cells). The two major subpopulations development and maturation of B and T of T lymphocytes are T helper (TH) cells lymphocytes. and T cytotoxic (TC) cells. The secondary lymphoid organs Immunity is the state of protection function to capture antigen and provide against foreign organisms or substances sites where lymphocytes interact with that (antigens). Innate immunity offers antigen and undergo clonal proliferation resistance to any microorganism or and differentiation into effector cells. foreign material. It has no immunological The lymphatic system drains the tissue memory. Acquired immunity resists spaces and interconnects many organized particular foreign agent. Moreover, lymphoid tissues. The spleen, lymphnodes acquired immunity improves on repeated and mucosal associated tissues (GALT and exposure to the agent. Mechanisms of SALT) are secondary lymphoid organs. innate immunity include physical barriers, Lymph nodes are specialized to trap antigen chemical mediators, phagocytosis and from regional tissue spaces, whereas the inflammation. Acquired (adaptive) spleen traps blood- borne antigens. immunity refers to the type of specific The cells that participate in the immunity that a host develops after immune response are white blood cells exposure to a suitable antigen. Two or leukocytes. All of the white blood cells branches or arms of acquired immunity develop from a common pluripotent stem are humoral (antibody mediated) cell in hematopoiesis. Lymphocytes are the immunity, and cellular (cell mediated) central cells of the immune system and are immunity. The important features responsible for acquired immunity. The of acquired immunity are memory, other types of white blood cells play ancillary specificity, diversity and discrimination roles such as engulfing and destroying between self and nonself. The humoral microorganisms, presenting antigens and immunity is best suited for elimination secreting cytokines. Basophills and mast of extracellular antigens. The cellular cells are non phagocytic granulocytes immunity is best suited for elimination of that play a role in allergic responses. intracellular antigens. Acquired immunity Eosinophils are motile phag ocytic cells. can be obtained actively or passively by Their phagocytic role is less important natural or artificial means.

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Immunogenicity is the ability of an molecule, the constant region contains one antigen to induce an immune response by of five basic heavy chain sequences (γ, α, either the humoral or cell mediated branch μ, δ and ε) and one of two basic light chain of the immune system. Antigenicity is the sequences (k or γ). There are three major ability of an antigen to interact specifically effector functions of immunoglobulins with free antibody and/or antigen binding are Opsonization, Complement activation receptors on lymphocytes. The foreignness, and Antibody-dependent cell-mediated molecular size, chemical composition and cytotoxicity (ADCC). susceptibility to antigen processing and In vitro antigen-antibody reactions presentation influence the immunogenicity (serological reactions) provide methods for of a substance. In addition, several properties the diagnosis and for the identification and of biological system that an antigen quantitation of antigens and antibodies. encounters affect its immunogenicity; these The reactions between antigen and include the genetic constitution of the host antibody occur in three stages viz, primary animal, the immunogen dosage and the stage, secondary stage and tertiary stage. route of administration and the presence or absence of adjuvants. Epitopes are the The interaction between a soluble regions or sites of the antigen that bind antigen and precipitating antibody to a specific antibody or T cell receptor. (precipitin) produces visible precipitate or Haptens are small molecules that can bind precipitation. Precipitation reactions can be to antibodies but cannot by themselves performed in liquids or gels. Most are useful induce an immune response. The conjugate primarily for quantitative comparison of formed by coupling a hapten to a large antibodies or antigens. Electrophoresis can carrier protein is immunogenic and elicits be combined with precipitation in gels a production of antihapten antibodies when technique called immunoelectrophoresis. injected into an animal. The cross reactivity The interaction between a particulate is the ability of a particular antibody or T cell antigen and agglutinating antibody receptor to react with two or more antigens (agglutinin) produces visible clumping or that possess a common epitope. Antibodies agglutination. In some cases, the antigen are immunoglobulins which are produced is membrane protein on a bacterial by B cells or plasma cells in response to cell or red blood cell. In other cases, antigenic stimulation. the antigen may be attached to a latex Antibodies are a group of glycoproteins particle or adsorbed on the surface of a present in the blood tissue fluids and red blood cell. Agglutination reactions mucous membranes of vertebrates. All are more sensitive and much faster than immunoglobulins have a basic structure precipitation reactions and can detect composed of four polypeptide chains (two 100 or 1000 fold lower levels of antigen lights and two heavy) connected to each other or antibody than can be detected with a by disulphide bonds. In any given antibody precipitation reaction.

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ICT CORNER Immunology

How are we protected from microbes?

STEPS: • Use the link or Scan the QR code given below. “Cells Alive-Immunology” will open. You can select any topic you wish. For example click “Making Antibodies” • ‘Making Antibodies’ page will open. You can go through How ‘Lymphocytes Produce Antibody’, ‘Antigen Processing’, etc…. • From the top of the page click on ‘Video’ and select ‘watch’ view video topics. Select ‘Cytotoxic T Cells’. • From the top select ‘Study’ and then ‘Quiz’ to answer the questions for the topic you choose.

Step1 Step2

Step3 Step4

URL: https://www.cellsalive.com/toc_micro.htm

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Evaluation c. Erythrocytes, eosinophils, basophils, monocytes, mast cells, Multiple choice questions platelets and T lymphocytes. 1. Who coined the term d. Erythrocytes, eosinophils, vaccine? neutrophils, basophils, monocytes, a. Jenner b. Pasteur mast cells and NK cells. c. Koch d. Roux 7. Which of the following is a correct 2. Who advanced the idea that immunity statement about NK cells? was primarily due to white blood a. They kill target cells by cells? phagocytosis and intracellular a. Metchinikoff b. Ehrlich digestion. c. Wright d. Kitasato b. They proliferate in response to 3. Which of the following does apply antigen. uniquely to secondary lymphoid c. They kill target cells in an organs? extracellular fashion. a. Presence of precursor B and T cells. d. They are a subset of b. Circulation of lymphocytes. polymorphonuclear cells. c. Terminal differentiation. 8. Which of the following cells play an important role in the development of d. Cellular proliferation. allergies? 4. Which of the following is the major a. Neutrophils b. Mast cells function of the lymphoid system? c. Monocytes d. Dendritic cells a. Acquired immunity. c. Absence of specificity b. Innate immunity. d. Activation by a stimulus c. Inflammation. 9. All of the following will act as d. Phagocytosis. opsonins except 5. Lymph nodes taken from neonatally a. Complement b. Antibody thymectomized mice have unusually few cells in the c. Acute – phase proteins a. Paracortex b. Cortex d. Lactoferrin c. Medulla d. Thymus 10. Which of the following is not the important feature of acquired 6. The myeloid progenitor gives rise to immunity? a. Erythrocytes, neutrophils, a. Phgocytosis b. Memory. eosinophils, basophils, monocytes, mast cells and platelets. c. Specificity b. Erythrocytes, eosinophils, d. Discrimination between self and basophils, monocytes, mast cells, non-self platelets and B lymphocytes.

