TECHNI QUES I N CELL BI OLOGY

( A LABORATORY MANUAL)

A NI L K UMAR, DI NESH PA NDEY SONU AMBWA NI G.K. GAR G

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TECHNIQUES IN

CELL BIOLOGY

ANIL KUMAR DINESH PANDEY SONU AMBWANI G.K. GARG

DEPT. OF MOLECULAR BIOLOGY & GENETIC ENGINEERING COLLEGE OF BASIC SCIENCES & HUMANITIES G.B. P.U.A. & T., PANTNAGAR-263 145 U.S. NAGAR, (INDIA)

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CONTENTS I. MICROBIAL TECHNOLOGY a. Maintenance of aseptic conditions and handling during microbial culture practices. b. Techniques to obtain pure culture of microorganism from mixed culture c. Inoculation of pure culture by streak plate and pour plate methods d. To perform simple/ direct staining and indirect/ negative staining and Gram’s staining of pure culture isolates e. Identification of unknown bacterial cultures by biochemical tests II. CELL/TISSUE CULTURE TECHNOLOGY a. Plant cell culture : i. Seed culture of Brassica/ Tobacco seeds ii. Callus culture or shoot regeneration from hypocotyls of Brassica or Tobacco seedlings b. Animal Cell culture : i. To culture human fibroblast cells III. BIOPROCESS ENGINEERING & ENVIRONMENTAL BIOTECHNOLOGY a. Immobilization of yeast cell by formation of agar beads b. Immobilization of yeast cell by formation of sodium alginate beads. c. Demonstration of fermenter and glucose fermentation process d. Bioremediation of heavy metals from industrial effluents IV. IMMUNOTECHNOLOGY a. To perform Haemagglutination test to determine blood groups. b. To detect pregnancy by the presence of hCG in urine by latex particles agglutination inhibition test c. Demonstration of gel diffusion/ouchterlony d. Widal slide test for the detection of Salmonella antibodies in human serum e. Lateral flow Immunochomatography Test Strip for the Qualitative Detection of Treponemal Antibodies in Syphilis patients f. Extraction of total proteins from wheat seeds g. To perform SDS polyacrylamide gel electrophoresis of the protein samples h. Western blotting

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V. DNA TECHNOLOGY a. Isolation of genomic DNA from plant , animal and plant cells b. Isolation of plasmid DNA c. Quantitation of genomic and plasmid DNA d. Restriction digestion

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TECHNIQUES IN CELL BIOLOGY Cells are small and complex. It is hard to see their structure, hard to discover their molecular composition, and harder still to find out how their various components function. What we can learn about cells depends on the tools at our disposal, and major advances have frequently occurred from the introduction of new techniques. To understand contemporary cell biology, therefore, it is necessary to know something of its methods/techniques. Thus, the course Techniques in cell biology has been introduced for the students of Biotechnology and related discipline. Cell biology is a specialized branch or biological sciences dealing with the structure, function and uses of microscopic organisms/cells, such as prokaryotic cells, Viruses and bacteria, Eukaryotic cells; actinomycetes, fungi, algae, protozoa, plant and animal cells. This course has been designed to acquaint students with cell culture techniques of microbes, such as microorganisms, plant and animal cells and to apprise them with the importance of microbes in our daily life and the vital roles they play in the ecology of life on earth. This course covers a wide spectrum of exercise such as microbiological techniques, Bacterial culture, plant tissue culture, Animal cell culture and immunological techniques. There is, as in all sciences, a need for basic equipment (instruments, tools, glassware and miscellaneous items) much of which can be found in any cell biology laboratory. Following is a list of basic requirements which a cell biologist/microbiologist require in the laboratory of microscopic examination, isolation or culturing and identification of a microbe as well as to study its structure, function and application. A. Instruments and Applications i. ii. iii. iv. v. vi. vii. viii. ix. Microscopes and immersion oil Photomicrographic camera Laminar flow safety hood Microbiological inoculation chamber Autoclave Pressure Cooker Incubators Refrigerator Oven

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x. xi. xii. xiii. xiv. xv. xvi. xvii. xviii. xix. xx. xxi. xxii. xxiii. xxiv. B. Tools i. ii. iii. iv. v. vi. vii. viii. ix. x. C.

Water bath Bunsen burner or spirit lamp Hot plate/heater Centrifuge pH meter Spectrophotometer Camera Lucida Quebec colony counter Balance Homogenizers for grinding of specimens constant temperature water bath Tripod with asbestos mat Haemocytometer Counter Stop watch

Transfer needle Inoculating loop Dissecting needles Forceps Scissors Ocular micrometer Stage micrometer Burette set up Thermometers Scale

Glass wares

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i. ii. iii. iv. v. vi. vii. viii. ix. x. xi. xii. xiii. xiv. D.

Petri dishes Conical flasks Culture tubes without screw caps Screw capped tubes for media Beakers Funnels Graduate cylinders Graduate pipettes Capillary pipettes Dropper bottles for staining reagents Screw-capped bottles for stock reagents Glass microscopic slides Depression (concave) slides Glass cover slips

Miscellaneous i. ii. iii. iv. v. vi. vii. viii. ix. x. xi. Culture media, Agar, peptone, Beef extract, yeast extract, plant and animal cell culture media Test tube rack Cotton plugs Wooden sticks, with and without cotton scabs Rubber bulk for pipettes Glass marking pen Tube for labels for sealing plates Stains and staining apparatuses Parafilms Aluminum foil Butter paper/bags

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xii. xiii. xiv. xv. xvi. xvii. xviii. xix. xx. xxi. xxii. xxiii. xxiv. xxv. xxvi. xxvii. xxviii. xxix. xxx.

Nail polish/wax for sealing microscopic mounts Disinfectant (savlon, phenyl etc.) Discard containers Facilities for disinfecting hands Distilled water Syringes and needless Bottling paper and lens tissue Tissue paper Rubber bands Muslin/cheese cloth Pipette can (tin or glass) Petri dish can Cotton or gauze masks Test tube cap Drawing pencil Match box Surgical gloves or rubber gloves Mask Apron

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USEFUL DATA OF CELL BIOLOGY Number of cells present in Human 1 cm3 volume contains Number of lymphocyte in Human Number of chromosome Size of DNA present in a chromosome DNA content pgm/Cell 7. Number of base pair in a DNA 8. 1 K. base pair Kilodt. 9. 1 x 10-12 gm or/ pgm DNA kbp 10. 1 kb pair can coded Amino acid 11. 260 nm absorbance give rise DNA 12. 1 µ g/ml DNA have phosphate 13. RNA content pgm/Cell 14. 1 kbp mRNA will coded 15. 1 AA 16. 10 kdt protein coded by pair 1. 2. 3. 4. 5. 6. DEFINITIONS 1. Sterilization Means complete destruction of all forms of life various method of sterilization are as follows: Day heat: One hr at 160°C to 180°C is hot air sterilizer Use – Glass ware, surgical instruments etc. Intermittent: 30 minutes in flowing steam 100°C for 3 consecutive days. Use – Media tissue, liquids, solids. Moist heat: 15-30 minutes at 121°C at 15 psi pressure Use – Glassware, media Filtration: Use – media, liquid samples 1014 Cells 109 Cells 1012 46 (44+2) 5 mm 6.4 5.8 x 109 660 9.1 x 105 333 50 µ g/ml 3.0 10 3.4 kdt 111 dt 270 base µ g

Radiations: Use – Surface sterilization

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Disinfection Means the destruction of pathogenic microorganism.

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Disinfectant Is the chemical substance which is used to destroy (kill) disease producing agent.

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Antiseptic Is an agent that opposes sepsis i.e. stops growth of microorganism.

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Antibiotic Are the substance produced by one microorganism to kill the other microorganism.

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Sanitizer Is an agent that reduces the microbial population to safe level.

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SUGGESTIONS & REGULATIONS General Suggestions and Information 1. 2. Wear a coat, smock, or apron to protect your clothing. Before each laboratory period, read over the exercises to be done and plan your work carefully, known how each exercise is to be done and what basic principles it is intended to convey. Each laboratory meeting will begin with a short discussion and instruction period. Don’t begin work until you have received your instructions. Ask questions when you do not understand the method and purpose of any experiment. Good laboratory technique depends primarily on knowing what you are to do. Properly record all observations at the time they are made laboratory examinations will cover both the information given by your laboratory instructor and that contained in the manual, as well as you own observations and deductions. Answer the questions following each exercise on the report sheet.

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Laboratory Regulations 1. 2. 3. Sponge off the top of your laboratory desk with germicide solution to both the beginning and the close of each laboratory period. Keep your desk free of non-essential materials at the time and at the end of the period leave it free of all materials and equipment. Place all solid waste material in the waste cans and all dirty glassware in the trays. Your laboratory grade will depend to some extend on your techniques, orderliness, and cleanliness. Because many of the microorganisms with which you will be working are potentially pathogenic it is imperative to develop aseptic techniques in handling and transferring them. Avoid any hand to mouth operation such as smoking, eating, or moistening labels with the tongue. Report immediately all accidents such as cuts, burns, or spilled cultures to your instructor, take all precautions to avoid such accidents.

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Make yourself familiar with the equipments incubators, centrifuges and microscopes etc. If you have problems, approach the course director or course instructors. Good laboratory work habits will help you and the course a ground success. Follow the following guide lines very strictly. These will protect you and you experiments.

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1. 2. 3. 4. 5. 6.

No eating, drinking or smoking in the lab. No storage of food in the lab. No mouth pipetting. Use appropriate pipetting aids that are available. Wear gloves for certain experiments. Work cleanly and in an organized manner. Wipe tissue culture hood bench with 70% ethanol. Most important, label every thing you use with your initial, date and name of the reagent, buffer or medium.

Procedures for Handling Cells and Medium Use sterile glassware and pipettes. When you open a glass bottle, before and after use flame the mouth in a Bunsen flame. Flame glass pipettes. Use bulbs to control pipettes. DO NOT MOUTH PIPETTE. This is to protect both you and the cells from contamination. NEVER INSERT a pipette which may have contacted cells back into your stock bottle of medium. Use of fresh pipette. Thus, to change the medium on a dish, the procedure is as follows: Pre-warm the medium and serum to 37°C in water bath. Wipe bottles with filter paper and transfer to the tissue culture hood (which you should have been wiped with ethanol, equipped with pipettes, beakers for waste etc.). Open bottles, flame tops, replace caps loosely so that they won’t fall off. Transfer desired quantity of serum to the medium or mix in separate (sterile) container with the help of a sterile pipette. Use rubber bulb for pipetting and flame the pipette before use. Move dish(es) of cells to the hood. Using sterile, flamed, but not plugged, Pasteur pipette, aspirate the medium flame the Pasteur pipette between dishes. Use a separate pipette for different cells, Don’t do too many dishes at a time.

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Using a sterile flamed pipette, transfer desired amount of medium and serum to the dish(es). Don’t reuse the pipette. Return dishes to incubator without shaking to avoid spill over of medium. Reflame tops of bottles and close tightly. Close pipette cans. Work only in designated tissue culture area. Wipe surfaces with ethanol before starting. Wipe bottles dry (e.g. if they have been standing in a water bath before moving them into tissue culture hood. Discard used medium, time/expired and especially contaminated when you open a new bottle of medium or serum, write the date on it and indicate how much has been removed. If you add anything to it, indicate this on the bottle. If there is only a small amount left in a bottle, discard it. After you have finished, remove your belongings; put them away or discard properly. Wipe the working surfaces with ethanol.

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Cleaning of Glassware It is extremely important to use clean glassware for culture purpose. The following procedure ensures clean surface for culture flasks, tubes, Petri dishes and other glassware. 1. Immerge all glassware in 2-5% detergent (eg. Extran/Merck) immediately following use for 20-24 hr. Heating considerably accelerate the cleaning process. To remove resistant dirt soak the glassware in a solution containing 100 ml of potassium dichromate in a gallon of concentrated sulfuric acid for 4 h. Rinse thoroughly in tap water. Rinse in hot water. Soak in distilled water for at least 3 h. Rinse in distilled water and dry in a hot air oven.

2.

3. 4. 5. 6.

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Sterilization 1. Sterilize surgical instruments, glassware, Petri dishes, pipettes, beakers etc. and aluminum foil after wrapping in aluminum foil for 2 h in a hot air oven at 200°C. Sterilize culture media, distilled water and other stable mixture in glass containers with cotton wool plug and caped with aluminum foil or caps in an autoclave at a pressure of 15 psi for 15 min. Solutions of unstable substances (eg. IAA) can be filter sterilized using appropriate Millipore filters and added to the culture medium after autoclaving. All sterile operations should be carried out in a Laminar Flow cabined to sterile room. The cabinets are available in different sizes. They are movable and eliminate the necessity of a separate room. Fins on the cabinets are often operated continuously and pre-filters replaced or cleaned bi-monthly. Alternatively a less expensive inoculation hood fitted with UV lamps can also be used.

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Sterilization and lab ware/material preparation: Now a days, a lot of tissue culture plastic ware is available as disposable pre-sterilized single use materials. In spite of these laboratory level, sterilization needs to be done for glassware, pipettes and for the purpose of decontamination of infected tissue culture use is listed in Table-I. Table-I Sterilization methods Method Time and Use Model of action Disadvantage temperature
Dry heat One hour at Glassware, 160°C to 180°C in Surgical, a hot air sterilizer Instrument etc. Thirty minutes in Media, flowing steam tissues, (100°C) for 3 Liquids, consecutive days Solids Oxidizes bacterial proteins Cannot be used and nucleic acid for media sterilization Heat shock the material for Tedious, the first 30 minutes and if consuming spores are present and they will germinate and the vegetative cell will be destroyed at the next cycle. Coagulates proteins microorganisms time

Intermittent

Moist heat

Fifteen to thirty Glassware minutes at 121°C media at 15 psi Ambient Media liquids samples

Filtration

of Some nutrients are not stable to these conditions, eg. lactose. Filters uniform pore from Pores permit virus and mycoplasma pore size of upto 0.22 µ . of flow through

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Toxic gases Ethylene sterilization under pressure

Radiation

oxide Large slight surface area pieces of particularly . a) Ultraviolet Media (UV) liquids b) Ionizing Y or solids X rays) pharmaceu ticals and surface sterilization

Lethal gas kills all life Flammable, forms; equipment plastic toxic ware.

Causes genetic mutations interferes with metabolism and respiration, and leads to death.

Does not penetrate glassware easily; requires access to a core or cathode ray tube, mutation inducing.

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MICROBIOLOGICAL TECHNIQUES INTRODUCTION Microorganisms are all round us and according to Louis Pasteur “Life would not along remain possible in the absence of microbes”. However, some micro-organisms cause disease in humans, other animals and plants. They were friends as well as adversaries. As co-habitants on the planet earth, micro-organisms plays significant role in the lives of other organisms. They are present every where in air, in water and soil. Close interaction between microbes and humans existed from time immemorial. Man was quick to tame them and use them to his advantage, for improving agriculture productivity, in controlling food shortage and producing safe and nutritious foods, to check diseases, as supplements to the depleting energy resources, for environmental explorations and of course as powerful tools of research to unravel the mysteries of the living organisms. Microorganisms were first discovered by Antony Van Leeuwenhock who called them “animalcules”. Louis Pasteur laid microbes in the spoilage in wine. Robert Koch was the first one to show that diseases are produced by micro-organisms. Soon a host of discoveries followed and several industries came up based on them yeast is used in the leavening of bread and in the manufacture of wine and beer. It is also used for fermenting sugar into alcohol, as well in the production of organic acids, amino acids, enzymes and vitamins. The discovery of penicillin by Alexander Flaming revolutionized modern medicine. The discovery of a host of antibiotic like streptomycin, tetracycline, erythromycin, chloramphenicol etc. soon followed which are now widely used for the control of diseases. Microbial enzymes are used worldwide for industrial purposes. This, in the closing yeast of this century, when the sciences of microbiology has seemingly reaches its pinnacle, it ourselves where has all this led us to? Has it resulted in a better world? A less hungry world? Have we controlled all diseases? Have we been able to improve the quality of life? Has it resulted in a cleaner and healthier planet? Or to the end of the world and to conquering new planets to inhab? Yes, a little of all this o doubt, and just enough perhaps to open new areas of investigations. Techniques now being available for manipulating living organisms in a genetic and biochemical sense have immense potential. The possibilities now before us for the construction of bacterial strains finely tuned to the tasks of human welfare give us a clue to the further possibilities. In this section we shall review briefly some of the principle microbiological techniques used to study micro-organisms their culture, aseptically handling, requirement for growth. Isolation, identification characterization preservation, mutant study and basic bacterial and phage genetics.

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Introduction to Microscopy Microscope: Microscope enables us to see microorganisms and their structure otherwise invisible to the naked eye. Microscopes are of two type: (A) Light Microscopy In which magnification is obtained by a system of optical lenses using light waves. (B) Electron Microscopy It uses a beam of electron in place of light to produce the image. Specimen examined by either transmission or scanning electron microscopy. Resolving power The ability to distinguish two adjacent points as distinct and separate. Numerical aperture The angle θ subtended by the optical axis and the outermost rays still covered by the objective is the measure of the aperture of the objective. N.A. + n sine θ Maximum N.A. for a dry objective is less than 1 oil immersion objective lens have an NA value of slightly greater than 1. Limit or Resolution The limit of resolution is the smallest distance by which two objects can be separated and still be distinguishable as two separate objects:
d= λ 2N A

TYPES OF MICROSCOPY BRIGHT FIELD MICROSCOPY In bright field microscopy the microscopic field is brightly lighted and the microorganisms appear dark because they absorb some of the light.

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DARK FIELD MICROSCOPY The effect produced by the dark field technique is that of a dark back ground against which object are brilliantly illuminated. PHASE CONTRAST MICROSCOPY Phase contrast microscopy is exactly valuable for studying living unstained cells and is widely used in applied and theoretical biological studies.

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LIGHT MICROSCOPY PRINCIPLES AND APPLICATIONS INTRODUCTION Microscope has been directly involved in emergence of several fields in science. The science of microbiology being with the latter of Antony von Leeuwenhock in 1967 to the Royal Society in England describing the minute organisms he observed with the help of magnifying lenses. Human eye has a limit within which only it can see two adjacent points separately. This power is known as the resolving power of the 0.2 mm. Most of the micro-organisms and cell components are very small and of micron size and can’t be seen by naked eye. So science was developing very slowly before the invention of microscopes. Several milestones were laid in the development of light microscopy and several new doctrines were developed with the help of it, the first major one being the cell theory put forward by Schleiden and Schwaan in 1938 which resulted in the formal birth of cell biology. Microscopes have been greatly helpful in identifying the course of several diseases and Louis pasture and Robert Koch were able to suggest the causes and remedies for different animal and human diseases. But the development of microscopy from the infancy stage of simple use of lenses to advanced equipments like video enhancement microscopy, florescence microscopy, etc. take a long time due to the lack of knowledge about the character and properties of light. The discovery of electromagnetic wave nature of light has resulted in sudden jump in the development of microscopy. This enabled scientists to develop new of microscopy. This enabled scientists to develop new instruments like electron microscope which is having very high magnification power to the tune of 2,00,000 times and very high resolving power of 1 nm where as for light microscope these powers are 1500 times and 0.2 µ m respectively. MICROSCOPY These of microscope to study the microorganisms, which are invisible to naked eye since their size is very small is known as microscopy. We are able to see an object when light from a source falls in the object and it reflects the light and when this light falls in the retina of our eye. But this is possible with the help of the lens we are having in the eye which focuses the rays in retina. The microscope is an optical system for magnification and illumination and it contain a lens or a series of lenses with a source of illumination. PRINCIPLES OF LIGHT MICROSCOPY LIGHT To know how microscope is working we must first know about the characteristics of light. Light is a form of energy and it has an electromagnetic wave character. It follows a straight line path but undergo deviation when it passes from a medium to another medium which is known as Refraction. It can also be reflected with a mirror and when an object is put on the path of

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light you can see dark and lighter bands is put the image of the object. This is due to the curvature of light rays and the phenomenon is known as diffraction. When 2 waves of light of different wavelength reach a point they interfere with each other and result in a new wave which will be according to the phase of the waves and this phenomenon is known as interference. Visible light when passes through a prism it get divided into light of different wavelengths and is known as dispersion. The above said qualities of light have a definite role to do in microscopy. Light has a fixed speed in a medium, this also affect the resoling power of a microscope. HOW IMAGES ARE FORMED Lenses are devices which cause a beam of light to coverage or diverge as passing through it (or a transparent medium bounded by atleast one spherical surface). According to nature of the surface of lenses there are different types of lenses like: (i) (iv) Biconvex Biconcave (ii) (v) Planocovex Planoconcave (iii) (vi) Concavoconvex Convexo concave

The image formed by a lens or a group of lenses depend upon the focal length of the lens, distance of object form the lens numerical aperture of the lens and refractive index of the lens. MAGNIFICATION Magnification power of a lens is its ability to increase the size of the image of the object. Generally it is measured as the ratio of size of image to the size of the object. Before going into details of magnification we must know two properties of the lens the focal length of power and refractive index. Focal length of a lens is the distance between optic centre and principal focus (the point of convergence of divergence of rays). The reciprocal of focal length in meters is known as power of the lens is expressed in diopters. As focal length increases the power decreases and vice-versa. Refractive Index (RI) of a lens is the ratio of the speed of light in vacuum to its speed in the lens medium. This ratio determines how much the light rays are bent while it enter and exit the lens medium from or into air. Magnification can be defined in another way also as the ratio of angle subtended at the eye by the image formed at the least distance of distinct vision to the angle subtended at the eye by the object kept at the same distance. The total magnification of a microscope is found by multiplying the magnifying power of the objective by that of the eyepiece. The lens system near to the objective lens, called objective produces a magnified real image which is further magnified by the eyepiece. The greatest useful magnification of a microscope is that which makes the smallest visible objects clearly visible. Theoretically it is possible to magnify the objects infinitely using more number of lenses.

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b)

In case of compound microscope = Magnifying power of eyepiece X power of objective

The total magnification Magnifying

NUMERICAL APERTURE AND RESOLVING POWER Resolving power is the ability to distinguish two adjacent objects as separate and distinct images rather than a single blurred image. The resolution power of human eye is 0.2 mm which means eye can not distinguish between 2 points which are distant by less than 0.2 mm. The resolution power is influenced by several factors. 1. Wavelength of the light used for illumination

Resolving power is inversely proportional to the wavelength of light used. If you use a light of smaller wavelength or higher frequency higher resolution can be obtained. 2. Numerical Aperture

Resolution power is directly proportional to the numerical aperture. Numerical aperture is defined as the function of the effective diameter of the objective lens in relation to its focal length and the light bending power or refractive index of the medium between the specimen and the objective. This property of an objective helps in resolving fine details. The higher the entire value of NA of optical system including condenser and objective lens, the greater is the resolving power. Numerical aperture also decides certain other things like light transmission i.e. the amount of light passed through an objective lens. It also decides working distance. As the N.A. increases working distance decreases. The flatness of the field of view i.e. uniform brightness and depth of field is also inversely related to N.A. You will face certain problems when high power is used since the refractive index of air is different to that of lens medium a lot of light rays are bent and many rays reflected from the specimen are refracted at a higher angle and they completely miss the objective. By interposing immersion oil which is having refractive index nearly same as glass we can decrease refraction and can give more resolution power and a clear image. Since for a dry lens n = 1 and since the absolute maximum value that θ can have is 90° the maximum value of NA must be: 1 x Sine 90 = NA Hence all objectives with an aperture greater than 1 must be of oil immersion type and greater the refractive index of the medium used for immersion the large the aperture can be.

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Resolving power is given by the formula
R sovling Pow e er = D eter iam of sm allest structure N erical aperture um visible

So the maximum resolution power possible by light microscopy is 0.22 µ m with oil immersion objective.

WORKING DISTANCE It is the distance between objective lens and the object. Focal length and working distance are directly related i.e. as focal length decreases or power increase the working distance also decreases and vice-versa. It is possible to use the oil immersion since the working distance is small due to very small focal length. Generally in compound microscopes 3 types of objectives are provided low power (10 x) high power (40 x) and 100 x oil immersion type.

HOW SPECIMEN’S DETAILS ARE VISIBLE In case of plant tissues just by taking a thin section of tissue and observing under microscope it is possible to see the cells. A thin section is essential to allow light to pass through the specimen. For getting more contrast to the specimen stains are used. According to the nature of the specimen different types of stains are used either they will enter the specimen and stain the cells completely or differentially or stain the surrounding and the cells remain colourless known as indirect staining. Now a days fluorescent chemicals are added as stain so that they will bind to cell and emit fluorescence which can be microscope effective staining and preparation of specimens are very important. Complex staining methods are there where tissues are treated with different stains for fixed time interval. This methods give best contrast e.g. Gram staining. TERMS IN MICROSCOPY ACHROMATIC A lens which brings light from 2 parts of the spectrum (red and blue wavelengths) to the same focus thus reducing chromatic aberration. It is the most common type used in microscope. If you use an achromatic with white light, colour fringes may appear on the margins (outer) of the image because this objective can’t bring all the wavelengths to an acceptable focus range. If monochromatic light is used as in phase contrast microscopy the image will be much better and sharper.

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BARREL FOCUS A microscope in which the body tube (barrel) moves and the stage is stationary during focusing. BRIGHT FIELD ILLUMINATION White light illuminates a transparent or translucent specimen that is either naturally colored or stained and which appears dark against a bright or white background. BODY TUBE LENGTH The distance between the inserted position of the objective and the top of the body tube-usually 160 mm. If objective and body length are mismatched it will lead to spherical aberration. COAXIAL Focusing arrangement where the find and coarse focus knobs and mounted together on a common axis. CONDENSER (SUBSTAGE) / ABBE CONDENSER It provides an even cone of light that illuminates the specimen. Light from the condenser coverage’s on the specimen, passes through it and diverges from an inverted illumination cone that is captured by the objective lens. Abbe condenser is most commonly used. Numerical aperture of condenser should be equal or more than the NA of the highest objective i.e. 1.25 to 1.32 for a 100 x objective. The resolving power of the optical systemcondenser, objective, ocular lens-is limited by the lowest NA value of its individual components. CHROMATIC ABERRATION Failure of a lens to bring light of different wavelengths to a common focus. DARK FIELD ILLUMINATION An optical technique in which the specimen is seen as a bright objective against a dark background.

DIOPTER ADJUSTMENT Adjustment ring on the eyepiece that find positions eye lens element to accommodate visual acuity, bringing an object into sharp focus, will not accommodate astigmatism. DIN

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Abbreviation for deutsche Industries Norman, an industry standard for optics, assuring you a high quality lens system. EYEPIECE IDAPHRAGM The round field of view that is seen when looking in a microscope whatever is placed on the diaphragm of eyepiece will be superimposed on and in focus with the specimen image. FIELD OF VIEW The area visible through the eye piece when the microscope is in focus. FLAT FILE OPTICS Optically corrected to eliminate field curvature. Flat field optics are particularly suitable for examination of photomicrography of large viewing areas. The prefix plain or plano is used before the name of the objective lens with its characteristic. IMMERSION OBJECTIVE It is used to achieve high magnification and high resolving power. The most commonly used is oil when NA of objective exceeds 1.0. It is placed on the cover slip. Only if the condenser and objective are oiled to slide you will achieve full N.A. IRIS DIAPHRAGM Leaf diaphragm mounted beneath or between within the condenser controls the size of illumination cone. KOEHILER ILLUMINATION The technique provides for a uniformly illuminated field from nonuniform light sources such as coiled filament lamps. MICROMETER DISC Glass disc with scale or grid mounted to the eye piece diaphragm and used for quantitative measurement of object. PLANOCONCAVE MIRROR Usually two sided (50 mm diameter) with one side flat and the other curved. Always use the plane surface with substage condenser. Use concave surface for very low NA objective when no condenser is used.

