SLOVAK UNIVERSITY OF TECHNOLOGY IN BRATISLAVA FACULTY OF CHEMICAL AND FOOD TECHNOLOGY INSTITUTE OF CHMICAL AND ENVIRONMENTAL ENGINEERING

General Biology & Microbiology Laboratories

REPORT ON TRAINING VISIT

In the frame work of the project

No. SAMRS 2009/09/02

“Development of human resource capacity of Kabul polytechnic university”

Funded by

Bratislava 2011 Assist. Prof. Ahmad Khalid “Nayab” SLOVAK UNIVERSITY OF TECHNOLOGY IN BRATISLAVA

FACULTY OF CHEMICAL AND FOOD TECHNOLOGY

DEPARTMENT OF CHEMICAL AND BIOCHEMICAL ENGINEERING

REPORT

ON MY ACADEMIC AND SIENTIFIC ACTIVITIES IN TRAINING COURSE AT THE SLOVAK UNIVERSITY OF TECHNOLOGY IN BRATISLAVA

PREPARED BY: AHMAD KHALID “NAYAB”

Assist. Professor of Chemical Technology Faculty of Kabul Polytechnic University

GUIDENCE BY:

1- Ing. Viera Illeova, Ph.D

2- Ing. Barbora Kaliňakova, Ph.D

2011

Preface

My name is Ahmad Khalid “Nayab”, and I am an assistant professor in Food Technology Department of Chemical Technology faculty of Kabul Polytechnic University in Afghanistan.

I attended a two month training stay from 25.04.2011 to 01.06.2011 in Slovak University of Technology in Bratislava, Slovakia.

Main purposes of my training stay was familiarity with new pedagogic methods, familiarity with general biology & microbiology laboratories, doing laboratory experiments in general biology & microbiology, and collecting new & scientific materials on general biology, microbiology, and chemical technology topics.

Activities I’ve done during this training stay are listed below:

• Familiarity with general biology & microbiology laboratories. • Doing laboratory experiments in general biology & microbiology laboratories. • Participating in 38th International Conference of Slovak Society of Chemical Engineering. • Collecting some new & scientific information on general biology, microbiology, and chemical technology topics.

I want to appreciate & thank Doc. Ing. Juma Haydary, Ph.D, and other professors of Slovak University of Technology in Bratislava for their cooperation with us.

I want to thank especially Ing. Barbora Kalinakova, Ph.D & Ing. Illeova Viera, Ph.D “my instructors in laboratory experiments in general biology and microbiology laboratories”, and appreciate their unsparing cooperation which caused that I learned a lot from them in biology and microbiology than what I was expecting to learn in these two months.

With Respect

Ahmad Khalid “Nayab”

Contents

Title Page Introduction 4 Chapter 1: General Techniques in Laboratory 1.1- Solution 5 1.2- Concentration 7 1.3- Dilution 11 1.4- Absorption Spectroscopy 11 1.5- Lambert Beer Law 12 Chapter 2: Microscope 2.1- Microscope 14 2.2- Optical Miscroscope 14 2.3- Digital Microscope 18 2.4- Electron Microscope 18 2.5- Microscopic Slide 20 Chapter 3: Aseptic Technique & Sterilization 3.1- Aseptic Technique 22 3.2- Laminar Flow Cabinet 22 3.3- Sterilization 23 Chapter 4: Microorganisms 4.1- Microorganisms 26 Chapter 5: Cultivation & Isolation of Microorganisms 5.1- Microbial Culture 31 5.2- Growth Meduim 32 5.3- Pour Plate Method for Isolation 34 5.4- Spread Plate Method for Isolation 36 5.5- Streak Plate Method for Isolation 38 Chapter 6: Microbial Growth Measurement 6.1- Microbial Growth 40 6.2- Direct Plate Counting 40 6.3- Using a Counting Chamber 41 Chapter 7: Enzyme Activity Assay 7.1- Enzyme 44 7.2- Enzyme Assay 50 Chapter 8: The Cell 8.1- The Cell 53 8.2- Anatomy 53 8.3- Subcellular Components 57 8.4- Structures Outside the Cell 61 8.5- Growth and Metabolism 64 References 4

Introduction

As I’ve learned different biological & Microbiological topics during this training stay, so I tried to collect some brief and theoretical information on topics what I’ve learned in this report.

I will prepare a book from laboratory works and experiments I’ve done in biology & microbiology laboratories and I will use it as a laboratory instructions book in our university in Afghanistan.

This report contains eight different chapters which are listed below:

Chapter 1: General Techniques in Laboratory

Chapter 2: Microscope

Chapter 3: Aseptic Techniques

Chapter 4: Microorganisms

Chapter 5: Cultivation & Isolation of Microorganisms

Chapter 6: Microbial Growth Measurement

Chapter 7: Enzyme Activity Assay

Chapter 8: The Cell

5

Chapter 1: General Techniques in Laboratory

1.1- Solution

In chemistry, a solution is a homogeneous mixture composed of two or more substances. In such a mixture, a solute is dissolved in another substance, known as a solvent. The solvent does the dissolving.

1) Types of solutions

Solutions are homogenous mixtures consisting of one solvent and one or more solutes. Homogenous means that the components and properties of the mixture are uniform throughout its entire volume. Usually, the substance present in the greatest amount is considered the solvent. Solvents can be gases, liquids, or solids. One or more components present in the solution other than the solvent are called solutes. The solution has the same physical state as the solvent. a) Gas

If the solvent is a gas, only gases are dissolved under any given set of conditions. An example of a gaseous solution is air (oxygen and other gases dissolved in nitrogen). Since interactions between molecules play almost no 6 role, dilute gases form rather trivial solutions. In part of the literature, they are not even classified as solutions, but addressed as mixtures. b) Liquid

If the solvent is a liquid, then gases, liquids, and solids can be dissolved. Examples are:

• Gas in liquid: o Oxygen in water. o Carbon dioxide in water is a less simple example, because the solution is accompanied by a chemical reaction (formation of ions). Note also that the visible bubbles in carbonated water are not the dissolved gas, but only an effervescence; the dissolved gas itself is not visible since it is dissolved on a molecular level. • Liquid in liquid: o The mixing of two or more substances of the same chemistry but different concentrations to form a constant.(Homogenization of solutions) o Alcoholic beverages are basically solutions of ethanol in water. • Solid in liquid: o Sucrose (table sugar) in water o Sodium chloride or any other salt in water forms an electrolyte: When dissolving, salt dissociates into ions.

Counter examples are provided by liquid mixtures that are not homogeneous: colloids, suspensions, emulsions are not considered solutions.

Body fluids are examples for complex liquid solutions, containing many different solutes. They are electrolytes since they contain solute ions (e.g. potassium and sodium). Furthermore, they contain solute molecules like sugar and urea. Oxygen and carbon dioxide are also essential components of blood chemistry, where significant changes in their concentrations can be a sign of illness or injury. c) Solid

If the solvent is a solid, then gases, liquids, and solids can be dissolved.

• Gas in solid: o Hydrogen dissolves rather well in metals, especially in palladium; this is studied as a means of hydrogen storage. • Liquid in solid: o mercury in gold, forming an amalgam 7

o Hexane in paraffin wax • Solid in solid: o Steel, basically a solution of carbon atoms in a crystalline matrix of iron atoms. o Alloys like bronze and many others. o Polymers containing plasticizers.

1.2- Concentration

In chemistry, concentration is defined as the abundance of a constituent divided by the total volume of a mixture. Four types can be distinguished: mass concentration, molar concentration, number concentration, and volume concentration. The term concentration can be applied to any kind of chemical mixture, but most frequently it refers to solutes in homogeneoussolutions.

1) Qualitative description

These glasses containing red dye demonstrate qualitative changes in concentration. The solutions on the left are more dilute, compared to the more concentrated solutions on the right.

Often in informal, non-technical language, concentration is described in a qualitative way, through the use of adjectives such as "dilute" for solutions of relatively low concentration and "concentrated" for solutions of relatively high concentration. To concentrate a solution, one must add more solute (for example, alcohol), or reduce the amount of solvent (for example, water). By contrast, to dilute a solution, one must add more solvent, or reduce the amount of solute. Unless two substances are fully miscible there exists a concentration at which no further solute will dissolve in a solution. At this point, the solution is said to be saturated. If additional solute is added to a saturated solution, it will not dissolve (except in certain circumstances, when supersaturation may occur). Instead, phase separation will occur, leading to either coexisting phases or a suspension. The point of saturation depends on many variables such as ambient temperature and the precise chemical nature of the solvent and solute. 8

2) Quantitative notation

There are four quantities that describe concentration: a) Mass concentration

The mass concentration ρi is defined as the mass of a constituent mi divided by the volume of the mixture V:

The SI-unit is kg/m3. b) Molar concentration

The molar concentration ci is defined as the amount of a constituent ni divided by the volume of the mixture V:

The SI-unit is mol/m3. However, more commonly the unit mol/L is used. c) Number concentration

The number concentration Ci is defined as the number of entities of a constituent Ni in a mixture divided by the volume of the mixture V:

The SI-unit is 1/m3. d) Volume concentration

The volume concentration φi (also called volume fraction) is defined as the volume of a constituent Vi divided by the volume of all consituents of the mixture V prior to mixing:

The SI-unit is m3/m3. 9

3) Related Quantities

Several other quantities can be used to describe the composition of a mixture. Note that these should not be called concentrations. a) Normality

Normality is defined as the molar concentration ci divided by an equivalence factor feq. Since the definition of the equivalence factor may not be unequivocal, IUPAC and NIST discourage the use of normality. b) Molality

The molality of a solution mi is defined as the amount of a constituent ni divided by the mass of the solvent msolvent (not the mass of the solution):

The SI-unit for molality is mol/kg. c) Mole fraction

The mole fraction xi is defined as the amount of a constituent ni divided by the total amount of all constituents in a mixture ntot:

The SI-unit is mol/mol. However, the deprecated parts-per notation is often used to describe small mole fractions. d) Mole ratio

The mole ratio ri is defined as the amount of a constituent ni divided by the total amount of all other constituents in a mixture:

If ni is much smaller than ntot, the mole ratio is almost identical to the mole fraction.