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11. Cell mediated immunity is brought 16. Antigenic sites with which antibodies about by react are called a. B cells b. T cells a. Immunogens b. Carriers c. NK cells d. Null cells c. Epitopes d. haptens 12. Vaccines induce immunity that is 17. Basic structural unit of an a. Naturally acquired active immunoglobulin molecule includes immunity. a. Identical λ light chains only. b. Naturally acquired passive b. One constant and three variable immunity. regions. c. Artificially acquired passive c. Two identical heavy and two immunity. identical light chains. d. Artificially acquired active d. A total of five domains. immunity. 18. J chain is a glycopeptides chain 13. Haptens associated with which of the following a. Require carrier molecules to be immunoglobulins? immunogenic. a. IgA b. IgG c. IgD d. IgE b. Interact with specific antibody, 19. Primary interactions between even if the haptens are monovalent. antigens and antibodies involve all of c. Cannot stimulate immune the following except which? responses without carriers. a. Van der Waals forces d. All of the above. b. Hydrophobic forces 14. The protection against small pox virus c. Electrostatic forces infection afforded by prior infection d. Covalent bonds with cowpox represents 20. When instead of the antigen, the a. Antigenic specificity antibody is adsorbed to carrier b. Antigenic cross reactivity. particles in test for estimation of c. Innate immunity. antigen, this technique is known as d. Passive protection. a. Indirect agglutination 15. An adjuvant is a substance that b. Direct agglutination a. Enhances the immunogenicity of c. Reverse passive agglutination haptens. d. Hemagglutination inhibition b. Increases the chemical complexity of the immunogen. Answer the following c. Enhances the immune response to 1. What is immunology? the immunogen. 2. Define the term vaccination. d. Enhances the immunologic cross 3. What are M cells? – reactivity.

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4. What is the role of primary lymphoid 22. Briefly explain the various stages organs and secondary lymphoid involved in phagocytosis. organs? 23. Write short notes on interferons. 5. Define hematopoiesis. 24. Write short notes on primary im- 6. What are pluripotent stem cells? mune response/secondary immune 7. What is acquired immunity? response? 8. What is immunological memory? 25. How do adjuvants function? 9. Define the term active/passive 26. Explain immunoglobulin structure immunity. and function? 10. What is immunogenicity? 27. Describe briefly the structure and 11. Define the term immunogen. function of thymus. 12. What are haptens? 28. Describe the structure and function of spleen/lymph node. 13. What is antigenicity? 29. Describe the characteristics of 14. Define epitopes. macrophages 15. Define the term antibodies. 30. Write the characteristics of 16. What is opsonization? B/T cells. 17. What is immunity/complement? 31. Briely explain the three major events 18. What is precipitation/agglutination? in the inflammatory response. 19. Write short notes on eosinophils/ 32. Briefly explain humoral immunity. neutrophils. 33. Write an account of cell mediated 20. Give a short account of natural killer immunity. cells. 34. Mention the properties of IgM. 21. How do intact mucous membranes 35. List out the general features of resist microbial invasion of the host? antigen-antibody reactions.

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Chapter 14 Microbial Genetics

Chapter Outline

14.1 Genetic Information is Stored in DNA 14.2 Structure of DNA 14.3 DNA Replication

Microbial genetics provides powerful tools for deciphering the regulation, as well as the functional and pathway organization of cellular processes. The genetic study of microbes has played a highly significant role in the developments of Molecular Biology, Recombinant DNA Technology and in the preparation of useful products. Microbial Genetics makes microbes beautiful, beneficial and bountiful.

Learning Objectives

After studying this chapter the student • To know Meselson and Stahl’s will be able, experiment. • To review the historical discoveries • To explain the steps of replication. that led to establishing DNA as the • To know the enzymes and their roles genetic material. involved in DNA replication. • To identify the role of genetic material. 14.1 Genetic Information is Stored • To recognize the contributions in DNA of Griffith, Avery, MacLeod, and Microorganisms are diverse in nature. McCarty, and Hershey and Chase. A particular bacterium can be identified • To explain the structure of DNA. based on certain characteristics. When a • To recognize the contributions bacterial cell grows and divides, it gives of Chargaff, Rosalind Franklin, rise to cells with similar characteristics. Maurice Wilkins, Watson and Crick. Have you ever pondered as to why some • To describe the Watson and Crick of the characteristics of progeny cells are model of DNA. similar and a few dissimilar? th • To compare structure of DNA and In the middle of the 19 century it RNA. was assumed that there was some particle present somewhere in the cell which

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was the controlling factor to carry the 1. Rough strain (R) – avirulent, characteristics from one generation to non-capsulated strain, forming rough another. colonies on culture media. Genetics, a branch of science aims to 2. Smooth strain (S) – virulent, understand the working of the controlling capsulated strain (resists phagocytosis), factor. The factor governing the transfer forming smooth colonies on culture of information is now very well known media. as Gene. Gene can be defined as a unit of Griffith injected live smooth strain into heredity which is transferred from parent mice which caused disease and killed to progeny. it. When he injected live rough strain Although there were experiments into mice, it did not cause disease and to prove the inheritance pattern due to mice remained alive. He heat killed the gene, there was no real understanding smooth strain and injected into mice, the of the molecular nature of the gene. mice remained alive. But the experiment Work of Frederick Griffith introduced gave surprising results when a mixture the transforming principle which was of harmless live rough strain and heat- further confirmed as Deoxyribonucleic killed smooth strain was injected acid (DNA) by experiments of Avery, Mac into mice. Griffith observed that the Leod and Mac Carty in 1944 followed by mice developed pneumonia and died. Hershey and Chase in 1952. Further, when he analysed the blood sample from dead mouse, he found that 14.1.1 Frederick Griffith’s Experiment it contained live smooth strain. This In 1928, British accidental discovery made Griffith to bacteriologist conclude that the rough strain changed Frederick Griffith (transformed) into smooth strain by (Figure 14.1) was taking up a substance which he called a trying to develop “transforming principle” from heat killed a vaccine against Figure 14.1: smooth bacteria. This phenomenon pneumonia. In his Frederick Griffith is called “Bacterial Transformation” experiments Griffith Griffith’s experiment is summarized in used two related strains of Streptococcus Figure 14.3. pneumoniae (Figure 14.2).

Figure 14.2: Rough and Smooth colonies of Streptococcus pneumoniae

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Live rough Live smooth Heat-killed Heat killed smooth strain and strain strain smooth strain live rough strain

In blood sample, living S cells are found that can reproduce, yielding more S cells

Mouse healthy Mouse dies Mouse healthy Mouse dies Figure 14.3: Summary of Griffith’s experiment

(Figure 14.4) used the extracts of HOTS heat-killed smooth bacteria and treated it with enzyme protease, RNase, DNase What did Griffith expect to happen to eliminate proteins, RNA and DNA to mouse when he injected it with live respectively. Each of the treated extracts rough strain and heat killed smooth were mixed with live rough bacteria and strain injected into mice. The mice injected with a mixture of DNase treated extract and 14.1.2 Oswald T. Avery, Colin live rough strain did not die. This partially Mac Leod and Maclyn Mc Carty’s proved that DNA was responsible for Experiment changing the rough strain of Streptococcus pneumoniae bacteria into smooth bacteria. Avery et al., experiment is summarized in Figure 14.5. Later Hershey and Chase’s experiment on T2 bacteriophage confirmed that genetic information is present in DNA. These important early experiments and many other lines of evidence have shown that DNA bears the genetic information of Figure 14.4: Avery et al., living cells and it is responsible for transfer Griffith’s experimental results led to the of characteristics from one generation to curiosity to explore the transforming another. This is true in all organisms, the principle. Avery and his colleagues notable exceptions being RNA viruses

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tract c ntaining r tein heat kille sm th strain Streptococcus pneumoniae

reate r tease ase ase ith

Li e r gh strain Li e r gh strain Li e r gh strain

M se ies M se ies M se alie

S cells resent S cells resent S cells absent there re trans rmati n there re trans rmati n there re trans rmati n cc rre rans rmati n cc rre rans rmati n i nt ccr cc rs in the absence cc rs in the absence rans rmati n es r teins O cc r IO Figure 14.5: Avery, Mc Cleod and Mac Carty’s experiment

Infobits

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The entire genetic content of a cell is known as its genome and the study of genomes is genomics. In eukaryotic cells, but not in prokaryotes, DNA forms a complex with histone proteins to form chromatin, the substance of eukaryotic chromosomes. A chromosome may contain tens of thousands of genes. Many genes contain the information to make protein product. DNA controls all of the cellular activities by turning the genes “on” or “off.”