PARCENTERED

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All the elements of the optical system are aligned on a single axis minimizing aberration. PERFOCALITY The property of microscope system when the subject stays in focus when the objective lenses are changed. PLANACHROMAT An achromat lens corrected for flatness of field. PHASE CONTRAST MICROSCOPY Optical techniques of revealing the structural features of microscopic transparent objects whose varying but invisible differences in thickness result in varying difference in the phase of transmitted light. This phase differences are converted to visible intensity differences when part of the transmitted light has its optical path changed by about 1/3rd wavelength. POLARIZER A transparent material which absorbs all vibrations of light passing through it except those in a single plane. RACK AND PINION The system gears used for raising or lowering the stage or barrel when focusing. RETRACTILE OBJECTIVE (XR) Spring loaded so that minimal damage is done to microscopic slides when objective is ranked down too far. Designated “XR” for higher magnification, low working distance objectives. SLIP (SAFETY) CLUTCH Mechanism built into the focus system that prevents gear stripping if the coarse focus is forcibly “ranked down” beyond its normal stop. SPHERICAL ABERRATION Failure of a lens system to image the central and peripheral rays at the same focal point. STAGE FOCUS A microscope style where the stage moves and the body tube is stationary during focusing. ZOOM LENS

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It is a lens system that provided for variable magnification capacity. The object stays in focus through the full magnification range. SIMPLE AND COMPOUND MICROSCOPES AND THEIR PARTS Simple microscope The hand lens or magnifying lens falls in this category and operates upon this principle. The image of an object is projected and enlarged but not inverted. The object should be placed at any point within the focus. We can observe only the gross from of the cells with this lens. Compound microscope The magnification of compound microscope is achieved in 2 stages. A real image of the object is first formed by the objective and an image of this image is formed by the eyepiece which is virtual and inverted and so cannot be used to be real under conditions of projection. COMPONENTS OF MICROSCOPE Stand It is mechanical frame to carry to optical system of instrument and is divided into 2 parts foot and arm. Foot is horseshoe shaped and has 3 contact pads on the underside to give a stable support as surfaces which might not be truly flat and sufficient to give balance to microscope in inclined portion. Arms carry three optical units and the focusing system. BODY TUBE On this the eye piece and the objectives are mounted and the distance between them can be adjusted. Coarse adjustment This is used to focus the image by rack and pinion. Fine adjustment After focusing with a screw and nut mechanism fine adjustment with about 70 threads to inch. The result is small movement of objective by the turn of the knob. Objective changer This is a rotating nosepiece carrying upto 4 objectives with an indexing spring mechanism to ensure its position in line with that of microscope.

Substage

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To hold the condenser. Mirror Double sided mirrors with one side plain and other concave is used to provide illumination. If concave is used due to desirable convergence accurate focusing of light on the specimen will not occur. Stage It is square or rectangular plate of great rigidity. It is usually treated with reagent resisting finish. Spring clips are provided to keep the specimen in firm contact to the stage surface. By rack and pinior it can be moved slowly go get every part in the field of view. It can also be removed to view Petri plates. Sales and verniers Two vernies and scale are used on running sough north and other east, west and north and other east-west and can be used as finding device. LENSES It is of two types objectives and eyepiece. Lenses has optical centre which is on the principal axis on the lens and all the rays passing through this point emerge without deviation. In a simple microscope with a single biconvex lens, 200-300 times magnification is possible. Objectives They are lenses near to object and produce inverted real image. They are of different types such as follows. a) Achromatic lenses

This is used to nullify spherical and chromatic aberrations. This is achieved by cementing together a convex and concave parts and one made of crown glass and other flint glass so that both errors are cancelled each other. Such lenses are also called doubled. They are of moderate NA, hence moderate resolving power and long working distance and flat field of view. b) Apochromatic

It is a combination of triplet of lenses made of suitable material so that the aberrations can be decreased by the maximum. They are perfect in colour so used in colour photomicrography.

c)

Fluorite

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Intermediate between achromatic and apochromatic objectives but nearer to latter. It is made of fluorite ground and polished. They can’t correct chromatic aberration perfectly. They have high NA. d) Flat field objectives

High NA is used in photomicrography and flat field is obtained with less NA so better resolution. e) Spring loaded objectives and parfocalled objectives which are explained earlier

Another classification is dry, water immersion and oil-immersion according to their use in air, water and oil respectively. Both high power and low power objectives can be used with uncovered objects but 4 mm objective is very much sensitive. EYEPIECE It is the lens near to eye and give second magnification and image is virtual. It is of different types such as: a) Huyghenian eyepiece

It consists of 2 plano convex lenses with a metal diaphragm in between them located at the focal plane of the top or eye lens. The bottom lens or field lens collect the rays of light coming up from the objective and brings them to focus in the plane as diaphragm. Pointer eyepiece is some variety of this provided with an adjustable pointer and compensation eyepiece is one with a correction for achromatism. b) i) ii) Holoscopic eyepiece Telaugic eyepiece: as one to correct the curvature of the field. Complex type: This is of compensation type but of special design with a large eyelens, a raised eye point and a rubber guard over the run of the eyepiece so that a spectacle wearer can easily rest his lens against the rubber guard and then see the whole of the field of view without moving his head. Rainsden and Kellner: Plane of the focus of eye lens lies just below the file lens external to the eyepiece. The allows easy positioning of reticules and hairlines and so used in micrometry. Projection eyepieces: They are hughenian type but with an achromatic lens mounded in an adjustable sleeve. This image can be projected and used in photomicrography.

iii)

iv)

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v)

Parfocal eyepieces: Eyepieces of tubular collars of different lengths so arranged to view the object in focus after focusing even if the eyepiece is changed.

As the eye piece power increases the size of field decreases. So for better resolving power and depth of field the highest power of objective is used with the lowest of eyepiece. CONDENSER Condenser is used to illuminate the object at the point on which the object is focused and to fill the field of view with uniform illumination. There are different types of condensers as: a) Sample abbe

It consists of 2 large diameter planocovex lenses of considerable thickness, air separated with an adjustable iris diaphragm mounded at the back of focal plane. b) Three lens abbe They consist of 2 plano-convex lenses. c) Achromatic and Aplanatic They do correction of chromatic and spherical aberration respectively. d) Oil immersion type

To get most number of rays concentrated on object distance between condenser and underside of slide is replaced by oil which prevent total internal reflection. e) Special condenser like macro for photomicrography

Dark ground for dark ground illumination, Trilux for phase contrast, Auxillary for Koehler illumination, etc. are also used. The effective aperture of any objective-condenser combination is the mean of N.A. of the objective and the aplanatic aperture of the condenser i.e. the six of cone, which is free from spherical aberration. The light source can be built in and it is of limited adjustability or preferably external and movable. HOW TO USE THE MICROSCOPE 1. Identify the objective lens to be used and put it in proper position and fully open the condenser and adjust the mirror to get the maximum light. Place a specimen slide on the stage and looking through the eyepiece turn the coarse focusing knob when a blurred image is obtained and

2.

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bring the specimen into fine focus with fine adjusting knob. Never focus downward while looking through the eyepiece. Adjust the iris diaphragm bright light to cover the whole field. 3. After adjusting with low power and then high power dry adjective or 100 x using the oil. Place a drop of oil at the centre of the specimen or cover glass, locate the specimen with low power and then use 100 x and focusing is done by the same way do not allow objective to touch the slide and just the diaphragm to get clean image. Each time after use, the oil is wiped off with tissue paper and moistened with xylol.

A finely focused image should be clearly visible without superimposition and amount of light will be optimum slides. After viewing the object keep the best possible from the dirt free and slides are washed and dry and keep the microscope covered with polythene cover. Keep the lens clean by wiping it with dirt free linen paper moistened with xylol. SPECIAL TYPES OF LIGHT MICROCOPY 1. DARKFIELD MICROSCOPY

It provides a useful means of looking at wet mounts of unstained specimens and detecting very small structures by reflected and detracted light. It is performed by illuminating the specimen with the hollow cone of light such that only the light detracted by object in the field of view is transmitted up the microscope tube to the eye or camera, the beam forming the cone of light focused on the specimen one at too low an angle to be captured by the objective. The result is a field of bright objects against dark background low power and high power adjectives can be used in a dark field mode by using an ordering condenser enquired with a filter carrier below the condenser mount and an angular cone of light is produced which can be focused on the object without any direct rays entering the objective. There must be good intensity of light and focal length should be shorter for high power this slides are needed so that condenser can them are high light intensity, oil immersion contact throughout, funnel stop in the objective, central mount and a special dark ground condenser. 2. PHASE CONTRAST MICROSCOPY

It is system of joining contrast in a translucent specimen without the help of stains and has the advantage of using high resolution optical components. It also needs high intensity of light. A green filter is used to reduce the unavoidable achromatic aberration. Phase contrast works because a phase annuls is inserted at the lower focal phase of the condenser and because of phase plate is incorporated in the back of focal plane of the objective. An image of the annulus will be thus formed on the phase plate. Since light beams are detracted by the specimen to go through all the objective field and some go directly through the specimen and the phase plate ring, both take part in forming the image. It is essential to centre the annulus plate in back focal plane of the objective. The specimen for phase microscopy

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must be as thin as possible and must be mounted in fluid or gel and under a cover slip to reduce heterogeneity of the background of the image. Light is passed through the cell depending on the refractive index of the cell. So different parts of various intercity pass light at different rate so this difference gives the phase difference. This is used to visualize living cells. 3. INTERFERENCE MICROSCOPY

It is used in finding out the structure of the cells. Interference microscope produces separate object and reference beams, rather than forming an image from the direct and diffracted elements of a single beam from a condenser annulus as in phase contrast microscopy. It gives internal morphological details of the cells. Using this we can see the movements involved in mitosis, cell migration etc. 4. FLUROESCENCE MICROSCOPY

In this the phenomenon of fluorescence is used which is the property displayed in certain substances whereby, if they are illuminated with short wavelength (UV) they are able to convert the short wavelength which is invisible to long wave, visible light. So when such substances are added to cells with the help of other protein like antibodies they specifically bind certain sites and when illuminated with UV they produce fluorescence which can be varied by the microscope as colored spots while other area remaining usual. It is widely used to detect molecules and give structure of the cell. The illumination can be different types like (i) transmitted light through a substage condenser (ii) dark field illumination and incident illumination. Flurescent microscope uses 2 set of filters to filter light falling on another filter is used which will permit only the longer wavelength to pass to the objective. Common dyes used for this are fluorescent and rhodamine which gives green and red colors respectively. 5. POLARIZED LIGHT

Light is a form of energy and contain a series of waves at right angles to the direction of travel. When this is passed through certain prisms the light become plane polarized all others being eliminated and they are called polarizer. This is used to find whether an object is optically active or not, by passing the plane polarized light through the solution of object and measuring the direction of turning of the light. This is with the help of analyzer. 6. PHOTOMICROGRAPHY

This is a method taking the picture of the image of the object with the help of camera attached to the eyepiece. Also video cameras can be attached to get live films of cell migration. There are 3 types of photomicrography. a) A roll film camera back and shelter together with an integral device that includes the ocular and beam splitting prism with a

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side viewing arm to allow accusing forms the working unit. The whole device rests on the microscope tube. b) An old fashioned light tight bellows is sometimes used. It is located on a stand on which microscope can also be placed. Also there is a plate carrier to place the file.

Success of photomicrography depends on specimen quality and focusing, good optics properly aligned and illuminated and choice of color filters and camera free of vibrations and good quality film and processing and proper panchromatic ASA 60-100 and for phase contrast microscopy ASA 300-400 is needed. Photomicrography is helpful in proper recording of the details of cells and microorganisms and their activities. 7. STEREOMICROSCOPY

In effect there are 2 microscopes of its own objective and eye piece and 2 microscopes have in inclination separation of 15°, the images presented to the eyes differ as they do in normal unaided vision and a true stereoscopic picture is seen i.e. 3 dimensional. These are also called binocular microscopes. For this flat field of view as large as possible is needed and a long working distance and distortion for clear images, maximum depth of field are needed so special optics are used and have images are erect giving natural sense. In zoom stereo microscopes there is an extra set of lenses zoom lenses by which you can vary magnification and field of coverage and focus of the lens system remains the same. This is also used to observe surfaces of materials and sections are not essential. As the value of this microscopy is in depth of focus and field of coverage which is best at low power objectives we use lowest possible objective and highest possible eyepiece to produce desired magnification. 8. CONFOCAL SCANNING MICROSCOPY

This used to view unsliced live specimens 2 dimensionally and with high power laser object is illumined at a particular point at a particular depth and is viewed. All other light sources are prevented, fluorescence emitted from illuminated material is collected and brought into an image at the entry point of a suitable light detector. Through a pinhole this light is focused and other portion become out of focus. But using electronic techniques like image processing we can get cleat pictures. Light sensitive video camera are used to detect dim light details which can’t be seen so it is possible to view camera produce digitalized image which can be recorded and interference contrast microscopy helps to view a single microtubule clearly which have a diameter of 0.025 µ m now-a-microscopy, high contrast and VIM-video enhancement microscopy, high contrast and VIM-Video intensification microscopy which amplifies low light images are available. LIMITATIONS OF LIGHT MICROSCOPY

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1.

There is maximum limit of resolving power with light. Light can’t resolve more than its wavelength 0.4-0.7 µ m in one of visible light. Because of wave nature light doesn’t follow exactly the idealized path (straight line) but travel through an optical system in slightly different routes so that they interfere with each other and cause optical diffraction effects. If 2 rays reading a point are not exactly in same phase they produce complex interference effects by mismatching of crests and troughs of the wave. At high illumination and magnification the evenly illuminated light of uniform wavelength appears as a set of parallel lines where as set of concentric rings. So the image formed will be blurred and theoretically with a NA of 1.4 and violet lights of (0.4 µ m) limit of resolution in 0.2 µ m. Although it is possible to enlarge the image as much as one wants it is never possible to resolve the two objects in the light microscope that are separated by less than about 0.2 µ m such objects will appear as one. Chromatic aberration: Since different wavelengths are focused at different points dispersion occurs and color fringes occurs and it hinder to view the object in color this corrected by using achromatic lenses. Spherical aberration also result in blurred image and this can also be corrected to an extent by using combination of lenses of adjusted refractive index this is possible. Also the field view is sometimes curved and this hinder the power of resolution. There is limitation for magnification also (upto 1500 times).

2.

3.

4. 5.

CONCLUSION Light microscopy has developed a lot from the times of Anton von Leuwenhock to the end of 20th century where by the help of electronic image processing fine quality pictures can be taken. But light microscopy has been loosing its glamour with the evolution of electron microscopy and its improved applications which give high magnification and resolution power. Even then light microscopy is widely used in the labs and in microbiology and cell biology studies and to view live specimen. Anyway the science of microscopy flourished through the advances in light microscopy.

REFERENCES 1. 2. 3.
Alberts et al. – Molecular Biology of the cell (1994) Garland Publishing Company, Newyork. p. 139-149. Casartelli, J.D.; Microscopy for students (1965) Mc-Graw Hill Publishing Company Ltd. London P 10131. Frielder David – Physical Biochemistry – Applications to Biochemistry and Molecular Biology (1976). W.H. Freeman and Company – Sanfransisco P 21-50.

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PHASE CONTRAST MICROSCOPY INTRODUCTION When a small biological object, such as a microbial cell is observed in the living state, it is normally suspended in an aqueous medium compressed into a thin layer between a slide and cover slop. The perception of such an object depends on the fact that it displays some object degree of contrast with the surrounding medium as a result of the fact that less light is transmitted through it than through the medium. This decreased light transmission is caused by two factors: light absorbed by the cell and light refracted out of the optical path of the microscope by a difference in refractive index between the cell and the surrounding medium. The degree of contrast can be greatly increased by staining procedures. Before staining, the ells are sometimes fixed, by treatments designed to minimize postmortem changes of structure. For light microscopy, heat is the most commonly used fixative. So to overcome all these difficulties and to observe the cells in living state, phase contrast microscope was developed by a German optical worker Prof. F. Zernike. Phase contrast microscopy Phase contrast microscopy is technique which enables us to see very transparent objects, which are almost invisible by ordinary transmitted light in clear detail and in good contrast to their surrounding and to detect very small differences in thickness or density within the object. This is achieved without altering the object by staining or other processes so that we can see living cells and tissues in their natural state. Principle Phase contrast microscopy is based on the fact that the rate at which light travels through the objects is inversely related to their refractive indices. Since the frequency of light waves is independent of the medium through which they travel, the phase of a light ray passing through an object of higher refractive index than the surrounding medium will be relatively retarded. These retarded waves are out of phase with the reminder of the light waves but the difference is not large enough to be detectable with ordinary light microscope. The phase microscope amplifies these phase differences and converts them into difference in light intensity thereby increasing the contrast between the cells and its environment. Phase Contrast If we have a very transparent object whose density or thickness is insufficient to create a phase difference of about one half of a wavelength between the direct and diffracted rays, the object can barely be seen and it is in such cases the phase contrast comes to the resume.

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The phase microscope differs from the conventional light microscope in having annular diaphragm in the substage condenser and a diffraction plate at the rear focal plane of the objective lens system. Annular diaphragm permits only a ring of light to pass upward through the condenser and the object. At this point is mounted the phase plate. This consists of a disc of glass in one face of which is an annular groove or depression. Any light passing through this groove as at AA, has less glass to penetrate than light passing through the remainder of the plate as at BBB. We see that rays passing through B, having more glass to penetrate will be retarded in relation to other rays passing through AA. As light rays pass through the area-surrounding object as well as through the object, some rays are retarded and thrown out of phase. Both the deviated and undeviated light waves pass as a cone of light into the objective lens system, where they pass through the diffraction plate. In phase contrast we form an image of the light source annulus at AA, the annular groove of the phase plate, and the rays producing the image are all the direct rays. They rays which are scattered by the object the diffracted rays, will pass through the remainder of the phase plate BBB. The retardation imposed by the phase plate, which is one quarter of a wavelength, added to the slight initial retardation impose by the object itself due to its optical density and thickness, usually about one quarter of a wavelength, amount to the desired one half of a wavelength retardation necessary to produce an image of good contrast when these two sets of rays meet and interfere with each other. This has been done by retarding the diffracted rays and so altering their phase relationship to the direct rays. In other words, we have obtained good contrast by a phase alterations. Hence the name of this technique: Phase Contrast. DARK FILED ILLUMINATION They are many objects which because of their transparency, cannot be seen easily by the method using transmitted light. In such cases, an increases in contrast between the object and its surrounding is of more help and dark ground illumination is used to provide this contrast. Principle When light impinges on a small object, some light is scattered, making the object appear luminous and thus visible against a dark background. It permits the detection of objects so small as to otherwise provide insufficient contrast. Working Dark field illumination employs a special type of condenser, which forms a Hollow cone of light. This cone of that no light ray directly enter the objective lens. As the apex of the cone strikes the object light rays are

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scattered or reflected from it into the objective lens of the microscope. This one sees a brightly illumines particle or cell against a dark background. Advantages By providing contrast, the dark field microscope, is useful in the study of living cells. With dark field illumination the presence of bodies, whose size is below the resolving power of the light microscope can be detected. This is because light emitted from an object can be seen even though the object itself is not discernible. This technique while it demonstrates the shape, size and presence or absence of motility of the object, shows only the outer envelop; no internal structure is visible by dark ground. Phase, contrast on the other hand, allows us to see the interior of these very transparent object. UV MICROSCOPY INTRODUCTION Scientists have come a long way since Anton Von Leeuwenhoek Ist sighted his animalcules. The formally unknown world of microbes is now accessible to the common man, through that indispensable aid-the microscope. Light Microscopy-both simple (as that used by von Leeuwenhock) and compound (used by latter scientists), reached its height of development in the latter part of the 19th century. The light microscope has two essential parts objective and the eyepiece. No major changes in the eyepiece resulted in a satisfactory improvement of the compound microscope but improvement on the objective side by oil immersion system and use of apochromatic lenses, helped reach the resolving limit of the microscope (0.2 µ ) by 1890.2 scientists responsible for this were- Carl zeiss and Ernest Abbe. Thus scientists had reached a major stands till at this point of time. It was the subsequent efforts made from them on that led to one of the most advanced forms of microcopy-ultraviolet and Fluorescent Microscopy. PRINCIPLE OF UV MICROSCOPY The major drawback of the light compound microscope is its resolving limit/limit of resolution. The only way in the 1900s that scientists could think of – to increase the resolving power and thus reduce the limit of resolution was to reduce the wavelength of light. This was because the value of N.A. i.e. numerical aperture was relatively stable at around 1.2 to 1.4 and could be changed to every little extent. Visible light lies in the range of 4000-7000 A 248° wavelength. Light of lower wavelength thus comes in the ultraviolet spectrum i.e. 150-3900 A°.

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Characteristics of UV Microscopy Ordinary glass starts to absorb heavily from the 340 nm range downwards. Virtually all transparent plastics have a similar transmission limit. Therefore, it becomes impossible to form an image with glass lenses for UV light. Materials like quartz (transparent for UV down to 200 nm), fluorite (transparent down to 185 nm) or lithium fluoride are in sue for construction. It has been shown possible to construct ultraviolet microscopes with objectives of short focal length and high N.A. In 1904, A Kohler was the I st to construct such a quartz objective. Light Source: Use has to be made to a light source emitting sufficient amount of radiant energy in the ultraviolet region. The specimens have to be mounted between quartz object slide (usually made smaller and less thick than ordinary glass slides, 25 x 37.5 x 0.5 nm) and a quartz cover glass which has to be thicker than glass ones (0.025 mm). The mounting medium and contingent immersion fluid should be transparent for UV light use is often made of anhydrous glycerin. Limitations of UV microscopy 1. 2. UV light as an imaging agent entails a series of complications-the need for quartz system makes it very expensive. Though there was a measurable gain in resolving power, the theoretically expected gain e.g. : of a factor of 2 for 275 nm instead of 550 nm) was seldom obtained due to factors like stray light, deficiencies in corrections in objectives, problems with contrast and illumination. Ultraviolet radiation can cause damage to the cornea of the eye. Suitable sources of radiation are difficult to make for light of wavelength below 250-350 nm ranges. The specimen itself shows progressive absorption and even quartz object slides can no longer be used. Air gradually absorbs much of the UV such that the microscope has to be placed in the vacuum system or in an atmosphere of nitrogen. Cells are highly sensitive for ultraviolet light of certain wavelengths.

3. 4. 5. 6. 7.

Advantages 1. Ultraviolet light in the region of 260-320 nm is subjected to strong selective absorption by certain biological substances.

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Caspersson showed that biologically important proteins (280-320 nm) and nucleic acids (265 nm) show natural absorption for UV light of the indicated wavelength. This is used as a major asset of UV microscope i.e. the revealing of distribution and quantitative analysis of materials which show selective absorption in 250-380 nm region i.e. UV micro-spectrophotometry and micro-photometry. 2. As contrasts in cells are related entirely to natural absorption of certain substances without any pretreatment of cells, it is possible to perform UV microscopy on unfixed cells.

Applications 1. 2. Microspectrometry and microfluorometry Microspectrophotometry and microphotometry for analysis of nucleic acids, proteins. This also has been applied for study of the metabolism of uric acid in certain yeasts.

Photomicrography can also be used to register images as these cannot be perceived by human eye. Images are now being perceived by image converters. FLUORESCENCE MICROSCOPY Phenomenon of Florescence Florescence can be defined as the phenomenon displayed when certain substances are struck by electromagnetic radiation absorb a part of it and re-emit it at a greater wavelength. The process of emission is a short one and takes only a few milliseconds during which atoms struck return to their ground state. In contrast to fluorescence phosphorescence, depending on other types of transition has a much longer emission period. This phenomenon is of no importance for microscopy. Molecular aspect of Fluorescence When a molecule is exposed to suitable radiation, it gets excited to a higher electronic level. If the ground state of the molecule is a singlet state then the excited is also a singlet state because singlet-singlet transitions are allowed transitions, whereas singlet triplet transitions are forbidden transitions. If his excited singlet state is a stable state, then there are much less chances for a secondary reaction. When conditions are such that the probability if intermolecular collisions is less and the excited molecule is unable to transfer its energy either to other molecules/convert it into kinetic energy, then the excited molecule relaxes through a radioactive process within 10-6 seconds and the absorbed energy is re-emitted in same/different wavelength. This phenomenon is termed fluorescence. This is instantaneous.

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Electronic level possesses several vibration levels, the excited molecule may or may not return to its original vibration level. If there is a change in the vibration level, the emitted light has a different wavelength. Phosphorescence is due to intersystem crossing when the excited molecule during vibration relaxation transits to the triplet state. Since this is a forbidden transition the molecule takes time to return: reason for its being a delayed fluorescence. Types of fluorescence a. b. Primary fluorescence/ auto-florescence- it occurs by natural properties of certain substances. Secondary, fluorescence, induced by substances with fluorescent properties which have been attached to certain components of other specimens egs. : acridine orange staining for DNA and RNA. Immuno florescence: A phenomenon of supreme importance in diagnostic microbiology today. This is a phenomenon in which an immunological agent i.e. the antibody previously conjugated to a florescent marker like fluorescing is used to detect specific antigens or sites, even microtubule structures.

c.

Fluorochromes There are certain chemical substances having fluorescent property used for the purpose of staining and as dyes. The advantage in using these chemicals is that they are needed in very, very low concentrations such as 1: 500-1; 100,000 with distilled water. Depending on their nature in water, they are divided into: 1. Alkaline fluorochromes: Acridine orange, auramine, acridine yellow, corphosphine, berberine sulphate, netural red, acriflavine hydrochloride etc. Acridine group is that containing most used fluorochromes. Neutral fluorochromes: Rhodamine B. Acidic fluorochromes: Primuline fluoroscein, sodium fluorescein, pepsin, erythrosine. AND EMISSION SPECTRA Emission Maxima (nm) 540 550 520 595, 710 OF

2. 3.

MAXIMA FOR ABSORPTION FLUORESENT AGENTS

Absorption Maxima (nm) Acridine orange Auramine Fluroescein isothiocyanate Lissamine rhodamine B. 267; 492 440 280, 325, 495 350, 575

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Fluorescence Microscope System Components: 1. Light source: Generally mercury discharge lamps which give out high intensity of UV fluorescent light. Incandescent filaments for blue light fluorescence. Light yield should be high. Ultimate yield in flux, of fluorescent light is only of the order of 0.1% of light emitted by light source. High pressure mercury lamps have powerful spectral line sin proximal ultraviolet especially at 366 nm and in visible blue violet near 405 and 435 nm. Recently, period xenon burners are also being used. 2. 3. Excitation filters/Ist Barrier filters: These are place at the entry of light and lets through only blue light of 450 and 490 nm absorbs the rest. Mirror: This can not be of the conventional design as light from the UV region is only partly reflected by silver. Therefore, specially designed mirror is used having aluminum covered with thin layer of silver. Now a days bean spitting mirrors, i.e. dichroic mirrors as developed by Ploem (1967) are used. This has an interference coating which has a high reflectance for the light passed by an excitation filter but is transparent for the fluorescence light of longer wavelength. Thus even this acts as a filter. 4. Barrier filter: Placed near eyepiece to remove stray fluorescent illumination. It only transmits light of longer wavelength emitted by the object/specimen. This can be of low absorption since the mirror also functions effectively as a barrier.

Confocal scanning microscope This is a recently developed form of microscopy which enables a direct way of visualizing a specimen in 3D. Electronic imaging methods make it possible to focus on a chosen plane in a thick specimen while rejecting light that comes from out of focus regions. From a series of such images-3D images are obtained. The microscope is used with fluorescence optics. But instead of illuminating the entire object, the optical system at any instant focuses a spotlight into a single point at specific depth in the specimen. A very bright pin point source of illumination is required-laser is used. Fluorescence emitted from the point is collected; brought to an image at the entry port of a suitable light detector. Pin hole aperture is placed at the detector, at the site that is confocal with illumination pin Hoe-precisely where rays from illuminated point come to focus. Thus this light converges on aperture-enters the detector.

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Out of focus light can not enter. To build a 2-D image, data from each point on the plane of focus is collected sequentially by scanning and are displayed. This is used to resolved structure of numerous complex 3-D objectsnetwork of cytoskelecal fibres in cytoplasm and arrangements of chromosomes and genes. Applications 1. Immuno fluorescence is flurescein antibody staining in diagnostic microbiology: In this technique fluorescent dyes are combined with antibodies i.e. substances that combine with specific microorganisms (antigens). These are labeled antibodies. These labeled antibodies are mixed with a suspension of eg: bacteria. The preparation is then examined by fluorescent microscopy. Those cells which have been linked to the antibody are now visible. This is the fluorescent antibody technique. Theoretically it is possible to identify single bacterial cells by this procedure eg: For poliomyelitis-Mahoney stain-fluorescein. This techniques permits detection of various aggregates smaller than Negri bodies. Even species of Salmonella typhosa can be identified from S. Virginia. 2. Fluorescent analogue cytochemistry In this techniques to introduce membrane impairment molecules into & living cell, whether antibodies or normal cell proteins-these are tagged with fluorescent label and microinjected into the cell. Purified protein coupled to fluorescent dye microinjected and fate of injected protein followed by fluorescence microscope as cell grows and divides eg. if tubulin is labeled with a dye that fluoresces red microtubule dynamics can be followed second by second. 3. Chromosome analysis in cytogenetics: When it is possible to localize certain substances in a cell, it is possible to detect minute quantities of the substance using fluorescent dyes. Reigler was able to demonstrate 10-5 gm DNA in single cell. 4. Microflurometry To measure fluorescence/total amount of fluorescent material in the cell eg: acridine orange to DNA 5. Flow system analysis: Large number of cells measured in few minutes/flow microfluorometry, flow in capillary across a beam of excitation light. Emitted pulses sensed by photomultiplier used in leukemia blood analysis.

