Microbiology

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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First Edition - 2018

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TN_GOVT_XI_Micro_Biology_FM.indd 2 17-04-2018 16:36:52 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

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TN_GOVT_XI_Micro_Biology_FM.indd 3 17-04-2018 16:36:52 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

Directions are provided to students to conduct activities Activity 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

TN_GOVT_XI_Micro_Biology_FM.indd 4 17-04-2018 16:36:53 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

TN_GOVT_XI_Micro_Biology_FM.indd 5 17-04-2018 16:36:53 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

TN_GOVT_XI_Micro_Biology_FM.indd 6 17-04-2018 16:36:53 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

TN_GOVT_XI_Micro_Biology_FM.indd 7 17-04-2018 16:36:53 • 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

TN_GOVT_XI_Micro_Biology_FM.indd 8 17-04-2018 16:36:53 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

TN_GOVT_XI_Micro_Biology_FM.indd 9 17-04-2018 16:36:53 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.

TN_GOVT_XI_Micro_Biology_FM.indd 10 17-04-2018 16:36:53 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.

TN_GOVT_XI_Micro_Biology_CH01.indd 1 17-04-2018 16:37:38 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

TN_GOVT_XI_Micro_Biology_CH01.indd 2 17-04-2018 16:37:38 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.

TN_GOVT_XI_Micro_Biology_CH01.indd 3 17-04-2018 16:37:38 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,

TN_GOVT_XI_Micro_Biology_CH01.indd 4 21-04-2018 11:23:29 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

TN_GOVT_XI_Micro_Biology_CH01.indd 5 17-04-2018 16:37:39 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

TN_GOVT_XI_Micro_Biology_CH01.indd 6 17-04-2018 16:37:39 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

TN_GOVT_XI_Micro_Biology_CH01.indd 7 21-04-2018 11:23:30 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

TN_GOVT_XI_Micro_Biology_CH01.indd 8 17-04-2018 16:37:40 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

TN_GOVT_XI_Micro_Biology_CH01.indd 9 17-04-2018 16:37:40 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

TN_GOVT_XI_Micro_Biology_CH01.indd 10 17-04-2018 16:37:40 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.

TN_GOVT_XI_Micro_Biology_CH01.indd 11 17-04-2018 16:37:40 12

TN_GOVT_XI_Micro_Biology_CH01.indd 12 17-04-2018 16:37:42 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#

TN_GOVT_XI_Micro_Biology_CH01.indd 13 17-04-2018 16:37:44 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?

TN_GOVT_XI_Micro_Biology_CH01.indd 14 17-04-2018 16:37:44 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

TN_GOVT_XI_Micro_Biology_CH02.indd 15 17-04-2018 16:38:44 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-raysUVIR Microwave Radio and TV

400 nm Visible light 700 nm Figure 2.2:Tr ansmissionThe electromagnetic spectrum-WhiteReflection light is a combinationRefraction of all colours of visible spectrum

Transmission Reflection Refraction

Diffraction Absorption Scattering

Figure 2.3: Interaction of light with matter

TN_GOVT_XI_Micro_Biology_CH02.indd 16 21-04-2018 11:23:19 Microscope resolution Microorganisms Objective is the important part in the are measured in microscope which is responsible to produce a micrometers and clear image. The resolution of the objective is nanometers. The most important. Resolution is the capacity of average bacterial cell is 0.001mm in a lens to separate or distinguish between small diameter. objects that are close together. The major factor in the resolution is the wave length of light used. The greatest resolution obtained with light of the shortest wave length, that F is the light at the blue end of the visible spectrum are in the range of 450 to 500nm. The highest resolution possible in compound

f light microscope is about 0.2µm. That means, the two objects closer together than 0.2µm Figure 2.4: Lens function are not resolvable as distinct and separate. The light microscope is equipped with three Generating an image with a lens or four objectives. The working distance of When an object is placed outside the an objective is the distance between the front focal plane (the plane containing the surface of the lens and the surface of the focal point of the lens), all the light rays cover glass or the specimen. Objectives with from the object are bent by the lens. large numerical apertures and great resolving The bent rays converge at the opposite focal point. At the focal point, the power have short working distances. light rays continue and converge with Numerical aperture nonparallel refracted light rays. The resultant reversed and magnified image Numerical Aperture (NA) is the value is formed in the plane of convergence representing the light gathering capacity (Figure 2.5). of an objective lens. NA was first described

Object

F Focal point F’ Focal point

Focal distance Focal plane Focal plane Real Biconvex lens image

Figure 2.5: Generating an image with a lens

TN_GOVT_XI_Micro_Biology_CH02.indd 17 21-04-2018 11:23:20 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.