The SI-unit is mol/mol. However, the deprecated parts-per notation is often used to describe small mole ratios. 10 e) Mass fraction

The mass fraction wi is the fraction of one substance with mass mi to the mass of the total mixture mtot, defined as:

The SI-unit is kg/kg. However, the deprecated parts-per notation is often used to describe small mass fractions. f) Mass ratio

The mass ratio ζi is defined as the mass of a constituent mi divided by the total mass of all other constituents in a mixture:

If mi is much smaller than mtot, the mass ratio is almost identical to the mass fraction.

The SI-unit is kg/kg. However, the deprecated parts-per notation is often used to describe small mass ratios.

Table of concentrations and related quantities

Concentration type Symbol Definition SI-unit other unit(s) mass concentration ρi or γi mi / V kg/m3 g/100mL (=g/dL) molar concentration ci ni / V mol/m3 M (=mol/L) number concentration Ci Ni / V 1/m3 1/cm3 volume concentration φi Vi / V m3/m3 Related quantities Symbol Definition SI-unit other unit(s) normality ci / feq mol/m3 M (=mol/L) molality mi ni / msolvent mol/kg mole fraction xi ni / ntot mol/mol ppm, ppb, ppt mole ratio ri ni / (ntot − ni) mol/mol ppm, ppb, ppt mass fraction wi mi / mtot kg/kg ppm, ppb, ppt mass ratio ζi mi / (mtot − mi) kg/kg ppm, ppb, ppt

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1.3- Dilution

Dilution is a reduction in the concentration of a chemical (gas, vapor, solution). It is the process of reducing the concentration of a solute in solution, usually simply by mixing with more solvent. To dilute a solution means to add more solvent without the addition of more solute. The resulting solution is thoroughly mixed so as to ensure that all parts of the solution are identical.

The same direct relationship applies to gases and vapors diluted in air for example. Although, thorough mixing of gases and vapors may not be as easily accomplished.

For example, if there are 10 grams of salt (the solute) dissolved in 1 liter of water (the solvent), this solution has a certain salt concentration/molarity. If one adds 1 liter of water to this solution the salt concentration is reduced. The diluted solution still contains 10 grams of salt/(0.171 moles of NaCl).

Mathematically this relationship can be shown in the equation:

Where:

C1 = Concentration/molarity 1

V1 = Volume 1

C2 = Concentration/molarity 2

V2 = Volume 2

1.4- Absorption Spectroscopy

Absorption spectroscopy refers to spectroscopic techniques that measure the absorption of radiation, as a function of frequency or wavelength, due to its interaction with a sample. The sample absorbs energy, i.e., photons, from the radiating field. The intensity of the absorption varies as a function of frequency, and this variation is the absorption spectrum. Absorption spectroscopy is performed across the electromagnetic spectrum.

Absorption spectroscopy is employed as an analytical chemistry tool to determine the presence of a particular substance in a sample and, in many cases, 12 to quantify the amount of the substance present. Infrared and ultraviolet-visible spectroscopy are particularly common in analytical applications. Absorption spectroscopy is also employed in studies of molecular and atomic physics, astronomical spectroscopy and remote sensing.

1.5- Lambert Beer Law

In optics, the Beer–Lambert law, also known as Beer's law or the Lambert–Beer law or the Beer–Lambert–Bouguer law relates the absorption of light to the properties of the material through which the light is traveling.

1) Equations

Diagram of Beer–Lambert absorption of a beam of light as it travels through a cuvette of width ℓ.

The law states that there is a logarithmic dependence between the transmission (or transmissivity), T, of light through a substance and the product of the absorption coefficient of the substance, α, and the distance the light travels through the material (i.e. the path length), ℓ. The absorption coefficient can, in turn, be written as a product of either a molar absorptivity (extinction coefficient) of the absorber, ε, and the concentration c of absorbing species in the material, or an absorption cross section, σ, and the (number) density N' of absorbers.

For liquids, these relations are usually written as:

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Whereas for gases, and in particular among physicists and for spectroscopy and spectrophotometry, they are normally written

where I0 and I are the intensity (or power) of the incident light and the transmitted light, respectively; σ is cross section of light absorption by a single particle and N is the density (number per unit volume) of absorbing particles.

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Chapter 2: Microscope

2.1- Microscope

A microscope (from the Greek: μικρός, mikrós, "small" and σκοπε ν, skopeîn, "to look" or "see") is an instrument used to see objects too small for the naked eye. The science of investigating small objects using such an instrument is called microscopy. Microscopic means invisible to the eye unless aided by a microscope.

There are many types of microscopes, the most common and first to be invented is the optical microscope which uses light to image the sample. Other major types of microscopes are the electron microscope (both the transmission electron microscope and the scanning electron microscope) and the various types of scanning probe microscope.

2.2- Optical Microscope

The optical microscope, often referred to as the "light microscope", is a type of microscope which uses visible light and a system of lenses to magnify images of small samples. Optical microscopes are the oldest design of microscope and were designed around 1600. Basic optical microscopes can be very simple, although there are many complex designs which aim to improve resolution and sample contrast. Historically optical microscopes were easy to develop and are popular because they use visible light so the sample can be directly observed by eye.

The image from an optical microscope can be captured by normal light- sensitive cameras to generate a micrograph. Originally images were captured by photographic film but modern developments in CMOS and charge-coupled device (CCD) cameras allow the capture of digital images. Purely digital microscopes are now available which just use a CCD camera to examine a sample, and the image is shown directly on a computer screen without the need for eyepieces.

Alternatives to optical microscopy which do not use visible light include scanning electron microscopy and transmission electron microscopy. 15

1) Components

Two Leica oil immersion microscope objective lenses; left 100x, right 40x.

All modern optical microscopes designed for viewing samples by transmitted light share the same basic components of the light path, listed here in the order the light travels through them:

• Light source, a light or a mirror (7) • Diaphragm and condenser lens (8) • Objective (3) • Ocular lens (eyepiece) (1)

In addition the vast majority of microscopes have the same 'structural' components:

• Objective turret (to hold multiple objective lenses) (2) • Stage (to hold the sample) (9) • Focus wheel to move the stage (4 - coarse adjustment, 5 - fine adjustment)

These entries are numbered according to the image on the right. a) Eyepiece (ocular)

The eyepiece, or ocular, is a cylinder containing two or more lenses; its function is to bring the image into focus for the eye. The eyepiece is inserted into the top end of the body tube. Eyepieces are interchangeable and many different eyepieces can be inserted with different degrees of magnification. Typical magnification values for eyepieces include 2×, 5× and 10×. In some high performance microscopes, the optical configuration of the objective lens and eyepiece are matched to give the best possible optical performance. This occurs most commonly with apochromatic objectives. b) Objective

At the lower end of a typical compound optical microscope there are one or more objective lenses that collect light from the sample. The objective is usually 16 in a cylinder housing containing a glass single or multi-element compound lens. Typically there will be around three objective lenses screwed into a circular nose piece which may be rotated to select the required objective lens. These arrangements are designed to be parfocal, which means that when one changes from one lens to another on a microscope, the sample stays in focus. Microscope objectives are characterized by two parameters, namely, magnification and numerical aperture. The former typically ranges from 5× to 100× while the latter ranges from 0.14 to 0.7, corresponding to focal lengths of about 40 to 2 mm, respectively. Objective lenses with higher magnifications normally have a higher numerical aperture and a shorter depth of field in the resulting image. Some high performance objective lenses may require matched eyepieces to deliver the best optical performance. c) Stage

The stage is a platform below the objective which supports the specimen being viewed. In the center of the stage is a hole through which light passes to illuminate the specimen. The stage usually has arms to hold slides (rectangular glass plates with typical dimensions of 25 mm by 75 mm, on which the specimen is mounted).

At magnifications higher than 100x moving a slide by hand is not practical. A mechanical stage, typical of medium and higher priced microscopes, allows tiny movements of the slide via control knobs that reposition the sample/slide as desired. If a microscope did not originally have a mechanical stage it may be possible to add one.

All stages move up and down for focus. With a mechanical stage slides move on two horizontal axes for positioning the specimen to examine specimen details.

Focusing starts at lower magnification in order to center the specimen by the user on the stage. Moving to a higher magnification requires the stage to be moved higher vertically for re-focus at the higher magnification and may also require slight horizontal specimen position adjustment. Horizontal specimen position adjustments are the reason for having a mechanical stage.

Due to the difficulty in preparing specimens and mounting them on slides, for children it's best to begin with prepared slides that are centered and focus easily regardless of the focus level used. 17 d) Light source

Many sources of light can be used. At its simplest, daylight is directed via a mirror. Most microscopes, however, have their own controllable light source - normally a halogen lamp. e) Condenser

The condenser is a lens designed to focus light from the illumination source onto the sample. The condenser may also include other features, such as a diaphragm and/or filters, to manage the quality and intensity of the illumination. For illumination techniques like dark field, phase contrast and differential interference contrast microscopy additional optical components must be precisely aligned in the light path. f) Frame

The whole of the optical assembly is traditionally attached to a rigid arm which in turn is attached to a robust U shaped foot to provide the necessary rigidity. The arm angle may be adjustable to allow the viewing angle to be adjusted.

The frame provides a mounting point for various microscope controls. Normally this will include controls for focusing, typically a large knurled wheel to adjust coarse focus, together with a smaller knurled wheel to control fine focus. Other features may be lamp controls and/or controls for adjusting the condenser. g) Magnification

The actual power or magnification of a compound optical microscope is the product of the powers of the ocular (eyepiece) and the objective lens. The maximum normal magnifications of the occular and objective are 10× and 100× respectively giving a final magnification of 1000×. h) Operation

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2.3- Digital microscope

A digital microscope is a microscope equipped with a digital camera allowing observation of a sample via a computer. Microscopes can also be partly or wholly computer-controlled with various levels of automation. Digital microscopy allows greater analysis of a microscope image, for example measurements of distances and areas and quantitaton of a fluorescent or histological stain.