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Reference 1. Alberts. B. Bray D., Lewis, J.; Raff. M.; Roberts K.; Watson J.D. Molecular Biology the cell : Garland Publishing Inc. 1994 – Third Edition. 2. James J. Light Microscopic Techniques. M.N. Medical Division, 1993. 3. Pelczer M.; Kreig; Chan. Microbiology. Tata Mc. Graw Hill, 1993 4. Rodina, R. Aquatic Microbiology, 1972. 5. Stanier R.; Ingrahm, Wheelis General Microbiology, 1987.

ELECTRON MICROSCOPY INTRODUCTION No one person can be singled out as the inventor of electron microscope. The invention is a product of large no. of inventions and discoveries in Physics. A culmination of many scientific advancements finally lead to the advent of Electron Microscope. Before its development several microscopes like light Microscopy, Fluorescent Microscopy, Phase contrast, Microscopy, Interference microscopes were invented. By these microscopes the maximum resolution that can be obtained is 0.2 µ m. Before this dimension however “Words inside the cell” seems to be particularly interesting. It was the dream of every biologist to get hold of a device with better power of resolution. U.V. radiation has slight advantage. Although the radiation is visible, the picture produced by it can be visualized by a radiation transformer (fluorescent layer, photo plate). However the increase in ‘resolution produced by this is relatively small. The resolution can be increase by a factor of 1000 by using electromagnetic waves – “X-rays”, rather than light. But the problem for this radiation to be used in microscopy is that there are no sufficiently strong deviating mirrors and lances elements one could not build a useful X-ray microscope for atomic resolution. This is because of lack of interaction to the rays with the specimen. In case of X-rays interaction is extremely weak. The same physical law that permits to transilluminate a human body would let a bacterium appear completely without contrast. Then came the concept of e-rays to be used in microbiology. As for as interactions are concerned e-rays are almost ideal. Since with medium voltage. Each …… is only a few thousand of nm. One need not worry about the restruction of resolution by diffractions. Furthermore e-ray can be influenced in the direction of their propagation by electrical and magnetic field. A source of e-rays provided to means. By this, our vision can be extended thousand fold. By this thus the simplest forms of life-viruses can be observed and even the building blocks the macromolecules. Thus electron microscopy has significance as it provides us with the opportunity of studying matter molecular level. HISTORICAL BACKGROUND

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The major events which contributed to the development of electron microscope are: (1) Discovery of electron by J.J. Thomson as negatively charged particles who termed them as “thermions” Charge of eMass of e= = -1.6 x 1019C -9.1 x 10-31 kg

(2) De Broglie’s hypothesis that moving charge particles like e- have wave nature similar to electromagnetic waves. (3) Bush gave a theoretical hypothesis that e-beams can be focused by employing electrostatic field and thus laid the foundation of Electron optics. (4) Knoll and Ruska Published the first result obtained with electron microscope by using magnetic radiation. Although the resolution obtained was inferior to that of light microscope, the real beginning in electron microscopy was accomplished, and the Ist electron micrograph of non-self illuminous specimen had been obtained. This Ruska built the Ist TEM: (5) Knoll demonstrated the feasibility of the SEM and three years later a prototype was built by Von Ardenne. (6) Siemens produced the Ist commercial TEM. (7) Rebertson described the trilaminar structure of cell membrane, seen for the Ist time in the electron microscope. BASIC NTHEORY OF ELECTRON MICROSCOPY (1) Wave nature of e-beam De Broglic in 1924 proposed that e-beam have wave nature:
= h 1 X m v .......... .1

h m v KE v e

= = = = = = =

Wavelength of matter wave Planks constant = 6.63 x 1034 J/S Mass of particle Particle velocity ½ mv2 = Ve ……..2 Pot. difference through which e-bean has been accelerated. charge on e.

Combining eq (1) and (2) we get

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=

12 .3 A° V

This shows that the bean is dependent upon the voltage through which has been accelerated. This high velocity e-owing to their small wavelength might provide suitable form of radiation of fulfill the requirement for high resolution microscopy. (2) Resolution Defined as ability to distinguish between two points or lines separated by as short a distance possible. Abbe gave an eq.
d = 0.612 0.612 = n sin α N A

= NA n d α = = = =

wave wavelength of image forming radiation Numerical aperture = n sin α Refractive index of medium Diameter of the image 1.2 aperture angle

This example expressed the magnitude of diffraction effect and provides numerical value of limit of resolution any aberration free optical system. Significance of d is that two points will just be resolvable if they are separated by a distance greater than d. Thus to obtain maximum value for resolution the limit or resolution or d should be as minimum as possible and to obtain minimal value of d … should be as small as possible and n and sin α should be large. But since, the values of sin α and n can’t be increased significantly beyond a certain value, the only factor that can be altered is … R.P. of eye = .25 mm, R.P. of light mic = .25 µ m, R.P. of e- = 2.5 A°. Thus R.P. of e.m. = 100 times of light mic microscope. Development of Magnetic lens Comparison between optical lens and magnetic lens Lenses are used to bend rays of light o a bean of e- so that they are deflected in predictable manner from their original path. Lenses do so because the transparent material of which they are made of cause slowing down of the velocity of light rays during its passage through the lens. The lens with shorter focal length is a “strong lens” Convex lenses are “Converging” or “Magnifying” or “Positive”. Concave lenses are “diverging” or negative lens.

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Unlike light lays in light microscope the electron does not change its velocity as it passes through the magnetic field and only gets deviated by a force acting on it owing to the surrounding magnetic field. In case of optical lenses refraction takes place at the interface between the lenses and the immersion medium which is air in case of light microscope. In case of magnetic lenses the e= get defected whole time while their passage through the magnetic field refraction is continuous and there is no sharp interface between the magnetic field (refracting medium) and the immersion medium (the vacuum within the electron microscopic column). Principle When a current is passed through an axillary would coil it behaves like a hollow bar magnet. Part of the magnetic field run inside the coil parallel o the axis, part is radiated out in specie outside the coil. The only useful part of B is that inside the coil which is parallel to axis and the external field of the solenoid is simply wasted. The efficiency of a simple solenoid lens was improved by enclosing the energizing coil in a sheath of soft iron. Soft iron has the property of concentrating the lines of force in magnetic field thus becoming itself magnetized by induction. As the current in the solenoid is increased, strength of B is increased and so the induced magnetism in soft iron sheath and hence strength of the lens increase or its focal length decreases. Focusing action of uniform magnetic field An e- enters the uniform portion of the magnetic field of a long solenoid at an oblique angle. When its reaches the point ‘O’ which lies on the axis of coil its trajectory will be determined by its vector components (horizontal and vertical). This simultaneous actie forces produce curricular forward motion i.e. a helical path which is tangent to the axis. If there had been no B than the ewould have moved optical counter part functions by forcing an e-which is diverging from the axis back into the axis. It the number of e-having same axial velocity, the pitch of their helices will be the same and all electrons passing through a given point will after one revolution come together at the same time. Hence in this way magnetic field can produce an erect image of an object placed in its path which rendered visible by fluorescent screen. Magnification M of light Mic = 1000 times M of Electron Mic = 1,00,000 = 1000 x Magnification by light microscope

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Different parts of Electron Microscope Electron Microscope consists of 3 systems (1) (2) (3) I. (i) Illuminating system Imaging system Image Translating system Illumination system has following parts Electron Gun The gun acts as an electrostatic lens and as has following parts. (a) Filament

It is a thermionic cathode, usually V shape tungsten wire and can be electrically heated to incandescence to act as electron source. (b) Shield

It is equivalent to a grid of triode and is an aperture cylinder. A small potential difference is maintained across filament and shield. It acts as a focusing electrode. (c) Anode

An aperture disc and attached to high positive terminal voltage. The emitted e-are accelerated in the space between to be filament and the anode by the potential difference across the gap. There are basically two methods of connecting the accelerating potential to the electron gun. In Non biased system a shield and filament are connected through a pair of Balancing resistors directly to – V terminal of high voltage supply. In Biased system the filament is connected to the high voltage supply through both a Bias and a pair of balancing resistors while the shield is attached directly to the high voltage supply. (e) Self Biased gun

In practice almost all microscopes currently manufactured have incorporated the biased system in their electron source and the type usually used in called the self biased gun. In such a system the shield aperture acts as a strongly converging lens. (2) Condenser lens

The basic function of the condenser lens is to focus the electron beam emerging from gun into the specimen to permit optimal illuminating conditions

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for visualizing and recording the image. This depends upon the condenser lens, focal length. The condenser lens of Electron Microscope is a relatively weak lens having a focal length of the order of few cms which can be adjusted over a considerable range of values.
αc = fc π S s −f c

α π fc S

c

= = = =

Condenser aperture angle radius of condenser aperture focal length of the condenser distance from the specimen to the lens

Intensity of illumination: As in case of light microscope, the intensity of illumination is directly proportional to the square of aperture angle. I I K α II. = = = = k α c2 Intensity of illumination an Instrument constant Condenser aperture angle

c

Imaging system has follows parts It consists of objectives lens and one or more projectors lens.

(i)

Objective lens

It forms the initial enlarged image of the illuminated portion of the specimen in a plane that is suitable for further enlargement of the projectors lens. Their f length is very short = 1.5-5 mm i.e. it is a stronger lens. The magnification of the image produced is 100. (2) Projector lens

The projector lens as the name implies serves to project the final magnified image on the screen or photographic emulsion. This lens has a construction similar to objective lens except that it has no provision to accommodate the specimen holder. The pre image obtained from the condenser lens acts as a object for projector magnification is 100. Projector lens has follows criteria: (1) (2) (3) Board range of focal length. The great depth of focus of the resulting high magnification system. A difference in manifestation and a less serious effect of its aberrations on resolution.

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(3)

Intermediate lens

May be employed in between objective and projector lens for further magnification. It has variable focal length. III. Image Translating System

The information the e microscopy provides in the form of variations of e-intensity. The e-image is converted into a visible light image by the following. (1) Fluorescent Observation Screen

Instantaneous and continuous transformation of the electron image into visible image is done by fluorescent materials which have the property of emitting light of greater wavelength when bombarded by e-beam. A mixture of Zn and Cd is generally used. (2) Photographic Recording

A photographic negative of the final electron image is produced. A print of image on the film is known as electron micrograph and serves as a permanent record. Operation of Electron Microscope Operating Adjustment The operating adjustment consists of following steps: Lens Condenser Objective Projector Photography Routine procedure followed is: (1) (2) (3) The field of view selected at low magnification and at low beam intensity and preliminary centering and focusing is carried out. The magnification is raised so that described level of resolution of detail in the e-image is obtained. The intensity of illumination raised but only up to adequate level for vertical focusing and reasonably short exposure. Usual E.M. adjustment Aperture diameter of condenser and condenser current controlled. Objective current control. Projector and/or Intermediate current control and/or pole piece interchange.

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(4)

The photograph is than taken without changing the condenser setting.

Methodology of Electron Microscopy Tissue to be studied is removed from the site and cut into small pieces within one or two min. cutting should be done rapidly. The size after slicing should be less than 1 mm. Immediately after slicing transferred to fixative. (2) In site fixation

Tissues can be fixed in situ in the living an aesthetic animal to minimize the effects of both cutting on unfixed tissue and of anoxia resulting from cutting off circulation. (3) Perfusion

Tissues that the too delicate to with stand anoxia like that on CNS are subjected to in-situ vascular perfusion of the tissue. a) Osmium tetraoxide fixation

OSO4 is widely used in majority of studies as fixative. It reacts with unsaturated lipids which is evident by the blackening of the tissues. OSO 4 acts to stabilize proteins of the cell by reacting at the polypeptide side groups of tryptophan and histidine thereby linking protein chains together. No. of factors are critical for good fixation. i) pH of fixative 7.2 – 7.6 is recommended ii) Buffering medium

Acetate veronal buffer of Michaelis was used before. Now S-collidine and phosphate are used. iii) Tonicity of fixative Optimum is 0.4°C. iv) Duration of fixation

Optimum is from 30-90 min. but it dependents upon the nature of specimen.

(B)

Formalin

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Useful for plant tissue for 10-15 min. (C) Glycerol dehyde

Very useful in studying cellular ultra-structure esp. organelles like microtubule. (D) Permanganate For study of membrane system in plants Dehydration Various dehydrating media employed Ethyl alcohol Acetone Propylene Embedding The various embedding medium used are : i) Methacrylate

Easy to handle with good rate of penetration through the tissue capacity to be cut to thickness in the range of 500-1000 A248° with relative case. ii) Water soluble medium

Three water soluble agents has been used – Glycol methacrylate, Aquon (eposey resins component) and Durcupan. Aq. Sol of these media are used immediately after fixation both as dehydrating agent and than in pure form as infiltration and imbedding agents. iii) Polyester resin are also used

Microtomy Improvements in the method of thin sectioning were needed to take full advantage of the increased resolving power of electron microscope. Principle of Microtomy In order to efficiently cut successive thin sections an ultramicrome must structurally meet following requirements. (1) (2) All of its movements must be free of vibrations of magnitude of the order of .01 rev. Should have very little static friction

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(3)

The specimen should pass the knife edge once only

Ultramicrotome consists of a horizontal bar to the front end of which is attached the specimen bolder. This bar is moved forward b advanced mechanism, knife is mounted in front of instrument and cuts sections by repeatedly moving the specimen past the knife edge. Knives can also be used for microtomy but must be able to fulfill following criteria: i) ii) iii) iv) v) The edge should have redices less than section thickness required. Resistant to chemical decomposition Hard and tough Made by homogenous material Should be physically stable. Glass knives and diamond knives are usually employed. Section thickness Contrast of e-microscope and section thickness are related as follows:
C≅ ed ∆ V ( αo ) 1 / 2

C d P V α°

= = = = =

Contrast object thickness in A° Effective density Voltage objective aperture angle

This dic. aperture angle and increase in object thickness increases contrast. However, as object thickness increases and voltage decrease chromatic aberration rather than contrast sets the limit of resolution. Therefore depending upon particular properties of both specimen and microscope compromise is made for maximum resolution. Kinds of Electron Microscopes (1) TEM = Transmission Electron Microscope TEM can only image a specimen thin enough to transmit a substantial proportion (50-90%) of the electrons indicent on it. The electrons transmitted by the specimen are focused to form a magnified image on a fluorescent screen. (2) SEM = Scanning Electron Microscope

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High velocity electrons strike the surface of a solid object in the vacuum. Electrons are either reflected of emitted from the surface. Reflected e-are called as “back-scattered primaries” are deflect through large angle by interadtion with atomic nuclei. “Secondary electron” of very low velocity are produced due to ionization of the specimen atoms by the incident primary atoms. A picture of the surface may be built upon a synchronous display tube in the light either of back scattered primaries or of emitted secondaries. Advantages of E.M. (1) (2) In the determination of particle size and shape. Some of the industrial problems have been studied like structure of textile fibres, purification of lubricating oils, composition of paints and paper, surface of metals, plastic etc. The concentration of colloidal particles in suspension can also be quantitatively measured. Used indirectly in the quantitative analysis of nucleation and crystal growth. In the field of medicine and pathology used in the study of virus and bacteria. In the determination of no. of protein molecules in virus. Due to shadow casting method one can get good outlines and profiles of viruses, bacteirophages and macromolecules.

(3) (4) (5) (6) (7)

Disadvantages of Electron Microscope (1) The intense electron beam acting for a considerable length of time destroys the specimen in many cases and beam can serve as reducing agent. The electron beam is unable to penetrate through metallic surface; hence study of metals is carried out by an indirect method called replica method.

(2)

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APPLICATION OF ELECTRON MICROSCOPE The electron microscope is an instrument which utilized electron as a source of illumination for observing objects at a great magnification. Because of its high resolving power, it is widely used for visualization of biological specimen. Various types of electron microscope had been developed, they are1231. SCANNING ELECTRON MICROSCOPE (SEM) TRANSMISSION ELECTRON MICRSOSCOPE (TEM) FREEZE-FRACTURE AND FREEZE ETCH. E. MICROSCOPY SCANNING ELECTRON MICROSCOPE (SEM)

Scanning electron microscopy combine the mechanism of electron microscopy and television, scanning electron microscope has been used to examine the surface of microorganisms in great detail. SEM became commercially available only in the early 1960’s after over the 3 decades of intensive researches by knoll, Von Ardene, Zworykin, Hayes and many others. SAMPLE PREPARATION FOR SEM (leave, pods, insects, fungi, grains, and other tissue) 1. Select appropriate location and size of the leaf, fix it immediately in 5% Glutaraldehyde in phosphate buffer (0.1 M, 7.3 pH). Apply, mild vacuum so as to remove air for effective, infiltration for fixative. Glutaraladehyde used as a strong protein cross linking fixative. To retain biological activity (e.g. Antigenicity) a weak cross linker paraformaldehyde can be used. Glutaraldehyde is a dialdehyde that react primarily with free amino group of proteins, resulting in cross linking of adjacent protein molecules. It provides excellent structural preservation, due to formation of methylene bridges, as followsCross linking by chemical fixation 2. 3. Sample are fixed for 7 hrs to over night and then washed thrice in the same buffer at 15 minute interval. Post fix the samples with aquous OsO4 for 4 hrs of more depending upon the sample. OsO4 cross links mainly with unsaturated fatty acid side chains 4. 5. Wash OsO4 with water thrice or more with 15 minute interval Dehydrate the tissue with 30-100% graded series of Acetone/ethanol or Amyl acetate.

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The purpose of dehydration is to replace all free water in the specimen with a fluid which is miscible with water. Ethyl alcohol is widely used because it does not harden the tissue and make it brittle for subsequent ultra thin sectioning and a certain amount of OSO4 remain with tissue. This is reduced by the alcohol to black, insoluble OSO 2 which from a fine granular and colloidal suspension 6. Dry the sample in a critical point drier suing liquid CO2 Liquid CO2 used to avoid distortions due to surface tension affects which cause the collapse of surface structure as in microvilli and Pseudopodia 7. 8. 9. The dried sample is mounted on a clean SEM stub having gun, Use silver or graphite dag to aid conduction. Coat the sample with graphite to a thickness of 40 nm followed by gold/gold palladium for a final thickness of upto zoom. Observe the sample at 5-15 KV and W.D. 15 mm.

WORKING OF SEM The electrons originate at high energy (20,000V) from a hot tungsten or lanthanum hex a boride cathode gun. These electrons are sharply focused, adjusted and demagnetized by an arrangement of magnetic fields and lenses which is analogous to the condenser of an optical microscope. Instead of forming a side file as in optical microscopes or transmission electron microscope, the electrons are made to form a needle sharp probe about 5.0 nm to 10 nm in diameter. The primary beam or probe acts only as exciter of image, forming secondary electron that emerges from the surface of the specimen. When the probe strikes secondary electron entering the detector, strikes a scintillator causing it to emit light flashes that a photomultiplier converts to an electrical current and amplifiers. The single is sent to a cathode ray tube and produces an image like a television picture which can be viewed or photographed. The number of secondary electron reaching the detector depends upon the nature of the specimen’s surface when the electron beam strikes a raised area, the number of secondary electrons to reach the detector is large. The contrast beam strikes a depressed area, numbers of secondaries appear lighter on the screen and depressions are darker. A realistic 3D image of microorganism’s surface”, with great depth of focus result. ADVANTAGE 1. 2. 3. 4. In SEM very thick specimen can be viaualize In SEM specimen preparation is easily In SEM picture can be seen in television In SEM a 3D image formed

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TRANSMISSION ELECTRON MICROSCOPY Transmission electron microscope is an microscope in which the image is formed form electrons that are transmitted through a thin specimen. The TEM has a practical resolution roughly 1,000 times better than the light microscope. TEM is used for the visualization of interval structure of the smallest cell. The distinctive feature of the TEM is the specimen preparation and nature of specimen. SAMPLE PREPARATION FOR TEM Fixation and Embedding 1. The sample is cut when immersed in 3% Gluteraldehyde prepared in 0.1 M phosphate buffer pH 7.3 into pieces of 2 to 3 mm cube size and the fixative is gently vacuum in filtrated into the tissue for 2-6 hrs or over night. Samples are then washed throughout in the buffer several times over a period of 2 to 3 hrs. Post fixation is carried out in 2% aqueous osmium tetra-oxide for 4 to 6 hrs. The post fixed samples turn black and brittle after sufficient time. The tissue is washed carefully several times with distilled water till all excess fixative is removed. The samples are dehydrated using a graded series of Acetone of ethanol over a period of 3 to 5 hrs (30-45 minute each is 30, 50, 70, 90 and 100% ethanol/Acetone) side chains. The samples are then infiltrated with a 1:1 mixture of dehydration agent: Exposyresin for about an hour. Various Epoxy resins are used to embed the samples. Commonly Araldite, Epson 812 or spur resin are used. The purpose of embedding is to form a solid matrix for support during subsequent procedures, such as sectioning. 7. The samples are then infiltrated over night to 48 hrs. with pure resin mixed finally placed in resin poured into flat silicon rubber moulds to from block Blocks are formed by placing the mounds is 70°C in cm oven for 24 to 48 hrs.

2. 3. 4.

5.

6.

8.

Ultra thin Sectioning / Microtomy For the study of animal and plant tissue with optical microscope the tissue are first soaked is a fixing solution then water, afterwards dehydrating

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fluid and paraffin solvents. They are then heated and cooled and forms small blocks. The blocks with the embedded tissue are cut into thin slices called a microtomy. Each thin section is mounted on a glass slide, paraffin is removed by some solvents and the tissue are then stained and examined with a microscope. A analogous procedure is used for making thin sections of bacteria and other micro-organisms for TEM. Sine electron have very low penetrating power, the section must be exceedingly thin of the order of 0.02 µ m for this an ultramicrotome is used. The embedding material instead of paraffin is epoxy resin, and after solidifying and warming, blocks are formed. These blocks are cut by the knife with the edge of diamond. SHADOWING This technique is particularly useful in studying virus morphology, bacterial flagella and plasmids. In this technique, it is coated with a thin film of platinum or other heavy metal by evaporation at an angle of about 45% from horizontal, so that the metal strikes the microorganisms on only one side. The area coated with metal scatters electrons and appears light in photographs while the uncoated side and the shadow region is dark. STAINING Since most of the constituent element in biological material are of low mass the contrast of these material is weak is can be greatly enhanced by staining with the salt of heavy metals (lead/tungsten/Uranium). These stains are non specific i.e., it stain all the components. The two most widely used section stain are uranyl acetate and lead citrate. NEGATIVE STAINING Negative staining is an excellent way to study the structure of viruses, bacterial gas vacuoles and other similar material. The specimen is spread out is a film with either phosphotungestic acid or uranyl acetate, heavy metal do not penetrate the specimen but renders the back ground dark while the specimen appears bright in photograph. FREEZE PARACTURE AND FREEZE ETCH ELECTRON MICROSCOPY Microorganisms prepared for ultramicrotome sectioning by chemical treatment and embedding in plastic may show distortions and unnatural changes due to the preparatory process. These can be eliminated to a great extent by freezing the specimen instantaneously in ice at tem of -198° or low there are two different methods. 1. 2. Freeze fracture Freeze etch.

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1.

Freeze Fracture

Freeze fracture electron microscopy provides a way of visualizing the interior or cell membranes. In this method fells are frozen at the temp of liquid N2 (-198° C) in the presence of Cryoprotectant (Ant freeze) to prevent distortion from ice crystal formation and then the frozen block is cracked with a knife blade. The fracture plane often passes through the hydrophobic middle of cell membrane. The resulting fracture forces are shadowed with platinum, the organic material is dissolved away and the replicates are floated off and viewed is the electron microscopes. (Shadowing is capable of providing high contrast surface views). Such replicas are decorated with small bumps called intra membrane practices, which represents large trans membrane proteins. The technique provides a convenient and dramatic way to visualize the distribution of such protein in the plane of a membrane. FREEZE ETCH E.M. Freeze etch electron microscopy can be used to examine either the extenior or interior of cells. In this special device to cover the sample against a copper block cooled with liquid helium and the frozen block is crack with a knife blade. But now the ice level is lowered around the cells by the sub limitation of ice in a vacuum as the tem. is revised (process called freeze drying) the parts of the cells exposed by this etching process are then shadowed as before to make a platinum replica. This technique exposes structures in the interior of the cell and can reveal their 3D str. Because of a metal shadowed replica rather than the sample itself is viewed under vacuum in the microscope both freeze fracture and freeze each microscopy can be used to study frozen unfixed cells, thereby avoiding the risk of artifacts caused by fixation. Reference
1. 2. 3. 4. 5. 6. 7. Introduction to biophysical methods of protein and Nucleic acid Research – J.A. GLASSEL & DEUTSCHER. Practical electron microscopy for Biologist – GEOFFREY A. MEEK Practical Biochemistry – K. WILSON AND JOHN WALKER Fundamentals OF Microbiology – FOR WISHER, HINSDILL, CRABTREE, GOOD HEART. Molecular Biology of the Cell _ BRUCE ALBERTS, BRAY, LEWIS MARTIN RAFF, ROBERTS, WATSON. Microbiology PRESCOTT. HARLEY, KLEIN An introduction to Microscopy – The odorve GEORGE ROCHOW AND EUGENE GEORGE ROCHOW.

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Experiment No. 1 Objective: Requirements: Observations: To study the compound microscope and observe the microbial slide. Slides, Microscope, Oil etc. Different types of microbial slides were observed under the 100 X power in microscope.

Sample Slide No. 1 1. 2. 3. 4.

BACCILLUS MEGATERIUM

Rod like cells are visible. Chains of rod cells are present and such type of arrangement of cells is called as bacillus from structure. In some cells oval shaped slightly lightish colour zone is present which is called as endospore. Cells are violet is colour. E. COLI

Sample Slide No. 2 1. 2. 3.

Cells are small rod shape No chain of cell is present Cells are pink is colour

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Sample Slide No.3 1. 2. 3. 4.

SARICINA LUTEA

Cells are oval shaped in group of 8 cells Only 4 cells are visible at a line. Cells are present in a 2 tear of 4 cells A outer sheath like, irregular covering is present where 4 cells are enclosed. STAPHYLOCOCCUS

Sample Slide No. 4 1. 2.

Cells are spherical some what rounded in shape called cocci. Bunches of cells are present and such a arrangement of cells are called staphylococcus arrangement. SACCHAROMYCES CEREVISEAE

Sample Slide No. 5 1. 2. 3.

Yeast cells are oval in shape. In some cells are bud like appearance present which is a mode of a sexual reproduction is a yeast. Smooth sheath like membrane is visible to surrounding the cells.

Sample Slide No.6 NOSTOC 1. 2. 3. Oval shape cells arranged in a chain. In each filament a single transparent cells is present which is layer than the remaining cells is heterocyst. Heterocyst is site of N2 fixation is these blue green algae.

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EXPERIMENT NO. 2 Objective: Requirements: Background: To determine the dimension of microorganisms by using micrometer Bacterial suspension, Microscope stage micrometer, stains etc. Bacteria are microscopic organism having size ranging from 0.2 µ m to 1 µ m. And they are so small and only visible by the use of microscope, so measuring the bacterial size, various methods are available and one of them is micrometer count which givens very accurate size of bacteria.

Observations:

Example Least count = 10/6 = 1.67 1. Long rod (bacillus) One bacillus cell covers 4 ocular divisions So its size = 4 x 1.67 = 6.68 µ m

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EXPERIMENT NO. 3 Objective: Requirements: Background: To observe motility of bacteria by hanging drop method. Bacterial suspension, slide, inoculating loop. E. Coli consists a peritrichoris flagella by which it moved in a zigzag pathway. In small organisms such as bacteria they have flagella which help it into movement in various direction is response of different chemical/physical or environmental stimulates. But in case of sarcinae, the movement is also observed but this is due to H 2O molecule and cell Brownian movement.