TN_GOVT_XI_Micro_Biology_CH02.indd 18 17-04-2018 16:38:44 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

TN_GOVT_XI_Micro_Biology_CH02.indd 19 17-04-2018 16:38:45 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 microscope slide the working principle of oil immersion 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 Lenses objective

Refracted Unrefracted light rays light rays lost to lens enter lens Immersion oil Glass cover slip Glass cover slip

Slide Slide

Specimen Light source Specimen Light source (a) Without immersion oil (b) With immersion oil

Figure 2.8: Oil Immersion Objective Working Principle

TN_GOVT_XI_Micro_Biology_CH02.indd 20 17-04-2018 16:38:45 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

TN_GOVT_XI_Micro_Biology_CH02.indd 21 17-04-2018 16:38:45 22

TN_GOVT_XI_Micro_Biology_CH02.indd 22 17-04-2018 16:38:47 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.

TN_GOVT_XI_Micro_Biology_CH02.indd 23 17-04-2018 16:38:48 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 with beam of light 10. Differentiate between Bright field and Dark 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

TN_GOVT_XI_Micro_Biology_CH02.indd 24 17-04-2018 16:38:48 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.

TN_GOVT_XI_Micro_Biology_CH03.indd 25 17-04-2018 16:39:19 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

TN_GOVT_XI_Micro_Biology_CH03.indd 26 17-04-2018 16:39:19 Cover slip

Glass slide

(a) Liquid containing micro organism

Inoculation loop

Hanging drop

Bacterial culture

Cavity slide

Cover slip (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.

TN_GOVT_XI_Micro_Biology_CH03.indd 27 17-04-2018 16:39:19 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+)

Negatively charged Stain containing cell surface positively charged chromophore

Stained bacterial cell Figure 3.2: Positive staining

TN_GOVT_XI_Micro_Biology_CH03.indd 28 17-04-2018 16:39:20 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–)

Negatively charged Stain containing cell surface positively charged chromophore

Stained bacterial cell

Figure 3.3: Negative staining Negative staining

TN_GOVT_XI_Micro_Biology_CH03.indd 29 17-04-2018 16:39:20 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) A pplication 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 the slide to study the bacterial morphology and 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.

TN_GOVT_XI_Micro_Biology_CH03.indd 30 21-04-2018 11:38:09 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

TN_GOVT_XI_Micro_Biology_CH03.indd 31 17-04-2018 16:39:21 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

TN_GOVT_XI_Micro_Biology_CH03.indd 32 17-04-2018 16:39:21 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

Step Microscopic appearance Chemical reaction of cell in cell wall

Gram positive Gram nagative

1. Application of Crystal violet (Primary stain)

2. Application of Iodine (Mordant)

3. Alcohol wash (Decolourization)

4. Application of Safranin (Counter stain)

Outer membrane Peptidoglyeon Cell membrane Figure 3.7: Steps, micrograph and chemical reaction of Gram Stained Bacteria

TN_GOVT_XI_Micro_Biology_CH03.indd 33 21-04-2018 11:38:10 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

Gram-Positive Gram-negative

Outer membrane Peptidoglycan Peptidoglycan

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

TN_GOVT_XI_Micro_Biology_CH03.indd 34 21-04-2018 11:38:10 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.

TN_GOVT_XI_Micro_Biology_CH03.indd 35 17-04-2018 16:39:22 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

TN_GOVT_XI_Micro_Biology_CH03.indd 36 21-04-2018 11:38:10 Common Bacteria with their Gram reactions

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

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

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 micr oscopy.

TN_GOVT_XI_Micro_Biology_CH03.indd 37 17-04-2018 16:39:22 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?

TN_GOVT_XI_Micro_Biology_CH03.indd 38 17-04-2018 16:39:23 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

TN_GOVT_XI_Micro_Biology_CH03.indd 39 17-04-2018 16:39:23 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.