Types of Nucleic acids

DNA RNA

dsDNA Genetic RNA Non-genetic ssDNA Example: E.coli RNA: Example: Lamda (λ) mRNA, rRNA, ØX174 phage phage dsRNA ssRNA tRNA Example: Example: Rheovirus TMV

which store genetic information in RNA. • The sugar present in DNA is The understanding of DNA’s role in deoxyribose sugar. heredity has led to variety of practical • The nitrogenous bases present in DNA applications including forensic analysis, are paternity testing and genetic screening. * Purines – Adenine (A), Guanine (G) 14.2 DNA Structure * Pyrimidines – Thymine(T), • DNA is a polymer of simple monomeric Cytosine (C) units, the nucleotides (Figure 14.6). • The nucleotide as a unit is formed by • Each nucleotide is made up of three * Glycosidic bond between components: nitrogenous base and sugar, 1. Nitrogenous base * Ester bond between phosphate 2. Sugar group and sugar • Each of the nucleotides is bonded 3. Phosphate group by a phosphodiester bond to form • Nucleotide without phosphate group is a polynucleotide chain (strand) known as nucleoside. (Figure 14.7a).

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cen S gar c c c h s hate O O O O O O c c c S c c c c c S gar ase OOO cle ti e D c c cen O O

c O O O

S racil h mine t sine c nl nl b th an c S gar ase cle si e O

c c S cen cle si e tri h s hate enine anine

Figure 14.6: Structure of nucleotide, nucleoside, deoxyribose, ribose and nitrogenous bases • Two polynucleotide chains join variety of sequences that can be made from together through hydrogen bonds the four nitrogenous bases is limitless, as between nitrogenous bases, to form is the number of melodies possible with double stranded DNA (Figure 14.7b). a few musical notes. RNA differs from • Two hydrogen bonds exists between DNA by having a ribose sugar instead of adenine and thymine and three deoxyribose and nitrogenous base Uracil hydrogen bonds between guanine and instead of Thymine. cytosine. 14.2.1 Watson And Crick Model of • DNA is coiled in the form of a double DNA Double Helix helix, in which both the strands of DNA In the early 1950’s, Rosalind Franklin and coil around an axis (Figure 14.7d & e). Maurice Wilkins used the powerful method • The further coiling of this axis upon of X-ray diffraction to shed more light on itself produces DNA supercoiling an the structure of DNA. From the X-ray important property of DNA structure. Diffraction pattern it was deduced that All DNA, whether large or small, possess DNA molecules are helical. In 1953 Watson the same sugar phosphate backbone. What and Crick (Figure 14.8) postulated a three distinguishes one DNA from another is dimensional model of DNA structure based the length of the polymer and distribution on Franklin’s X-ray crystallographic studies. of four bases along the backbone. The In recognition of their work leading to

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a ln cle ti e chain b strans c laer e arn bne b h rgen ble a central a is bns heli Figure 14.7: Structure of a single polynucleotide chain of DNA, hydrogen bonding between two DNA strands, Double helix around axis the double helix model, Nobel prize was awarded in 1962 to Watson, Crick and Wilkins. According to Watson and Crick model (Figure 14.9), • The DNA consists of two helical polynucleotide chains wound around the same axis to form a right handed helix. • The Purine and Pyrimidine bases of both strands are stacked inside the double helix. Figure 14.8: Watson and Crick • Each nitrogenous base of one strand is and three hydrogen bonds are present paired in the same plane with a base of between G and C (G C). the other strand. • The hydrogen bonds provide chemical • According to Watson and Crick stability essential to hold the two rule Adenine base pairs with chains together. Thymine and Guanine base pairs • The specific A equal to T and G with Cytosine. equal to C base pairing is the basis • Two hydrogen bonds are present for the complementarity concept. between A and T (symbolised as A=T) This complementarity concept is very

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ƍƍ iameter

Min r re One c m lete t rn

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itr gen s base air nti arallel c m lementar Stran s ƍ ƍ ats n an rick mel ƍ ƍ entral a is

Figure 14.9: Watson and Crick DNA model, Antiparallel dsDNA

important in the process of DNA Chargaff measured the quantity of the replication and gene expression. bases in DNA and noticed that the number • The pairing of two strands creates a of Adenine is equal to the number of major groove and minor groove on the Thymine and the number of Guanine is surface of the duplex. equal to the number of Cytosine residues. Hence the sum of Purine residues equal to • The two strands are antiparallel, that is the sum of the Pyrimidine residues. their 5ʹ, 3ʹ phosphodiester bonds run in opposite directions. Quantitatively A=T or A/T = 1 C=G or C/G = 1 • The vertically stacked bases are 3.4 Aº A+G = T+C apart. • Each complete turn of double helix con- HOTS tain base pairs, which is 34 Aº units long.

14.2.2 Erwin Chargaff’s Rule If percentage of adenine in one of the In the late 1940s Erwin Chargaff and his DNA strand is 20. Can you determine colleagues found that the four nucleotide the percentage of other bases. If yes bases of DNA occur in different ratios in how? the DNA of different organisms. Erwin

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14.2.3 Alternative Forms of DNA Table 14.1: Properties of different forms DNA is a remarkably flexible molecule. of DNA Considerable rotation is possible around A form B form Z form a number of bonds in sugar-phosphate backbone and thermal fluctuations can Helical Right Right Left produce bending, stretching and unpairing sense handed handed handed of the strands. Watson and Crick model Diameter a 26Aº a 20 Aº a 18 Aº of DNA is called as B-DNA or B-form. However DNA can exist in A or Z form. Base pairs 11 10 12 In 1979 Alexander Rich discovered Z form per helical (Figure 14.10). Recently, several alternative turn forms of DNA have been discovered C-form, Distance 2.6 Aº 3.4 Aº 3.7 Aº D-form and E-form. The B-form of DNA is between the most stable structure and is therefore adjacent the standard point of reference in any study bases of the properties of DNA (Table 14.1).

Bacterial genome size

• Bacterial genomes are typically expressed in Mb • The length of Bacterial genomes are typically in the mm range and therefore 1000X bigger than the typical bacterial size. • The mass of Bacterial genomes are typically in 10−3 pg(picogram) range.

Conversions

1 Kb = 10 3 bp 1 bp ≈ 0.33nm 1 pg = 10−12 g (base pairs) 1 kb ≈ 0.33μm 1pg = 978 Mb 6 1 Mb = 10 bp 1 Mb ≈ 0.33mm Number of base pairs = 9 9 1 Gb = 10 bp 1 Gb ≈ 0.33m mass in pg × (0.978 × 10 ) 1 kb ≈ 10−6 pg 1 Mb ≈ 10−3 pg 1 Gb ≈ 1 pg

Bacteria Virus, organelles Eukaryotes Genomes Small(Mb) Tiny (Kb) Large (Gb) Gene Density High High Low Example E.coli 5000 genes Bacteriophages Homo sapiens 10-100 genes 25000 genes

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14.3 DNA Replication DNA is a marvelous device for the stable storage of genetic information. DNA replication is a process in which copies of DNA molecules are faithfully made. Here double stranded DNA molecule is copied to produce two identical dsDNA molecules. Replication is an essential process because whenever a cell divides, the two new daughter cells must contain the same genetic information Figure 14.10: Forms of DNA in DNA as parent cell. DNA replication occurs during the S (DNA synthesis) phase and precedes cell division. HOTS Watson and Crick proposed the hypothesis of semiconservative replication. Write the base sequence of According to them each DNA strand serves complementary DNA and RNA strand as a template for the synthesis of a new for the following. strand, producing two new DNA molecules 5ʹGCGCAATATTTCT3ʹ each with one old strand and one new strand. (Figure 14.11).