Low-powered digital microscopes, USB microscopes, are also commercially available. These are essentially webcams with a high-powered macro lens and generally do not use transillumination. The camera attached directly to the USB port of a computer, so that the images are shown directly on the monitor. They offer modest magnifications (up to about 200×) without the need to use eyepieces, and at very low cost. The lack of illumination optics limits their use in a similar manner to stereo microscopes.

2.4- Electron Microscope

An electron microscope is a type of microscope that uses a particle beam of electrons to illuminate the specimen and produce a magnified image. Electron microscopes (EM) have a greater resolving power than a light-powered optical microscope, because electrons have wavelengths about 100,000 times shorter than visible light (photons), and can achieve better than 50 pm resolution and magnifications of up to about 10,000,000x, whereas ordinary, non-confocal light microscopes are limited by diffraction to about 200 nm resolution and useful magnifications below 2000x.

The electron microscope uses electrostatic and electromagnetic "lenses" to control the electron beam and focus it to form an image. These lenses are analogous to, but different from the glass lenses of an optical microscope that forms a magnified image by focusing light on or through the specimen. In transmission, the electron beam is first diffracted by the specimen, and then, the electron microscope “lenses" re-focus the beam into a Fourier-transformed image of the diffraction pattern for the selected area of investigation. The real image thus formed is magnified by a factor ranging from a few hundred to many hundred thousand times, and can be viewed on a detecting screen or recorded using photographic film or plates or with a digital camera. Electron microscopes are used to observe a wide range of biological and inorganic specimens including microorganisms, cells, large molecules, biopsy samples, metals, and crystals. Industrially, the electron microscope is primarily used for quality control and failure analysis in semiconductor device fabrication. 19

The advantages of electron microscopy over X-ray crystallography are that the specimen need not be a single crystal or even a polycrystalline powder, and also that the Fourier transform reconstruction of the object's magnified structure occurs physically and thus avoids the need for solving the phase problem faced by the X-ray crystallographers after obtaining their X-ray diffraction patterns of a single crystal or polycrystalline powder. The transmission electron microscope's major `disadvantage' is the need for extremely thin sections of the 20 specimens, typically less than 100 nanometers. Biological specimens typically require to be chemically fixed, dehydrated and embedded in a polymer resin to stabilize them sufficiently to allow ultrathin sectioning. Sections of biological specimens, organic polymers and similar materials may require special `staining' with heavy atom labels in order to achieve the required image contrast.

1) Applications

Semiconductor and data storage Research

• Circuit edit • Electron beam-induced • Defect analysis deposition • Failure analysis • Materials qualification • Materials and sample preparation Biology and life sciences • Nanoprototyping • Nanometrology • Diagnostic electron microscopy • Device testing and • Cryobiology characterization • Protein localization • Electron tomography Industry • Cellular tomography • Cryo-electron microscopy • High-resolution imaging • Toxicology • 2D & 3D micro-characterization • Biological production and viral • Macro sample to nanometer load monitoring metrology • Particle analysis • Particle detection and • Pharmaceutical QC characterization • Structural biology • Direct beam-writing fabrication • 3D tissue imaging • Dynamic materials experiments • Virology • Sample preparation • Vitrification • Forensics • Mining (mineral liberation analysis) • Chemical/Petrochemical

2.5- Microscope Slide

A microscope slide is a thin flat piece of glass, typically 75 by 25 mm (3 by 1 inches) and about 1 mm thick, used to hold objects for examination under a microscope. Typically the object is placed or secured ("mounted") on the slide, and then both are inserted together in the microscope for viewing. This 21 arrangement allows several slide-mounted objects to be quickly inserted and removed from the microscope, labeled, transported, and stored in appropriate slide cases or folders.

Microscope slides are often used together with a cover slip or cover glass, a smaller and thinner sheet of glass that is placed over the specimen. Slides are held in place on the microscope's stage by slide clips or slide clamps.

1) Dimensions and types

A standard microscope slide measures about 75 mm by 25 mm (3" by 1") and is about 1 mm thick. A range of other sizes are available for various special purposes, such as 75 x 50 mm and for geological use, 46 x 27 mm for petrographic studies, and 48 x 28 mm for thin sections. Slides are usually made of common glass and their edges are often finely ground or polished.

Microscope slides are usually made of glass, such as soda lime glass or borosilicate glass, but specialty plastics are also used. Fused quartz slides are often used when ultraviolet transparency is important, e.g. in fluorescence microscopy.

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Chapter 3: Aseptic Technique & Sterilization

3.1- Aseptic Technique

Aseptic technique refers to a procedure that is performed under sterile conditions. This includes medical and laboratory techniques, such as with microbiological cultures. It includes techniques like flame sterilization. The largest example of aseptic techniques is in hospital operating theatres.

3.2- Laminar Flow Cabinet

A laminar flow cabinet or laminar flow closet or tissue culture hood is a carefully enclosed bench designed to prevent contamination of semiconductor wafers, biological samples, or any particle sensitive device. Air is drawn through a HEPA filter and blown in a very smooth, laminar flow towards the user. The cabinet is usually made of stainless steel with no gaps or joints where spores might collect.

23

Such hoods exist in both horizontal and vertical configurations, and there are many different types of cabinets with a variety of airflow patterns and acceptable uses. NSF49 is the commonly accepted regulatory standard for these cabinets.

Laminar flow cabinets may have a UV-Cgermicidal lamp to sterilize the shell and contents when not in use. It is important to switch this light off during use, as it will quickly give any exposed skin sunburn and may cause cataracts. These lights are not commonly used any more, due to the harmful nature of UV- radiation to humans and its relatively low decontamination efficiency.

3.3- Sterilization

Sterilization (or sterilisation, see spelling differences) is a term referring to any process that eliminates (removes) or kills all forms of life, including transmissible agents (such as fungi, bacteria, viruses, spore forms, etc.) present on a surface, contained in a fluid, in medication, or in a compound such as biological culture media. Sterilization can be achieved by applying the proper combinations of heat, chemicals, irradiation, high pressure, and filtration.

The term has evolved to include the disabling or destruction of infectious proteins such as prions related to Transmissible Spongiform Encephalopathies 24

Proper autoclave treatment will inactivate all fungi, bacteria, viruses and also bacterial spores, which can be quite resistant. It will not necessarily eliminate all prions.

1) Chemical sterilization

Chemicals are also used for sterilization. Although heating provides the most reliable way to rid objects of all transmissible agents, it is not always appropriate, because it will damage heat-sensitive materials such as biological materials, fiber optics, electronics, and many plastics. Low temperature gas sterilizers function by exposing the articles to be sterilized to high concentrations (typically 5 - 10% v/v) of very reactive gases (alkylating agents such as ethylene oxide, and oxidizing agents such as hydrogen peroxide and ozone). Liquid sterilants and high disinfectants typically include oxidizing agents such as hydrogen peroxide and peracetic acid and aldehydes such as glutaraldehyde and more recently o-phthalaldehyde. While the use of gas and liquid chemical sterilants/high level disinfectants avoids the problem of heat damage, users must ensure that article to be sterilized is chemically compatible with the sterilant being used. The manufacturer of the article can provide specific information regarding compatible sterilants. In addition, the use of chemical sterilants poses new challenges for workplace safety. The chemicals used as sterilants are designed to destroy a wide range of pathogens and typically the same properties that make them good sterilants makes them harmful to humans. Employers have a duty to ensure a safe work environment (Occupational Safety and Health Act of 1970, section 5 for United States) and work practices, engineering controls and monitoring should be employed appropriately.

2) Sterile filtration

Clear liquids that would be damaged by heat, irradiation or chemical sterilization can be sterilized by mechanical filtration. This method is commonly used for sensitive pharmaceuticals and protein solutions in biological research. A filter with pore size 0.2 µm will effectively remove bacteria. If viruses must also be removed, a much smaller pore size around 20 nm is needed. Solutions filter slowly through membranes with smaller pore diameters. Prions are not removed by filtration.

25

Filters can be made of several different materials such as nitrocellulose or polyethersulfone (PES). The filtration equipment and the filters themselves may be purchased as pre- sterilized disposable units in sealed packaging, or must be sterilized by the user, generally by autoclaving at a temperature that does not damage the fragile filter membranes. To ensure sterility, the filter membranes need testing for punctures made during or prior to use. For best results, pharmaceutical sterile filtration is performed in a room with highly filtered air.

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Chapter 4: Microorganisms

4.1- Microorganisms

A microorganism (from the Greek: μικρός, mikrós, "small" and ργανισμός, organismós, "organism"; also spelt micro-organism, micro organism or microörganism) or microbe is an organism that is unicellular or lives in a colony of cellular organisms. The study of microorganisms is called microbiology, a subject that began with Anton van Leeuwenhoek's discovery of microorganisms in 1675, using a microscope of his own design.

Microorganisms are very diverse; they include bacteria, fungi, archaea, and protists; microscopic plants (green algae); and animals such as plankton and the planarian. Some microbiologists also include viruses, but others consider these as non-living. Most microorganisms are unicellular (single-celled), but this is not universal, since some multicellular organisms are microscopic, while some unicellular protists and bacteria, like Thiomargarita namibiensis, are macroscopic and visible to the naked eye.

Microorganisms live in all parts of the biosphere where there is liquid water, including soil, hot springs, on the ocean floor, high in the atmosphere and deep inside rocks within the Earth's crust. Microorganisms are critical to nutrient recycling in ecosystems as they act as decomposers. As some microorganisms can fix nitrogen, they are a vital part of the nitrogen cycle, and recent studies indicate that airborne microbes may play a role in precipitation and weather.[4]

Microbes are also exploited by people in biotechnology, both in traditional food and beverage preparation, and in modern technologies based on genetic engineering. However, pathogenic microbes are harmful, since they invade and grow within other organisms, causing diseases that kill people, other animals and plants. 27

1) Classification and structure

Evolutionary tree showing the common ancestry of all three domains of life. Bacteria are colored blue, eukaryotes red, and archaea green. Relative positions of some phyla are shown around the tree.