Observations:

Example: Prepare a bacterial hanging drop slide and motion of bacteria can be observed Sample I (Bacillus sp.) Cilia are present on surface it exhibits a long to & fro motion, this is due to cilia. Sample II (E. Coli) It also exhibit a ciliary movement because of cilia, one present on all over the surface Brownian movement is absent. Sample III (Sarcinae sp.) Random zigzag movement is present.

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Microbial Technology
a. Maintenance of aseptic conditions and handling during microbial culture practices b. Techniques to obtain pure culture of microorganism from mixed culture c. Inoculation of pure culture by streak plate and pour plate methods d. To perform simple/ direct staining and indirect/ negative staining and Gram’s staining of pure culture isolates e. Identification of unknown bacterial cultures by biochemical tests

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Contents 1. Bacterial Staining 2. Nutritional requirements for growth of bacteria 3. Study of Bacterial Growth 3.1 3.2 3.3 3.4 3.5 Doubling time Bacterial Growth curve Synchronous culture Continuous growth culture Growth Measurements 3.5.1 3.5.2 3.5.3 3.5.4 3.5.5 3.5.6 Direct counts Colony Counts Most Probable number Biomass Measurement Light Scattering (Spectroscopy) Chemical Estimation

3.6 Total Viable count 3.7 Heat stable count 3.8 Octyl viable count 4. Growth of bacteria under Environment stress 4.1 Effect of Temperature 4.2 Effect of pH 4.3 Effect of osmotic pressure 4.4 Preservation of Laboratory Cultures 5. Koch,s postulates of bacterial and fungal pathogens 6. Culture sensitivity using different disinfectants and antibiotics 7. Biochemical Characterization of isolated microorganisms 8. Mutations and Mutants 9. Quantitation of viruses

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1. STAINING TECHNIQUE INTRODUCTION Fixed, stained preparations are most frequently used for the observation of the morphological characteristics of bacteria. Stains are used to make characteristics of bacteria contrast with back ground and to make differentiation of cells and localized of cellular materials. The advantages of this procedure are that: 1. The cells are made more clearly visible when they are coloured. 2. Differences between cells of different species and within the same species can be demonstrated by use of appropriate staining solutions (differential of selective staining). The essential steps in the preparation of a fixed, stained smear are: (i) Preparation of the film or smear. (ii) Fixation and (iii) Application of one or more staining solutions. Microbiological stains A large number of coloured organic compounds (dyes) are available for staining microorganisms. These compounds are generally rather complex in terms of molecular structure. On this basis they may be classified into groups such as Triphenylmethane dyes, Oxazine dyes and thiazine dyes. Type of Stains Stains are of two types: (a) Natural stains: Stains which are used in histological and general studies of cells. (b) Synthetic stains: Stains which are used in Molecular biological studies. Synthetic stains are dyes having a benzene ring and auxaphore or chromophoric groups. A more practical classification for the cytological point of view is based on the chemical behaviour of the dye, namely. Acid, Basic or Neutral An acid (or anionic) dye is one in which the charge on the dye ion is negative. E.g. Nigrosine, Eosin, India ink.  A basic (or cataiornic) dye is one in which the charge carried by the dye ion is positive. E.g. Methylene blue, crystal violet  A neutral dye is a complex salt of a dye acid with a dye base e.g. Eosinate of methylene blue.  Acid dyes generally stain basic cells components and basic dyes generally stain acidic cell components. Basic Principle behind staining of microorganisms The process of staining may involve ion-exchange reactions between the stain and active sites at the surface of or within the cell. e.g. the coloured ions of the dye may replace other ions on cellular components. Contain chemical grouping of cell proteins or nucleic acids may be involved in salt formation with positively charged ions such as Na+ or K+.

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Thus these peripheral areas of the cell carry a negative charge in combination with positively charged ions. E.g. (Bacterial cell) (Na+) Basic Terminology Mordant It is a substance which increases the affinity or attraction between the cell and the dye. Decolourizing agent It is the substance which removes the dye from a stained cell Counter stain It is a basic dye of a different colour from the initial one. The purpose of the counter stain is to give the decolourized cells a colour contrast different from that of the initial stain. Staining of bacteria Following methods are commonly used. 1. Simple stain technique The coloration of bacteria by applying a single solution of stain to a fixed smear is termed simple staining the fixed smear is flooded with a dye solution for a specified period of time after which this solution is worked off with water and the slide bottle dye. The cells are usually staining uniformly. However, with same organisms, particularly when methylene blue is used, some granules in the interior of the cell may appear, more deeply stained than the rest of the cell, indicating a different type of chemical substance. Procedure for simple staining A small amount of bacteria is placed in a droplet of water on a glass slide, then it is air dried. The slide is passed through a flame in a process called heat fixing which fixes the cells to the slide, kills most organisms and prepares them for staining. Now the slide is flooded with a basic dye such as crystal violet or methylene for a minute or so. The positively charged dye is attached to the bacterial cytoplasm, which has a negative charge and staining taken place. This is effective for negative cells; the stains do not easily penetrate spores. Flow chart Simple Staining Bacterial Suspension

Smear Air Fixation Heat Crystal Violet

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Wash with water

Dry Observe under 100 × 2. Negative Stain Technique This technique works in a manner opposite to simple stain technique. Bacteria are mixed on a slide with an acidic dye such as cargored or black stain, nigrosine. The mixture is smeared on the face of the slide and allowed to air dry. Because the stain carries a negative charge it is repelled by the bacteria, which also has a negative charge the stain gathers around the cell. Since a chemical reaction has not taken place, and because heat fixing has been avoided, the cells appear less shriveled or distorted. They often appear larger than stained cells and more natural. Flow chart Negative staining Nigrosine Smear Fixation by air only Observe under 100 X 3. Gram stain technique A Danish scholar, Christain Gram in 1884 designed a differential staining procedure, which differentiates between two kinds of bacteria. Gram positive and gram negative bacteria. This procedure is called gram stain technique. Different steps of Gram stain technique are as follows. A thin smear of the bacteria is prepared on the slide. To the smear crystal violet solution is applied for 30 seconds. The slide is then gently rinsed in clear water and iodine solution is applied for 30 seconds. This is them rinsed off. Then 95% ethyl alcohol is applied and this is renewed until all but the thickest parts of the smear have ceased to give off the dye. This usually takes from 20 seconds to one minute. Microscopic examination of the slide will reveal that Gram positive bacteria retain the violet iodine combination (Retaining of blue purple colour even after alcohol wash). Where as Gram negative ones look the blue purple colour after alcohol wash and will be of original colour. The species which retain the stain are called gram positive where as that which yield the stain to alcohol are called gram negative. A counter stain a dye of same contrasting colour may be used to clearly visualize the gram negative cells. The generally used counter stain are

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Eosin (red.), Safranin (red) Brilliant green or Bismarck brown. Each of these colours the Gram negative species. Flow chart Gram Staining Smear Air Fixation Heat Crystal Violet (30 Sec.) Washing with water (K/K1+I2) Lugals iodine (30 sec) (mordant) Washing with water Alcohol (25-40 sec.) most important step. Washing (Basic dye) Safranine (40 sec.) Washing Dry Gram negative (purple)

Observe under 100 ×

Gram negative (Pink)

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Experiment No.4 Objective: To perform simple/ direct staining and indirect/ negative staining Requirements: Slides, Microscope, spirit lamp, inoculating loop, bacterial culture, stains etc. Background: In staining we used dyes to study the cell morphology. Bacterial cell wall is slightly negatively charged structure. So we used a basic dye for staining. Crystal violet is a basic dye which contains a positive charge. It gives a colour basic group. Bacteria contain a slightly negative charge on surface. Thus on treatment with basic dye it takes a colour. While negative stain also contain a negative charge so on treatment of bacteria with acidic negative stain it will not gives a colour to bacteria. But the back group of bacteria will observed as black colour and bacteria are seen due to white transparent structure. Type of staining (i) Simple staining (ii) Differential staining (iii) Structural staining (iv) Biochemical staining Procedure: I. Simple staining (Direct)

Bacterial suspension Dry Smear Air Fixation Heat Crystal violet (30 sec.) Wash Observe under 100 X
Observations:

Conclusion:

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Procedure: II. Negative/ Indirect staining Bacterial suspension Nigrosine Smear Air dry only Observe under 100 X Observations:

Conclusion:

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EXPERIMENT NO. 5 Objective: To perform gram staining Requirements: Bacterial culture, inoculating loop, sprit lamp, crystal violet, alcohol, water, lugal’s iodine, safranine, slide, etc. Background: Gram staining is differentail staining. The stains are those which enable one to differentiate two different groups of bacteria is a mixture for instance Gram positive and Gram negative. This method was devised by a Danish Scholar CHRISTIAN GRAM in 1884. Gram stain is differential stain and it clearly differentiates a gram +ve bacteria from a gram –ve bacteria. Gram positive bacteria contains a very low amount of lipid (10-15%) and Gram –ve contain a enter lipo protein membrane so lipid content are very high that’s why on treatment of bacterial cell from alcohol which is a organic solvent, dissolve all lipid from the membrane and make a holes in the wall that all stain will comes out and bacteria and bacteria will not take a stain. Procedure: Bacterial suspension Smear Air Fixation Heat Crystal violet (30 sec.) (Basic dye) Washing with H2O (Mordants) Lugal’s iodine (30 sec) Washing with H2O (Removed excess stain) Alcohol (25 – 40 sec) Washing with H2O (Basic dye) Wash with H2O Dry Observed under 100x Gram +ve (purple) Gram –ve (Pink) Saffranine

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Observations:

Conclusion:

Some examples: Gram staining for E. coli, S. lutea and Bacillus megaterium were performed. E. coli Pink S. lutea purple B. megatherium Gram – ve Gram +ve Gram +ve

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EXPERIMENT NO. 6 Objective: To perform bacterial spore staining. Requirements: Slides, Microscope, spirit lamp, inoculating loop, bacterial culture, stains etc. Background: 1. It is highly resistant due to thick spore wall the high resistance of spore is due to dipcolinic acid (DPA). 2. The vegetative cell is killed at 600C + spore not even at 1000C 3. Spore requires special stain malachite green Procedure: Smear Fixation by heat Put a piece of filter paper on smear, keep the slide over a steam bath and add malachite green at regular intervals so that filter paper is not dried Wash Safranin Dry Observe under 100 X Observations:

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2. NUTRITIONAL REQUIREMENTS FOR GROWTH OF BACTERIA (Principles of Microbial Nutrition) Every organism requires certain substances, drawn from their environment, for the synthesis of their cell materials and for the generation of energy. These substances are termed as nutrients. The nutrients preparation on or in which a culture (i.e. a population of microorganisms) is grown in the laboratory is called a culture medium. A culture medium must therefore contain specific requirements of the microorganisms for which it is designed i.e. all necessary nutrients required for the sustenance of life. Although all the micro-organisms have the same basis requirements but there is a diversity as to the use of organic and inorganic and composition, depending on the species to be cultivated. Thousands of different media have been proposed for their cultivation. Some media contain solutions of inorganic salts and may be supplemented with one or more organic compounds while other media contain complex ingredients such as extracts or digests of plant and animal tissues. DESIGN OF CULTURE MEDIUM SHOULD BE BASED ON SCIENTIFIC PRINCIPLES I.E. THE PRINCIPLES OF NUTRITION • All the required metabolic elements can be supplied as nutrients in the form of the cations of inorganic salts. • Specific mineral requirements, for example diatoms and certain algae have a specific silicon requirement, supplied as silicates. • Specific chemical form of carbon, nitrogen sulphur and oxygen has been provided depending on the need. • Now variety of nutritional patterns known to exist in the living world. • Nutritional classification is based on two parameters the nature of the energy source and the principle carbon source. Nutritional Classification With respect to energy source, there are two classes. Phototrophs Use light as a energy source i.e. photosynthetic organisms. Chemotrophs: Use chemical energy source with respect to carbon source. Autotrophs CO2 as a principle carbon source. Heterotrophs Organic carbon source There are four major nutritional categories by means of these criteria. (i) Photoautotrophs Light as energy source CO2 as principle C-source. These include photosynthetic organisms: Higher plant, algae, many photosynthetic bacteria. (ii) Photoheterotrophs Light as energy source, organic compounds as principal C-source e.g. Purple and blue-green bacteria. (iii) Chemoautotrophs Chemical energy source CO2 as principal C-source.

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Energy is obtained by the oxidation of reduced inorganic compounds such as NH3, NO2-H2, reduced form of sulfur CH2S of light, grown in mineral media strictly. (iv) Chemoheterotrophs Chemical energy source, organic substances as principal C-source e.g. all metazoan animals, protozoa, fungi and bacteria. The role of oxygen in nutrition Oxygen is universal components of cells and is always provided in large amounts in the major nutrient (e.g. water and organic compounds). Many organisms also require molecular oxygen (O2). Obligately aerobic Organisms that are dependent on aerobic respiration for the fulfillment of their energetic needs and for which molecular O2 functions as a terminal oxidizing agents. Obligately anaerobic Do not require molecular oxygen to obtain energy. Molecular O 2 is a toxin substance which either kills them or inhibits their growth. Facultative anaerobes Some micro-organisms able to grow either in the presence or absence of O2 e.g. lactic acid bacteria (Anaerobic to aerobic in presence of O 2, fermentative to respiratory), yeast (Aerobic to anaerobic, respiratory to fermentative, energy yielding metabolism). Microaerophilic Some micro-organisms grow best at partial pressure of O2 below 0.2 atomosphere. Nutritional categories among micro-organisms Two principal nutritional classes among organisms. Autotrophs: Which can completely use in-organic nutrients e.g. plants. Heterotrophs: Which require organic nutrients e.g. animals. Osmotrophs: Take up all nutrients in dissolves form Phagotrophs Take up solid food particles by the mechanisms termed phagocytosis. The terms obligate and facultative are often used to indicate the absence (or presence of nutritional versatility). Additional pair of terms Prototrophs If a bacterium can grow in a minimal medium that is, it an synthesize all necessary organic substances such as amino acids, vitamins and lipids from simple precursors the bacterium is called a prototroph i.e. it can derive all carbon requirements from the principal (source) Auxotroph If any organic substance other than a carbon source must be added for growth to occur, the bacterium is called an auxotroph i.e. it requires one or more organic nutrients in addition to principal carbon source e.g. growth factors.

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For example, if the amino acid praline is required in the growth medium. The bacterium is praline auxotroph; the genetic symbol for a bacterium with this phenotype is pro- A bacterium that doest not require praline would be designated pro+. The Methods of Microbiology Cultures Growing micro-organisms under more or less well defined conditions. Pure or Axenic culture A culture that contains only one kind of microorganisms. Mixed culture A culture that contains more than one kinds of micro-organisms Two membered culture If it contains only two kinds of micro-organisms and maintained deliberately in association with one another Preparation of pure culture involves not only the isolation of a given micro-organisms from a mixed natural microbial population but also the maintenance of the isolated individual and its progeny in a artificial environment. In microbiology, we follow two kinds of operations. i) Isolation Separation of a particular micoroganisms from the mixed population that exist in nature. ii) Cultivation The growth of microbial population in artificial environments (culture media under laboratory conditions). The terms obligate and facultative are often used to indicate the absence (or presence of nutritional versatility). Additional pair of terms Prototroph If a bacterium can grow in a minimal medium that is, it an synthesize all necessary organic substances such as amino acids, vitamins and lipids from simple precursors the bacterium is called a prototroph i.e. it can derive all carbon requirements from the principal (source) Auxotroph If anyorganic substance other than a carbon source must be added for growth to occur, the bacterium is called an auxotroph i.e. it requires one or more organic nutrients in addition to principal carbon source e.g. growth factors. For example, if the amino acid praline is required in the growth medium. The bacterium is praline auxotroph; the genetic symbol for a bacterium with this phenotype is pro-A bacterium that doest not require praline would be designated pro+. The methods of Microbiology Cultures Growing micro-organisms under more or less well defined conditions. Pure or axenic culture A culture that contains only one kind of microorganisms Mixed culture A culture that contains more than one kinds of microorganisms Two membered culture

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If it contains only two kinds of micro-organisms and maintained deliberately in association with one another Preparation of pure culture involves not only the isolation of a given microorganisms from a mixed natural microbial population but also the maintenance of the isolated individual and its progeny in a artificial environment. In microbiology, we follow two kinds of operations. i) Isolation The growth of microbial population in artificial environments (culture media under laboratory conditions). These two operations are generally used for the study of virus, bacteria, fungi, algae, protozoa and even small invertebrate animals as well as isolation of cell or tissue lines derived from higher plants and animals (tissue culture). Three kinds of containers namely a test tube, a flask or a petridish are most commonly used to cultivate microorganisms. Inoculum The microbial material used to seed or inoculate a culture vessel with special precaution to maintain the purity of the culture. Inoculum is introduced on a metal wire or loop which is rapidly sterilized just before its use by heating in a flame. Plating: In lab jargon, a petridish containing a solid medium is called a plate and the act of depositing bacteria on the agar surface is called plating. Aseptic technique Inoculation of sterile medium with a pure culture of micro-organisms without contamination is commonly referred to as aseptic technique. Growth Media or Culture Media Construction of culture media The primary goal of construction of culture media is to provide a balanced mixture of the required nutrients in optimum concentration as well as favourable environment for attaining good growth. Two basic requirements for construction of media are: ii) The culture medium must contain those nutrients essential for the growth of a given microbial culture. iii) The medium must at the same time provide suitable surroundings for growth that is, the proper pH, osmotic pressure, atmospheric oxygen and so on. Requirement for carbon Photosynthetic organism of bacteria uses oxidized form of carbon (CO2) as the sole principal source of cellular carbon. Reductive CO2 = --------------- Organic cell constituents Process (Net input of energy) All other organisms obtain carbon principally from organic nutrients and they must supply the energetic requirements of the cell. Organic substrates thus have a dual nutritional role as they serve as a source of carbon and as source and energy are carbohydrate and its derivatives, fatty acids, dicarboxylic acids, other organic acids, primary alcohols (ethanol, propanol,

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butanol), Amino acids, other nitrogenous compounds and nitrogen-free ring compounds. Microorganisms can use single organic compound and variable number of additional organic compounds for their purely biosynthetic function. These addition organic compounds termed as growth factors. Requirement for growth factor (s) These must be provided as nutrients. Any compounds that an organism requires as a precursor or constituent of its organic cell material and can not synthesize from simpler carbon sources are collectively known as growth factors. They fall into three classes (in terms of chemical structure and metabolic function). 1) Amino acids-required as constituents of proteins 2) Purines and pyrimidines-required as constituents of nucleic acids 3) Vitamins-a diverse collection of organic compounds of certain enzymes that form parts of the prosthetic groups are active centres. Requirement for Nitrogen and Sulphur Nitrogen and sulphur occur in the organic compounds principally in reduced form as amino acids and SH groups. • Photosynthetic organisms and non-photosynthetic organisms (bacteria and fungi) assimilate nitrates and sulfates as a source of nitrogen and sulfur. • Some microorganisms are unable to bring about reduction and this must be supplied in the reduced form as ammonium salts. Sulfur is provided as sulfide or organic compound containing a –SH group e.g. cysteine. • N and S requirements can be met by organic nutrients that contain two elements in reduced organic combination (amino acids or more complex protein degradation products such as peptones). Such compounds also provide carbon and energy sources. • Some bacteria can also utilize the most abundant natural N2 source and assimilated by N2 fixation. It involves preliminary reduction of N2 → NH3 e.g. Rhizobium and other N2-fixing bacteria. Requirement for favorable conditions The control of pH A given medium may be suitable for the inhibition of growth. Microbial decomposition and utilization of compounds cause alterations in pH and may become inhibitory. Thus, to prevent excessive change in ion concentration, buffers are used e.g. phosphate buffers, bicarbonate buffers etc. Avoidance of mineral precipitate In culture medium mineral precipitation can be avoided by the addition of chelating agents e.g. EDTA. Control of O2 For obligate aerobic bacteria, O2 is an essential nutrient. It is therefore necessary to aerate the medium of liquid cultures by continuous passage of a stream of air through culture.

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The anaerobes which are killed by molecular O2 can be cultivated by using following techniques: - Use of pre-reduced media by inclusion of reducing agents as cysteine, thioglycollates, Na2S or sodium ascorbate which can be protected from exposure to air. - By evacuation of O2 and refilling with an inert gas e.g. N2. - By chemical destruction of O2 - By combination of both methods Provision of CO2 For photo autotrophs and chaemoautotrophs CO2 is required for their growth which may be provided by diffusion of CO2 from the atmosphere into the culture medium and by incorporation of NaHCO3 in the medium. Provision of light For phototrophic organisms (e.g. algae, photosynthetic bacteria) light is an essential requirement. This may be provided by: - Adequate illumination combined by control of temperature. - Direct exposure to sub light should be avoided. - Discontinuous illumination - Fluorescent light has advantages. - 750-1000 nm wave length of light is suitable for purple and blue green bacteria. Essentially all culture media are one or the other of two forms - Liquid or broth media and solid media. On the basis of their composition there are two main types of culture media. A) Synthetic medium A medium composed of entirely of chemically defined nutrients of special substances of known composition. It is also known as chemically defined culture medium. Synthetic medium are of four types. Enriched medium Used for a general purpose medium for a wide variety of microorganisms Selective media Used for a selected microbe the selective media are those which permit the growth of some specific group or type of organisms while preventing the growth of others, thus facilitating bacterial isolation. The selective action is brought about by the addition of certain chemicals in the medium e.g. the dye crystal violet is selectively bacteriostatic for grampositive bacteria and is added into a selective medium and is added into a selective medium for examination of enteries pathogens which are predominantly gram-negative e.g. Mannitol-salt agar. Differential medium Used for differential isolation of microbes. A differential medium is that which will cause certain colonies to develop differentially (i.e. differentiated) from other organisms present by producing a characteristic change in the bacterial growth and/or the medium surrounding the colonies. These media are used for distinguishing among morphologically. These media are used for distinguishing among morphologically and biochemcially related groups of organisms e.g. blood agar.

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Assay medium Used for the assay of vitamins, amino acids and antibiotics. Roulin’s solution and Martin’s rose Bengal medium. B) Complex medium (Natural or empirical culture media) that contains ingredients of unknown chemical composition. It is used for the cultivation of wide range or microorganisms. The natural media include milk, urine, diluted blood, vegetable juices, meat extracts and infusions, peptone (digested protein rich in amino acids that provide nitrogen and carbon). Now-a-days, group of living cells as tissues or callus or an organ are being used for viruses and rickettsias. Those media whose chemical composition is partially known are called semi-synthetic media. Any medium which contains agar becomes a semisynthetic medium potato dextrose agar. Czapek-Dox agar, oatned agar, beef peptone and nutrient. These agar media are some of the semi-synthetic media. Several media are available for use of microbiology (Table summarizes properties of some of the commonly used media for growing various microorganisms). Minimal medium If the growth medium contains no organic compounds other than a carbon source (such as sugar), it is called a minimal medium. A typical minimal medium contains the ions, Na+, K+, Mg++, Fe++, NH3++, Cl-, phosphate buffered to neutral pH and SO4-2, and a source of carbon such as glucose or glycerol. Other metal ions are also required but in such small quantities that they are usually provided as contaminants in the other salts used to prepare the medium. The best source of carbon for most bacteria is glucose than with any other single carbon source. Classification 1. Media may be grouped principally as follows: Those media that require living cells or tissues the latter get parasitized by them organisms to be cultured. Those media that do not require living cells or tissues. The media attain divided to two: a) Non-synthetic Media A medium in which the exact chemical composition of each of the constituent is not certainly known is referred to as non synthetic medium. Potato dextrose agar (GM-25) Soil Extract Agar (SM-1) Oat Meal Agar (GM-24) Malt extract agar (GM-19) Waksman medium (GM-40) b) Synthetic media A medium is which pure only chemicals in definite concentrations are used is called synthetic medium. These media are useful for nutritional and metabolic studies. Czakek’s Dox medium (GM-9) Richard’s medium (GM-27)

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Media may also be divided into 4 categories on the basis of consistency i.e. their physical state; they are: a) Liquid media b) Semisolid media c) Liquefiable solid media d) Solid media. b) Liquid Media These media are used in liquid form e.g. Nutrient broth Skimmed milk Peptone solution etc. Agar are some of the semi-synthetic media. c. Semisolid media These media contain a smaller amount (0.5% or less) or agar which imparts a “custard consistency” e.g. cystine Trypticase Agar medium d Liquefiable solid media This medium is also called ‘solid reversible to liquid medium’ these media are prepared by adding suitable amount of gelatin or agar to the liquid medium and remain solid when cool but become liquid when warm. e.g. Nutrient agar medium Nutrient Gelatin medium Potato dextrose agar medium e. Solid media These media always remain solid e.g. Potato slices Coagulated Blood serum Coagulated Egg Trypticase “Soy-Agar Medium” 3. Media classified on the basis of their application or function as follows: a. Cultivation media b. Storage media c. Enrichment media d. Differential media e. Assay media f. Maintenance media a. Cultivation media Medium which is used for the general cultivation of bacteria e.g. e.g. Nutrient broth Nutrient Agar b. Storage media Medium in which bacteria are stored in “Stock culture” condition for longer periods to provide a source of viable culture are called storage medium. e.g. Yeast Extract Mannitol Agar Medium

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c. Enrichement media Enrichment medium is that in which nutritional environment is adjusted in such a manner as to enhance selectively the growth of a certain bacterial type with in a given mixed inoculums. e.g. Addition of extracts of plant-animal tissues to nutrient broth of nutrient agar media provides add d. Differential media e. Assay media Certain media of prescribed composition have profound influence on the bacterial cell with respect to formation of enzyme, toxin antibiotics are other products. e.g. Pyridoxine deficient growth medium f. Maintenance media These media are different from growth media and are required to maintain the viability and physiological characteristics of bacteria. DEFINITION OF TERMS 1. Inoculum Small amount of microbial culture used to inoculate a culture vessel for the growth of the microorganisms in the culture under define condition. 2. Subculture/ Passaging Transferring culture from one vessel to another under aseptic condition. 3. Culture Growing microorganisms under desired condition. 4. Axenic culture = pure culture Growth of single microorganisms in the desired culture medium 5. Mixed culture Many microorganisms in a culture medium 6. Cultivation Growth of the isolated single microorganisms under desired conditions.