Infobits

Max Delbruck suggested that there could be three possible ways in which Semiconservative + DNA could replicate. Semiconservative – DNA replication that produce two copies of double Conservative + stranded DNA (dsDNA) each containing one old strand and one new strand.

Conservative – DNA replication that Random dispersive + produces two daughter ds DNAs, one of which consists of two original strands Figure 14.11: Semiconservative, whereas the other daughter DNA consists conservative and dispersive replication of two newly synthesised strands. Dispersive – DNA replication in which the original dsDNA undergoes fragmentation, the fragments synthesize complementary structure both of which assemble to form two replicas.

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14.3.1 Meselson and Stahl’s Experiment gradient centrifugation (Caesium Mathew Meselson and chloride (CsCl) centrifugation). Franklin Stahl in 1957 gave Expected results experimental evidence for • The heavy isotope 15N containing semiconservative replica- DNA, will reach equilibrium in a tion process. gradient point closer to the bottom of Steps the tube. 1. E.coli cells were grown for many • 14N containing DNA will reach generations in a medium containing equilibrium at a gradient point closer radioactive isotope (heavy isotope) of to the surface of the tube. 15 nitrogen source N. Observed Results 2. After many generations all nitrogen • After first generation the isolated DNA containing molecules in E.coli cells, occupied an intermediate density band. including nitrogen bases of DNA contained 15N. • After second generation two bands 3. The cells labeled with 15N were then were observed, one at intermediate transferred to a medium containing density and the other at lighter density 14 only 14N (light isotope). Hence all corresponding to the N position in subsequent DNA synthesised during the gradient. replication contained 14N. These experimental results and other 4. Cell samples were removed at periodic experiments repeated by Meselson time intervals from the growth medium. and Stahl with prokaryotes suggested 5. From each of the above samples, DNA that semiconservative mechanism of was isolated and subjected to density replication is universal (Figure 14.12).

re licati n is semic nser ati e

Semic nser ati e nser ati e is ersi e r bacteria in One ban

St ck c it re arental entri ge sam le in s I ea

rans er cells t gr r ne generatin One ban

r mintes

entri ge sam le First in s I bri re licati n

r r secn generati n in ban s

Light r mintes Sec n re licati n entri ge sam le bri in s I

Figure 14.12: Meselson’s and Stahl’s experiment

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14.3.2 Enzymes Involved in DNA Initiation Replication • DNA replication is initiated at DNA replication in E.coli requires many replication origin known as oriC (245 enzymes and proteins, each performing a base pairs in E.coli). specific task. The entire complex is called • Dna A protein molecules bind to the the DNA replicase system or replisome. The origin of replication. major enzymes and proteins involved with • Helicase (DnaB ) denatures the DNA their functions are tabulated in Table 14.2. helix by breaking the hydrogen bonds between base pairs. Table 14.2: Enzymes involved in DNA replication • Many molecules of SSB (single stranded binding) proteins bind cooperatively Enzyme Function to single stranded DNA, stabilizing Helicase Unwinds DNA the separated strands and preventing DNA gyrase Relieves stress created by renaturation. unwinding SSB protein Binds to single stranded • Gyrase (topoisomerase) releases the DNA and stabilises it topological stress produced by helicase. Primase Synthesis of RNA primer • RNA primers are synthesised by DNA pol I Excision of primers primase. and filling of gaps with The separated polynucleotide strands nucleotides are used as templates for synthesis of DNA pol III New strand elongation complementary strands. The area of the DNA ligase Joins the nick DNA opened by helicase for DNA synthesis is referred to as the replication fork. At the Infobits replication fork there are four strands of DNA, two are conserved and two are newly • First in vitro synthesis of DNA synthesised. Replication may occur in either with a template was carried out by a unidirectional or bidirectional manner Kornberg in 1959. (Figure 14.13) from each origin. Bidirectional • First in vitro synthesis of DNA replication can be explained as two replication without template was carried out forks moving in opposite directions around by H.G. Khorana in 1965. the circular chromosome. Both forks move along the double helix away from the origin of replication in opposite directions and 14.3.3 Events in DNA Replication in E.coli around the circular chromosome.

1. Initiation Elongation 2. Elongation • DNA synthesis proceeds in a 5ʹÆ3ʹ direction( read as 5 prime to 3 prime). 3. Termination • One strand is synthesised continuously DNA replication is depicted in and is known as leading strand. Figure 14.14.

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Origin Origin re licati n M ement re licati n re licati n rk M ement re licati n rk

Figure 14.13: Unidirectional and bidirectional replication

ƍ ƍ ƍ el ƍ S nthesi e stran ƍ ƍ ƍ ƍ irecti n ƍ e licati n rk ƍ ƍ Oka aki ragment em al rimers Lagging stran Lea ing stran ƍ ƍ ƍ ƍ ƍ ƍ ƍ ƍ ƍ ƍ ƍƍƍ ƍ ƍ ƍ ƍ a s bet een ka aki ragments Oka aki ragment ille b l merase

DA ƍ ƍ ƍ ƍ ƍ ƍ O ƍ ƍ ƍ ragments seale b Ligase ƍ O O ƍ O ƍ ƍ ƍ ƍ O ƍƍ O A O ADA Figure 14.14: a) DNA replication b) Removal of RNA primers c) Action of Ligase and • Other strand is synthesised Nobel Prize for discovering DNA discontinuously and is known as polymerase in 1956. lagging strand. • There are three known enzymes in • The enzymes that are able to Escherichia.coli viz., DNA polymerase synthesise new DNA strands on I, II and III. a template strand are called DNA • All of the known DNA polymerases polymerases. Kornberg was awarded can extend a deoxyribonucleotide

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chain from a free 3ʹOH end, but none Okazaki fragments named after R. Okazaki. can initiate synthesis. Okazaki fragments range in length from a • Synthesis of DNA requires nucleoside few hundred to a few thousand nucleotides triphosphates or nucleotides - depending on the cell types. Each okazaki deoxyadenosinetriphosphate (dATP), fragment must be initiated by the action deoxythymidinetriphosphate (dTTP), of primase. Once an Okazaki fragment has deoxycytidinetriphosphate (dCTP), been completed its RNA primer is removed deoxyguanosinetriphosphate (dGTP). and replaced with DNA by DNA polymerase When a nucleoside triphosphate bonds I and the nick is sealed by DNA ligase. to sugar in a growing DNA strand, it The fidelity of DNA replication is loses two phosphates. maintained by (1) base selection by the • RNA primer synthesised during polymerase, (2) a 3ʹÆ5ʹ proofreading initiation is removed and replaced exonuclease activity that is part of most with DNA by DNA polymerase I DNA polymerases, and (3) specific repair systems for mismatches left behind after • Sealing of the nick by DNA ligase which replication. catalyses the formation of phosphodiester bond between a 3ʹ hydroxyl end of one Infobits DNA fragment and 5ʹ phosphate at the end of another strand. RNA dependent DNA polymerases, The elongation phase of replication also called reverse transcriptases, were includes two distinct but related first discovered in retroviruses, which operations convert their RNA genomes into • Leading Strand Synthesis double-stranded DNA as part of their life cycle. These enzymes transcribe • Lagging Strand Synthesis the viral RNA into DNA, a process Leading strand synthesis begins with the that can be used experimentally to synthesis of short (10 to 60 nucleotide form complementary DNA. long) RNA primer at the replication origin. Deoxyribonucleotides are added to this Termination primer by DNA polymerase III. Leading Eventually, the two replication forks of strand synthesis then proceeds continuosly, the circular E.coli chromosome meet at a keeping pace with the unwinding of DNA at terminus region called Ter (for terminus). the replication fork. The continuous strand The Ter sequence function as binding or leading strand is one in which 5ʹÆ3ʹ sites for protein Tus (Termination synthesis proceeds in the same direction as utilization substance). The Tus-Ter replication fork movement. complex can arrest a replication fork Lagging strand or discontinuous strand from only one direction. When either is one in which 5ʹÆ3ʹ DNA synthesis replication fork encounters a functional proceeds in the direction opposite to the Tus-Ter complex, it halts. The other fork direction of fork movement. This strand is halts when it meets the first (arrested) synthesised as short fragments, known as fork (Figure 14.15).