Microorganisms can be found almost anywhere in the taxonomic organization of life on the planet. Bacteria and archaea are almost always microscopic, while a number of eukaryotes are also microscopic, including most protists, some fungi, as well as some animals and plants. Viruses are generally regarded as not living and therefore are not microbes, although the field of microbiology also encompasses the study of viruses. a) Prokaryotes

Prokaryotes are organisms that lack a cell nucleus and the other membrane bound organelles. They are almost always unicellular, although some species such as myxobacteria can aggregate into complex structures as part of their life cycle.

Consisting of two domains, bacteria and archaea, the prokaryotes are the most diverse and abundant group of organisms on Earth and inhabit practically all environments where some liquid water is available and the temperature is below +140 °C. They are found in sea water, soil, air, animals' gastrointestinal tracts, hot springs and even deep beneath the Earth's crust in rocks.[28] Practically all surfaces which have not been specially sterilized are covered by prokaryotes. The number of prokaryotes on Earth is estimated to be around five million trillion trillion, or 5 × 1030, accounting for at least half the biomass on Earth. 28 b) Bacteria

Bacteria are practically all invisible to the naked eye, with a few extremely rare exceptions, such as Thiomargarita namibiensis.[30] They lack membrane-bound organelles, and can function and reproduce as individual cells, but often aggregate in multicellular colonies. Their genome is usually a single loop of DNA, although they can also harbor small pieces of DNA called plasmids. These plasmids can be transferred between cells through bacterial conjugation. Bacteria are surrounded by a cell wall, which provides strength and rigidity to their cells. They reproduce by binary fission or sometimes by budding, but do not undergo sexual reproduction. Some species form extraordinarily resilient spores, but for bacteria this is a mechanism for survival, not reproduction. Under optimal conditions bacteria can grow extremely rapidly and can double as quickly as every 10 minutes. c) Archaea

Archaea are also single-celled organisms that lack nuclei. In the past, the differences between bacteria and archaea were not recognised and archaea were classified with bacteria as part of the kingdom Monera. However, in 1990 the microbiologist Carl Woese proposed the three-domain system that divided living things into bacteria, archaea and eukaryotes. Archaea differ from bacteria in both their genetics and biochemistry. For example, while bacterial cell membranes are made from phosphoglycerides with ester bonds, archaean membranes are made of ether lipids.

Archaea were originally described in extreme environments, such as hot springs, but have since been found in all types of habitats.[35] Only now are scientists beginning to realize how common archaea are in the environment, with crenarchaeota being the most common form of life in the ocean, dominating ecosystems below 150 m in depth.[36][37] These organisms are also common in soil and play a vital role in ammonia oxidation.[38] d) Eukaryotes

Ostreococcus is the smallest known free living eukaryote with an average size of 0.8 µm

Most living things which are visible to the naked eye in their adult form are eukaryotes, including humans. However, a large number of eukaryotes are also microorganisms. Unlike bacteria and archaea, eukaryotes contain organelles such as the cell nucleus, the Golgi apparatus and mitochondria in their cells. The nucleus is an organelle which houses the DNA that makes up a cell's 29 genome. DNA itself is arranged in complex chromosomes.[39] Mitochondria are organelles vital in metabolism as they are the site of the citric acid cycle and oxidative phosphorylation. They evolved from symbiotic bacteria and retain a remnant genome. Like bacteria, plant cells have cell walls, and contain organelles such as chloroplasts in addition to the organelles in other eukaryotes. Chloroplasts produce energy from light by photosynthesis, and were also originally symbiotic bacteria.

Unicellular eukaryotes are those eukaryotic organisms that consist of a single cell throughout their life cycle. This qualification is significant since most multicellular eukaryotes consist of a single cell called a zygote at the beginning of their life cycles. Microbial eukaryotes can be either haploid or diploid, and some organisms have multiple cell nuclei (see coenocyte). However, not all microorganisms are unicellular as some microscopic eukaryotes are made from multiple cells. e) Protists

Of eukaryotic groups, the protists are most commonly unicellular and microscopic. This is a highly diverse group of organisms that are not easy to classify. Several algae species are multicellular protists, and slime molds have unique life cycles that involve switching between unicellular, colonial, and multicellular forms. The number of species of protozoa is uncertain, since we may have identified only a small proportion of the diversity in this group of organisms. f) Animals

Mostly animals are multicellular, but some are too small to be seen by the naked eye. Microscopic arthropods include dust mites and spider mites. Microscopic crustaceans include copepods and the cladocera, while many nematodes are too small to be seen with the naked eye. Another particularly common group of microscopic animals are the rotifers, which are filter feeders that are usually found in fresh water. Micro-animals reproduce both sexually and asexually and may reach new habitats as eggs that survive harsh environments that would kill the adult animal. However, some simple animals, such as rotifers and nematodes, can dry out completely and remain dormant for long periods of time. g) Fungi

The fungi have several unicellular species, such as baker's yeast (Saccharomyces cerevisiae) and fission yeast (Schizosaccharomyces pombe). 30

Some fungi, such as the pathogenic yeast Candida albicans, can undergo phenotypic switching and grow as single cells in some environments, and filamentous hyphae in others. Fungi reproduce both asexually, by budding or binary fission, as well by producing spores, which are called conidia when produced asexually, or basidiospores when produced sexually. h) Plants

The green algae are a large group of photosynthetic eukaryotes that include many microscopic organisms. Although some green algae are classified as protists, others such as charophyta are classified with embryophyte plants, which are the most familiar group of land plants. Algae can grow as single cells, or in long chains of cells. The green algae include unicellular and colonial flagellates, usually but not always with two flagella per cell, as well as various colonial, coccoid, and filamentous forms. In the Charales, which are the algae most closely related to higher plants, cells differentiate into several distinct tissues within the organism. There are about 6000 species of green algae.[49]

31

Chapter 5: Cultivation & Isolation of Microorganisms

5.1- Microbial Culture

A microbiological culture, or microbial culture, is a method of multiplying microbial organisms by letting them reproduce in predetermined culture media under controlled laboratory conditions. Microbial cultures are used to determine the type of organism, its abundance in the sample being tested, or both. It is one of the primary diagnostic methods of microbiology and used as a tool to determine the cause of infectious disease by letting the agent multiply in a predetermined medium. For example, a throat culture is taken by scraping the lining of tissue in the back of the throat and blotting the sample into a medium to be able to screen for harmful microorganisms, such as Streptococcus pyogenes, the causative agent of strep throat. Furthermore, the term culture is more generally used informally to refer to "selectively growing" a specific kind of microorganism in the lab.

Microbial cultures are foundational and basic diagnostic methods used extensively as a research tool in molecular biology. It is often essential to isolate a pure culture of microorganisms. A pure (or axenic) culture is a population of cells or multicellular organisms growing in the absence of other species or types. A pure culture may originate from a single cell or single organism, in which case the cells are genetic clones of one another.

For the purpose of gelling the microbial culture, the medium of agarose gel (agar) is used. Agar is a gelatinous substance derived from seaweed. A cheap 32 substitute for agar is guar gum, which can be used for the isolation and maintenance of thermophiles.

5.2- Growth Medium

A growth medium or culture medium is a liquid or gel designed to support the growth of microorganisms or cells, or small plants like the moss Physcomitrella patens. There are different types of media for growing different types of cells.

There are two major types of growth media: those used for cell culture, which use specific cell types derived from plants or animals, and microbiological culture, which are used for growing microorganisms, such as bacteria or yeast. The most common growth media for microorganisms are nutrient broths and 33 agar plates; specialized media are sometimes required for microorganism and cell culture growth. Some organisms, termed fastidious organisms, require specialized environments due to complex nutritional requirements. Viruses, for example, are obligate intracellular parasites and require a growth medium containing living cells.

1) Types of growth media

The most common growth media for microorganisms are nutrient broths (liquid nutrient medium) or LB medium (Lysogeny Broth). Liquid media are often mixed with agar and poured into Petri dishes to solidify. These agar plates provide a solid medium on which microbes may be cultured. They remain solid, as very few bacteria are able to decompose agar. Bacteria grow in liquid cultures often form colloidalsuspensions.

The differences between growth media used for cell culture and those used for microbiological culture are because cells derived from whole organisms and grown in culture often cannot grow without the addition of, for instance, hormones or growth factors which usually occur in vivo.[4] In the case of animal cells, this difficulty is often addressed by the addition of blood serum or a synthetic serum replacement to the medium. In the case of microorganisms, there are no such limitations, as they are often unicellular organisms. One other major difference is that animal cells in culture are often grown on a flat surface to which they attach, and the medium is provided in a liquid form, which covers the cells. In contrast, bacteria such as Escherichia coli may be grown on solid media or in liquid media.

An important distinction between growth media types is that of defined versus undefined media. A defined medium will have known quantities of all ingredients. For microorganisms, they consist of providing trace elements and vitamins required by the microbe and especially a defined carbon source and nitrogen source. Glucose or glycerol is often used as carbon sources, and ammoniumsalts or nitrates as inorganic nitrogen sources. An undefined medium has some complex ingredients, such as yeast extract or casein hydrolysate, which consist of a mixture of many, many chemical species in unknown proportions. Undefined media are sometimes chosen based on price and sometimes by necessity - some microorganisms have never been cultured on defined media.

A good example of a growth medium is the wort used to make beer. The wort contains all the nutrients required for yeast growth, and under anaerobic conditions, alcohol is produced. When the fermentation process is complete, the combination of medium and dormant microbes, now beer, is ready for consumption. 34

5.3- Pour Plate Method for Isolation

The pour plate technique can be used to determine the number of microbes/mL or microbes/gram in a specimen. It has the advantage of not requiring previously prepared plates, and is often used to assay bacterial contamination of foodstuffs. The principle steps are to

1) prepare/dilute the sample 2) place an aliquot of the diluted sample in an empty sterile plate 3) pour in 15 mL of melted agar which has been cooled to 45o C, swirl to mix well 4) let cool undisturbed to solidify on a flat table top 5) invert and incubate to develop colonies.

Each colony represents a "colony forming unit" (CFU). For optimum accuracy of a count, the preferred range for total CFU/plate is between 30 to 300 colonies/plate.