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Experiment No. 7 Objective: To prepare nutrient broth, nutrient agar, agar slant, agar stab and potato dextrose agar (P.D.A.) Requirements: Measuring flask, distilled water, balances, peptone, beef extract, potato, agar agar, pH paper, autoclave etc. Procedure: 1. Nutrient broth Peptone 0.5% Beef extract 0.3% Final pH 7.0 2. Nutrient agar Peptone 0.5% Beef extract 0.3% Agar-agar 2% 3. Potato dextrose agar 200 gm of peeled potatoes cut into chips + 500 ml distilled water, boil for 30 min and make up the volume upto 1000 ml. Add 20 gm dextrose + 20 gm agar-agar powder and sterilize at 15 lb/sq inch for 20 minutes. Page 138 Preparation of nutrient media

Observations:

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Experiment No. 8 Objective: To perform streak plate in order to obtained pure colonies of bacteria. Requirements: Petridishes, nutrient agar, inoculating loop, bacterial culture, incubator, laminar flow, spirit lamp etc. Procedure: - Melt sterilized agar medium in a boiling water bath and cool to 45 0C: pour the medium on to Petri dishes at least 12-15 ml is each: distribute the medium throughout the dishes uniformly and let the medium solidify. Do all this in aseptic conditions. - Take an inoculation needle, sterilize it in flame and then cool by jabbing it on to the agar. - Take a small amount of the mixed culture at the tip of the needle and start streaking on the surface of the medium. - Streak several petridish-media from the same sample in different fashions. - Incubate petridishes at 300C for two days in an inverted position. This results in the growth and multiplication of the inoculated microorganisms in the form of isolated colonies. - After 2 days of incubation, you will observe that the growing colonies are more and more sparsely distributed towards the streak and. Pick up well separated colonies and transfer than to agar slants as pure culture. Observations:

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Experiment No. 9 Objective: Inoculation of pure culture of E. coli and Bacillus species in Nutrient broth. Requirements: Nutrient broth, bacterial culture, conical flask, laminar flow, spirit lamp, inoculating loop. Procedure: 1. 10 ml of nutrient broth (sterilized) is taken is a sterilized conical flask. 2. 1 ml of culture of E. coli and Bacillus species are aseptically added to the 10 ml nutrient broth is laminar flow in front of flame. 3. Conical flask is corked and properly labeled. Page no 116 Inoculation procedure 1 and 2 Observations:

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Experiment No. 10 Objective: To perform the pour plate and serial dilution and count TVC Requirements: Nutrient agar, sterilized test tube and water, bacterial culture, petridish, etc. Procedure: Make dilution of isolated bacteria by serial dilution method upto 10-6 and then from last 3 dilutions 10-1, 10-5, 10-6. Make plates by pour plating. Observations:

Examples: 1. Dilution 10-6 1st plate 2nd plate Mean No. of cells 2. Dilution 10-5 1st plate 2nd plate Mean No. of cells 3. dilution 10-4 1st plate 2nd plate Mean No. of cells 18 colonies 20 colonies 19 19 × 106 cells/ml 61 56 57.5 57.5 × 105 cells/ml 256 colonies 288 colonies 277 277 × 104 cells/ml

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Experiment No. 11 Objective: To perform selective and differential plating Requirements: Beaker, distilled water, reagent autoclave, cotton, aluminum foil etc. Procedure: 1. Differential Medium (bromocresol purple carbohydrate medium) Tryptone 10 gm Lactose 5 gm Yeast 5 gm K2HPO4 2 gm Indicator 2 ml Agar-Agar 20 gm distilled water 1000 ml 2. Selective media (a) E.M.B. Agar Peptone 10 gm Lactose 5 gm Sucrose 2 gm K2HPO4 2 gm Eosin yellow 0.4 gm Methylene blue 0.06 gm Agar 20gm Distilled water 1000 ml (b) Endo Agar Peptone 10 gm Lactose 10 gm K2HPO4 3 gm Na2SO4 2.5 gm Basic fuschin 0.5 gm Agar 20 gm Distilled water 1000 ml Observations:

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3. Study of Bacterial Growth Introduction Microoraganisms exerts a great impact on natural ecosystem & human affairs. Their role in transformation of organic matter and effect of ecosystem was recognized only after 19th century. The current chemical state of the elements on the earth’s outer surface is, to a considerable extent, a consequence of chemical activities of living organisms. The quantity of various gases in atmosphere and many other compounds on the earth’s surface represents the net balance between their rates of formations and utilization in biological & Geological processes. Such transformations occur in all regions of Biosphere. The composition of Biosphere is in more or less in static state, maintained by a cyclic turnover of chemicals necessary for life, powered by continuous input of Energy from the sun. The various steps in the cyclic turnover of elements are brought about by different types of organisms. Thus continued existence of any particular group of organisms depends on the chemical transformations carried out by others. A break in the cycle at any point would cause devastating effects on all life forms. All organisms participate in various steps of the cyclic conversions, but contribution of microorganisms is particularly important, both quantitatively & qualitatively. There is over whelming contribution of microorganisms to these processes of nature, which reflects the unequity of Microorganisms, their significant contribution to the bulk of living material i.e. their biomass, their high rates of growth and metabolism. Their collective ability to degrade a vast variety of naturally occurring organic matter into inorganic forms, which is again utilized by photosynthetic organisms. Only minority of microbial species are pathogenic, i.e. they can proliferate in human tissues causing infection and several disease. The discovery that bacteria can act as a specific agents of infectious disease was made through the study of Anthrax, a serious infection of domestic animals that is transmissible to humans, and these discoveries led to isolation, cultivation and characterization of the causative agents for the major infectious disease to man. Microbial processes have been used by human since prehistoric times in the preparation of food, drinks, and textile. The increase in knowledge about microorganisms, led to the improvement in many of them, but also to the development of entirely new industries based on the use of microorganisms that had previously not been exploited by humans. It will not be an exaggeration to state that there is no life possible without microoganisms. They are closely associated in major field like Agriculture, Industries (like food processing), pharmaceutical etc. , lot of research work is

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continuing with the help of various species of microorganisms, one of the emerging field is Biotechnology. But because of the very small size of microorganisms it is difficult to quantify and qualify them. Therefore special techniques are developed for this purpose, the first the foremost thing to do in this respect is the isolation of microorganisms in pure culture. Once it has been isolated in pure culture every aspect about them can be estimated? The following discussion is an attempt to explain the methods used for the measurement of microbial growth, together with various phases and modes of growth. 3. Bacterial Growth Each group of organisms has to adapt itself during its evolution not only on the nonliving environment, but also to the environment sometimes involves the acquisition of special metabolic capacities that endow their possessor of progeny with the unique ability to occupy a particular physicochemical niche, and existing into a unique physiochemical Niche is a highly effective means of meeting the challenges of biological competition. Thus, the microorganisms have vast diversity. From different natural environment different types of organisms can be isolated by using specialized techniques in pure culture and their growth in the pure culture could be monitored by applying. a) Optimal conditions pH = 7.0 temp = 370C and constant supply of nutrients b) By applying stress 3.1.1 Chemical stress Growth in presence of antibiotics, and thereby isolating mutant’s resistant to a particular antibiotic or drug. 3.1.2 Physical stress Growth of bacterial culture after they are given exposure to ultra violet radiation for various time intervals. Finally, to isolate U.V. radiation resistant strain???????????????????. 3.1.3 Environmental Stress  Effect of temperature  Effect of pH  Effect of osmotic pressure (by varying salt concentration)  Change in concentration of Glucose  Growth in the absence of oxygen (isolation of anaerobic bacteria) When we provide microorganisms optimal conditions of growth i.e. nutrients, physiological pH, temperature etc. They are in a state of balanced growth i.e. the rate of increase of all the components of the population is same (e.g. proteins, DNA, RNA etc.), or we could say that bacteria follows a definite growth kinetics once they are adapted to particular environmental conditions. The Bacterial culture undergoing a balanced growth, mimic a 1st Order chemical reaction. E.g. of other 1st order reactions are a) Nuclear reaction

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b) Human population growth I the 1st order reaction, the rate of chemical reaction in directly proportional to the concentration of the reactants or amount of substrate present at any given time. For bacterial growth, the substrate will be total no of viable bacterial cells. The chemical equation will be of the form: dN ---- α No. dt where, No. = no. or concentration of bacterial cells (viable) present at any given time. dN ---- = Rate of increase of bacterial cells in the culture media dt dN ---- = K No. – Equation (1) dt where K = constant of proportionality upon intetration of equation (1), we get

dN --- = K dt dt or log e N = Kt + C – Equation (2) (1/x dx = log x + C) Initially at t = 0 No. of bacterial cells = No. Putting value of t and no. in Equation 2 we get Log eN = 0+C = C = log e N = 0+ C = C = log e N Substituting the value of (C) in equation (2), we get log ↓ e N = Kt + log e No. N Or log e --- Kt ----- (3) No↓ ( log x – log y – log x/y) or K = 2.303/t log 10 N / No. ----- (4) (loge X = 2.303 log 10x) where K = rate constant for bacterial growth = from equation (3) we could write N --- = eKt No. kt Or N = No. e - ----- (5)

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Here, the equation (5) show’s that the no. of bacterial cells increases exponentially with respect to time, therefore, if we plot no. of bacterial cells vs. time, the curve to be obtained should be an exponential curve for the bacterial growth. 3.2 Doubling time Also known as mean doubling time or generation time. It is defined as the time required by the bacterial cell to double its concentration or amount. At time t1 = no. of cell present are no. And at time t2 = number of bacterial cells doubles its original concentration i.e. 2 No. Then t2 – t1 = tg Generation time of doubling time. Putting values of initial and final concentration of bacterial cells in equation (4). 2.303 N Or K = ------ log 10 ---t No. N = 2 No. t = tg 2.303 2 N Or K = ------ log ------tg No. 2.303 K = ------ log 102 tg 0.693 K = -------tg 0.693 or tg = -------…… (5) tg tg = generation time or mean doubling time. Proceed to calculate (1) The growth rate constant (K) of the culture. (2) The mean doubling time = tg 2.303 N equation (4) K = ------- log ----tg No Where t = Time taken by bacterial cells from increasing their concentration from No. to n cells. N = May be taken as the concentration of cells/ml But in the actual performance of this experiment different types of curve’s will be encountered with several phases. Two curves (A) and (B) depict the different phases of microbial growth in Nutrient media which depends on the nature of bacteria. Particularly whether bacteria are spore former or non spore former. Curve A

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Curve B

The curve A & B are drawn by plotting the growth with respect to time for two different types of organisms in which their initial concentration in the culture media is represented by No. This behavior is explained as follows: 3.3 Bacterial growth curve 1) Region A – B : log phase 2) Region B – C : Exponential growth phase or logarithmic phase (log phase) 3) Region C – D : Stationary phase 4) Region D – E : Decline or death phase (only in curve B) Between each phase there is a transition phase in the curved region also called as Transitional periods. When a culture proceeds from one phase of growth to another it does not implies that all the cells are in identical physiological conditions (i.e. some cells are remaining in original state of growth). Some time is needed for the one logging behind to catch up with the others which have entered a new phase. 3.3.1 Log period (phase) Bacterial cells that are transferred from a natural source or from a culture which was previously under the stationary phase, undergo a change of chemical composition, before they are capable of initiating growth. This period of adjustment is called log phase. The period of adjustment implies that the bacterial cells are undergoing some metabolic changes. For e.g. bacterial cell in this new environment may be deficient in enzymes or co-enzymes which are needed for optimal operation of cells chemical machinery. If cells are picked from natural environment where they were facing biological competition with other microorganisms they will take some time to be adjusted in new environment. The organisms are metabolizing but there is log in cell division. This log phases is extremely variable in duration and its length is directly related to the duration of the proceeding stationary phase. Greater is the stationary phase of the culture, greater will be the log phase when the organism is grown in fresh medium. 3.3.2 Log phase Once the bacterial cells get adapted to the growth conditions, it divides steadily at a constant exponential rate. Different strains of bacteria or different

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species have got different growth rates and generation time (or mean doubling time). As given by equation (5)] N = No ekt The cells growing according to given equation are said to be in state of exponential growth or logarithmic growth. It is also known as log phase. Log e N = kt + log e No. Kt Log10 N = ---------- + log10 No. ---(6) 2.303 This equation is similar to equation of straight line. Y = mx + c On plotting the graph between log N and time we obtain a straight line whose slope is K/2.303.  During log phase, the cells are in actively dividing conditions, as such their machinery is operating at a maximum potential, hence these enormous structural and physiological changes in cells are harvested at different times. This phase is however the most uniform phase and is commonly used for studies of microbial metabolisms. To obtain better results, the experiments should be performed only from a culture which is in logarithmic phase of growth. 3.3.3 Stationary phase Microbial population seldom maintains the exponential growth at high rates for log. The reason for this behaviour is obvious when one consider, the consequence of exponential growth e.g. after 48 hr of exponential growth, a single bacterium weighting about 10-12 g. Having doubling time of 20 min. the progeny produced has the mass = 2.2×1031 g. This is nearly 4000 times the weight of earth.  The growth of bacterial population is normally limited by two factors (1) either by exhaustion of available nutrients or (2) by accumulation of toxic products of metabolism as a result the growth declines and stops after a transitional phase, giving the characteristics horizontal line on the graph. Bacterial population remains constant with time.  The transition between the exponential phase and stationary phase involves a period of unbalanced growth during which various cellular components are synthesized at unequal rates, consequently cells in the stationary phase have a chemical composition that is different from the cells is the exponential phase and it depends on growth limiting factors.  The cells in stationary phase are small as compared to cells in exponential phase.  (cell division continues after increase in mass has stopped) and they are more resistant to adverse physical (heat, pH, radiation) and chemical agents. 3.3.4 Death Phase Bacterial cells which area held in a non growing state for a longer time eventually die. Death results from a no. of factors. Important one’s are: a) depletion of essential nutrients or the cellular reserves of energy. b) Accumulation of inhibitory products or toxins.

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 Like growth death is also an exponential function and hence death is an exponential decrease in the number of viable cells with time. The death of bacterial cells is highly variable depending on.  Environment and particular organisms, for Enteric bacteria die very slowly, while vegetative cells of certain Bacillus spp. die rapidly.  In spore forming bacteria (curve A) the death of all the cells don’t take place because one the vegetative cells find some hindrance for growth they form spores, which are metabolically inactive and can survive or exist under adverse circumstances. Vegetative cells Endospore Spore (metabolically inactive) 3.4 Synchronous growth culture So far, growth pattern of population’s of bacteria have been described but from such studies we are unable to conclude about the growth behaviour of individual cells, because the distribution of the cell size (and hence the cell age) in most bacterial cultures is completely random. Information about growth behaviour of individual bacteria can be obtained by the study of the synchronous cultures. These cultures are composed of cells that are all at the same stage of cell cycle, and measurements made on such cultures are equivalent to measurements made on individual cells. 3.5 Continuous Growth culture If nutrients are not renewed the growth remains exponential for only a few generation and after that stationary phase arrives. Such types of cultures are called batch cultures. Microbial population can be maintained in a state of exponential growth over a long period of time by using a system of continuous culture. Here, the growth chamber is connected to a reservoir of sterile nutrient medium, once the growth has been initiated; fresh medium is continuously supplied from the reservoir. The volume of the liquid in the growth chamber is maintained constant by allowing excess volume to be removed, continuously through a siphon overflow. The condition to be maintained is Rate of production of cells = rate of loss of cells through growth through overflow 3.6 Chemostat and Turbidostats Continuous culture system can be operated as chemostats or as turbidostats. In a chemostat the flow rate of nutrient media from reservoirs is set at a particular value and the rate of growth of the culture adjusts to this flow rate. 3.7 Growth Measurement Bacterial growth in a culture can be measured on the basis of different properties, some of the basic principles that are most commonly employed for bacterial cells enumerations are as follow: 3.7.1 Direct counts 3.7.2 Colony counts 3.7.3 Most probable numbers

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3.7.4 Biomass measurement 3.7.5 Light scattering (i.e. by absorption spectroscopy) 3.7.6 Chemical estimation of cellular components 3.7.1 Direct counts These culture methods measure viable cells that grow in the media provided. There are a number of methods that measure directly all cells in a sample, living or dead. These direct methods are not as precise as culture methods, but they are less time consuming. The few common methods are given below: 3.7.1.1 Microscopic Enumeration In this methods are measured volume of a sample is spread over a given area of a slide. By making a microbial count in a known area and multiplying by the appropriate factor, we can calculate the number of organisms in the original sample. There are special counting chambers e.g. the Petroff-Hausere slide that has cavities of known depth and volume, marked off into squared areas. The no. of organisms in an area is counted and the total number of bacteria in the sample can be calculated from the proportion of the total volume known to be contained in the area. The thickness of the fluid filling the squares is measured by focusing on bacteria attached on top and bottom interfaces with the help of micrometer scale in the microscope. In bred method a known volume of sample is spread over an area of one sq. cm on a slide and the film is dried, fixed and stained. The number of microorganisms in a microscopic field are then determined and multiplied with the area, same method is applied in Hawksly chamber. 3.7.1.2 Electronic Enumeration Bacterial cells or particles may also be counted electronically the help of counter. A sample is passed through a small orifice and from the measured change in electrical resistance, not only the no. of particles, but also their size can be determined. However, since this method measures any particle in a sample, it is applicable only to aqueous suspensions of microorganisms. Besides this there are certain other problems as well like production of pulse comparable to noise produced in the instrument causing conclusion, failure of cells to separate promptly from each other division, clogging of orifice etc. 3.7.1.3 Flow cytometery It is a common instrument in hospitals, laboratories etc. a small diameter stream of cells is encased in the stream of the fluid that is narrower. The flowing stream is examined with laser light of different frequencies and angles and the output of measurement circuits is used to detect when a particle passes through. The design permits analysis of biomass. 3.7.2 Colony counts The most common method of measuring the cell numbers is a plate or colony count. This method is based on the theoretical relationship of one bacterial cell giving rise to single colony and on the assumption that the no. of colonies that develop on the agar plate corresponds to the original bacterial count. There are various methods for counting these viable cells, or colony forming units (CFU). 3.7.2.1 Dilution count

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It used a serial dilution of a sample from which the dilutions are inoculated into a series of tubes of liquid medium instead of being plated out. On incubation, all tubes containing cells that have grown will be turbid, and some tubes in which no growth takes place will be clear. From the highest dilution of a sample that shows growth, the population is measured. Distilled or tap water alone, usually should not be used because it is osmotically hypotonic to all bacterial cells. A common general purpose diluents is phosphate buffered saline with a protein usually gelatin or peptone. 3.7.2.2 Spread plate method In this method, sample is pipetted onto the surface of solidified agar medium in a petridish and the cells are distributed with a spreader. Optimum dilution (so that countable colonies appear on agar surface) and temperature for isolated colonies and growth is needed. All bacterial colonies are on the agar surface. 3.7.2.3 layered plate method In this method an additional layer of agar medium is poured over a softagar layer, containing the cells so that all the colonies are subsurfaced. 3.7.2.4 Pour plate method In this method optimally diluted sample of bacterial cell is pipetted into an empty sterile petriplate. Then molten agar at temperature of about 450C is poured onto the sample, the contents are mixed by swirling and then the agar is cooled to harden. The colonies of bacteria are formed after incubation which are not in the same plane of agar surface. 3.7.3 Most Probable number The concentration of viable cells can be roughly estimated by the MPN method, also called the fraction negative method. It involves the mathematical influence of the viable count from the fraction of multiple cultures that fail to show growth in a series of dilution tubes, containing a suitable inoculum. It involves making a number of dilutions in growth medium, and taking into considerations the one’s not showing growth. The distribution of cells show poission ration. The mean no. plated at this dilution can be calculated from the formulae. Po = e-m where m = mean number Po = ratio of number of tubes with no. growth to the total number of tubes. 3.7.4 Biomass measurement A measure of the mass of bacterial cell constituents is frequently used as the unit, for measurement of metabolic activity of morphological or chemical constituents, i.e. “biomass” and cell numbers promote two basic parameters of bacterial growth. 3.7.4.1 Wet weight A nominal weight of bacterial cells originally in liquid suspensions is obtained by weighting a sample in a pan, after separating and washing the cells by filtration or centrifugation. A method for obtaining the actual wet weight of the cells themselves is to correct the nominal wet weight of a packed cell pellet by subtracting the experimentally determined weight of

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diluents in interspatial space. Use of nonionic polymeric solute e.g. dextrans is needed. 3.7.4.2 Dry weight The dry weight of cell from a given volume of growth medium may be determined as a measure of cell mass for such measurements. The culture is harvested either by centrifugation or filtration, the cells are washed (to make them free from medium), and their weight are determined after drying. 3.7.4.3 Water content The amount of cell water in fully hydrated cells is equal to the difference between the wet weight and the dry weight of cells. 3.7.4.4 Cell volume It is determined by placing a standard quantity of liquid culture in a calibrated centrifuge tube and measuring the volume of wet pellet after centrifugation. 3.7.4.5 Wet and dry densities The densities of bacterial cells may be obtained as either the wet density, based on the total amount of solid and water content’s or the dry density based on solid content. Both densities are expressed in weight/ volume. The wet density may be obtained by dividing the cell wet weight by cell dry volume. 3.7.5 Light Scattering They are techniques most generally used to monitor the growth of pure culture. They can be performed quickly are non-destructive. 3.7.5.1 Turbidimetric method It requires used of spectrophotometer to measure turbidity. In this technique the sample is taken in a cuvate and the light of particular wavelength is made incident light absorbed by a solution at a given wavelength is related to the thickness of the absorbing layer (i.e. path length) and the concentration of absorbing species. These two relationships are integrated into Beer Lambart Law. I0 Absorbency = log ---- = ecl. I I0 = I = are incident and transmitted light intensity respectively e = Molar extinction coefficient c= concentration of solute e = path length The assumption that is made is that the incident light is parallel and monochromatic and the solvent molecules are randomly oriented. The expression log I0/I is called absorbance and also designated as A. For bacterial culture before finding the concentration of cells in nutrient media. 1. Preparation of a blank, which is used as a reference to calibrate the spectrophotometer. Use distilled water as a blank. 2. Then calibrate the instrument for 100% transmittance, after adjusting the zero knob to zero when switch is not pressed.

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3. Take the % transmittance for the nutrient broth containing no bacterial cells (i.e. an autoclaved one). Finally calculate the % transmittency for the bacterial culture (after proper shaking so hat there is a random distribution of bacterial cells). 4. Then subtract two optical density readings, to find O.D. for bacterial cells at a particular concentration. O.D. = 2 – log T Where, O.D. = optical density or absorbance T= Transmittency of light (which is measured by the photo cell directly) The graph of optical density of absorbance vs. time can be plotted to obtain the curve for bacterial growth in the culture. 3.7.5.2 Nephelometry Although most of the light scattered by bacteria is nearly in the forward direction in the transmitted light is measured, instruments that measure the light scattered at 900C from the primary beam have also bean used to measure bacterial concentration of scattered light at right angles are called as Nephelometers. Rest all the principle’s used in a Nephelometer is same as those of a turbidimeter. 3.7.6 Chemical Estimates Chemical estimates of cell masses are made by measuring the amount of some components characteristics of cells, for example cellular nitrogen, protein, DNA, RNA or phosphorus. Under standardized condition, the amount of these components provides a reasonably accurate determination of total protoplast in a culture. 3.8 TOTAL VIABLE COUNT It gives the total no. of organisms that are living in a given culture. This can be calculated by using the procedures already discussed. The most common is plating methods, either pour plating or surface spreading of media with optimal dilution. Total viable count is the counts the total no. of vegetative cells, endospores and spores of bacterial, because endospores and spores are capable of germinating under favourable conditions. Total viable count = Number of (vegetative cells + endospores + spores) Present in the culture media

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Experiment No. Objective: Turbidometric estimation of Microorganisms Requirements: culture plates, test tube, photo colorimeter, tissue paper etc. Procedure: 1. Make a quantitative plate count of a culture of bacteria, plating 10-6, 107 and 10-8 dilutions of the culture; incubate the plates at 370C for 48 hrs. 2. Take 5 tubes, each containing 5 ml of sterile nutrient broth, use four of these tubes to make four serial 1:2 solution of the culture. a. Transfer 5 ml of culture to one of the tubes of sterile broth and mix cell. b. Transfer 5 ml of this first dilution to a 2d tube of sterile broth and mix well. c. Transfer 5 ml of this 2 d dilution to a 3d tube of broth, and after mixing transfer 5 ml of the culture dilution to a 4th tube of nutrient broth the obtain final solution. 3. Use of photo colorimeter, with the 5th tube of sterile broth inserted in the instrument. Set the galvanometer to read 100% transmittancy. Then determine the optical density of the undiluted 48 hrs culture of bacteria, as well as the four serially diluted cultures. Record the optical density of each tube. 4. After determining the plate counts of these cultures plot the optical densities of the various dilutions against the corresponding actual bacterial count. Observations: S.No. Dilutions used OD SPC CFU/ml

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Experiment No. Objective: To Determine the Bacterial Growth Requirements: purified bacterial culture, spectrophotometer etc. Procedure: 1. Select a particulars organism, Bacillus or E. coli or any bacterial strain, and inoculate this into a sterile 100 ml nutrient media in a 250 ml flask by powering 2 ml of the original culture. 2. Incubate at 370C in incubator. 3. Take the optical density reading at 0, 5, 1, 2, 4, 12, 24, 48, 72, 96 and 120 hours. 4. Take the control reading before inoculating the nutrient broth. 5. Plot these O.D. against time on a graph (O.D. & bacterial cell concentration). Observations: S.No. Incubation time OD

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3.9 HEAT STABLE COUNT Yeast, moulds, as well as some bacteria form spores under adverse conditions, which hampers their growth. The spores of yeasts and moulds are primarily reproductive spores, and several to many spores are produced per yeast cell or mould plant. In contrast only one bacterial spore in produced per cell. The bacterial spores are heat resistant (also endospores), whereas the spores of yeast and moulds are not, also the vegetative bacterial cells are susceptible to heat and are killed. When the inoculum from bacterial culture containing vegetative cells, spores and endospores is heated in a water bath to about 800C, only vegetative cells will be killed. And when the heated incoulum (after cooling) is plated to obtained viable count, the colonies that appear in the plate are those of spores and endospores. 2.10 OCTYL VIABLE COUNT Octyl alcohol kills the vegetative cells and endospores but not bacterial spores. So by treating the culture with (0.5%) octyl alcohol and then by subsequent dilution and plating we can count the spores. Octyl viable count = no. of (spores) + No. of Endospores. Total no. of Endospores = Heat stable count + octyl viable count & Total no. of vegetative cells = total viable count – octyl alcohol count

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Experiment No. Objective: To Determine the HSC, TVC & OSC. Requirements: Procedure: (1) TVC 1. Take 1 ml of culture from broth culture and add it to 9 ml of sterile water to prepare 10-1 dilution, like wise prepare dilution up to 10-6 2. Inoculate 0.1 ml culture by using pour plate or surface spreading method. 3. Take the petriplates having 30-300 colonies and determine the count. (2) HSC 1. Heat the culture in a tube at 800C for 5 minutes. 2. Prepare serial dilution from the heat treated culture 3. Inoculate these dilutions to nutrient agar 4. Inoculate the plates at 370C 5. Count number of colonies present in a plate and multiply with the dilution factor. 35 colonies in 10-4 dilution in original culture Total no. of cells = 35×104 (3) OSC 1. Treat the culture with 1% octyl alcohol (1.1 ml octyl alcohol rest water in 100 ml). Add 1 ml of octyl alcohol to 9 ml culture. 2. Prepare serial dilutions and inoculate in the nutrient plates and incubate it for 24 hours. 3. Count the number of colonies and nulgiply it with the dilution factor as described in above HSC procedure. Observations:

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4. GROWTH OF BACTERIA UNDER ENVIRONMENTAL STRESS Microbes like all form of life, are profoundly influenced by their surroundings. Environmental effects fall into three general categories: Physical : The effect of temperature of extreme pressure, U.V. irradiation etc. Chemical : The need for food and response to poisons (Antibiotics etc.) Biological : The influence of coexisting species (essentially chemical) There is a particular optimum set of environmental conditions for each living species but there is a wider range of tolerance in the microbial kingdom as a whole than in higher from of life. Although vast majority of microbial forms require the same conditions that are beneficial to most plants or animal cells, still some extremist microbial populations exist in the world which survives in extreme types of climatic conditions. The series of exercises in this section will illustrate the response of different bacterial strains; to various environmental conditions in continual fight to control. Destructive and useful microorganisms, the most powerful use have been the control of microbes environment. In the preservation of various commodities useful in life we have used temperature, osmotic pressure, pH, oxygen deprivation to check microbial growth. But their great adaptability and versatility makes the control of microorganisms difficult. 4.1 Effect of temperature Different types of bacteria have different requirements at which they can grow and the temperature at which maximum growth of bacterial cell is observed is called as optimal temperature. The temperature which hinders or stops the growth of bacteria are called extreme temperature. The rate of chemical reaction depends on temperature and given by Arrhenius equation (Fig. 1.). -H Log 10V = ------------ + C 2.303 RT V = reaction velocity H = activation energy R = gas constant T = temp. in 0K. • This equation is applicable to bacterial systems also. But it is observed that bacteria obey this equation for some intermediate temperature only, as the extreme temperature approaches, there is an abrupt fall in growth rate (Fig. 2). This is caused by the thermal inactivation for proteins (enzymes) and possibly other cell structures like membrane. As the temperature is raised above the optimum temperature for growth, metabolic activity markedly increases, but at the same time the rate of enzyme and protein break down (due to protein denaturation) markedly increases, resulting in the damage and finally death of cells. As the temperature is lowered, the enzyme activity and thus the growth of the cell is lowered as predicated by Arrhenius equation. Bacteria will continue to grow until the system freezes, however most of bacteria stops growing at a temperature well above the freezing point. Every microorganism

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has a precise minimum temperature of growth, below which growth will not occur however long the period of incubation. A. generalized arrhenius plot of Arrhenius plot of growth rate the relationship between velocity of E.coli, B→C range of temp. of a chemical reaction and temperature where the growth curve is linear above B or blow C the temperature is extreme. The numerical value of the cardinal temperatures (minimum, optimal and maximum), and the range of temperature over which growth is possible varies widely among bacteria. On the basis of the temperature range of growth, bacteria are divided into four broad groups. a. Extremophiles or hyperthermphiles (which grow above 900C) b. Thermophiles: which grow at temperature above 550C. c. Mesophiles : They grow at mid range of temp (20-450C).

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Experiment No. Objective: To observe a growth rate of a microorganism and prepare a growth curve. Requirements: Bacterial culture, spectrophotometer pipettes, test tube, tissue paper etc. Observations:

Sl. No. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

Time

Hrs.