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Parental strand Origin Replication forks Daughter strand

Termination of replication

Replication proceeds in both directions

Figure 14.15: Termination of replication in a circular DNA 14.3.4 Eukaryotic DNA Replication Infobits Replication in Eukaryotic cells is more complex. The DNA molecules DNA molecules exists in circular form in in Eukaryotes are considerably larger prokaryotic microorganisms, viruses and than those in bacteria and are organized in organelles of eukaryotic organisms. into complex nucleoprotein structures However not all DNA molecules are (chromatin). The essential features of circular. The chromosomes of eukaryotic DNA replication are same in eukaryotes organisms and of many viruses consists and prokaryotes. However, some of linear DNA molecules. There are interesting variations do occur. Initiation three general methods of replication of of replication in all eukaryotes requires DNA molecule a multisubunit protein. Multiple origins 1. Theta (θ) mode of replication are probably a universal 2. Sigma (σ) mode feature in eukaryotic cells. Like bacteria, 3. Linear mode

The two essential functions of genetic material are replication and expression. Genetic material must replicate accurately so that progeny inherit all of the specific genetic determinants (the genotype) of the parental organism. A gene is a DNA sequence that encodes a protein, rRNA, or tRNA molecule (gene product). Gene expression usually involves transcription of DNA into messenger RNA and translation of mRNA into protein. Genetic information encoded in DNA is expressed by synthesis of specific RNAs and proteins, and information flows from DNA to RNA to protein. Expression of specific genetic material under a particular set of growth conditions determines the observable characteristics (phenotype) of the organism. Bacteria have few structural or developmental features that can be observed easily, but they have a vast array of biochemical capabilities and patterns of susceptibility to antimicrobial agents or bacteriophages. These latter characteristics are often selected as the inherited traits to be analyzed in studies of bacterial genetics.

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eukaryotes have several types of DNA Erwin Chargaff rules states that A = T polymerases (Example: DNA polymerase and G = C. Watson and Crick postulated α [alpha] and DNA polymerase δ [delta]). that DNA consists of two antiparallel Some have been linked to particular chains in a right-handed double-helical functions, such as the replication of arrangement. Complementary base pairs, mitochondrial DNA. The termination A = T and G C, are formed by hydrogen of replication on linear eukaryotic bonding within the helix. The basepairs chromosomes involves the synthesis of are stacked perpendicular to the long special structures called telomeres at the axis of the double helix. DNA can exist in ends of chromosome. several structural forms. Two variations of the Watson-Crick form, or B-DNA, are HOTS A-DNA and Z-DNA. Replication of DNA occurs with very What will happen if single stranded high fidelity and at a designated time in the binding proteins are not present cell cycle. Replication is semiconservative, during replication of DNA? each strand acting as template for a new daughter strand. It is carried out in three phases: initiation, elongation, and Summary termination. Many lines of evidence show that DNA The reaction starts at the origin and bears genetic information. Frederick usually proceeds bidirectionally. DNA Griffith showed transformation of is synthesized in the 5ʹÆ3ʹ direction by bacteria. Avery, Mac Leod, Mc Carty DNA polymerases. At the replication fork, experiment and further experiment by the leading strand is synthesized continu- Hershey Chase confirmed that DNA is the ously in the same direction as replication transforming principle. fork movement; the lagging strand is syn- There are two types of nucleic thesised discontinuously as Okazaki frag- acid: RNA and DNA. Nucleic acids are ments, which are subsequently ligated by polymers of nucleotides, joined together DNA ligases. by phosphodiester linkages between the Most cells have several DNA 5ʹhydroxyl group of one pentose and polymerases. In E. coli, DNA polymerase the 3ʹhydroxyl group of the next. The III is the primary replication enzyme. nucleotides in RNA contain ribose, and Replication of the E. coli chromosome the common pyrimidine bases are uracil involves many enzymes and protein and cytosine. In DNA, the nucleotides factors. Replication is similar in contain deoxyribose sugar, and the eukaryotic cells, but eukaryotic common pyrimidine bases are thymine chromosomes have many replication and cytosine. The primary purines are origins. adenine and guanine in both RNA and DNA.

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ICT CORNER Bacterial DNA extraction

Lets separate DNA from bacteria

STEPS: • Scan the QR code • Click DNA extraction on the left tab • Select student lab notebook and click open Bacterial Extraction Protocol • Press return and read students protocol on the left table • Click producer and follow the steps

OBSERVATIONS : • Select base pair interactions at the right side and join nitrogenous base pairs as in DNA.

Step1 Step2 Step3

URL: http://labcenter.dnalc.org/labs/dnaextraction/ dnaextractiond.html

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Evaluation a. Age b. Nutritional State Multiple choice questions c. Changing environment 1. The genetic material of d. None virus is 8. The bond between adenine and a. DNA thymine in a DNA double helix is b. RNA c. a or b a. Hydrogen d. None b. Double hydrogen 2. is used to denature RNA c. Vander Waal’s d. Triple hydrogen a. DNase b. Protease 9. Watson and Crick DNA model is c. Nuclease a. A form d. RNase b. B form 3. In DNA molecule, the sugars c. Z form a. Bond to nitrogenous bases by d. D form hydrogen bonds 10. In the first generation of Meselson and b. Bond to nitrogenous bases by glycosidic bonds Stahl’s experiment, the results showed c. Bond to phosphate by hydrogen a hybrid band of DNA containing bonds both 14N and 15N. Which of the d. Bond to phosphate by glycosidic following is the best interpretation of bonds these results? 4. Which of the following is not present a. The results are consistent with in DNA semi-conservative replication a. Adenine b. The results support conservative b. Guanine replication c. Uracil c. The results support both semi d. None conservative and conservative 5. A nucleoside contains replication d. Neither dispersive nor conservative a. Sugar b. Nitrogenous base replication can take place. c. Both a and b 11. A form of DNA is d. Only b a. Left Handed helix with 6. Glycosidic bond is present between 20 nucleotide pairs per turn a. Phosphate and sugar b. Right handed helix with b. Sugar and nitrogenous base II nucleotide pairs per turn c. Nitrogenous bases c. Right handed helix with d. All the above 12 nucleotide pairs per turn 7. According to Chargaff the base d. Left handed helix with composition of DNA in a given 11 nucleotide pairs per turn species does not change with