One disadvantage of pour plates is that embedded colonies will be much smaller than those which happen to be on the surface, and must be carefully scored so that none are overlooked. Also, obligate aerobes may grow poorly if deeply imbedded in the agar.

35

1) EQUIPMENT:

15 mL sterile Plate Count Agar (PCA)*, in capped 16 x 150 mm test tubes, melted and cooled to 45oC Hot Block, 45oC (or water bath), 3" deep to equal agar depth sterile capped 16 x 150 mm test tubes 0.1, 1.0 and 2.0 mL pipets, sterile petri dishes, empty and sterile flame colony counter with magnifying glass

2) POUR PLATE TECHNIQUE:

1. Write out details of preparing and plating your specimen(s): Construct a table in your notebook with a line for each plate:

• the identity/source of the specimen (notebook entries should be detailed). • the dilution of the specimen expected to contain between 30-300 CFU/0.1-1.0 mL and how you will prepare it • the volume of diluted specimen you will plate (usually 0.1 to 1.0 mL)

Label the bottom of the empty, sterile plates your initials, seat number, date and the above data.

2. Dilute specimen to yield approximately 30 to 300 CFU per aliquot to be plated (from 1).

3. Inoculate labeled empty petri dish with the aliquot of diluted specimen (from 1)

4. Pour 15 mL of melted Plate Count Agar (45o C) into the inoculated petri dish.

5. Cover and mix thoroughly by gentle tilting and swirling the dish. Do not slop the agar over the edge of the petri dish.

6. Place on a flat surface undistrubed for about 10 minutes to allow the agar to completely gel. In this illustration, the agar is completely gelled and the surface is "smooth as glass."

7. Invert and incubate at 37o C for 24-48 hours.

8. Count, record, calculate: Count all colonies (note that the embedded colonies will be much smaller than 36 those which happen to form on the surface). A magnifying colony counter can aid in counting small embedded colonies. Record the data. Calculate CFU/mL or CFU/g. Enter results in your table.

CFU/ mL = CFU/plate x dilution factor x 1/aliquot

On the plate shown, milk was diluted 1 to 100 (10 2), 1.0 mL of the dilution was plated and 40 colonies formed. Therefore the count per mL in the milk was:

40 colonies x 102 x 1/1 = 4 x 103/mL

* For 600 mL of NA + 1% glu: 9 g agar, 4.8 g nutrient broth, 6 g dextrose. Dissolve ingredients at 95oC, repipet into 16 x 150 mm tubes, cap, autoclave, 15 lb, 15 min. Cool to 45oC before using. Plate Count Agar may also be used.

5.4- Spread Plate Method for Isolation

The number of bacteria in solution can be readily quantified by using the spread plate technique. In this technique, the sample is appropriately diluted and a small aliquot transferred to an agar plate. The bacteria are then distributed evenly over the surface by a special streaking technique. After colonies are grown, they are counted and the number of bacteria in the original sample calculated.

The end point of our analysis is the number of colony forming units per mL (CFU/mL) since we are counting the number of colonies rather than the actual number of bacteria. We are assuming that the each viable bacteria in the suspension will form an individual colony, which is a valid assumption if we do all the techniques properly. CFU/mL is actually a more useful determination than counting all the bacteria under a microscope because in many bacterial populations some significant number will be dead cells and thus of no interest.

Diluting the bacteria. Bacteria commonly grow up to densities around 109 CFU/mL, although the maximum densities vary tremendously depending on the species of bacteria and the media they are growing in. Therefore, to get readily countable numbers of bacteria, we have to make a wide range of dilutions and assay all of them with the goal of having one or two dilutions with countable numbers. We do this by making serial 10-fold dilutions (see serial dilutions section if this is an unfamiliar concept) of the bacteria that cover the whole probabey range of concentrations. We then transfer 0.1 mL of each dilution to an agar plate, which in effect makes another 10-fold dilution since the final units are CFU/mL and we are only streaking 0.1 mL. 37

Inoculating the plate. Streaking in this technique is done using a bent glass rod. 0.1 mL of bacterial suspension is placed in the center of the plate using a sterile pipet. The glass rod is sterilized by first dipping it into a 70% alcohol solution and then passing it quickly through the Bunsen burner flame. The burning alcohol sterilizes the rod at a cooler temperature than holding the rod in the burner flame thus reducing the chance of you burning your fingers. When all the alcohol has burned off and the rod has air-cooled, streak the rod back and forth across the plate working up and down several times. Unlike streaking for isolation, you want to backtrack many times in order to distribute the bacteria as evenly as possible. Turn the plate 90 degrees and repeat the side to side, up and down streaking. Turn the plate 45 degrees and streak a third time. Do not sterilize the glass rod between plate turnings. Cover the plate and wait several minutes before turning it upside down for incubation. This will allow the broth to soak into the plate so the bacteria won't drip onto the plate lid.

Counting bacteria. Colonies are most readily counted using a plate counter. The plate counter has a light source and a magnifying glass making colonies easier to see. If at all possible, you don't want to count plates with more than 300 or less than 30 colonies. In the former case, the colonies will be running together and in the latter there are too few to allow statistically accurate counts. Once 38 you count the colonies, multiply by the appropriate dilution factor to determine the number of CFU/mL in the original sample.

5.5- Streak Plate Method for Isolation

Read these entire instructions thoroughly and watch a demonstration before you attempt the procedure yourself. Each student choose either E. coli or S. epi. Prepare a previously poured agar plate by labeling with name, date, and organism. Use a grease pencil to make "map" for a streaking pattern. Mark sections from behind the agar plate. (Do not remove the top until sample is ready for streaking). There are many methods for streaking. The most effective method depends on the concentration of bacteria within a particular inoculum as well as each individual technician’s dexterity. A typical pattern will be demonstrated which works with the samples used in this teaching lab.

39

Although we are practicing with pure cultures, the streak plate technique is designed to isolate species from mixed cultures. As the bacteria in a mixed sample are spread apart from each other and allowed to grow into colonies separated across the agar surface, an isolated colony can then be used as a source of inoculum for a pure culture. We will try this with an unknown mixture from an environmental culture.

1) PROCEDURE

1. Using aseptic technique, remove a loopful of bacteria from a pure culture. 2. Lift the lid of the Petri dish out of the way, but keep it angled over the dish. 3. Place the loop gently on the agar surface in section 1. (Never put enough pressure to dig into the agar surface!) 4. Gently sweep the loop back and forth across the agar surface, spreading the sample out in section 1. 5. Heat-sterilize the loop. 6. Allowing the loop to cool, touch it to a sterile part of the agar in section 2. 7. Sweep the loop across the agar surface from section 2 into section 1, then back to 2, then back to 1, etc. Take care not to double back over any streaks in section 2. Stop when you run out of area in section 2, and lift the loop. 8. Heat-sterilize the loop. 9. Allowing the loop to cool, turn the dish so that section 3 is accessible. 10. Place the loop in a sterile section of section 3, then gently stroke the agar surface, returning several times into section 2. 11. Do not double back over any streaks in section 3. Continue on in section 3 even though there is not space available for access to section 2. Above all, do not re-enter section 1. 12. Re-cover the dish, heat-sterilize the loop, invert, and incubate.

The goal of the above procedure is to dilute the original sample by heat- sterilizing. As you streak out the bacteria in section 1, they are separated from each other. By carefully streaking back into 1 from 2, you pull back into 2 from 1 only a small amount. As you streak from 2 to 3, you are pulling out even fewer. Your isolated colonies after incubation should be found in section 3. This is where single bacterium landed and multiplied into a clone of identical cells: a colony.

Invert the plates in the rack provided. Incubate the rack of plates in a minimum of 48 hours; retrieve and observe.

40

Chapter 6: Microbial Growth Measurement

6.1- Microbial Growth

Requirements for microbial growth:

– Two subgroups physical and chemical.

– Physical include: Temperature, pH and osmotic pressure.

– Chemical include: Sources of carbon, nitrogen, oxygen, hydrogen, sulfur, phosphorous and trace elements.

6.2- Direct Plate Counting

When one decides to count the number of cells in a sample, the issues of viable numbers, and the total numbers should come to mind. There are many methods used for counting cell numbers, some of which count total cell numbers (live cells + dead cells), and others that count only viable, or live cells. 41

Direct plate counting is a method used to count the number of viable cells in a sample. By the very nature of the procedure, the dead cells are unable to be included in the count.

Once the cells to be counted have been isolated, they are to be diluted due to the fact that too many cells will cause the Petri plate to be so densely populated with colonies, that they would be impossible to count. After the cells have been diluted, they are incubated on an agar medium until colonies form. It is at this time that the cells may be counted. The image on the left shows a soil dilution plate containing bacteria and fungi. This plate is countable, but on the difficult side.

6.3- Using a Counting Chamber

For microbiology, cell culture, and many applications that require use of suspensions of cells it is necessary to determine cell concentration. One can often determine cell density of a suspension spectrophotometrically, however that form of determination does not allow an assessment of cell viability, nor can one distinguish cell types.

A device used for determining the number of cells per unit volume of a suspension is called a counting chamber. The most widely used type of chamber is called a hemocytometer, since it was originally designed for performing blood cell counts.

To prepare the counting chamber the mirror-like polished surface is carefully cleaned with lens paper. The coverslip is also cleaned. Coverslips for counting chambers are specially made and are thicker than those for conventional microscopy, since they must be heavy enough to overcome the surface tension of a drop of liquid. The coverslip is placed over the counting surface prior to putting on the cell suspension. The suspension is introduced into one of the V- shaped wells with a pasteur or other type of pipet. The area under the coverslip fills by capillary action. Enough liquid should be introduced so that the mirrored surface is just covered. The charged counting chamber is then placed on the microscope stage and the counting grid is brought into focus at low power. 42

It is essential to be extremely careful with higher power objectives, since the counting chamber is much thicker than a conventional slide. The chamber or an objective lens may be damaged if the user is not not careful. One entire grid on standard hemacytometers with Neubauer rulings can be seen at 40x (4x objective). The main divisions separate the grid into 9 large squares (like a tic- tac-toe grid). Each square has a surface area of one square mm, and the depth of the chamber is 0.1 mm. Thus the entire counting grid lies under a volume of 0.9 mm-cubed.