O.D.

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Experiment No. Objective: To study the effect of temperature on Bacterial Growth Requirements: Bacterial culture, spectrophotometer pipettes, test tube, tissue paper etc. Procedure: 1. Incuabte one whole set of 5 tubes with the culture of the organisms in the sterile nutrient broth medium. 2. Incubate 5-tubes of each culture at 0, 5, 28, 37 and 500C. 3. Observe the growth by measuring the OD. Take nutrient broth as the blank and measure the growth in interval of two hour. 4. Plot the O.D. reading vs. time in a group for each organisms at different temperature. 5. Care should be taken to use nutrient broth without any organisms (i.e. sterile) as the blank and all readings of OD are to be taken with respect to it. 6. Phychrophile : they grow at 00C. Observations: Hrs. 0 2 4 8 16 24 34 00C 50C RT 280C 370C 500C

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4.2 Effect of pH Most organisms have an optimum H+ ion concentration for growth, however some of them can grow over a fairly wide region of pH. This pH conditions are also effected by the metabolic activities of the bacteria for ex yeast decreases the pH of the medium by production of alcohol. While some putrefacture bacteria decomposes protein and produces basic amino acid thereby increasing the pH of surroundings. Since pH limits the kinds and type of microorganisms capable of growing under a given conditions. Its control has a considerable practical importance. We know that a optimum pH is required for maintenance and function of enzyme any change in pH effects the enzyme activity and since enzyme activity is responsible of metabolism carrying in the organisms change in pH affects growth and in extreme concentration of pH the organisms is unable to survive because of the deactivation of enzymes. Thus, pH concentration like temperature could be defined by maximum, minimum and optimal pH concentration.

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Experiment No. Objective: To study the effect of pH on Growth Requirements: Bacterial culture, spectrophotometer pipettes, test tube, tissue paper etc. Procedure: 1. Prepare nutrient broth culture of the given strain. 2. Prepare nutrient broth, maintained at different pH, 5, 6, 7, 8 and 9 in separate flasks. 3. Inoculate 2.5 ml of inoculum into 22.5 ml of nutrient medium. 4. Keep in incubator at 340C. 5. Observe OD at different time intervals i.e. 24 hours, 48, 72, 96 and 120 hr. 1. Plot the graph of O.D. vs time for bacterial culture maintained at different pH. Observations: Sl. No. 1 . 2 . 3 . 4 . 5 . 6 . 7 . Result: Time Hr pH 5 O.D. pH 6 O.D. pH 7 O.D. pH 8 O.D. pH 9 O.D.

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4.3 Effect of Osmotic Pressure on bacterial growth At level optimal for metabolic activity, even when the so molarity of surrounding environment varies over a relatively wide range. Most bacteria with a rigid cell wall do not have to regulate their internal osmotic pressure with precision because cell wall has a capability of withstanding high osmotic pressure. Bacterial cell usually maintain their internal osmotic pressure well above the of the media. This causes flow of water inside the cell, a condition essential for diffusion of nutrient substances and maintenance of outward pressure called turgor pressure. When the osmotic concentration of the medium is considerably lower than that of the cell (a hypotonic medium), excessive diffusion of water into cell causes lyses of cells due to increased turgor pressure. Gram positive are resistant to lyses due to rigid cell wall, but gram negative bacteria cell lysis occurs. This phenomenon is called plasmolysis and if medium is of high concentration i.e. a hypertonic medium. Water will leave the cell and the volume cytoplasm decreases. Causing damage to the membrane in Gram positive bacteria, this cause the cell membrane to pull away from the wall, the cell is said to be plasmolyzed. Whereas in Gram negative bacteria which have got only outer membrane contracts along with the plasma membrane, and this damages the membranes. Bacteria vary widely in their osmotic requirements. Some are able to grow in very dilute solutions and some in solution saturated with salt. Microorganisms that can grow in high osmolarity are called osmophiles. In terms of their salt tolerance bacteria can be divided into four broad categories. 1. Non halophiles : 0.0-4 (% g/100 ml) salt concentration) 2. Marine organisms : 0-5 (% g/100 ml) 3. Moderate halophiles : upto 20.5 (% g/100 ml) (salt concentration) 4. Extreme halophiles : upto 36 (% g/100 ml) (saturated) Organisms Concentration (%, w/v) in Ratio of intracellular to growth medium of extra cellular conc. Of NaCl KCl Na+ K+ Nanhalophiles Staphylococcus aureus 0.9 0.19 0.7 27 Salomella oranienbung 0.9 0.19 0.9 10 Moderate halophiles Mirococcus 5.9 0.02 0.3 120 Halodenitrification 5.9 0.02 0.7 55 Vibro costicolus Extreme halophiles Sarcina morrhuae 23.4 0.24 0.8 64 Halobacterium salinarium 23.4 0.24 0.3 140 Osmotic tolerance – The ability of an organism to grow in a media with wide variety of osmolarities is by the adjustment of internal osmolarity so that it always exceed, that of the medium. This in accomplished by different phenomenon, each of which has a specific biochemical basis. A hypotonic solution A hypertonic solution The osmotic pressure can be varied by the change in salt concentration.

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Experiment No. Objective: To study the effect of salt concentration on bacterial growth Requirements: Bacterial culture, spectrophotometer pipettes, test tube, tissue paper etc. Procedure: 1. Prepare three sets of five test tubes which are having luria broth having different concentration of NaCl. [NaCl] → 1.0%, 0.5%, 5%, 10%, 25% 2. Inoculate one whole set of five tubes 3. Incubate the culture at 370C 4. Observe the growth by measuring the O.D. in spectrophotometer at 24, 48, 72, 96 and 120 hrs. 5. Plot the O.D. readings vs. time on a graph paper for various salt concentrations. Observations: S.No. Incubation period OD

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4.4 PRESERVATION OF LABORATORY CULTURES A microbial culture may be preserved in order to avoid the necessity of subculturing or of transferring it to fresh media at frequent intervals. To be suitable any means of preservation should permit maximum survival of the population with little or no genetic and physiological change. In principle all methods depends upon bacteriostasis - That is maintenance of culture without growth or reproduction. Most methods employ refrigeration or desiccation to bring about such dormancy. Short term preservation can be accomplished in a no. of different ways depending aerobes may be preserved for months by refrigeration on agar slope. With some facultative anaerobic preservation as refrigerates agar-stab culture is more satisfactory. Agar-slope (slant) → an agar slant is prepared and then the pure culture of organism is inoculated by streaking process with the help of needle. During incubation growth of bacteria takes place. The exclude oxygen further and to prevent evaporation, a seal of sterile mineral oil may be applied over the agar slant. After which the slant culture is refrigerated. Strict anaerobes are cultured and preserved in a strongly reducing medium such as thioglycollate broth or chopped meat medium. If acid is metabolizing product of the organism being preserved the medium employed should be well buffered. Although these refrigerated cultures prepared on the slant remains viable for several month refrigeration methods are not suitable for long-term preservation because such stocks must periodically be recultured for three is possibility that cultures refrigerated for long periods may be frozen & dead. Unfortunately many microbial species do not survive ordinary freezing or dehydration. The common method for long term preservation of microbes is the freeze drying technique called as Lyophilization. In this process the microbial culture is suspended in a sterile milk blood serum or some equally protective substance in a glass vial or tube quick-frozen with a mixture of dry ice and alcohol then thoroughly desiccate while in the frozen state. Finally the vial containing the dried culture is sealed. Some bacteria which form spores, their spores can be harvested by centrifugation and then they can be preserved. When these spores are provided with favourable environmental, condition for growth they again germinate & we get colonies.

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5. A DEMONSTRATION OF KOCH’S POSTULATES OF BACTERIAL AND FUNGAL PATHOGENS Introduction When a pathogen is found on a diseased plant, the pathogen is identified by reference to special manuals, if the pathogen is known to cause such a disease and the diagnostician is confident that no other causal agents are involved. Amongst the syndrome of symptoms which is the ball mark of a particular disease one often sees a micro-organism which is presumed to be the pathogen. To determine with certainty that this micro-organism is the cause of the disease rather than some incidental contaminant it is necessary to examine critically its relationship with the host plant. This dilemma was first recognized in studies on microbial pathogens of man. In 1876 Robert Koch provided the first experimental proof of disease causation of applying a set of rules which have since come to be known as Koch's postulates. Koch's considered that these rules must be satisfied before any micro-organism can be regarded as pathogen. The rules may be summarized as involving five step- wise operations, outlines below: 1. The suspected pathogen must be consistently associated with the some symptoms. 2. The organism should be isolated into culture, away from the host. This preludes the possibility that the disease may be due to malignant tissues or other disorders of the host itself. 3. The organism should then be re-inoculated into a healthy host. 4. Symptoms should then develop which are identical to those observed in the original out break of disease. 5. The causal agent should be re-isolated from the test host into pure culture and should be shown to be identical with the micro organism initially isolated. Even today these rules are still relevant, although we now appreciate that they can not be rigidly applied in their original form to all types of pathogens. The most important exceptions in plant pathology are those pathogens which can not be grown in artificial culture i.e. the viruses and some biotrophic fungi. The problem of isolating viruses from their host plants is generally overcome by using indicator plants. These are alternative hosts which develop symptoms which are specific for a particular virus. Healthy specimens of the original host may then be re-inoculated. In addition, electron microscopy of plant sap or of purified crystalline samples of the viruses, coupled with serological techniques may be employed to investigate the types of virus present at each step of the infection procedure. The procedure of application of Koch's rule to non culturable fungal pathogens presents less serious problems because they produce spores. These can be removed from the host and used in re-inoculation experiments. In many instances spore morphology is also a valuable aid to the identification of the inoculated and re-isolated pathogens. Disadvantages Difficulties in satisfying these postulates may be experienced in cases were symptoms result from mixed infections or when dealing with previously, undiscribed disease agent. Not long ago, few pathologists would have

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predicted the existence of pathogens such as viroids, which scarcely conform to our preconceptions of a successful parasite. Koch's rules are possible to implement, although not always easy to carry out, with such pathogens as fungi, bacteria, parasitic higher plants, nematodes, some viruses, some viroids and the spiroplasmas. These organisms can be isolated and cultured or can be purified, and can then be introduced into the plant to see if them cause the disease. With the other pathogens, however such as some viruses, mycoplasmas, fastidious vascular bacteria and protozoa, culture or purification of the pathogen is not get possible, and the pathogen often can not be reintroduced into the pathogen into the plant to reproduce the disease. Thus, with these pathogens, koch's rules cannot be carried out and their acceptance as the pathogens of the disease with which they are associated 1S more or less tentative. In most cases, however,

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Experiment No. Objective: To demonstrate Koch’s postulates in bacterial pathogens. Requirements: Procedure: 1. Isolate the micro-organism, i.e. the pathogen from the diseased part of the plant. For bacterial disease cut the damaged part and then wash with water. 2. Then place it in distilled water in an Petri plate and then cut or piece it with sterilized glass rod the micro organism will get collected in sterilized distilled water. 3. Observe the suspension for pathogen under electron microscope. 4. Then grow the culture by streaking in the agar medium. And then incubate it. 5. After incubation observe for colonies and see whether the pathogens are some as they were in suspension. 6. Then pick a colony from the agar plate then make it suspension in distilled water then take a host plant which is diseased free. 7. Then make an injury in the host plant or by giving an injection of that suspension make the inoculation into the plant. 8. Make the plant susceptible to that disease. After few days see for any symptoms of the disease. 9. When symptoms occur visualize the symptoms same for as that of the original from which it was isolated. 10. Then again isolate the pathogens from the disease plant and observe under microscope the pathogens must be identical to that of the original one which are isolated earlier. For visualizing in animals 1. Take a mouse that was killed by bacteria injected into its peritoneum. Open the abdomen of the mouse aseptically by washing the exterior surface with ethanol and by using scissors sterilized with ethanol. 2. Insert a sterile loop into the peritoneal cavity to obtain the micro organism and streak one blood-agar plate to isolate to pure culture. At the same time, make a smear and gram stain the material obtained by activity. Incubate the blood agar plate at 37°C for 24 hrs. 3. Inoculate a tube of strep-base broth with a colony of the predominant microorganism growing on the blood agar plate. Also streak two entire blood agar plates with this same micro organism to obtain confluent growth and aseptically apply sensitivity discs to one plate. Incubate the tube and plates at 37°C. 4. Flood the blood agar plate which does not have any sensitivity discs with 2 ml sterile saline. Using a sterile glass spreader scrape the cells from the plate into a sterile test tube. 5. Then inject one labeled mouse with 0.2 ml of the isolated culture. Inject a second mouse with 0.2 ml of the isolated culture plus 0.1 ml of the antibiotic to which the culture demonstrated sensitivity on the disc plate. 6. Reinject your second mouse with 0.1 ml of the antibiotic for two successive days. Observe the mice over a 24-74 hr period and record

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the time when a mouse dies. Stain the growth from the peritoneum of any mouse that dies to determine if it is similar to isolated one. Observations:

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6. DETERMINATION OF CULTURE SENSITIVITY USING DIFFERENT DISINFECTANTS AND ANTIBIOTICS Introduction Disinfection generally refers to the use of; germicidal chemical agents to destroy the potential infectivity of a material. . Micro organisms which cause disease and destroys food materials should be controlled; many chemicals both inorganic and organic are toxic to micro organisms and to control the microbes responsible for deterioration and disease, and thousand of these chemicals have been discovered. Disinfectants are lethal to all sorts of cells. Because of this non specificity it is not surprising that bacteria develop little resistance to these agents. Classification as a disinfectant however, is some what arbitrary for at a high enough concentration a great variety of compounds will inhibit bacterial growth. Indeed whether a given substance is a meat or a poison to bacteria is often simply a matter of concentrations. Thus oxygen, various salts, fatty acids, some amino acids and glycerol, in high enough concentrations may be batceristatic or even actively bactericidal to bacteria. Disinfectants act directly on cell structures and thus do not require specific metabolic activities on the part of the microbe. Disinfection also means the freeing of an article from some or all of its burden of live pathogenic microorganisms which might cause infection during its use. Uses Disinfection, rather than sterilization is attempted in circumstances in which sterility is unnecessary or sterilizing procedures are impracticable, yet there is still some value of obtaining a partial or complete removal of non sparing pathogens. It is highly impracticable to apply sterilizing procedures to bedpans, baths, wash-basins, furniture, eating utensils, bed-cloths and other somites that might spread infection. ANTIBIOTICS An antibiotic is a substance which destroys or interferes with the reproduction of micro organisms. It is now possible to produce some antibiotics by a semi-synthetic process. It has revolutionized the treatment of infection, they carry the risk of causing side-effects or of encouraging the growth of resistant micro organism. They should not be used unnecessarily when several infective agents are available. Bacterial resistance develops because antibiotics kill the most susceptible strains, leaving the most resistant strains to multiply. Gram negative bacteria can also in some circumstances transmit resistance to other types of bacteria. To diminish the development of resistant strains, antibiotics should always be given in adequate doses for an adequate period and should be given in combination, if the emergence of resistant bacterial strains is likely. Prophylactic antibiotics should be given only when specifically indicated for instance to prevent rheumatic fever or bacterial endocarditis.

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Experiment No. Objective: To determine the sensitivity of the culture using different disinfectants and antibiotics. Requirements: Procedure: Many chemicals, both inorganic and organic are toxic to micro organisms and to control the microbes responsible for deterioration and disease thousand of these chemicals have been discovered. The) chemical agents simply inhibit microbial activities and growth or lethal and kill the micro organisms. Inhibitory chemicals are usually termed antiseptics, the lethal are known as disinfectants. And by using antibiotics which are responsible for killing the specific micro organism we can control the destruction of food and control the diseases, with are deadly to human beings. 1. Prepare a nutrient agar and autoclave it so as to sterilize the nutrient medium. 2. Melt the agar and pour in Petri plates and then cool it to 45°C, and then inoculate the plates with micro organism one with Escherichia coli and other with Bacillus 3. Then cut the filter papers into small circles and put into number of disinfectants and antibiotics that are available. Allow it to drain and place into the inoculated plate at an distance of 2" with the help of sterilized forceps. 4. Then incubated the plate. Then record the relative effectiveness of each antibiotics and disinfectants by measuring the diameter of the inhibition zone. Observations:

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Experiment No. Objective: Effect of various antibiotics on bacterial growth. Requirements: Observations:

Plate NO. 1 (Bacillus pseudomonas)

Plate No. 2 (Bacillus)

Plate No. 3

S – Streptomycine P – Penicillin Results:

O – Oxoteracycline Ch – Chloramphicol

+ = 5 mm K – Kanamycin

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Experiment No. Objective: Impact of different disinfectants on killing bacteria Requirements: Introduction: Use of antibiotics, antiseptics, disinfectants or immunization is required to control the growth or check the growth of many pathogenic microorganisms. Antiseptics are the chemicals which avoid sepsis or microbial contamination. Disinfectants are the chemicals which when used at low concentrations avoid the growth or entry or pathogen in a place. Antibiotics are the substances produced by microorganisms and which act against another kind or microorganism. Disinfectants are usually used for treatment of inanimate objects whereas antiseptics are used at low concentrations for treatment of animals objects. Examples of disinfections: NaoH (2%), Na2Co3 (4%), Calcium oxide, CaCl2, KMno4, phenol, Dettol. Formalene, H2O2, HgCl2 (1:10000) Copper sulphate. Examples of antiseptics: Aniflamin (1:1000) Crystal violet (1:100), Alcohol (95%). Antibiotics: Streptomycin, Penicillin etc. all these compounds disrupt and disturb internal mechanism of microorganism. So their metabolic activities are affected drastically. When these chemicals are applied on a bacterial lawn, inhibition zone are formed on it depending on ability of these compounds to inhibit microbial growth. Stronger the chemical against a particular organism, more will be the diameter of inhibition cone formed by it on bacterial lawn. Procedure: Nutrient agar was made and placed in 2 plates then a bacterial lawn was made on agar plates when they were cooled and agar was solidified. Bacterial lawn was made near the flame. Then we cut the discs of filter paper with the help of cutter and there discs were dipped in following four disinfectants (1) Savlon (2) Formalin (3) Dettol (4) KMnO4 These discs were placed on solid surface of one agar plate. We 0.1 M solution of sodium azide was made and diluted to obtain 10-1, -2 10 , 10-3 and 10-4 concentrations. Filter paper discs dipped in these dilutions were placed on second agar plate. These plates were kept in incubator but we did not get results because. 1. We placed plates in incubator which has temperature of 25º C only. 2. Bacterial lawn was not adequately made on agar plates. So this experiment was repeated and this time we took following four disinfectants for sensitivity test. 1. Dettol 2. Savlon 3. Mercurochrome 4. KMnO4 use of different disinfectants for use of different concentration of sensitivity test Mecurochrome for sensitivity test We performed this experiment in 2 parts (1) To observe the sensitivity of different disinfectants. (2) To observe the sensitivity of one disinfectant concentrations.

at

different

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We first made circular discs of filter paper and autoclaved them in a blank Petri plates. Next we prepare nutrient agar plate and place 4 discs dipped in 4 different disinfectants over the media after making a bacterial lawn over the agar plate. In order to test the sensitivity of one disinfectant at different concentrations Mercurochrome was diluted to obtain 10-1, 10-2, 10-3 concentration and them sterilized discs were dipped in different concentrations and placed over a agar plate after making a bacterial lawn from pure culture with the help of bent glass rot. The plates were incubated at 37º C for 24 hrs.

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7. CULTURING OF ANAEROBIC BACTERIA Introduction The present atmosphere of the earth contains about 20% (v/v) of the highly reactive oxygen. Many of the microorganisms are dependent on the oxygen as a source of nutrient. The aerobes are dependent on O 2, the facultative anaerobes use O2 if it is available, but they also can grow in its absence, the anaerobes can not utilize oxygen. Anaerobes are of two types the obligae anaerobes for which O2 is toxic and the aerotolerant anaerobes which are not killed to exposure to O2. many obligate aerobes cannot grow in oxygen concentrations greater than atmospheric i.e. >20%v/v). Indeed some obligate anaerobes require O2 i.e. about 2-10%/v/v). Such type of bacteria are known as microaerophiles. The effect of atmospheric oxygen on microbial growth is closely related to the oxidation-reduction potential of the micro organism and culture medium. All bacteria contain certain enzymes capable of reacting with O2. The oxidations of flavoprotein by O2 invariably results in the formation of a toxic compound H2O2. These oxidations produce small quantities of an even more toxic free radical superoxide or O2. The enzymes which convert them to water and oxygen are not present in the anaerobic organism. The oxidation reduction potential is a means of expressing the degree of oxidation or reduction of a compound or an environment. A compound having a high ratio of Hydrogen to oxygen i.e. higher than water is highly reduced and has a negative oxidation reduction potential, and a highly oxidized material has a positive oxidation reduction potential. Anaerobic bacteria can be grown by eliminating free oxygen from the environment or by establishing a low oxidation-reduction potential by providing sufficient reducing materials. A considerable range of oxygen tolerance exists among the “Strict anaerobes”. Some grow in air to a barely visible extent, others require the complete exclusion of free oxygen. The protection anaerobic cultures from free oxygen can be accomplished by the following methods. 1. Expel the gas from culture media by boiling and then preventing its reintroduction by adding relatively solid barrier such as a layer of vesper or mineral oil. 2. We can use sodium thioglycolate (HScH2 COOHa) compound which reacts with oxygen and maintains enzymes in the cell in a reduced condition. The Brewer plate is especially designed to exclude oxygen and at the same time to permit the development of surface colonies of anaerobes. 3. Replacement of air by an inert gas in a widely used method and offers a number of advantages. Agar plated or tubes to be incubated are placed in an anaerobic incubator. The incubator is evaluated and carbon dioxide, hydrogen or nitrogen is ruin in. 4. The burning of phosphorous is another chemical method for oxygen removal.

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8. BIOCHEMICAL CHARACTERIZATION OF ISOLATED MICROORAGANISMS INTRODUCTION An organism that has been isolated and studied must be given a name; otherwise reference to it could not be made. Also, it is highly desirable that the name applied to an organism by one person be understood by others. Systematics bring order from chaos and to organize the many kinds to bacteria into a coherent, understandable and useful system with three inter related topics. 1. Classification The arranging of bacteria with similar phenotypic characteristics (Numerical taxonomy) or genotypic characteristics (the base composition of DNA; its determination and significance, nucleic and hybridization, nucleic acid sequence) into groups. 2. Nomenclature The naming of bacteria according to internationally agree upon principles. 3. Identification The comparing of unknown organisms, with bacteria that have already been classified, for the purpose of determining the identities (or) names of the unknown organisms. During the early years of bacteriology organisms were classified entirely on the basis of morphology. The morphological characters employed are: (i) Size and shape of nn organism (ii) Arrangement of the cells (iii) Presence (or) absence of well defined capsules. (iv) Presence (or) absence of spores. (v) Size, shape and position of the spore in the cell. (vi) Presence, number and arrangement of flagella. (vii) Presence 9or) absence of characteristics granules. (viii) Acid fastness, Gram reaction and other differential staining procedures. Classification based on phenotypic characters (Numerical taxonomy) Numerical taxonomy It is based on quantification of similarities and differences among organisms. This was first suggested by Michel Adansonand is known as Adansonian taxonomy. The underlying assumption is that, provided each phenotypic character is given explicit weighing, it should be possible to express numerically the taxonomic distances between organisms, in terms of number of characters examines. For any pair or organisms, the calculation of similarity can be made in two different ways. The similarity coefficient (Sj) does not take into account characters negative for both organisms, being based only on positive matches; the matching coefficient (Ss) includes both positive and negative matches in the calculation. The data can then be transposed into a dendrogram, as a basis for determining taxonomic arrangement in terms of numerical relationship.

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Numerical taxonomy does not have the evolutionary cornotations of phylogenetive taxonomy, but it provides an objective and stable basis for the construction of taxonomic groupings. Classification based on genotypic characters DNA Base composition Overall nucleotide base composition (moles percent g+c) for various stains resolve many taxonomic problems i.e. strains having significantly different DNA base composition do not belong in the same species. Nucleic acid hybridization These techniques allowed the entire genome of one bacterial strain to be compared with that of other strains in terms of nucleotide base sequence. A transition from native to denatured state is called denaturation. The temperature at which DNA is half denatured is known as melting temperature (Tm). At temperature near Tm, only duplexes between strands with a high degree of complimentarily persist. When DNA preparations from who related strains are mixed and treated in this manner, hybrid DNA is formed. This technique is useful at species level and less useful at Genus level. Nucleic acid sequencing It deduced broader phylogenetic relationship among bacteria. The rate of change of sequence of genes encoding 1675 r RNA is much less than that of the bulk of the genome. The major requirement of systematic is to learn about the bacteria as much it is possible. Classification based on the above characteristics is therefore inadequate and that more characteristics are necessary. Biochemical reactions were, therefore introduced into the newer classifications (eg: Production of indole and H2S, reduction of Nitrate- Nitrite – Ammonia - Nitrogen; fermentation of carbohydrates). As the present time biochemical reactions are important in the classification of bacteria.

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IDENTIFICATION OF UNKNOWN BACTERIAL CULTURES BIOCHEMICAL TESTS Microorganism like all living things may modify their environment to some extent and utilize chemicals in solution as sources of energy and as building blocks for growth and reproduction. All activities of cells are mediated by enzymes, and in the complex chemical reactions of life the microorganisms employs a large number of individual enzymes whose activities interlock. The chemical end products of some of these enzymatic actions may be measured, or in other cases the disappearance of certain substances from the medium can be detected. By making a series of different tests a pattern of activity can be established (which in turn reflects the enzymatic make up of the micro organism) and the pattern aids in the identification and differentiation of the microorganism from closely related species. BIOCHEMICAL TESTS FOR BACTERIA The tests described in this section are a fairly limited selection of tests which are of general usefulness in the characterization and identification of bacteria. Some organisms require the use of a medium specially adapted to particular nutritional (or) osmotic requirements. In other cases, a selective medium may be employed which not only allows the isolation of the organism under investigation but also incorporates one (or) more biochemical tests. It is important to check that each batch of a medium is satisfactory by using a strain of bacterium known to give positive reaction. In addition, where appropriate, an uninoculate tube or plate is to be incubated with each test in order to detect false positive reactions due to impurities in or a deterioration of medium or reagents. A. REACTIONS INVOLVING PROTEIN, ANIMOACIDS AND OTHER NITROGEN COMPOUNDS, INCLUDING TESTS FOR PROTEOLYTIC ACTIVITY Many bacteria can degrade a variety of products like proteins and utilize the resulting peptides and amino acids to synthesize cell protein and to provide a potential energy source for the cell. Microorganisms vary from species to species with regard to their proteolytic ability and this feature can be used to characterize a given species. Although amino acids serve primarily as the basis they are also utilized by the cell for other purposes. Amino acids may be degraded to yield energy to the cell and they give a variety of end products for example NH3, indole and H2S are formed. Amino acids may be altered chemically to yield essential cell components, including other amino acids. The following exercises illustrate types or protein degradations and some of the diverse ways in which microorganisms can utilize amino acids. Since the pattern of amino acid degradation is characterization of a given genus (or) species, there biochemical patterns may be used in microbial characterization. 1. GELATIN HYDROYSIS The protein, gelatin is obtained by the hydrolysis of collagen- a component of connective tissue and tendons of animals. Gelatin is convenient as a substrate to test for proteolytic enzymes in microorganisms. Water solution of gelatin of the concentrations used in media for this exercise are liquid at room temperature and solidifying in an ice bath. If the

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gelatin has been hydrolyzed by the action of the microorganism being tested, the medium will remain liquid. Medium: Nutrient broth with the addition of 10-15% gelatin, final pH 7.2, sterilize by autoclaving for 20 min at 115°C. Procedure 1. Inoculate four tubes of nutrient gelatin with E.coli, Bacillus subtiles, Streptococcus faecalis, and Proteus vulgaris. 2. Incubate at 37°C together with a sterile tube of nutrient gelatin, which will serve as a control. Thest at 2 days and upto7 days (or) until a positive reaction is obtained. 3. To examine for hydrolysis, chill the tubes in ice water. The control tube and tubes in which no hydrolysis has taken place will solidity. Hydrolyzed gelatin will remain fluid. Precautions 1. Care should be taken not to agitate the tubes while their contents are liquid. 2. If liquefaction of the gelatin is proceeding slowly from the surface down, as it often does, agitation can minimize the hydrolyzed with the unhydrolyzed gelatin to give a combination that solidifies giving negative and erroreous results. Recording result The liquefaction of gelatin by bacteria is the result of the action of an enzyme known as gelatinase. It is an extracellular enzyme concerned with the hydrolysis of the in diffusible protein prior to intracellular utilization. - If the gelatin remains liquid, it indicates that the organism under examination secreted a gelatinase into the culture medium. - The extracellular nature of the enzyme may be demonstrated by filtering a bacterial culture and adding some of the filtrate to a tube of gelatin medium. The presence of the enzyme results in liquefaction of the gelatin. - The presence of a fermentable carbohydrate may result in a protein sparing action. Under these conditions a test for gelatin liquefaction will most likely be negative even though the organism under examination is capable of hydrolyzing the protein. Therefore, non-carbohydrate media should be employed to demonstrate. The test is of value in identifying and classifying bacteria. 2. CASEIN HYDROLYSIS Casein is the principal protein of milk. It exists as colloidal suspension. Which gives milk its opaque whiteness. Many bacteria are equipped with enzyme that hydrolyze thin protein into more soluble and transparent derivatives. Protein breackdown, sometime called peptonization, is a useful reaction in the identification of microbial species. Casein is a protein capable of reacting with both acids and basis. Some bacteria serete a rennin like enzyme capable of hydrolyzing casein to soluble paracasein and a peptone like compound. The soluble paracasein then reacts with the calcium salts in solution to give a precipitate of paacasein (or) calcium paracaseinate. The clear liquid surrounding the curd of paracasein is known as whey. This may be diagrammed as follows:

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Casein + rennin -----------------------------------------Peptone-like body soluble paracasein + Calcium salts Para casein (Calcium paracascinate) bacteria which ferment lactose very rapidly produce sufficient acid to precipitate or curdle the casein. The clear supernatant liquid that separates from the clot is known as when the acid produced is sufficient to stop further growth of the bacteria. Medium: Milk agar, which consists of agar with the addition of skim milk to 10 percent, sterilized by autoclaving for 20 min at 115 o C. A mole opaque milkagar may be made by mixing 10 ml hot sterile skim milk, immediately before pouring lthe plates. Test reagent Mercuric chloride solution, 1 percent hydrochloric acid or 1 percent tannic acid solution. Procedure 1. 2. 3. Pour two plates to skim-milk agar. Inoculate one plate with Escherichia Coli and the other with Bacillus subtites, using short shokes of the loop over a limited area. Incubate at 37oC until the next laboratory period.