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12. DNA polymerase is required for the 18. In DNA adenine is complementary synthesis of base for and cytosine a. RNA from DNA is the complementary for b. DNA from RNA a. Guanine, thymine c. DNA from DNA b. Uracil, guanine d. RNA from RNA c. Thymine, guanine 13. Okazaki segments are d. Thymine, uracil a. Segment of a chain of nucleotides removed during replication of Answer the following DNA 1. Define gene b. Segment of a chain of nucleotides 2. DNA is not always the genetic material, formed during replication of DNA what are the exceptions? c. Segments of gene which undergo 3. Define Nucleotide. recombination 4. List any two difference between DNA d. Segments of DNA capable of and RNA replication 5. In what sense are the two strands of 14. DNA replication is aided by DNA antiparallel. a. DNA Polymerase only 6. What is a nucleoside? b. Both DNA polymerase and 7. Depict Erwin Chargaff rule by an primase only equation. c. DNA ligase only 8. Give examples of nitrogenous bases. 9. Draw the structure of Deoxyribose. d. RNA Polymerase. 10. State Watson and Crick rule. 15. The semi-conservative mode of DNA 11. List 2 characteristics of Z DNA replication was proved by 12. Define replication. a. Beadle and Tatum 13. What is a template DNA? b. Meselson and Stahl c. Watson and Crick 14. Explain semiconservative replication. d. H.G Khorana 15. List major events in replication 16. Enzymes involved in unwinding of 16. Write two main events during initiation of replication. DNA at replication are 17. What is the role of topoisomerase? a. Ligases b. Helicases 18. What do you understand by leading c. Endonucleases strand/lagging strand? d. DNA Polymerases 19. What is RNA primer? 17. DNA replication is semiconservative 20. Name two enzymes that make primers for DNA synthesis because the strand will 21. What is the origin of replication? become half of the molecule 22. Label the following diagram of Griffith. a. RNA, DNA b. Template, finished c. Sense, mRNA d. Condon, anticodon

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23. Point out the mistake in the following 37. What was the motive of Avery scheme: and colleagues for conducting the Extracts of smooth strain + RNase experiment. Mouse Alive 38. Differentiate between R and S strains of 24. Differentiate between right handed and Streptococcus pneumoniae. left handed DNA forms with any three 39. Describe the various characteristics salient features. of the Watson and Crick double helix 25. What are the types of DNA polymerases model for DNA present in E.coli? Write their functions. 40. Discuss the various bonds present in 26. Explain replication fork. DNA double helix 27. Explain bidirectional replication. 41. Discuss various forms of DNA double helix structure. 28. Explain continuous replication 42. Diagramatically explain the DNA 29. Define okazaki fragments. double helix structure 30. Why are RNA primers required 43. Draw a four base pair segment of a DNA 31. Describe what is meant by the molecule, including each nucleotide antiparallel arrangement of DNA and associated bonds involved in the 32. Why is one strand of DNA synthesised maintenance of the double helix. discontinuously? 44. Diagrammatically represent the results 33. How is the faithfulness of DNA of Meselson and Stahls experiment. replication maintained? Write the name 45. Explain how Meselson’s and Stahl ruled of the enzymes with its function. out dispersive model of replication. 34. Outline the experiment of Griffith. 46. Tabulate the enzymes involved in DNA 35. Relate the experiments of Griffith and replication with their functions Avery. 47. Explain Elongation of DNA during 36. Discuss Avery, Mc Cleod’s experiment. replication

Student Activity 1. Fun with beads – students will understand the concept of polymer – polynucleotide and different sequences of DNA by preparing a chain of 20 beads of four different colours. 2. Prepare a model of DNA 3. Supercoiling of DNA – students will hold the ends of the rubber band and twist it. The two ends will be joined to feel the stress of coiling relieved due to supercoiling. 4. On paper replicate the following segment of DNA 5ʹATCGGCTACGTTCAC3ʹ 3ʹTAGCCGATGCAAGTG5ʹ Show the direction of replication of the new strands and explain what the lagging and leading strands are? Explain how this is semiconservative replication. Are the new strands identical to the original segment DNA?

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Glossary microorganisms to remove an environmental pollutant. 1. Acute disease: A disease in which 14. Biotechnology: The industrial application symptoms develop of microorganisms, cells, or cell rapidly but lasts for components to make a useful product. only a short time. 15. Caramel: Sugar or syrup heated until 2. Antibiotic: An organic substance it turns brown, used as a flavouring or produced by one organism that in colouring agent in food or drink. low concentrations kills or inhibits 16. Clone: Cells which have descended the growth of other organisms. from a single parent cell organisms 3. Antibiotic Susceptibility Test: It is having identical copies of DNA usually carried out to determine which structure which is obtained by antibiotic will be most successful in replication. treating a bacterial infection in vivo. 17. Coagulation: The action or process of 4. Antiserum: A blood derived fluid a liquid, especially blood, changing to containing antibodies. a solid or semi-solid state. 5. Aseptic techniques: Laboratory tech- 18. Coal-tar dyes: Liquid produced by niques to minimize contamination. distilling coal containing benzene naphthalene, phenols, aniline and 6. Assimilation: The absorption and many other organic chemical. digestion of nutrients by any biological system. 19. Coliforms: Aerobic or facultatively anaerobic, Gram negative, non 7. Axenic: Pure cultures of micro endospore forming, rod shaped bacteria organisms, which are not contaminated that ferment lactose with acid and gas by any foreign organisms. formation within 48 hours at 350°C. 8. Bacterial transformation: The 20. Colony: A Colony is defined as a production of a new phenotype as a visible mass of microorganism all result of introduction of novel genetic originating from a single mother cell. material. 21. Competition: A rivalry between two 9. Bactericide: A substance capable of or more species for limiting factor in killing bacteria the environment. 10. Base Stacking: Stacking implies 22. Cover slip: A small, thin piece of glass vertical interactions between bases as used to cover and protect a specimen they sit on top of one another. on a microscope slide. 11. Bio-augmentation: The use of 23. CsCl Density gradient centrifugation: pollutant acclimated microbes or DNA is mixed with CsCl and genetically engineered microbes for centrifuged at very high speeds in bioremediation. an ultracentrifuge for many hours. A 12. Bioreactor: A fermentation vessel linear gradient of CsCl is produced with with controls for environmental the highest density at the top and the conditions; temperature and pH. heaviest at the bottom. As CsCl gradient 13. Bioremediation: The use of forms, the DNA comes to equilibrium

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in the gradient where its density equals 36. Fluorescent antibody (FA) technique: the density of the surrounding CsCl. A diagnostic tool using antibodies 24. Denaturation of DNA: Separation or with fluorochromes and viewed unwinding of dsDNA strands into through a fluorescence microscope; single strands. also called immunofluorescence. 25. Denature: To deprive something of its 37. Fluoresence: The property of absorbing natural character and properties. light of short wave length and emitting light of longer wave length. 26. Depyrogenation: Removal of pyrogens from solutions mostly from 38. Folliculitis: An infection of hair injectable pharmaceuticals. follicles, often occurring as pimples. 27. Dermatomycosis: A fungal infection 39. Fulminating: A condition that of skin. develops quickly and rapidly increases in severity. 28. Diatomaceous earth: A soft, crumbly, porous sedimentary deposit formed 40. Furuncle: A pus filled, painful from the fossil remains of diatoms. infection of a hair follicle. 29. DNA amplification: The production of 41. Gene: A unit of heredity which is multiple copies of a sequence of DNA. transferred from parent to progeny. 30. Electromagnetic spectrum: The range 42. Genetic code: The mRNA codons and of wavelengths or frequencies over the amino acids they encode. which electromagnetic radiation 43. Genetics: The study of heredity and extends. variation of inherited characteristics. 31. Enzymes: A molecule that catalyzes 44. Genome: One complete copy of the biochemical reactions in a living genetic information in cell. organism, usually a protein. 45. Genomics: Study of genes and their 32. Exudate: Low molecular weight functions. metabolites that enter the soil from 46. Genotype: The genetic makeup of an plant roots. organisms. 33. Fermentation: The enzymatic 47. Gestation: The development of degradation of carbohydrates in something over a period of time. which the final electron acceptor is an 48. Heavy isotope: A stable atom in which organic molecule, ATP is synthesized there are more neutrons than in the by substrate level phosphorylation, normal isotope of the element, giving and oxygen is not required. it a greater mass. For example, 15N is 34. First line of defense: Includes physical the heavy isotope, 14N the common and chemical barriers that are always form. ready and prepared to defend the 49. Histology: The study of microscopic body from infection. structure of tissues. 35. Flake: A small flat thin piece of which 50. Horizontal gene transfer: The transfer has broken away or been peeled from of genes between two organisms in a larger piece. the same generation.