Suspensions should be dilute enough so that the cells or other particles do not overlap each other on the grid, and should be uniformly distributed. To perform the count, determine the magnification needed to recognize the desired cell type. Now systematically count the cells in selected squares so that the total count is 100 cells or so (number of cells needed for a statistically significant count). For large cells this may mean counting the four large corner squares and the middle one. For a dense suspension of small cells you may wish to count the cells in the four 1/25 sq. mm corners plus the middle square in the central square. Always decide on a specific counting patter to avoid bias. For cells that overlap a ruling, count a cell as "in" if it overlaps the top or right ruling, and "out" if it overlaps the bottom or left ruling. 43

Here is a way to determine a particle count using a Neubauer hemocytometer. Suppose that you conduct a count as described above, and count 187 particles in the five small squares described. Each square has an area of 1/25 mm-squared (that is, 0.04 mm-squared) and depth of 0.1 mm. The total volume in each square is (0.04)x(0.1) = 0.004 mm-cubed. You have five squares with combined volume of 5x(0.004) = 0.02 mm-cubed. Thus you counted 187 particles in a volume of 0.02 mm-cubed, giving you 187/(0.02) = 9350 particles per mm- cubed. There are 1000 cubic millimetres in one cubic centimetre (same as a millilitre), so your particle count is 9,350,000 per ml.

Cells are often large enough to require counting over a larger surface area. For example, you might count the total number of cells in the four large corner squares plus the middle combined. Each square has surface area of 1 mm- squared and a depth of 0.1 mm, giving it a volume of 0.1 mm-cubed. Suppose that you counted 125 cells (total) in the five squares. You then have 125 cells per 0.5 mm-cubed, which is 250 cells/mm-cubed. Again, multiply by 1000 to determine cell count per ml (250,000).

Sometimes you will need to dilute a cell suspension to get the cell density low enough for counting. In that case you will need to multiply your final count by the dilution factor. For example, suppose that for counting you had to dilute a suspension of Chlamydomonas 10 fold. Suppose you obtained a final count of 250,000 cells/ml as described above. Then the count in the original (undiluted) suspension is 10 x 250,000 which is 2,500,000 cells/ml.

44

Chapter 7: Enzyme Activity Assay

7.1- Enzyme

Enzymes (pronounced / nza mz/) are proteins that catalyze (i.e., increase the rates of) chemical reactions. In enzymatic reactions, the molecules at the beginning of the process are called substrates, and they are converted into different molecules, called the products. Almost all processes in a biological cell need enzymes to occur at significant rates. Since enzymes are selective for their substrates and speed up only a few reactions from among many possibilities, the set of enzymes made in a cell determines which metabolic pathways occur in that cell.

Like all catalysts, enzymes work by lowering the activation energy (Ea‡) for a reaction, thus dramatically increasing the rate of the reaction. As a result, products are formed faster and reactions reach their equilibrium state more rapidly. Most enzyme reaction rates are millions of times faster than those of comparable un-catalyzed reactions. As with all catalysts, enzymes are not consumed by the reactions they catalyze, nor do they alter the equilibrium of these reactions. However, enzymes do differ from most other catalysts by being much more specific. Enzymes are known to catalyze about 4,000 biochemical reactions. A few RNA molecules called ribozymes also catalyze reactions, with an important example being some parts of the ribosome. Synthetic molecules called artificial enzymes also display enzyme-like catalysis.

Enzyme activity can be affected by other molecules. Inhibitors are molecules that decrease enzyme activity; activators are molecules that increase activity. Many drugs and poisons are enzyme inhibitors. Activity is also affected by temperature, chemical environment (e.g., pH), and the concentration of substrate. Some enzymes are used commercially, for example, in the synthesis of antibiotics. In addition, some household products use enzymes to speed up biochemical reactions (e.g., enzymes in biological washing powders break down protein or fat stains on clothes; enzymes in meat tenderizers break down proteins into smaller molecules, making the meat easier to chew).

1) Control of activity

There are five main ways that enzyme activity is controlled in the cell. 45

1. Enzyme production (transcription and translation of enzyme genes) can be enhanced or diminished by a cell in response to changes in the cell's environment. This form of gene regulation is called enzyme induction and inhibition (see enzyme induction). For example, bacteria may become resistant to antibiotics such as penicillin because enzymes called beta-lactamases are induced that hydrolyse the crucial beta-lactam ring within the penicillin molecule. Another example are enzymes in the liver called cytochrome P450 oxidases, which are important in drug metabolism. Induction or inhibition of these enzymes can cause drug interactions. 2. Enzymes can be compartmentalized, with different metabolic pathways occurring in different cellular compartments. For example, fatty acids are synthesized by one set of enzymes in the cytosol, endoplasmic reticulum and the Golgi apparatus and used by a different set of enzymes as a source of energy in the mitochondrion, through β-oxidation.[80] 3. Enzymes can be regulated by inhibitors and activators. For example, the end product(s) of a metabolic pathway are often inhibitors for one of the first enzymes of the pathway (usually the first irreversible step, called committed step), thus regulating the amount of end product made by the pathways. Such a regulatory mechanism is called a negative feedback mechanism, because the amount of the end product produced is regulated by its own concentration. Negative feedback mechanism can effectively adjust the rate of synthesis of intermediate metabolites according to the demands of the cells. This helps allocate materials and energy economically, and prevents the manufacture of excess end products. The control of enzymatic action helps to maintain a stable internal environment in living organisms. 4. Enzymes can be regulated through post-translational modification. This can include phosphorylation, myristoylation and glycosylation. For example, in the response to insulin, the phosphorylation of multiple enzymes, including glycogen synthase, helps control the synthesis or degradation of glycogen and allows the cell to respond to changes in blood sugar.[81] Another example of post-translational modification is the cleavage of the polypeptide chain. Chymotrypsin, a digestive protease, is produced in inactive form as chymotrypsinogen in the pancreas and transported in this form to the stomach where it is activated. This stops the enzyme from digesting the pancreas or other tissues before it enters the gut. This type of inactive precursor to an enzyme is known as a zymogen. 5. Some enzymes may become activated when localized to a different environment (e.g. from a reducing (cytoplasm) to an oxidizing (periplasm) environment, high pH to low pH etc.). For example, hemagglutinin in the influenza virus is activated by a conformational 46

change caused by the acidic conditions, these occur when it is taken up inside its host cell and enters the lysosome.

2) Industrial applications

Enzymes are used in the chemical industry and other industrial applications when extremely specific catalysts are required. However, enzymes in general are limited in the number of reactions they have evolved to catalyze and also by their lack of stability in organic solvents and at high temperatures. Consequently, protein engineering is an active area of research and involves attempts to create new enzymes with novel properties, either through rational design or in vitro evolution. These efforts have begun to be successful, and a few enzymes have now been desiged "from scratch" to catalyze reactions that do not occur in nature.

Application Enzymes used Uses

Production of sugars from starch, such as in making high-fructose Food processing corn syrup.[91] In baking, catalyze Amylases from fungi and breakdown of starch in plants the flour to sugar. Yeast fermentation of sugar produces the Amylases catalyze the carbon dioxide that release of simple sugars from raises the dough. starch. Biscuit manufacturers Proteases use them to lower the protein level of flour.

To predigest baby

Baby foods Trypsin foods 47

They degrade starch and proteins to Enzymes from barley are produce simple sugar, released during the amino acids and mashing stage of beer peptides that are used production. by yeast for fermentation.

Widely used in the Brewing industry brewing process to Industrially produced substitute for the barley enzymes natural enzymes found in barley.

Split polysaccharides Amylase, glucanases, and proteins in the proteases malt.

Improve the wort and Germinating barley used for Betaglucanases and beer filtration malt arabinoxylanases characteristics.

Low-calorie beer and Amyloglucosidase and adjustment of pullulanases fermentability.

Remove cloudiness Proteases produced during storage of beers.

Increases fermentation Acetolactatedecarboxylase efficiency by reducing (ALDC) diacetyl formation.[92]

Fruit juices Cellulases, pectinases Clarify fruit juices. 48

Rennin, derived from the Manufacture of stomachs of young cheese, used to ruminant animals (like hydrolyze protein calves and lambs) Dairy industry Now finding Microbially produced increasing use in the enzyme dairy industry

Is implemented during the production of

Roquefort cheese to

Lipases enhance the ripening of the blue-mould Roquefort cheese cheese.

Break down lactose to Lactases glucose and galactose.

To soften meat for

Meat tenderizers Papain cooking

Amylases, Converts starch into amyloglucosideases and glucose and various glucoamylases syrups.

Starch industry Converts glucose into fructose in production of high fructose syrups from starchy materials. These Glucose isomerase syrups have enhanced Glucose Fructose sweetening properties

and lower calorific values than sucrose for the same level of sweetness. 49

Degrade starch to lower viscosity, aiding Paper industry sizing and coating paper. Xylanases reduce bleach required for decolorising; Amylases, Xylanases, cellulases smooth Cellulases and ligninases fibers, enhance water

drainage, and promote ink removal; lipases A paper mill in South reduce pitch and Carolina lignin-degrading enzymes remove lignin to soften paper.

Biofuel industry Used to break down cellulose into sugars

Cellulases that can be fermented (see cellulosic ethanol)

Ligninases Use of lignin waste Cellulose in 3D

Used for presoak Primarily proteases, conditions and direct produced in an liquid applications extracellular form from helping with removal bacteria of protein stains from clothes

Biological detergent Detergents for machine dish washing

Amylases to remove resistant starch residues

Used to assist in the Lipases removal of fatty and 50

oily stains

Used in biological

Cellulases fabric conditioners

To remove proteins on

Contact lens cleaners Proteases contact lens to prevent infections

To generate oxygen from peroxide to

Rubber industry Catalase convert latex into foam rubber

Dissolve gelatin off scrap film, allowing

Photographic industry Protease (ficin) recovery of its silver content.