Recording result Colonies of organisms that digest casein (proteolytic microbes) will be surrounded by clear zones. Areas in which the casein has not attacked will remain slightly opaque.

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3. UREASE TEST The genus proteins may be distinguished from some other gram negative rods by its ability to produce large amounts to the enzyme urease. The hydrolysis of urea by urease releases NH3 as follows: NH2 O=C NH2 Since the production of ammonia raises the pH of the medium urease activity may be detected by the change of the phenol redindicator to purple. 1. 2. Medium User broth Peptone 1gm; NaCl -5 Glucose 1 gm; KH2PO4 -2gm; phenol red – 0.012 gm; Urea – 20gm; pH – 6.8 – 6.9; Filter, sterilize the aseptically transfer to tubes. Recording result The presence of a purple color is a positive test. 4. HYDROGEN SULPHIDE PRODUCTION TEST The activity of some bacteria on sulfur containing amino acids frequently results in the liberation of H2S. common examples of this reaction are found in the “aroma” of rotten eggs and in the blackening of certain spoiled canned foods. In the latter instance, the blackening results from a reaction between the H2S formed by bacteria and the metal. Production of H2S by bacterial cultures can be demonstrated in the laboratory if sulfide producing cultures are grown on media containing salts of metals such as bismuth and iron. The darkening along the line of the stab is caused by the formation of the metal sulfide. Cystine and methionine are two sulfur containing amino acids present in proteins. Practically all the information available on the effect of bacteria on these acids has been obtained from cystine or its reduction product cysteins. Some organisms are capable of attacking cystine with the liberation of hydrogen sulfide, others are unable to do so. The test is of value in the identification and classification of bacteria. Cystein does not occur in all proteins. Under anaerobic conditions the cysteine is first reduced to two molecules of cysteine. Then the cysteine is decomposed to hydrogen sulfide, ammonia, acetic acid and formic acid. Inoculate a tube of urea broth with unknown culture. Incubate along with a control tube at 37oC for 24 hours. ------ 2 NH3 + CO2

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COOH CHNH2 CH2 –S-SCystine COOH. CH NH2. CH2SH + HCOOH Cysteine Acetic Formic acid

COOH CHNH2 CH2

COOH + H2 CHNH2 CH2SH Cysteine

2H2O

H2S + NH3 + CH3COOH +

acid

Under aerobic conditions cysteine is dissimilated as follows COOH. CH NH2. CH2SH + O HOOC . CO . CH2SH + NH3 CYSTEINE HOOC. CO .CH2SH H2S + Other products H2S reacts with heavy metals to produce coloured compounds. The metal salts are incorporated in solid media. The presence of H 2S is detected by a darkening of the media. Medium Thiosulfate iron medium: Beef extract -3.0 gm; Peptone -30.0 gm; Peptonised iron 0.2gm; Sodium thiosulfate – 0.025gm; Agar -30 gm. Procedure 1. 2. 3. Prepare step inoculation as Escherichea coli and proteus vulgaris in two tube of thiosulfate from medium. Inoculate the stab cultures at 37oC for 48 hours. Observe for H2S production

Recording result Production and liberation of hydrogen sulphide causes the blackening of the lead acetate paper. 5. INDOLE PRODUCTION TEST Indole is a nitrogen containing compound formed from the degradation of the amino acid tryptophan by various bacteria. The importance of the indole test has in the fact that only certain bacteria form indole and that it can be readily detected chemically. Thus the degradation of tryptophan is suitable differentiating reaction.

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Pure tryptophan is not ordinarily used in the list medium. Instead tryptone – a digestion product of certain products is used as the substrate, since it contains considerable amounts of tryptophan. Medium Peptone water (tryptone-1-2%; sodium chloride-0.5%; final pH 7.2) dispensed in 5 ml amounts in test tubes and sterilized b autoclaving for 15 minutes at 121oC. Tryptone is the peptone to be recommended for this test, since it is rich in tryptophan, one of the amino acids destroyed by the usually methods of preparing peptones.

Test Reagent Kovac’s reagent is to be prepared, being rather more sensitive due to the higher solubility of day complex in the amyl alcohol layer. Procedure 1. 2. 3. 4. Inoculate two tubes of 1% typtone broth one with Escherichia coli and other with streptococcus. Inoculate a tube of 1% typtone btoth + 1% glucose with Escherichia coli. Incubate at 37oC until the next laboratory period. Test each tube for the presence of indole, using the method of Kovac,s.

Recording result To the culture fluid (about 6ml) add .05ml kovac’s reagent. Mix well by rotating thee tube between the hands. The alcohol layer will separate from the aqueous layer upon standing and reddening of the alcohol layer with in few minutes indicates that indole is present. 6. MICROBIAL REACTIONS IN LITHUS MILK The fermentation of the lactose produces acid, which lower the pH. As the acidity increases, the casein is curdled (acid curd) and is accompanied by the sequeezing out of a liquid (whey) from the curd. If the organism forms gas during fermentation of milk sugars, the gas may form bubbles in the curd, sometimes to the extent that the curd becomes furrowed or even term to shreds. This is knows as the “stromy fermentation” of milk. A differed type of curd is formed by some organisms which produce a rennin like enzyme, whose action is to coagulate the casein. As this reaction occurs at neutral pH, the curd is often called a “sweet curd” Generally such bacteria are also proteolytic and through the proteolytic

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digestion of the casein results in the formation of soluble products and thus in the increasing transparency of the milk. Procedure 1. 2. 3. Filtered B.C.P. milk and transfer it into three test tubes 10ml each. Inoculate the three test tubes with Baccilus, E. coli and Pseudomonas. Incubate at 37oC. B.C.P. milk turn from purple to yellow in all the three test tubes. B. REACTIONS INVOLVING CARBOHYDRATE AND OTHER CARBON COMPOUNDS 1. AMYLASE PRODUCTION TEST [STARCH HYDROLYSIS] Starch is a complex carbohydrate of the polysaccharide type. A qualitative test for starch is the appearance of a blue color upon the addition of a solution of iodine. When starch is hydrolysed, (decomposed with the addition of water) the cleavage products – dextrine, maltose and glucose to list them in descending order of complexity – do not give cold reaction, this principle is used in testing for starch hydrolysis in this exercise. An organism”s ability to hydrolyze starch is dependent upon the presence of the enzyme amylase. Since not all microorganisms produce amylase, the ability to hydrolyze starch can be used aid in the identification of a given species. a. Medium Starch hydrolysis may be tested with solid or liquid media, although starch agfaris perhaps more convenient. This medium consists of nutrient agar with addition of 0.2-1% soluble starch. The best results are obtained by preparing layer plates which are prepared by pouring 10ml of nutrient agar into each plate, allowing it to set, and then verifying this with 5ml of starch agar. Test Reagent Gram’s iodine solution as used for Gram’s stain. Procedure 1. Prepare two starch-agar plates and inoculate them by streaking with short strokes over a limited area with cultures of Escherichia coli and Bacillus sublites. Since the attempt here is α-amylase + starch dextrin + α- maltose

Recording result

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not to isolate colonies, the streaks may be confined to the large center area of the plate. Avoid streaking all the way to edge so that you will leave an uninoculated control cone to provide an area of contrast after the plate has been developed with iodine. The very centre of a Petri plate has a hump. Do not restrict your sticks to this area alone. 2. Incubate at 37oC until the following laboratory period. Reading result Flood plates with 5-10 ml of iodine solution. Unhydrolysred starch forms a blue color with the iodine. Areas of hydrolysis therefore appear as clear cones and are the result of pro amylase activity. Record the width of any clear cone in mm from the edge of the colony to the limit of clearing. Reddish brown cones around the colony indicate partial hydrolysis of starch (to dextrins) which is the result of α- amylase activity. 2. FERMENTATION OF CARBOHYDRATES Among the common products of carbohydrate breakdown by microorganism are gases (for example CO2, and H2). The types of products formed, and the proportion of each, depends upon the species of micro-organism as well as the particular carbohydrate being dissimilated. Medium The formation of acids can be readily detected by the pH indicator in microbial growth media. In this experiment, media containing the chemical bromocresol purple (BCP) which is purple at pH 6.8 and yellow at pH 5.2 are used. In broth, gas formation may be detected through the use of a vaspor seal (or) an inverted vial. (a Dusham tubs). Gas production in agar medium is accompanied by the formation of gas pockets, which appear as cracks in the agar. This exercise illustrates a simple method, used to detect acid and gas formation. Procedure 1. 4 tubes each of the following media are taken. a. Glucose broth with fermentation tubes. b. Sucrose broth with fermentation tubes. c. Lactose broth with fermentation tubes. B.C.P. B.C.P. B.C.P. indicator indicator indicator containing containing containing a a a Durham Durham Durham

d. Inoculate a different tube of each medium with E. coli, Streptococcus facales and Bacillus subites. The 4th tube in each set is a control tube and is not inoculated. Note: Be sure to label each tube and be sure to place the cap (or) plug back on the same tube it came from.

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2. (a) (b) (c)

Using the culture of E. coli provided, inoculate: A tube of glucose BC. P. broth covered with a vaspor (or) agar seal. A glucose BC. P. agar-shake culture Incubate all tubes at 37oC At the next laboratory period observe the culture for acid and gas production, and record the results in the table on the report sheet; some bacteria –for example, leuconostoc-produce only small amounts of gas that cannot be detected by the Durham tube technique the gas being lost from the surface of the medium. By the use of vaspar seal technique, this problem is avoided, and small amounts of gas can be detected.

Recording result

3.

CITRATE UTILIZATION TEST The ability to utilize citrate as a sole source of carbon and energy can be used to distinguish certain gram negative rods.

Medium Simmon’s citrate agar contain citrate as its only carbon and energy source. Growth on this medium is a positive test for citrate utilization. Certain organisms that give a positive test increase the pH, changing the bromothymol blue indicator in the medium from the green to blue. Requirements: Bacterial culture of Bacillus, E. coli, pseudomonas; Simmons Citrate agar. Composition of Simmons citrate agar - per liter – MgSo 4 0.2mg; NaH2PO4 1.0gm; k2HPO4 1.0gm. sod. Citrate 2.0gm; NaCl 5.0gm; Agar 15.0gm; Bromothymol blue 0.08gm. Procedure: 1. 2. 3. Inoculated a slant of simmons citrate agar with bacterial cultures. Inoculated at 37oC for 48 hours. Observed the growth.

Recording result Utilization of citrate and growth on citrate agar results in an alkaline reaction, so that the bromothymol blue indicator in the medium changes from the green to bright blue. When no growth occurs and citrate is not utilized, the color of the medium remains unchanged. Bacillus, E. coli, Pseudominas are not citrate utilizing bacteria.

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C.

MISCELLANEOUS TESTS

1. CATALASE TEST Catalase is an enzyme found in most bacteria. It catalyzes the breakdown of Hydrgen peroxide which release tree oxygen gas. Catalane + hydrogen peroxide water + molecular oxygen. In many cases, gases can be readily seen as a white growth if a few drops of 3% H2O2 are added to a microbial colony (or) to a broth culture. Catalase negative organisms tend to be anaerobic since cation terminal respiratory enzyme reset with atmospheric oxygen to from H2O2 which is tonic to the living cell. Perhaps the enzyme catalase is essential for the aerobic growth of most micro organisms. The enzyme catalase contains the hemeporphyrin structure. This porphyrin ring structure is characterized not only of catalase but also of the cytochrome respiratory electron carrier found in aerobic forms of life and in the chlorophyll of all photosynthetic cells. Medium Nutrient broth nutrient agar. Test Reagent Hydrogen peroxide (10 volume concentration) Procedure 1. 2. Inoculated the three bacterial cultures (Bacillus, E. coli, and Pseudomonas) nutrient broth and incubate at 37oC. Prepared three nutrient agar slants in test tubes and streaked the 3 bacterial cultures in 3 different slants and incubated at 37oC. Transfer a bit of growth from each slant to slide, added few drops of Dissolve the colony. Then add 3% H2O2 and observe. Take three samples in test tube from nutrient broth add 3% H2O2 and observe.

3. 4.

Recording result Effervescence, caused by the liberation of free oxygen as gas bubble, indicates the presence of catalase in the culture under test. References 1. 2. 3. 4. 5. Microbes in action Fundamental principles in Bacteriology – A. J. Salle Bacterial metabolism- H.W. Dowelle Methods is microbiology General microbiology – Stanier.

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BIOCHEMICAL CHARACTERIZATION OF BACTERIA A. GELATIN HYDROLYSIS TEST Peptone Beef extract Gelatin Tryptone Yeast extract K2HPO4 Soluble starch 0.5% 0.3% 4% 10 gm 10 gm 5 gm 3gm

Microorganism inoculated in the medium after sufficient time it is seen that medium is solidified or net. If medium solidified then there is on organism synthesized proteolytic enzyme gelatinase which hydrolyses the gelatin. B. CASEIN HYDROLYSIS TEST Skimmed milk Agar HO4 Casein -M. O Calcium paracaseinate is formed by which around microorganism white appearance will be lost. (Kanin also hydrolysed the casein but in this case curd like appearance is seen). C. H2S PRODUCTION TEST Beef extract Peptone Peptonized iron Sodium thiosulfate Agar Distilled water D. INDOLE PRODUCTION TEST Tryptone NaCl pH 1-2% 0.5% 7.2 3gm 3gm 0.2gm 0.25gm 3.0GM 1000ml paracasein + Ca+2 -Calcium paracaseinate 2% 2%

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E.

UREASE TEST Peptone NaCl KH2PO4 Phenol red Urea pH Distilled water Na2 C=0 NH2 -----H2O Urease 2 NH3 + CO2 1gm 1gm 4gm 0.01gm 20gm 6.8 – 6.9 1000ml

Microorganism in which urease enzyme synthetized gives the positive test urease hydrolyses the urea by which ammonia is formed. Due to increase of ammonia pH is also changed (incl) and thus to this high pH the red colour of phenol red into purple colour. F. CITRATE UTILIZATION TEST Simond’s Agar MgSO4 NaH2PO4 K2H2PO4 Sodium citrate NaCl Agar Bromomethyl 0.2 gm 1 gm 1 gm 2 gm 5 gm 15 gm 0.08 gm

Citrate utilization elevates the pH, they produced alkaline substances. Green colour changes to blue.

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Experiment No. Objective: To identified the organism producing H2S gas. Requirements: Beef extract, peptone, peptonized iron sodium thio sulfate, Agar, Distilled water. Principle: The activities of some bacteria frequently result in liberation of H2S. common example for this reaction are found in the “Aroma” of rotting eggs and is blackening of certain spoiled common food. Blackening occurs due to the reaction between H2O and compared CH2SH 2H H2N – CH - COOH Cystine Observations: ----H2N – CH + H2 2 ST

Result:

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Experiment No. Objective: To find out the presence of catalase in bacteria. Requirements: Principle: Catalase is an enzyme that is present in an aerobic bacteria. It catalyses a break down of H2O2 in to H2O H2O2 ---------H2O + O2 H2O2 is a toxic for living organism and generally all aerobic bacteria continues this enzyme. Presence of this enzyme in bacteria is easily measurable. Take a bacteria culture into a test tube (1 ml) and a 2-3 drop of 3% H2O2 and if bacteria contains this enzyme then inginous broth is obtained in the medium due to liberation of O2. Observations:

Fresh formation Sample I Sample II Sample III Sample IV Yes Yes Yes Yes

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8. MUTATIONS AND MUTANTS Introduction Any permanent sudden and heritable change in the sequence of bases in the bacterial genome is called mutation. Mutations occur very rarely, one cell in 108 cells carries a detectable mutation in any given, but exposure to certain powerful mutagens eg nitro so guanidine increase the frequency of cells in a population that assay a mutation in any given gene about 105 fold. Classification of Mutations based on genotype I. Macrolesions Mutations may involve a change that extends over a number of base pairs or even a of genes. A macrolesion include variety of changes in DNA. A. B. C. D. E. Deletions: Inversions: Insertion: Translocation: another The complete elimination of a segment of DNA. The inverting of a segment of DNA. Introduction of a new segment of DNA Within an existing Sequence. Movement of a segment of a segment of DNA to site in the genome. II. Micro lesions (Point Mutation) Mutations may involve a change in only a single base pair of the cell’s DNA A) Base pair substitutions If a micro lesion involves a change in only a single base pair to another. 1. Transition: Purine base of a pair is changed to another purine base. Purine base of a pair is changed to a pyrimidine 2. Translocation: base. B) Frameshift Duplications: The tandem repetition of a segment of DNA.

If the microlesion involves loss or gain of a base pair it is called a frame shift mutation because it changes the reading frame of all codons of the gene or operon distal to the point of mutation.

MUTAGENS

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Mutagens are chemical or physical agents that increase the frequency at which mutations occur in growing bacterial culture. Physical Agents These include various types of radiation. X=- rays cause breaks in chromosomes that reform in a variety of ways, causing macrolesions. Ultraviolet is absorbed by DNA and the energy so released causes dimerization. Between adjacent pyrimidine residues on the same DNA strand. The occurrence of these pyrimidine dimmers exposing a short region of single stranded DNA on the opposite strand. The presence of these single stranded regions induces the formation of a general DNA repair mechanism termed the SOS system. This is a rapidly acting but inaccurate DNA mutations that follow U. V. irradiation. Chemical Agents I. A. Mutagens that associate with or become incorporated into DNA. Mutagens that become incorporated into DNA. Eg 2- aminopurine – causes transition mutations. B. Intercalating agents Bg. Mutagens that become associated with DNA eg. ICR – 191 – causes frame – shift mutations. Intercalating agents are plain molecules and can insert between the stacked pairs of bases in the core of the DNA molecule. Such incorporation distorts the backbone of the double helix in such a way that frame shift mutations can occur when the distorted helix is replicated. II. A. Mutagens that react with DNA Alkylating agents Base analogue- Bg 5. Brornowail

These are most powerful known chemical mutagens which add methyl or ethyl groups to the heterocyclic nitrogen atoms of the bases. By these a variety of different types of mutations result. e.g. They cause transition, transfusion and frame shift mutations. NH eg. Nitrosoguanidine B. eg. O = N-N-C-NH-NO2 CH3 Other DNA modifiers 1) hydroxylamine (HONH2) reacts specifically with cytosine converting it to 6- hydroxylaminouracil which pairs with adenine, causing G/C to A/T transitions. 2) Nitrous acid is less specific in its action and reacts with all bases (A,G and C) that contain amino groups, thereby converting the

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amino groups to hydroxyl groups. This causes both A/T to G/C and G/C to A/T transition mutations. Phenotypic Consequences of Mutations Many mutations inactivate indispensable gene product and therefore kill the cell. But many others inactivate gene products that are not essential under all conditions of growth and these are not essential under all conditions of growth and these are not lethal to the cell. Cells carrying later type of mutations can be maintained in culture and they differ phenotypically from their unmutant parents (wild type strains). Other mutations that alter the target protein of an antibiotic can render the cell resistant to the antimicrobial agent. Still other mutation strains might have lost some nonessential capacity, such as motility. Mutant Methodology A mutation alter or eliminates the functioning of a particular gene particular gene product; by observing the effect of genotypic change on the cell’s phenotype, one can deduce the cellular functions of the gene product. This process is called mutant methodology. Mutation Rate The mutation rate can be defined as the probability that any one cell will Mutate during a defined interval of time. Mutation rate is thus the number of mutations per cell per generation.

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Experiment No. Objective: To study the effect of U.V. light on % survival of bacteria. Introduction: Mutagens are chemical or physical agents that increase the frequency at which mutation occur during growth of a culture. Among the physical agents various types of radiations are powerful mutagens. α -rays cause breaks in chromosomes that reform in variety of ways causing most types of macro lesions. Radiation damage is conveniently generated by a U.V. light source such as 15 or 25 W germicidal lamps. τ - irradiations from a 60CO source is also convenient method for DNA damage. Sunlight is normally injurious to most bacteria. This effect is primarily due to the U.V. portion of the spectra which increase the rate of mutation. U.V. light is observed by the DNA and the energy so released causes dimerisation between adjacent pyrimidine residues on the same DNA strand. The occurrence of these pyrimidine dimmers triggers a repair mechanism that excises the pyrimidine dimmers excising a short region of single stranded DNA on the opposite strand. The presence of these regions induces the formation of a general DNA repair mechanism, termed SOS system. It is a relatively inaccurate DNA repair system that fills in the gaps opposite to the single stranded regions. This error prone repair, introduces the mutations that follow U.V. irradiation. The irradiated bacterial cell cannot reproduce and hence dies. The maximum absorption of U.V. by DNA is at the wavelength of 254nm. Maximum mutagenicity also occurs at 254 nm. Suggesting that U.V. induced mutations process is mediated directly by absorption of U.V. by purines and pyrimidines. The two major products of U.V. absorption by pyrimidine are pyrimidine hydrates and pyridine dimmers. Many evidence suggest that thymine dimerization is probably the major mutagenic effect of U.V. Products of U.V. irradiation Hydrotes cause mispairing of bases during replication.Cross linking of adjacent thymine molecules to form thymine dimmers, which block replication.

Requirements: Apparatus and materials – U.V. light apparatus, patri plates, pure culture of bacteria (E. Coli) Chemicals – Nutrient agar of composition – Peptone = 0.5 gm Yeast extract = 0.3 gm Agar = 1.5 gm Distilled water =100 ml

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Procedure: Make 10-6 dilution of the culture Pour 1 ml of this dilution into 5 small sterile Petri plate Label these plates 0 sec, 5 sec, 10 sec, 15 sec, 20 sec Expose these plates with their cover removed under UV light for the interval of time marked on them. Add proper amount of melted and tempered agar and mix it Incubate all plates at 37oC for 48 hr Count and record the colonies and calculate the percentage of survivors in the irradiated sample. Observations:

General Comments: The virtues of radiation treatment are several fold: 1. 2. The dosage is easily controlled by varying the exposure time and intensity of the source. Radiation generates a wide variety of lesions across a genetic target and consequently a broad spectrum of mutational events including base substitutions, frameshifts and deletions. There is no hazardous chemical to handle or dispose off. A drawback of radiation mutagenesis is that the lesions generated by U.V. are not efficiently fixed as mutations unless the cells are capable to carrying out SOS repair. Precautions: U.V. radiation is a potent mutagen and human carcinogen. To avoid the exposure to U.V. radiations, wear U.V. opaque glasses and cover hands and fore arms.

3.

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Experiment No. Objective: To study the effect of Photoreactivation. Introduction: Although certain wavelengths of visible light are beneficial still sunlight is normally injurious to most bacteria. This effect is primarily due to rays of the ultraviolet spectrum, particularly wavelength between 2400 Å and 3000 Å. 2650Å is also the peak of absorption of UV light by DNA and radiation of this wavelength affects mutation rates, bacterial transformations and U.V. radiation appears to act on genetic material to produce pyrimidine dimmers on the same DNA strand or between different DNA strands. The pyrimidine bases absorb the energy of the U.V. light in the energized state link with adjacent pyrimidine bases interfering with the proper replication and function of the DNA molecule. Therefore irradiated bacterial cell cannot reproduce and die. The bactericidal effect of exposure of ultraviolet light can be educed by immediate subsequent exposure to certain wavelength of visible light (3650-4500 Å). This reversal of killing action of ultraviolet light is called photo reactivation. In this visible light activates an enzyme that cleaves the pyrimidine dimmers, which result from U.V. irradiation, allowing DNA to resume its normal functioning. Exposed to visible light immediately after being exposed to visible light will be many times of a population exposed solely to U.V. light. However there will always be some cells whose damaged cannot be repaired by this process of photo reactivation. Procedure: 1) Plate 10-6, 10-7 and 10-8 dilutions of a 24 hour culture on agar from the colony counts these plates you will determine the average number of cells per milliliter in the original culture. Place 2 ml from the 10-6 dilution flank into each of four small, sterile Petri plates. Label these plates 5 sec, 10 sec., 15 sec and 20 sec. Place each plate with the cover removed, under the U.V. light for the length of time marked on it. Immediately following its exposure to the U.V. light, prepare a 10-7 dilution of each sample by transferring a 0.1 ml aliquot to a sterile Petri plate adding the proper amount of method and tempered agar and mixing by suruling. Label each new plate with word irradiated, the time of exposure, and the dilution. Transfer a 1.0 ml sample from each plate irradiated in step 3 to a different sterile test tube; label each with the time of irradiation.

2) 3) 4)

5)

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6)

Place the four tubes 6 inches away 500 watt bulb for 30 minutes. If the light source you are given does not have a fan attached for cooling, place the tubes in a beaker of ice. Plate 10-7 dilutions of each sample exposed to light, label each plate with a word photo reactivated, the time of U.V. irradiation and 10-7. Incubate all plates at 30oC for 48 hours. Following incubation count and record he number of colonies on each plate. Calculate the percentage of survivors in the irradiated and photo reactivated samples.

7) 8) 9)

10) Plot the number of survivors versus time for the irradiated and photoreactivated samples.

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Selection of spontaneous mutants Mutant is an organism whose genotype differs from the wild type. Mutations are the raw material of evolution, reducing the changes on which selection can act. The process of generating mutations is called mutagenesis. Mutations sometimes arise spontaneously. This is called spontaneous mutagenesis. Mutations can also be induced by mutagens which may be chemicals or physical factors. Because of the low rate of appearance of mutation bacterial cells, special methods must be used to select them out & screen them. (A) Selection: is a condition that allows specific mutants to grow but not the parent cells. Genetic selections are from a population of cells eg. antibiotic resistant mutants can be selected by simply planting large no of bacteria on solid medium containing the antibiotic. only the resistant mutants can form colonies. Screen: In screening for mutants on media on which both the mutant and the parental cells grow, the phenotype of the mutation can distinguished by the phenotype of parental cells. Some bacteria are naturally resistant to certain antibiotics; others can acquire resistance by mutations. There are two main ways in ways which bacteria develop antibiotic resistance. 1) 2) Acquiring an enzymatic activity that directly inactivates the antibiotic. Acquiring a mutation that modifies the target site of the antibiotic, which prevents the antibiotic from interfering with the normal function of the target site.