TN_GOVT_XI_Micro_Biology_CH04.indd 40 17-04-2018 16:41:53 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

TN_GOVT_XI_Micro_Biology_CH04.indd 41 17-04-2018 16:41:53 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. Double jacket chamber

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

TN_GOVT_XI_Micro_Biology_CH04.indd 42 17-04-2018 16:41:53 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

TN_GOVT_XI_Micro_Biology_CH04.indd 43 17-04-2018 16:41:53 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 s terilization: ii) Steaming at 100°C (Tyndallization): To check the efficiency of moist heat It is a process discovered by John Tyndall th sterlization, the indicator commonly used is 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.

TN_GOVT_XI_Micro_Biology_CH04.indd 44 17-04-2018 16:41:53 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

Release valve Pressure gauge

Safety valve Cover Cover tightening nuts Body

Jacket Bucket Stand

Heating electrode Leg

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

TN_GOVT_XI_Micro_Biology_CH04.indd 45 21-04-2018 11:23:06 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

TN_GOVT_XI_Micro_Biology_CH04.indd 46 17-04-2018 16:41:55 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 transferred Sample to be to culture medium filtered (b) Membrane filter LM 2.5 m retains cells

To vacuum

Incubation

Colonies

(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

TN_GOVT_XI_Micro_Biology_CH04.indd 47 17-04-2018 16:41:55 Figure 4.6: Types of depth filters

Outside Safety glass Exhaust viewscreen HEPA fiter Blower Supply HEPA filter Light

High-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

TN_GOVT_XI_Micro_Biology_CH04.indd 48 17-04-2018 16:41:56 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)

TN_GOVT_XI_Micro_Biology_CH04.indd 49 17-04-2018 16:41:56 50

TN_GOVT_XI_Micro_Biology_CH04.indd 50 21-04-2018 11:23:10 Summary c. Hot-air sterilization Physical methods of microbial control d. Pasteurization include heat, radiation, drying and f iltration. 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

TN_GOVT_XI_Micro_Biology_CH04.indd 51 17-04-2018 16:41:58 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.

TN_GOVT_XI_Micro_Biology_CH04.indd 52 17-04-2018 16:41:58 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.

TN_GOVT_XI_Micro_Biology_CH05.indd 53 17-04-2018 16:42:21 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

TN_GOVT_XI_Micro_Biology_CH05.indd 54 17-04-2018 16:42:21 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.

TN_GOVT_XI_Micro_Biology_CH05.indd 55 17-04-2018 16:42:22 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

TN_GOVT_XI_Micro_Biology_CH05.indd 56 17-04-2018 16:42:23 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

TN_GOVT_XI_Micro_Biology_CH05.indd 57 17-04-2018 16:42:25 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

TN_GOVT_XI_Micro_Biology_CH05.indd 58 17-04-2018 16:42:25 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

TN_GOVT_XI_Micro_Biology_CH05.indd 59 17-04-2018 16:42:26 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

TN_GOVT_XI_Micro_Biology_CH05.indd 60 17-04-2018 16:42:26 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

Molten Bacterial agar Suspension at 45ºC-50ºC

Mix, Incubate

Colonies grow on surface and within agar Figure 5.10: Pour plate method

TN_GOVT_XI_Micro_Biology_CH05.indd 61 17-04-2018 16:42:26 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.

Spread plate method

12Sample (0.1 mL) poured Spread sample evenly 3 Bacterial colonies onto solid medium over the surface grow on the surface of the medium Bacterial dilution

Incubation

Figure 5.11: Spread plate method

TN_GOVT_XI_Micro_Biology_CH05.indd 62 17-04-2018 16:42:26 • 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

Heavy confluent growth Discrete colonies Initial Second set of 1 inoculum streaks 4

Heavy growth 2 Third set of Fourth set of Incubation 3 streaks streaks Light growth

D E

Figure 5.12: Steps in streak plate isolation method

TN_GOVT_XI_Micro_Biology_CH05.indd 63 17-04-2018 16:42:26 Form

Punctiform Circular FilamentousIrregular RhizoidSpindle (lens)

Elevation

Flat Raised Convex Pulvinate Umbonate

Margin

Entire Undulate Filamentous Lobate Erose Curled (even) (wavy) (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.

TN_GOVT_XI_Micro_Biology_CH05.indd 64 17-04-2018 16:42:26 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

TN_GOVT_XI_Micro_Biology_CH05.indd 65 21-04-2018 11:23:05 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

TN_GOVT_XI_Micro_Biology_CH05.indd 66 17-04-2018 16:42:28 67

TN_GOVT_XI_Micro_Biology_CH05.indd 67 17-04-2018 16:42:29 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

TN_GOVT_XI_Micro_Biology_CH05.indd 68 17-04-2018 16:42:30 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

TN_GOVT_XI_Micro_Biology_CH05.indd 69 17-04-2018 16:42:30 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

TN_GOVT_XI_Micro_Biology_CH05.indd 70 17-04-2018 16:42:30 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.