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51. Hypersensitivity: An altered, enhanced 63. Latent infection: A condition in immune reaction leading to pathological which a pathogen remains in the host changes it is also called allergy. for long periods without producing 52. Hypotonic environment: disease. Environment with higher water 64. Lymph: A colourless fluid containing concentration and less solutes. white blood cells, which bathes 53. Immunodiffusion test: A test the tissues and drains through consisting of precipitation reactions the lymphatic system into the carried out in an agar gel medium. bloodstream. 54. Immunoelectrophoresis: The 65. Lysis: Destruction of a cell by the identification of proteins by rupture of the plasma membrane, electrophoretic separation followed resulting in a loss of cytoplasm. by serological testing. 66. Lysozyme: An enzyme capable of 55. Immunotherapy: Making use of hydrolyzing bacterial cell walls. immune system to attack tumour 67. MHC: Major histocompatibility cells, either by enhancing the normal complex – The genes that code for immune response or by using toxin – histocompatibility antigens; also bearing specific antibodies. known as human leucocyte antigens. 56. Incubation: The microbial growth are 68. Microaerophile: An organism that obtained by placing the medium in grows best in an environment with the bacteriological incubator. less molecular oxygen (O2) than is 57. Indicator: Device or an organisms normally found in air. providing specific information on the 69. Molasses: It is a viscous product state or condition of something, in resulting from refining sugar cane or particular. sugar beets into sugar. 58. Inflorescence: The arrangement of 70. Monomer–A small molecule that the flowers on a plant. collectively combines to form 59. Inoculation loop: They are made of polymers. platinum or nichrome wire. They are 71. Mucigel: Mucilage or complex used to make smears. polysaccharide forming a layer 60. Inoculum: The material used to intro- around plant roots. duce an organism into a certain medium 72. Mycorrhiza (“fungus root”): The for growth or culture medium in which association, usually symbiotic, of microorganisms are implanted. specific fungi with the roots of higher 61. In vivo: Process taking place in a plants. living organisms. 73. Neutralism: A lack of interaction 62. Ionizing radiation: Radiation between two organisms in the same consisting of particles, X-rays, or ecosystem. gamma rays with sufficient energy 74. Nick: It is discontinuity in a to cause ionization in the medium dsDNA molecule where there is through which it passes. no phosphodiester bond between

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adjacent nucleotides of one strand. 86. PCR: Polymerase chain reaction, a 75. Non-ionizing radiation: Any type of technique using DNA polymerase electromagnetic radiation that does to make multiple copies of a DNA not carry enough energy per quantum template in vitro. (photon energy) to ionize atoms or 87. Pellicle: scum on the surface of the molecules—that is, to completely liquid medium. remove an electron from an atom or 88. Phagocytes: Cells capable of ingesting molecule. bacteria or other substances. 76. Normal microbiota: The 89. Phagocytosis: Ingestion of bacteria or microrganisms that colonize a host other substances by phagocytes. without causing disease; also called 90. Phylogenetic: Relating to the normal flora. evolutionary development and 77. Occupational health: The branch of diversification of a species or group of medicine dealing with the prevention organisms, or of a particular feature of and treatment of job related injuries an organism. and illnesses. 91. Plasmolysis: Loss of water from a cell 78. Ophthalmic: Relating to the eye and in a hypertonic environment. its diseases. 92. Pluripotent: A cell that can 79. Osmotic lysis: Rupture of the plasma differentiate into a many different membrane resulting from movement types of tissue cells. of water into the cell. 93. Polynucleotide: Chain of nucleotides. 80. Osmotic pressure: The force with 94. Pore: A minute opening in a surface, which a solvent moves from a solution especially the skin or integument of of higher solute concentration. an organism, through which gases, 81. Oxidation: The removal of electrons liquids, or microscopic particles may from a molecule. pass. 82. Oxidation reduction: A coupled 95. Predation: A relationship between reaction in which one substance is two organisms whereby one organism oxidized and one is reduced also (predator) engulfs and digests the called redox reaction. second organism (prey). 83. Pathogenecity: The ability of a 96. Prevalence: The fraction of a microorganism to cause disease by population having a specific disease overcoming the defence of host. at a given time. 84. Pathogenesis: The manner in which a 97. Progeny: Offspring, decendant of a disease develops. cell. 85. Pathologist: A scientist who studies 98. Protein sequencing: The practical the causes and effects of disease. process of determining the amino acid He examines laboratory samples of sequence of all or part of a protein or body tissue for diagnostic or forensic peptide. purposes.

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99. Protocooperation: An association 110. Sutures: A stitch or row of stitches of mutual benefit to two or more holding together the edges of wound species but without the cooperation or surgical incision. or without being obligatory for their 111. Template: A structure that would allow existence or the performance of some molecules to be lined up in a specific order function. and joined, to create a macromolecule 100. Pustule: A small pus filled elevation with a unique sequence and function. of skin. 112. Three-dimensional: having or 101. Renaturation/Annealing: Process in appearing to have length, breadth, which ssDNA or ssRNA pair to form and depth. double stranded DNA. 113. Topological stress: stress created 102. Salmon-GAL (6 chloro 3- indolyl –β – D due to over winding or repeated galactopyranoside): It is a chromogenic interwinding of DNA during substrate capable of detecting LacZ replication. gene encoded β galactosidase. 114. Topography: the arrangement of the 103. Semi-transparent: Partially admitting natural and artificial physical feature the passage of light through its of an area. substance. 115. Toxigenic: (especially of a bacterium) 104. Serial dilution: Series of step wise producing a toxin or toxic effect. dilutions normally done in sterile 116. Turbo blower: It is a fan that blows water which is done to reduce the air. microorganism population to 117. Vaccine: A preparation of manageable numbers. killed, inactivated, or attenuated 105. Serological methods: Methods for microorganisms or toxoids to induce identifying microorganisms based on artificial immunity. its reactions with antibodies. 118. Vacuoles: A space or vesicle within 106. Smear: A thin spread of bacterial the cytoplasm of a cell enclosed by a suspension from a clinical spemicen membrane and typically containing or from a culture on a glass slide. fluid. 107. Spectrophotometer: An apparatus for 119. Vegetative cells: A bacterial cell measuring the intensity of light in a growing actively under favorable part of the spectrum, especially as conditions. transmitted or emitted by particular 120. Virulence: The degree of a substances. pathogenicity of a pathogenic 108. Stab culture: A long straight wire microorganism. dipped in culture is punctured into 121. X-ray diffraction method: Technique a solid medium usually to see the used for determining the atomic and motility. molecular structure of a crystal, in 109. Strain: A population of cells all of which the crystalline atoms cause a which arise from a single pure isolate. beam of incident X rays to diffract Strain is a subtype of a microbe. into many specific directions.