Used to manipulate DNA in genetic Molecular biology engineering, important in pharmacology, agriculture and Restriction enzymes, medicine. Essential for DNA ligase and restriction digestion polymerases and the polymerase chain reaction. Molecular biology is Part of the DNA double helix also important in forensic science.

7.2- Enzyme Assay

Enzyme assays are laboratory methods for measuring enzymatic activity. They are vital for the study of enzyme kinetics and enzyme inhibition. 51

1) Enzyme units

Amounts of enzymes can either be expressed as molar amounts, as with any other chemical, or measured in terms of activity, in enzyme units.

2) Enzyme activity

Enzyme activity = moles of substrate converted per unit time = rate × reaction volume. Enzyme activity is a measure of the quantity of active enzyme present and is thus dependent on conditions, which should be specified. The SI unit is the katal, 1 katal = 1 mol s-1, but this is an excessively large unit. A more practical and commonly used value is 1 enzyme unit (U) = 1 μmol min-1. 1 U corresponds to 16.67 nanokatals.

Enzyme activity as given in katal generally refers to that of the assumed natural target substrate of the enzyme. Enzyme activity can also be given as that of certain standardized substrates, such as gelatin, then measured in gelatin digesting units (GDU), or milk proteins, then measured in milk clotting units (MCU). The units GDU and MCU are based on how fast one gram of the enzyme will digest gelatin or milk proteins, respectively. 1 GDU equals approximately 1.5 MCU.

3) Types of assay

All enzyme assays measure either the consumption of substrate or production of product over time. A large number of different methods of measuring the concentrations of substrates and products exist and many enzymes can be assayed in several different ways. Biochemists usually study enzyme-catalysed reactions using four types of experiments:

• Initial rate experiments. When an enzyme is mixed with a large excess of the substrate, the enzyme-substrate intermediate builds up in a fast initial transient. Then the reaction achieves a steady-state kinetics in which enzyme substrate intermediates remains approximately constant over time and the reaction rate changes relatively slowly. Rates are measured for a short period after the attainment of the quasi-steady state, typically by monitoring the accumulation of product with time. Because the measurements are carried out for a very short period and because of the large excess of substrate, the approximation free substrate is approximately equal to the initial substrate can be made. The initial rate experiment is the simplest to perform and analyze, being relatively free from complications such as back-reaction and enzyme degradation. It is therefore by far the most commonly used type of experiment in enzyme kinetics. 52

• Progress curve experiments. In these experiments, the kinetic parameters are determined from expressions for the species concentrations as a function of time. The concentration of the substrate or product is recorded in time after the initial fast transient and for a sufficiently long period to allow the reaction to approach equilibrium. We note in passing that, while they are less common now, progress curve experiments were widely used in the early period of enzyme kinetics.

• Transient kinetics experiments. In these experiments, reaction behaviour is tracked during the initial fast transient as the intermediate reaches the steady-state kinetics period. These experiments are more difficult to perform than either of the above two classes because they require rapid mixing and observation techniques.

• Relaxation experiments. In these experiments, an equilibrium mixture of enzyme, substrate and product is perturbed, for instance by a temperature, pressure or pH jump, and the return to equilibrium is monitored. The analysis of these experiments requires consideration of the fully reversible reaction. Moreover, relaxation experiments are relatively insensitive to mechanistic details and are thus not typically used for mechanism identification, although they can be under appropriate conditions.

Enzyme assays can be split into two groups according to their sampling method: continuous assays, where the assay gives a continuous reading of activity, and discontinuous assays, where samples are taken, the reaction stopped and then the concentration of substrates/products determined.

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Chapter 8: The Cell

8.1- The Cell

The cell is the functional basic unit of life. It was discovered by Robert Hooke and is the functional unit of all known living organisms. It is the smallest unit of life that is classified as a living thing, and is often called the building block of life. Some organisms, such as most bacteria, are unicellular (consist of a single cell). Other organisms, such as humans, are multicellular. Humans have about 100 trillion or 1014 cells; a typical cell size is 10 µm and a typical cell mass is 1 nanogram. The largest cells are about 135 µm in the anterior horn in the spinal cord while granule cells in the cerebellum, the smallest, can be some 4 µm and the longest cell can reach from the toe to the lower brain stem (Pseudounipolar cells). The largest known cells are unfertilised ostrichegg cells, which weigh 3.3 pounds.

In 1835, before the final cell theory was developed, Jan Evangelista Purkyně observed small "granules" while looking at the plant tissue through a microscope. The cell theory, first developed in 1839 by Matthias Jakob Schleiden and Theodor Schwann, states that all organisms are composed of one or more cells, that all cells come from preexisting cells, that vital functions of an organism occur within cells, and that all cells contain the hereditary information necessary for regulating cell functions and for transmitting information to the next generation of cells.

The word cell comes from the Latincellula, meaning, a small room. The descriptive term for the smallest living biological structure was coined by Robert Hooke in a book he published in 1665 when he compared the cork cells he saw through his microscope to the small rooms monks lived in

8.2- Anatomy

There are two types of cells: eukaryotic and prokaryotic. Prokaryotic cells are usually independent, while eukaryotic cells are often found in multicellular organisms. 54

1) Prokaryotic cells

Diagram of a typical prokaryotic cell

The prokaryote cell is simpler, and therefore smaller, than a eukaryote cell, lacking a nucleus and most of the other organelles of eukaryotes. There are two kinds of prokaryotes: bacteria and archaea; this share a similar structure.

Nuclear material of prokaryotic cell consists of a single chromosome that is in direct contact with cytoplasm. Here, the undefined nuclear region in the cytoplasm is called nucleoid.

A prokaryotic cell has three architectural regions:

• On the outside, flagella and pili project from the cell's surface. These are structures (not present in all prokaryotes) made of proteins that facilitate movement and communication between cells; • Enclosing the cell is the cell envelope – generally consisting of a cell wall covering a plasma membrane though some bacteria also have a further covering layer called a capsule. The envelope gives rigidity to the cell and separates the interior of the cell from its environment, serving as a protective filter. Though most prokaryotes have a cell wall, there are exceptions such as Mycoplasma (bacteria) and Thermoplasma (archaea). The cell wall consists of peptidoglycan in bacteria, and acts as an additional barrier against exterior forces. It also prevents the cell from expanding and finally bursting (cytolysis) from osmotic pressure against 55

a hypotonic environment. Some eukaryote cells (plant cells and fungi cells) also have a cell wall; • Inside the cell is the cytoplasmic region that contains the cell genome (DNA) and ribosomes and various sorts of inclusions. A prokaryotic chromosome is usually a circular molecule (an exception is that of the bacterium Borrelia burgdorferi, which causes Lyme disease). Though not forming a nucleus, the DNA is condensed in a nucleoid. Prokaryotes can carry extrachromosomal DNA elements called plasmids, which are usually circular. Plasmids enable additional functions, such as antibiotic resistance.

2) Eukaryotic cells

Diagram of a typical animal (eukaryotic) cell, showing subcellular components. Organelles: (1) nucleolus, (2) nucleus, (3) ribosome, (4) vesicle, (5) rough endoplasmic reticulum (ER), (6) Golgi apparatus, (7) Cytoskeleton, (8) smooth endoplasmic reticulum, (9) mitochondria (10) vacuole, (11) cytoplasm, (12) lysosome, (13) centrioles within centrosome

Eukaryotic cells are about 15 times wider than a typical prokaryote and can be as much as 1000 times greater in volume. The major difference between prokaryotes and eukaryotes is that eukaryotic cells contain membrane-bound compartments in which specific metabolic activities take place. Most important among these is a cell nucleus, a membrane-delineated compartment that houses the eukaryotic cell's DNA. This nucleus gives the eukaryote its name, which means "true nucleus." Other differences include:

• The plasma membrane resembles that of prokaryotes in function, with minor differences in the setup. Cell walls may or may not be present. 56

• The eukaryotic DNA is organized in one or more linear molecules, called chromosomes, which are associated with histone proteins. All chromosomal DNA is stored in the cell nucleus, separated from the cytoplasm by a membrane. Some eukaryotic organelles such as mitochondria also contain some DNA. • Many eukaryotic cells are ciliated with primary cilia. Primary cilia play important roles in chemosensation, mechanosensation, and thermosensation. Cilia may thus be "viewed as sensory cellular antennae that coordinate a large number of cellular signaling pathways, sometimes coupling the signaling to ciliary motility or alternatively to cell division and differentiation." • Eukaryotes can move using motile cilia or flagella. The flagella are more complex than those of prokaryotes.

Table 1: Comparison of features of prokaryotic and eukaryotic cells Prokaryotes Eukaryotes Typical bacteria, archaea protists, fungi, plants, animals organisms ~ 10–100 µm (sperm cells, apart from the Typical size ~ 1–10 µm tail, are smaller) nucleoid region; no Type of nucleus real nucleus with double membrane real nucleus linear molecules (chromosomes) with DNA circular (usually) histone proteins RNA-/protein- coupled in RNA-synthesis inside the nucleus synthesis cytoplasm protein synthesis in cytoplasm

Ribosomes 50S+30S 60S+40S Cytoplasmatic highly structured by endomembranes and very few structures structure a cytoskeleton flagella and cilia containing microtubules; flagella made of

Cell movement lamellipodia and filopodia containing flagellin actin one to several thousand (though some

Mitochondria none lack mitochondria)

Chloroplasts none in algae and plants single cells, colonies, higher multicellular Organization usually single cells organisms with specialized cells Binary fission Mitosis (fission or budding)

Cell division (simple division) Meiosis

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Table 2: Comparison of structures between animal and plant cells Typical animal cell Typical plant cell Organelles • Nucleus • Nucleus o Nucleolus (within o Nucleolus (within nucleus) nucleus) • Rough endoplasmic • Rough ER reticulum (ER) • Smooth ER • Smooth ER • Ribosomes • Ribosomes • Cytoskeleton • Cytoskeleton • Golgi apparatus • Golgi apparatus (dictiosomes) • Cytoplasm • Cytoplasm • Mitochondria • Mitochondria • Vesicles • Plastids and its derivatives • Lysosomes • Vacuole(s) • Centrosome • Cell wall o Centrioles

8.3- Subcellular components

The cells of eukaryotes (left) and prokaryotes (right)

All cells, whether prokaryotic or eukaryotic, have a membrane that envelops the cell, separates its interior from its environment, regulates what moves in and out (selectively permeable), and maintains the electric potential of the cell. Inside the membrane, a saltycytoplasm takes up most of the cell volume. All cells possess DNA, the hereditary material of genes, and RNA, containing the information necessary to build various proteins such as enzymes, the cell's primary machinery. There are also other kinds of biomolecules in cells. This article lists these primary components of the cell, then briefly describe their function. 58

1) Membrane

The cytoplasm of a cell is surrounded by a cell membrane or plasma membrane. The plasma membrane in plants and prokaryotes is usually covered by a cell wall. This membrane serves to separate and protect a cell from its surrounding environment and is made mostly from a double layer of lipids (hydrophobic fat- like molecules) and hydrophilicphosphorus molecules. Hence, the layer is called a phospholipid bilayer. It may also be called a fluid mosaic membrane. Embedded within this membrane is a variety of protein molecules that act as channels and pumps that move different molecules into and out of the cell. The membrane is said to be 'semi-permeable', in that it can either let a substance (molecule or ion) pass through freely, pass through to a limited extent or not pass through at all. Cell surface membranes also contain receptor proteins that allow cells to detect external signaling molecules such as hormones.