TN_GOVT_XI_Micro_Biology_CH05.indd 71 17-04-2018 16:42:30 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

TN_GOVT_XI_Micro_Biology_CH06.indd 72 17-04-2018 16:42:57 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 2+ Magnesium (Mg ) 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.

TN_GOVT_XI_Micro_Biology_CH06.indd 73 17-04-2018 16:42:57 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

TN_GOVT_XI_Micro_Biology_CH06.indd 74 17-04-2018 16:42:58 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.

TN_GOVT_XI_Micro_Biology_CH06.indd 75 17-04-2018 16:42:58 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

TN_GOVT_XI_Micro_Biology_CH06.indd 76 17-04-2018 16:42:58 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  (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

TN_GOVT_XI_Micro_Biology_CH06.indd 77 17-04-2018 16:42:59 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 H2O H2O H2S

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

TN_GOVT_XI_Micro_Biology_CH06.indd 78 17-04-2018 16:42:59 Stationary phase

Logarithmic Death growth (decline) phase phase

Lag phase Logarithm of viable cells

0 5 10 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

TN_GOVT_XI_Micro_Biology_CH06.indd 79 17-04-2018 16:42:59 Fresh medium Reservoir of sterile medium

Control valve

Value controlling Air Flow of medium supply Air filter Outlet for Culture spent vessel medium Light source Photo cell

Receptacle Turbidostar

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,

TN_GOVT_XI_Micro_Biology_CH06.indd 80 17-04-2018 16:42:59 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

TN_GOVT_XI_Micro_Biology_CH06.indd 81 17-04-2018 16:42:59 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

TN_GOVT_XI_Micro_Biology_CH06.indd 82 17-04-2018 16:43:00 Obligate Obligate Facultative Aerotolerant aerobes anaerobes anaerobes anaerobes Microaerophiles

A B C D E

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

TN_GOVT_XI_Micro_Biology_CH06.indd 83 17-04-2018 16:43:00 84

TN_GOVT_XI_Micro_Biology_CH06.indd 84 17-04-2018 16:43:01 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

TN_GOVT_XI_Micro_Biology_CH06.indd 85 17-04-2018 16:43:04 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

TN_GOVT_XI_Micro_Biology_CH06.indd 86 17-04-2018 16:43:04 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.

TN_GOVT_XI_Micro_Biology_CH06.indd 87 17-04-2018 16:43:04 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.

TN_GOVT_XI_Micro_Biology_CH07.indd 88 17-04-2018 16:43:15 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

TN_GOVT_XI_Micro_Biology_CH07.indd 89 17-04-2018 16:43:15 100 pm 1 nm 10 nm 100 nm 1 µm 10 µm 100 µm1 µm

Eye

Light Microscope

Electron Microscope

Proteins

Lipids Small Organelles Atom Molecules Virus Bacteria Eukaryotic 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

TN_GOVT_XI_Micro_Biology_CH07.indd 90 17-04-2018 16:43:16 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.

TN_GOVT_XI_Micro_Biology_CH07.indd 91 17-04-2018 16:43:17 Coccus Coccobacillus

Bacillus Vibrio

Spirillum

Spirochete

Different Bacterial Shapes

COCCI COCCI

Single Diplococci Streptococci Tetrads Staphyiococci Sarcinae

BACILLI

Single Diplobacilli Streptobacilli Palisade

SPIRILLA

Single

Figure 7.3: Shapes and arrangement of bacteria

TN_GOVT_XI_Micro_Biology_CH07.indd 92 17-04-2018 16:43:18 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.

Flagellum

Hook

Filament

Outer membrane

Basal Body Rod

Peptidoglycan portion of cell wall

Figure 7.4: Structure of bacterial flagella

TN_GOVT_XI_Micro_Biology_CH07.indd 93 17-04-2018 16:43:18 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.

TN_GOVT_XI_Micro_Biology_CH07.indd 94 17-04-2018 16:43:18 Flagellum Fimbriae Sex pilus

Flagella Fimbriae

Pilus

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 of Bacillus 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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