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References Pearson Education. 15. Precotts Microbiology: Willey, Sherwood, 1. Medical Microbiology: Mims.c, th Dockrell.H.M, Goering.R.V., Ivan Roit., Woolverton (2014), Mc Graw-Hill, 9 Waklin.D., Zukerman.M (2005). Third Edition. edition, Elsevier Mosby, London. 16. Microbial Ecology: Atlas and Bartha (1997), 2. Medical Microbiology: Mackie and Pearson Publishers. McCartney (1983) Churchill Livingston, 17. Plant Pathology: R.S Mehrotra (1998), Tata ELBS Edition. McGraw-hill Publishing Company Limited, 3. Microbiology: Davis.B.D, Dulbecco.R., New Delhi. Eisen.H., Ginsberg.H., (1980). Third edi- 18. Brock Biology of Microbiology: Michael tion, Harper International Edition. T. Madigan and John M. Martinko (2006), 4. Jawetz, Melnick, & Adelberg’s Medical Eleventh Edition. Pearson Prentice Hall. Microbiology: Brooks.G.F., Butel.J.S., 19. Essentials of Molecular Biology: Pamela Morse.S.A. (1998), 21st Edition. Prentice Hanaratty and George M. Malacinski, 4th Hall International Inc. Edition. 5. Ganong’s Review of Medical Physiology: 20. Microbial Genetics - David Freifelder Barrett.K.E.,Barman.S.M., Boitano.S.C., (1935) Boston, MA : Jones and Bartlett, and Brooks.H.L. 25thEdn. McGraw Hill c1987. Education India Private Limited. New 21. Priciples of Microbiology: Ronald Atlas Delhi. (2015), 2nd Edition. Mc Graw Hill India. 6. Microbiology: Michael J. Pelczar, E.C.S 22. Textbook of microbiology: R. Anantharayan Chan, Noel E Krieg( 1993), 5th edition, Mc and C.K. Jayaram Paniker(2005). VII Graw-Hill. Edition, Orient Longman Private Limited. 7. Environmental Microbiology: Raina 23. Microbiology: A Systems approach- M.Maier,Ian L.Pepper,Charles P.Gerba Marjorie Kelly,Cowan, III Edition nd (2009), 2 Edition, Acadamic press. 24. Microbiology: Essentials and applications- 8. Soil Microbiology: Prof. N.S. Subha Larry Mackane/Judy Kancle,II Edition. Rao (2009), Fourth Edition of Soil 25. Microbiology: Daniel Lim II edition Microorganisms and Plant Growth, Oxford 26. Foster & IBH Publishing Co. Pvt. Ltd, New Delhi. 27. Microbial Genetics - David Freifelder 9. Environmental Microbiology: K. Vijaya (1935) Boston, MA : Ramesh (2004), MJP Publishers. 28. Jones and Bartlett, c1987. 2nd edition 10. Soil Microbiology: R.R.Mishra (2014), CBS Narosa Publications. Publishers and distributors Pvt. Ltd. 29. Microbiology : A systems approach – 11. A Textbook of Microbiology. Dr. R.C. Marjorie Kelly, Cowan (2011), III Edition, Dubey and Dr. D.K. Maheshwari (1999). McGraw - Hill Publications. Revised edition. Chand and company Pvt. Ltd. New Delhi. 30. Microbiology : Essentials and applications – Larry Mackane/Judy Kandel (1996), II 12. Practical Microbiology: Dr. R.C. Dubey Edition, McGraw – Hill Publications. and Dr. D.K. Maheshwari(2002). Revised edition. Chand and company Pvt. Ltd. New 31. Microbiology : Daniel V.Lim (1998), II Delhi. edition, McGraw – Hill Publications. 13. Industrial Microbiology: A. H. Patel (2012), 32. Microbiology : An evolving Science Joan 2nd Edition, Mac Millan publishers India L. sonczewskl,John W. Foster (2113). 3rd Ltd. Edition, W.W. Norton Publications. 14. Microbiology: Gerard J. Tortora, Berdell R. 33. Kuby Immunology. Janis Kuby (1997). 3rd Funke, Christine L. Case (2004). 8th Edition. Edition. W. H Freeman and Company.

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Microbiology Weblinks

Chapter - 1 Chapter – 9 Web link: http://www.britannica.com / Can microbes clean up oil biography/Alexender-Fleming https://youtu.be/a_HWLFzgQiM Chapter - 2 Composting Working of compound microscope https://youtu.be/VNgFXvL9ZH8 https://youtu.be/cmzWDkOYTjM Chapter – 10 Chapter – 3 soil horizons types Gram Staining https://youtu.be/OEvLuucpYw8 https://youtu.be/L9bats-vGDY Chapter – 11 Endospore Staining Nitrogen fixation https://youtu.be/o1uYmUW4qe8 https://www.youtube.com/ Chapter – 4 watch?v=qzh7ZzJQJ84 Quick review of sterilization Late blight of potato https://youtu.be/ZDmP14twN8g https://youtu.be/2Y77KEYuw_g Chapter – 5 Chapter – 12 Streak Plate Bacterial meninigitis http://youtu.be/NDMNGnxCZ1Q https://youtu.be/HhWjA1xq3Ig Bacterial colony description Chapter – 13 https://youtu.be/gH--8YWdyyk Agglutination Reaction Chapter – 6 https://www.youtube.com/ Photosynthesis watch?v=3W67OH3v2lU https://youtu.be/1Dn_zdAZN0I Coomb’s Test https://www.youtube.com/ Chapter – 7 watch?v=sUHsX3xrlFM Bacterial flagellum Chapter – 14 https://youtu.be/PIOfMifowP4 Structure of DNA Chapter – 8 https://youtu.be/F5JazhVvlm4 Classification of microbes Difference between prokaryotic and https://youtu.be/W2nNIRUs6Wo eukaryotic DNA Taxonomy and Classification https://youtu.be/0CoZT6hYemk https://youtu.be/yCMDHd44ekQ

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Microbiology – Class XI List of Authors and Reviewers

Domain Experts Manimegali K PGT, Govt Model HSS, Dr. Sundararajan T Saidapet, Chennai HOD (Retd) Dr.ALM PG IBMS, University of Madras, Chennai Pameela Fathima H PGT, JGGHSS, Thiruvottiyur, Reviewers Thiruvallur District Dr. Arunagirinathan N Parvin Zeenath Anwar Assistant professor (Retd), Department PGT, St Mary’s Girls, HSS, Microbiology, Presidency College Perambur, Chennai-11 Dr. Carol D Assistant professor Academic Coordinator Loyola College, Nungambakkam, Chennai Angeline Ruby G Authors Assistant Professor SCERT, Chennai-6 Dr. Niren Andrew S Professor Department of Microbiology, Madras Christian College, Chennai ICT Coordinators Rajesh Kumar N Dr. Linnett Naveena M B.T. Asst., CCMA GGHSS School, Coimbatore. Assistant professor Ethiraj College, Chennai Janakiraman M B.T. Asst., PUMS, Mattayampatti, Salem. Mary Sheela J Assistant professor Ethiraj College for Women, Chennai Gajalakshmi S PGT, KBC GGHSS, Redhills, Thiruvallur District Ramachandran N PGT, GHSS, Kovur, Kancheepuram District

Art and Design Team Chief Co-ordinator and Creative Head Srinivasan Natarajan

Illustration Jaishree A Pugazharasi N

Layout Winmac Solutions, Chennai

In-House QC - Gopu Rasuvel - Tamilkumaran - Jerald Wilson - Ragupathi - Karthik Kalaiarasu

Wrapper Design This book has been printed on 80 G.S.M. Kathir Arumugam Elegant Maplitho paper.

Printed by offset at: Co-ordination Ramesh Munisamy

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