2) Cytoskeleton

Bovine Pulmonary Artery Endothelial cell: nuclei stained blue, mitochondria stained red, and F-actin, an important component in microfilaments, stained green. Cell imaged on a fluorescent microscope.

The cytoskeleton acts to organize and maintain the cell's shape; anchors organelles in place; helps during endocytosis, the uptake of external materials by a cell, and cytokinesis, the separation of daughter cells after cell division; and moves parts of the cell in processes of growth and mobility. The eukaryotic cytoskeleton is composed of microfilaments, intermediate filaments and microtubules. There is a great number of proteins associated with them, each controlling a cell's structure by directing, bundling, and aligning filaments. The prokaryotic cytoskeleton is less well-studied but is involved in the maintenance of cell shape, polarity and cytokinesis.

3) Organelles

The human body contains many different organs, such as the heart, lung, and kidney, with each organ performing a different function. Cells also have a set of "little organs," called organelles, that are adapted and/or specialized for carrying out one or more vital functions. Both eukaryotic and prokaryotic cells have organelles but organelles in eukaryotes are generally more complex and may be membrane bound. 59

There are several types of organelles in a cell. Some (such as the nucleus and golgi apparatus) are typically solitary, while others (such as mitochondria, peroxisomes and lysosomes) can be numerous (hundreds to thousands). The cytosol is the gelatinous fluid that fills the cell and surrounds the organelles. a) Cell nucleus – eukaryotes only - a cell's information center

The cell nucleus is the most conspicuous organelle found in a eukaryotic cell. It houses the cell's chromosomes, and is the place where almost all DNA replication and RNA synthesis (transcription) occur. The nucleus is spherical and separated from the cytoplasm by a double membrane called the nuclear envelope. The nuclear envelope isolates and protects a cell's DNA from various molecules that could accidentally damage its structure or interfere with its processing. During processing, DNA is transcribed, or copied into a special RNA, called messenger RNA (mRNA). This mRNA is then transported out of the nucleus, where it is translated into a specific protein molecule. The nucleolus is a specialized region within the nucleus where ribosome subunits are assembled. In prokaryotes, DNA processing takes place in the cytoplasm. b) Mitochondria and Chloroplasts – eukaryotes only - the power generators

Mitochondria are self-replicating organelles that occur in various numbers, shapes, and sizes in the cytoplasm of all eukaryotic cells. Mitochondria play a critical role in generating energy in the eukaryotic cell. Mitochondria generate the cell's energy by oxidative phosphorylation, using oxygen to release energy stored in cellular nutrients (typically pertaining to glucose) to generate ATP. Mitochondria multiply by splitting in two. Respiration occurs in the cell mitochondria. Organelles that are modified chloroplasts are broadly called plastids, and are involved in energy storage through photosynthesis, which uses solar energy to generate carbohydrates and oxygen from carbon dioxide and water.[citation needed]

Mitochondria and chloroplasts each contain their own genome, which is separate and distinct from the nuclear genome of a cell. Both organelles contain this DNA in circular plasmids, much like prokaryotic cells, strongly supporting the evolutionary theory of endosymbiosis; since these organelles contain their own genomes and have other similarities to prokaryotes, they are thought to 60 have developed through a symbiotic relationship after being engulfed by a primitive cell.[citation needed] c) Endoplasmic reticulum – eukaryotes only

The endoplasmic reticulum (ER) is the transport network for molecules targeted for certain modifications and specific destinations, as compared to molecules that float freely in the cytoplasm. The ER has two forms: the rough ER, which has ribosomes on its surface and secretes proteins into the cytoplasm, and the smooth ER, which lacks them. Smooth ER plays a role in calcium sequestration and release. d) Golgi apparatus – eukaryotes only

The primary function of the Golgi apparatus is to process and package the macromolecules such as proteins and lipids that are synthesized by the cell. It is particularly important in the processing of proteins for secretion. The Golgi apparatus forms a part of the endomembrane system of eukaryotic cells. Vesicles that enter the Golgi apparatus are processed in a cis to trans direction, meaning they coalesce on the cis side of the apparatus and after processing pinch off on the opposite (trans) side to form a new vesicle in the animal cell.[citation needed] e) Ribosomes

The ribosome is a large complex of RNA and protein molecules. They each consist of two subunits, and act as an assembly line where RNA from the nucleus is used to synthesise proteins from amino acids. Ribosomes can be found either floating freely or bound to a membrane (the rough endoplasmatic reticulum in eukaryotes, or the cell membrane in prokaryotes). f) Lysosomes and Peroxisomes – eukaryotes only

Lysosomes contain digestive enzymes (acid hydrolases). They digest excess or worn-out organelles, food particles, and engulfed viruses or bacteria. Peroxisomes have enzymes that rid the cell of toxic peroxides. The cell could not house these destructive enzymes if they were not contained in a membrane- bound system. These organelles are often called a "suicide bag" because of their ability to detonate and destroy the cell.[citation needed] g) Centrosome – the cytoskeleton organiser

The centrosome produces the microtubules of a cell – a key component of the cytoskeleton. It directs the transport through the ER and the Golgi apparatus. 61

Centrosomes are composed of two centrioles, which separate during cell division and help in the formation of the mitotic spindle. A single centrosome is present in the animal cells. They are also found in some fungi and algae cells.[citation needed] h)Vacuoles

Vacuoles store food and waste. Some vacuoles store extra water. They are often described as liquid filled space and are surrounded by a membrane. Some cells, most notably Amoeba, have contractile vacuoles, which can pump water out of the cell if there is too much water. The vacuoles of eukaryotic cells are usually larger in those of plants than animals.

8.4- Structures outside the cell wall

1) Capsule

A gelatinous capsule is present in some bacteria outside the cell wall. The capsule may be polysaccharide as in pneumococci, meningococci or polypeptide as Bacillus anthracis or hyaluronic acid as in streptococci.[citation needed] Capsules are not marked by ordinary stain and can be detected by special stain. The capsule is antigenic. The capsule has antiphagocytic function so it determines the virulence of many bacteria. It also plays a role in attachment of the organism to mucous membranes.[citation needed]

2) Flagella

Flagella are the organelles of cellular mobility. They arise from cytoplasm and extrude through the cell wall. They are long and thick thread-like appendages, protein in nature. Are most commonly found in bacteria cells but are found in animal cells as well.

3) Fimbriae (pili)

They are short and thin hair like filaments, formed of protein called pilin (antigenic). Fimbriae are responsible for attachment of bacteria to specific receptors of human cell (adherence). There are special types of pili called (sex pili) involved in conjunction.[citation needed] 62

8.5- Growth and metabolism

Between successive cell divisions, cells grow through the functioning of cellular metabolism. Cell metabolism is the process by which individual cells process nutrient molecules. Metabolism has two distinct divisions: catabolism, in which the cell breaks down complex molecules to produce energy and reducing power, and anabolism, in which the cell uses energy and reducing power to construct complex molecules and perform other biological functions. Complex sugars consumed by the organism can be broken down into a less chemically complex sugar molecule called glucose. Once inside the cell, glucose is broken down to make adenosine triphosphate (ATP), a form of energy, through two different pathways.

The first pathway, glycolysis, requires no oxygen and is referred to as anaerobic metabolism. Each reaction is designed to produce some hydrogen ions that can then be used to make energy packets (ATP). In prokaryotes, glycolysis is the only method used for converting energy.

The second pathway, called the Krebs cycle, or citric acid cycle, occurs inside the mitochondria and can generate enough ATP to run all the cell functions.

Within the nucleus of the cell (light blue), genes (DNA, dark blue) are transcribed into RNA. This RNA is then subject to post- transcriptional modification and control, resulting in a mature mRNA (red) that is then transported out of the nucleus and into the An overview of protein synthesis. cytoplasm (peach), where it undergoes translation into a protein. mRNA is translated by ribosomes (purple) that match the three-base codons of the mRNA to the three-base anti-codons of the appropriate tRNA. Newly synthesized proteins (black) are often further modified, such as by binding to an effector molecule (orange), to become fully active.

63

1) Cell Division

Cell division involves a single cell (called a mother cell) dividing into two daughter cells. This leads to growth in multicellular organisms (the growth of tissue) and to procreation (vegetative reproduction) in unicellular organisms.

Prokaryotic cells divide by binary fission. Eukaryotic cells usually undergo a process of nuclear division, called mitosis, followed by division of the cell, called cytokinesis. A diploid cell may also undergo meiosis to produce haploid cells, usually four. Haploid cells serve as gametes in multicellular organisms, fusing to form new diploid cells.

DNA replication, or the process of duplicating a cell's genome, is required every time a cell divides. Replication, like all cellular activities, requires specialized proteins for carrying out the job.

64

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