(B)

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Experiment No. Objective: To develop antibiotic resistant mutant Requirements: Bacterial suspension, Tetracycline, Agar yeast extract, peptone, streaking needle. Principle: Antibiotic susceptible strains can be made mutant by various means. The mutant can be developing by growing the culture at different concentration of antibiotic. The concentration that can grow at higher conc. Of antibiotic can be selected as the mutant. Procedure: 1. 2. 3. 4. 5. 6. Prepare the agar gradient plate and solidify. Pour the antibiotic mixed agar (50 µ g/ml) on the already prepared agar plate. Grow the bacterial culture by streaking on the agar plate. Incubate it 37oC for 48-72 hrs. Select the bacterial colonies that grow at higher. Concentration of the antibiotic culture the selected bacterial colony.

Observations:

Result:

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Experiment No. Objective: Selection of antibiotic resistant spontaneous mutants using gradient plate method. Requirements: Principle If a mutant cell, by virtue of the change that it has undergone, is somehow especially suited to the environment in which it is formed its growth may outstrip that of its parent culture and it may thus become dominant. The spontaneous mutants that are resistant to antibiotics are readily detected since they grow in concentrations of the antibiotics that would inhibit growth of normal organisms. Requirements: Pure culture of E. Coli, oxytetracy cline, nutrient agar, Petri plate bent glass rod. Procedure: We employ “gradient plate” method to isolate oxytetracycline resistant mutants. 1) Prepare a gradient agar plate a) Till the sterile Petri dish by placing a glass rod or wood stock, approximately 1/16 inch in diameter, under one side. Pour a tube of melted trypticase soy agar into the Petri and allow the agar to harden, slanting the plate just enough to cover the entire bottom. b) After the agar has solidified, place the dish in the normal horizontal position. To a scanned tube melted agar (taken 100 ml) add 0.1 ml of oxytetracyline. Mix the tube well and pour the melted agar on the surface of the gradient agar plate. 2) Pipette approximately 0.5 ml E. coli culture on the agar surface. Using a sterile glass spreader, streak the cultures over the agar surface. Glass spreader is sterilized by dipping 95% alcohol, flaming and cooling on the sterile surface of the gradient plate. Incubate at 37oC for 24 hours.

3)

Observations:

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Experiment No. Objective: Transformation of DNA into competent E. coli cells. Introduction: Transformation is the form of recombination in which free DNA from a donar cell is taken up by a recipient cell and integrated into the latter’s genome without the two cells ever coming into contact. Those recipient strains that are capable of incorporating free DNA are called competent. A natural competent is one which can act as recipient and such bacteria are capable of undergoing natural transformations. Some bacteria can be as exposure of cells to hi concentrations of divalent cations. Such transformation systems are called artificial transformation. Transformation is the only mechanism of genetic exchange among prokaryotes that is chromosomally encoded. Inspite of this many baceria lack natural transformation eg E. coli. The genetic exchange in transformation is permanent and this method is employed in lab to bring recombination; but it may be accruing innature as well. Competence has been demonstrated only in some strains of a few bacterial genera, and then only in cells at a specific stage of growth in nutritionally minimal media. In this exercise we will extract DNA from a tryptophan – requiring, histicline – synthesizing strain of Baccilus subtilus. This free DNA will then be used to transform an auxotrophic strain of Baccilus substlus, which is unable to synthesize the amoni acid histidine, a histidine synthesizing strain. Procedure: A. DNA extraction 1) 2) Centrifuge the tryptophan- requiring culture of Bacillus subtilus strain 168 provided. Aseptically d:, scard the supernatant and resuspend the cell pellet in 5 ml of sterile saline- citrate solution. Pour the solution into a 25 x 150 mm test tube. Add 0.5 ml of lysozyme solution to test tube. Min well. Shake gently at room temperature for 15 min. and observe clearing or lysis. If lysis is not complete, add another 0.25 ml of lysozyme. When lysis appears to be complete, add 5 ml of 4 M NaCl to the test tube, mix gently and filter the solution through a membrane filter (0.45 µ m pore size), collecting the filtrate in asterile test tube. Test 0.1 ml of filtrate (containing DNA) for sterility, by streaking on a tryptose – blood agar plate. Table the tube and refrigerate it. Incubate the plate at 37 oC for 24 hours.

3) 4)

5)

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B. Transformation 1) To prepare competent cells, inoculate 1 ml of washed suspension of a 5-hour culture of Bacillus subtilus (his, trp) into a sterile 1.25 ml flask containing 4 ml of transformation media (a minimal medium containing trp and his) Aerate this flask for by shaking for 1 hour. Pipette 0.9 ml of these competent cells into 3 sterile tubes. Label the three tubes A, B and C. To tubes A and B add 0.1 ml of the sterile DNA sample from a previous period. In addition add 0.1 ml of DNA are (2 mg/ml) to tube B to tube C add 0.1 ml of sterile saline – citrate as a DNA less control. Aerate all tubes by shaking for 1 hour. Label three minimal-plus – tryptophan agar plates A, B and C. transfer 0.1 ml aliquots from tubes A, B and C to plates A, B and C respectively. With a sterile bent glass rod spread each aliquot over the agar surface of a plate. In addition make 1/10 and 1/100 dilutions of the contents of tube A and transfer 0.1 ml aliquots of these dilutions of minimal – plus – tryptohan agar plates labeled A -100 and A-1000. again using a sterile bent glass rod, spread these samples over the surface of the agar plates. Incubate the plates at 37oC for 24-48 hour. Count the number of transform ants at each dilution.

2) 3) 4)

5) 6)

7)

Observations:

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Experiment No. Objective: Genetic recombination of two strains of E. coli by conjugation. Introduction: Genetic recombination is the exchange of genetic material either by exotic means as well as by a straight forward conjugation process. Conjugation is a process by which DNA can be transferred form a donar cell to a recipient cell by cell to cell contact. Conjugation has been demonstrated primarily in enteric gram negative bacteria- including Escherichia, salmonella etc. and has been most studied in E. coli. There are two mating types in E. coli genetically determined by the presence or absence in the cell of a small DNA element called the F (fertility) factor. a) b) Cells tacking the F – factor can only act as recipient: called F (or female) Cells carrying F – factors act as donars and are “male” cells. These are of two types [ F+ cell – F particle is detached from the cells chromosome Hf cells – F – particle is part of the chromosome.] In F+ cell only V- factor is transferred in conjugation, with the F - cell being converted to a V+ cell in the process. In conjugation if Hfr is one of the participants, chromosome 1 DNA is transferred to the recipient cell. Complete chromosome transfer is rare Vfactor is not transferred: indicating that the F- particle is attached at the distal end of the chromosome being transferred. In this exercise we will observe genetic recombination resulting from the conjugation of two strains of E. coli mutants1) Strain C- 600 is an F- strain that requires threonine (T-) and Leucine (L-) for growth; this is unable to ferment lactose (Lac -) and is resistant to streptomycine (Ss). Strain Hfr – 235 DONAR: does not require threonine (T +) or leucine (L+) for growth, is capable of fermenting lactose (Lac +) and is sensitive to streptomycine (Ss). The genetic crossing of these mutants should result in recombinant like the wild type E. coli from which the mutant strains arose with respect to growth requirements. Procedure: You are provided with culture of E.coli, strains C- 600 and Hfr235, that have been grown in a complete nutritional medium, aseptically, washed several times by centrifugation to remove nutrients, and concentrated by resuspending in sterile saline to 1/20 of the original volume.

2)

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1)

With a sterile 1.1 ml pipette, transfer 1.0 ml of C- 600 suspension to a sterile wasermann tube and 0.1 ml to the surface of a minimal plus- streptomycin agar plate. Spread the 0.1 culture on the agar plate with a bent glass rod as folowa: a) Dip rod in alcohol, pass quickly through a flame, and allow alcohol to burn off. b) After cooling the spreader, using a circular motion, spread the inoculum evenly over the entire surface of agar. Label this plate NAC-600.

2)

Remove 0.2 ml of Hfr- 23suspensinon, trasnsferring 0.1 ml of Co600 suspension and pipette remaining 0.1 ml onto the surface of a second minimal – plus – streptomycin agar plate. Using the glass the inoculum over the entire plate. Label this second plate NA Hfr235. Mix the tube containing the two strains by rotating gently. Violent mixing will interrupt genetic transfer. With the sterile 1 ml pipette further mix by gently drawing the suspension into the pipette one allowing it to flow out. Allow this mixture to rest on the bench for 3060 minutes. Remove some of the mixture with the same pipette and add 0.1 ml of it to a third minimal plus streptomycin agar plate spread in the same manner as above. Label this plate MAC- 600 plus Hfr-235. Invert and incubate the plates at 37oC.

3)

4)

Observations:

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9. QUANTITATION OF VIRUSES BY PLAQUE ASSAY Background: There are many methods for the enumeration of viruses. Plant viruses are typically enumerated by the plant leaf local lesion assay: a suspension of viruses is applied to the surface of a leaf along with an ambrosia material that tears small holes in the walls of plant cells. Each vision that enters a host cell initiates a local infection that spreads to surrounding cells, creating a region of infection that becomes discolored and thus easily recognized. Before the development of techniques for culturing animal cells invitro, animal viruses were usually enumerated by the pock assay performed in developing chicken embryos: a sample containing viruses is injected into a fluid compartment of the egg. These viruses absorb to cell in one of the interval membranes of the developing embryo, producing regions of infection termed pocks that can be recognized as being thickened and discolored. Another important method of enumeration of viral particles is by Plaque Assay. The viruses are ultra microscopic – too small to the viewed with light microscope. They are particulate, not cellular; being more or less macromolecules composed primarily of a nucleic acid genome either DNA of RNA and protein. They are obligate parasites and use hosts machinery for replication. Basically the viral particle. Of version, is a nucleic acid core surrounded by a protein coat or capsid composed of protein subunits or cashmeres? The viruses would have gone unnoticed during the early development of microbiology except for the fact that they are infections agents, evident from the symptoms of the disease they caused. Even today, when are see a viruses with electron microscope, we still depend for study primarily on the symptoms of experimental virus infections. Bacterial viruses are called bacteria phages. As the bacterial cells increase the viral particles are formed; the cells eventually disrupt and release thee viruses into the medium. The plaques represent areas of phage reproduction and lyses of the infected bacterial cells in the area.

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Experiment No. Objective: To quantitative viruses by plaque assay. Introduction: Most commonly, animals and bacterial viruses are enumerated by infecting host cells that growing in their layer on a medium partially solidified by agar. An infected cell in such a culture establishes a lock, spreading infection where, depending on the infecting virus, cells either die or grow abnormally slowly. These infected zones termed plaques differ in appearance from the surrounding cell layer. Plaques formed by phages are usually clear, circular regions in a turbid layer of cells termed a lawn; plaques formed by animal viruses are sometimes visualized following application of a dye that stains line cells, but not those killed by the viral infection. Requirements: Apparatus and material Test tubes, pipette bacterial culture (E. coli), virus lysate, Petri plates. Chemical: TYG broth, TYG agar Constituent: Tryptone Yeast extract Glucose Agar Distilled water Procedure: Took virus Lysated Prepared its dilution ranging from 10-1 to 10-7 Prepared its dilution ranging from 10-5, 10-6 and 10-7 and pipette into molten tubes of top layer agar (3.5 ml each) By pipette we added .2 ml of overnight E. coli culture to each of the 6 tubes. Poured the content of all six tubes into freshly made TYG agar plates and spread it. 5 gm 3 gm 1 gm 15 gm 1 liter, pH = 7

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Incubated at 37oC overnight Prepared control plate by inoculating only E. coli on TYG agar Incubated at 37oC overnight Observations:

General Comments It is wise to plate each dilution in duplicate in case one of them smears because of excess moisture. The most reliable liter is determined from assay plates they contain between 50-400 plaques. At the time of determining the phage lysate liter, it is wise to take some of the lysate and sheak on a non selective L agar plate for enquiring that the lysate is free from all viable donor bacteria.1

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Experiment No. Objective: Quantitation of viruses by Plaque assay (Bacterial lysis by bacteriophage) Principle: Bacterial viruses are enumerated by infecting host cells that are growing in a thin layer on a medium partially solidified by agar. In infected bacterial cell in such a culture establishes a local spreading infection where cells either die or grow very slowly. These infected zones are called plaques, formed by phages are usually clear, circular regions in a turbid layer of cells termed a lawn. This technique of counting phage is called plaque assay. Theory: If 108 bacteria are spread on a rich plate, the colonies formed are so close to each other that appear as a confluent, turbid layer of bacteria called a lawn. Or the bacteria can be mixed into a small volume of warm, slightly dilute, liquid agar, which is then poured into the surface of the solid medium. The liquid known as top agar or soft agar, hardens, providing a uniform lown of bacteria. If a phage is present in the top agar, it can adsorb to one of the bacteria in the agar; the infected bacterium lysis and releases about 100 phage, each of which adsorb to nearby bacteria. These bacteria in turn release a burst of phage which can infect other bacteria in the cinity. These multiple cycles of infection continue and after several house phage will have destroyed all of the bacteria at a single localized area in the agea, giving rise to a clear, transparent circular region in the turbid confluent layer. This region is called plaque. As one phage forms one plaque: number of bacterial virus added can calculated from the number of plaques. The efficiency of plating (EOP) is the fraction of phage particles that can form a plaque. It is one for many phages but can less that 1 for phages that make very small plaques. If phage lysates are stored, EOP may decrease, as a result of accumulated chemical damage of penetration of phage proteins. Storage of phage at 4oC often reduces the loss of phage viability. Addition of cations, glycerol and proteins also protects phage from damage.

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CONTENTS ANIMAL (Mammalian) CELL CULTURE I. II. Introduction Cell culture techniques. A. B. C. D. E. F. III. A. B. C. IV. A. B. Cell biology Development of cell culture technique Handling of mammalian cell culture lies. Small scale culture of animal cells. Suspension culture. Anchorage dependent cells. Primary cell culture. Genetic engineering Monoclonal antibodies Introduction Components of cell culture media 1. 2. C. D. Water Basal media

Cell culture: Organism and products.

Cell culture media

Animal sera as medium supplements Defined media 1. 2. 3. 4. Carrier proteins Insulin Growth and attachment factor Miscellaneous Quality control Sterility

E.

Preparing and handling of media 1. 2.

V.

Process design and operculum A. B. C. D. E. Introduction Suspension culture High density suspension culture Entrapped – cell suspension reactors. Micro carrier culture

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F. VI. A. B. C. D. VII. A. B. VIII. I.

High density perfusion reactor Introduction Cell removal Concentration Purification Biohazards associated with the cell culture. Regulatory environment.

Down stream processing

Regulatory aspects of products from Animal cell culture

References

INRODUCTION

The culture of animal cells has been a rather difficult field of biology. Developments in several areas of biotechnology have brought remarkable changes in this in the last few years. The discovery of bimolecular with great potential for therapeutic or diagnostic benefit has provided commercial motivation for rapid development of cell culture technology. At the same time, advanced analytical techniques and methods for manipulation of cell at the genetic level, along with an improved understanding of cell biology at all levels, have given researchers new tools to help bring the benefits of these discoveries to the public. This chapter will provide an over view of current animal culture technology with an emphasis on animal biotechnology. Developments in plant cell culture and organ culture have also generated considerable interest. II A. CELL CULTURE TECHNIQUES CELL BIOLOGY

A discussion of cell culture technology should begin with the animal cell in form and function, have been “designed’ by millions of years of evolution. Malian cells have a fragile membrane, a high cell density and an intricate circulatory system for supplying essential nutrients and removing waste products, light control of the physical environment (temp, pH etc) and control system for regulation by externally provided hormones and growth factors individual cells are typically differentiated to serve specific functions within a tissue or organ. This differentiation will suppress most of the available genetic information and commit the cell to its particular course. Aside from the use of animal cells in viral vaccine manufacture, the main benefits of cell culture as a manufacturing system are derived from the ability to assemble and than excrete large and complex proteins. These complex proteins are often modified after translation by glycosylation or carboxylation at specific locations. Bacteria do not possess the machinery for such modification; yeast and other eukaryotic organisms do have some

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capabilities in this regard but produce molecules different from the native proteins. The animal cell culture now becomes the only practical method for producing significant quantities of fully functional proteins. Even among mammalian cells, the structure or post-translational modifications of a given protein may vary when produced in different cell lines, the correlation of functional activity with changes must be examined on a case-by-case basis. B. DEVELOPMENT OF CELL CULTERE TECNIQUE Early work with cultured cells focused on normal or “Primary” tissues and cell lines. Cell lines are generally isolated by grinding or chopping on animal tissue, washing carefully and soaking a trypsin or collagens (if necessary) to dissociate the tissue matrix (BASHOR, 1979). Ideally the cells can be “cloned” or diluted such that a collection of cells can be “cloned” or diluted such that a collection of cells can be recovered that has grown from an individual cell. Many normal cells do not grow well under limiting dilution conditions; as a result this procedure can select a cell line with characteristics that differ markedly from the average properties of cells in the starting tissue. Most normal animal cells can only reproduce for a limited number of generations and do not survive in suspension culture; these features limit their usefulness as producing cell lines. Those cells that do become established in long-term culture (immortal cell lines) typically exhibit chromosomal abnormalities and sometimes fail to respond to hormonal signals and growth factors. C. HANDLING OF MAMMALIAN CELLS LINES A well managed and thoroughly documented system for cataloguing and handling cell lines is critical for meeting regulatory requirements and establishing a reliable process. A sterile sample of the producing cell lines should be preserved in vials or ampoules in liquid nitrogen to form a master seed lot. One of the containers from the master seed lot should then be expended and used of form a working seed lab. The frozen seed lots and copies of the documentation should be maintained in at least two separate locations. Both the master and working seeds lots should be tested as necessary to ensure the integrity of the manufacturing process. Cell cultures can contain aerobic or anaerobic microbial contaminants, mycoplasma, fungal or viral contaminants. Cross contamination of cell lines is also a major problem. The table below list procedures available for testing and characterization of mammalian cells. METHODS FOR ANALYSIS OF CELL LINES Contamination Test Result Bacterial, fungal Method Microscopy, culture Nutrient Positive broth Presence of cells colonies turbidity in broth broth, Mycoplasma

Mycoplasma

Nutrient immunofluorescence

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morphology, immune reaction hybridization to mycoplasma DNA Antibody production test coculture with sensitive Cell Detection of antibodies Viral: MAP testing pathologic effect enzyme assay Cytopathicity Detecon o9f reverse Retrovirus transcriptase.

CELL LINE IDENTIFICATION TESTS Karyatyping Isozyme analysis Characterization of products (esp. antibodies) Nucleic acid hybrization Immunofluorescent detection of cell surface antigens. D. SMALL SCALE CULTURE OF ANIMAL CELLS Once a cell line of interest has been isolated and a seed lot established, various techniques are available for culturing the cells at laboratory scale. Cell cultures researchers recognized at an early stage that tumor cell lines could be cultured simply by injecting the cultured cells into a susceptible host animal. This technique is still important for the production of antibodies form murine hybridomas. In vivo antibody production begins with the injection of about 106 hybridoma cells into the peritoneal cavity of mouse. This procedure works best if the mouse has been primed a week of so previously by the injection of a mineral oil such as pristanc (HOOGANRAAD and WRAIGHT, 1986). After a few weeks 10 ml aliquots of ascites fluid can be with drawn at regular intervals, containing rather high concentrations of immunoglobulin. This in vivo method suffers form poor reproducibility and contamination by murine proteins and there is still proential for economy of scale when larger amount of products are needed. SUSPENSION CULTURE In vitro cell culture techniques depend on the type of the cell to be grown. Anchorage dependent cells require different techniques than cells that have been adapted to suspension culture. Suspension culture is conducted in “Semi- continuous mode” (repeated batch culture with exchange of the bulk of the medium on the regular schedule) under conditions that allow continual cell growth. This repetitive sub culturing procedure is common at all scales i. e. wheat her in flasks or to full size production reactors. Cell cultures should be maintained when possible under conditions that do not stress that cells. This can cause selection of a subpopulation of cells that may have low productivity or other undesirable

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features. Stress covers a widevariety of potential problems. The most typical being oxygen or nutrient limitation, accumulation of toxic metabolites in the culture medium, changes in pH or over dilution. Non agitated cultures in closed flasks usually show a reduction in net growth rate and then a cessation of growth (called stationary phase at populations approaching 1.5 x 106 cells/Ml. semi-continuous culture will proceed most smoothly, however if the medium is replaced well before 80% of the maximum concentration is reached. At small scales (10 to 50 ml of culture fluid), adequate nutrient and oxygen supplies are easily maintained. The method of choice at this scale is generally the tissue culture flask or T- flask: this is a rectangular prism with a neck raised or inclined at an angle to keep liquid out of the cap – when typical sodium Bicarbonate buffer (system) is used; one can control the culture pH with in a tolerable range using an atmosphere enriched to about 5% in carbon dioxide. The valium of the vessel occupied by the liquid is generally kept below 20% in order to allow sufficient gas exchange through the gas liquid interface. At volume large than about 30 ml, cell concentrations and productivity can be improved through agitation of the culture medium. Spinner flasks are routinely used; these are cylindrical glass vessels featuring a suspended magnetic sire bar on a survival and side arms for addition or removal of inoculums or medium components. Up to 50% of the flask volume can be filled with the culture medium. Spinner flasks can be used for routine work in volumes up to about 10 liters, about aeration the liquid surface become limiting. F. ANCHORANGE- DEPENDENT CELLS Anchorage – dependent cell lines are also maintained in Tflasks at small scale; a wider size range of T-flasks is used routinely because spinner flasks do not provide the necessary surface area for cell attachment. The maintenance of anchorage dependent cells requires new techniques and more careful attention to the condition of the cells. Cells provided with a compatible surface will attach, physically spread into a flattened conformation and then begin to reproduce and spread across the available surface. The attachment process depends on numerous biological and physical factors such as the cell line (some cells produces their own attachment factors), cell density and culture history, presence of attachment factors in the medium, mixing conditions and especially the characteristics of the surface (BARNGROVER 1986).Glass, polycarbonate, and treated polystyrene are the materials in widest use as surfaces for the culture of anchorage dependent cells. As the cells approach conflnce (complete physical occupation of the provide surface) growth rates slow and sometimes stop entirely. Many cell types, however continue to reproduce after confluence is reached, forming multiple lays or shedding cells (which in some cases remain viable) in to the medium. Sub culturing of anchorage dependent cells requires removal from the surface and reattachment after dilution into fresh medium. A solution

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containing trypsin and EDTA is generally used to detach cells from surfaces. Trypsin can have damaging effects on the cells. So this exposure should be kept to the minimum, necessary for the cell detachment. Anchorage dependent cell can be expanded to larger culture volumes through the use of roller bottles. Roller bottles are typically plastic, filled to about 20% of the total volume with culture fluid. The bottle is inoculated and placed on a apparatus that slowly rolls the bottle, alternately submerging the cells and exposing them to CO2 up to 500 ml with other small scale technologies the manufacture of biological products with roller bottles becomes limited as material requirements increase. The culture of anchorage-dependent cells can be expanded to still larger volume through the use of micro carriers. PRODUCTS OF MAMMALIAN CELL TECHNOLOGY PRODUCT TYPE VACCINES: Rabies Rubella Mumps infection Polio MONOCLONAL ANTIODIES Diagnostic kits In vitro therapy body In vitro diagnosis Detection of disease targeting Tissues Diagnosis of disease, cancer Immunity purification biomolecules. INTERFERONS treatment INTERLEUKINS IL-1, IL-2 GROWTH FACTORS G-M-colony stimulating factor Immune potentiator Anticancer, immune potentiators. Purification Prevention of infection. of and of certain Pregention of viral USE OF PRODUCT

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Earythroprotein Fibroblast growth factor Epidermal growth factor tissue BLOOD REGULATION PROTEINS Tissue plasminogen activator Prourokinase Auriculin

Promotes blood cell Formation Prometes tissue growth Promotes Growth Dissolves blood clots Dissolves blood clots. Blood pressure control. skin and

Microcarriers can be added to spinner flasks, roller bottles, or other small scale vessels to increase the available surface area. CELL CULTURE ORGANISMS AND PRODUCTS This section will describe some of the products of commercial importance and the cell lines normally used for production. The production technology of choice can be influenced considerably by the special requirements of the desired products. A. PRIMARY CELL CULTURE

The manufacture of cell culture products from the primary cells is still of considerable commercial importance. Much of the cell culture technology in current use was developed for the large – scale manufacture of final vaccines from animal cells; the use of primary cells is still being extended to the manufacture of various biological products excreted from normal cr transformed primary cells (HOPPS 1985). Vaccines for prevention of human and animal viral infection are generally produced by cultivating a large volume of susceptible cells, infecting the cells with the pathogenic virus of interest, and collecting the newly produced virus. If the virus is pathogenic, it is then inactivated in some manner that retaina the immunogenic character of the viral proteins. Human vaccines include poliomyelitis, influenza, measles. And mumps: veterinary vaccines such as foot and mouth disease (FMD). Vaccines are also produced in cell culture. Interferon, actually a class of infection fighting molecules, is one of the first cell culture products manufactured in commercial quantities. Alphainterferon is produced in namalwa cells (Human-B-lymphoblast) in suspension culture at scales up to 8000 liters the largest mammalian cell culture vessels. Plasminogen activators – have thrombolytic capability and are useful in allowing reperfusion of blood vessels after a heart attack.

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-

Tissue plasminogen activator (t-PA) has been produced using human normal and melanoma cells, and recombinant CHO cells. Other molecules with thromblytic activity such as prourokinase can also be produced by tumor cell lines.

-

Numerous other substances which are produced by mammalian cell lines are being investigated as possible commercial products. A partial list includes interleukins, stimulating factors and growth factors, hormones such as erythropoietin and enzymes. GENETIC ENGINEERING Genetic engineering techniques can be used to produce molecules of interest in mammalian cells, though genetic engineering of mammalian cells presents several difficulties comparable to prokaryotic bacteria. One can use genetic engineering techniques to over produce a molecule already manufactured by the cell. A more common situation is the use of viral vectors with a well characterized cell line for example. Chines hamster- ovary cells (CHO) to produce the material of interest. The benefits of working with a cell line with a well-known gnome and physiological properties are substantial. There are no guarantees that CHO cells will perform the same glycosylation and post-translational modifications as the original primary cells. A recent development is the potential for the use of cell culture to make novel molecules. This is designed through the use of “protein engineering ” techniques such as replacement, of a portion of the coding DNA, site directed mutagenesis, or design of proteins that include active sites combined from two or more other proteins. Such techniques have not yet provided only visible examples of improved products; however the technology is advancing rapidly and is a major focus of academic and commercial efforts. C. MONOCLONAL ANTIBODIES Antibodies produced by murine hybridoma have been reached considerable Commercial importance despite the fact that the hybridoma technology was developed as recently as 1975 (KOHLER and MILSTEIN, 1975,1976). Hybridomas are the product of a fusion between a B-cell that provides the ability to grow in suspension and reproduce indefinitely. Techniques for generation and selection of hybidomas of various species have been described by OSTERHAUS and UYTDEHAAG, 1985. typical antibody titers range from 20 µ g/ml up to 200 µ g/ml. At present, the most important application of monoclonal antibodies is the manufacture of in vivo diagnostic kits : monoclonal antibodies provide rapid response and specificity compared to most traditional analytical techniques. Antibodies can also be used for in vivo diagnostic or therapeutic use. These applications usually involve coupling of the antibody to an imaging agent (e. g. indium-11-for radiological imaging) or a cytotoxic agent.

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It is possible that many of the problems with in vivo administration of murine antibodies can be over come through the use of human – human or human- mouse hybridomas. at line present, human hybridoma technology is poorly developed. Cell lines producing certain antibodies are difficult to be produced (developed) and manipulate and the antibody yields have been low relative to murine hybridomas. The ability of monoclonal antibodies to find specific molecules has also generated considerable interest in immunoaffinity purification techniques. The widespread application of this technique will depend on improvements in durability and the ability to revse the fixed antibody. IV CELL CULTURE MEDIA A. INTRODUCTION The application of cell culture to the large-scale production of biological products has been limited, partly by slow development of suitable reactors but also by problems encountered in developing low cost defined media able to support cell growth. Bacterial media utilize fairly basic compounds to support cell growth and product formation: nutritional needs are very flexible apart from requiring balanced amounts of the required ions and elements. The needs of animal cells are far more complex and variable. The ideal cell culture medium would be a define formulation that provides all the nutrients and factors in serum relevant to cell growth and product formation. Unfortunately, some of the biochemical factors affecting cell metabolism are unknown, unavailable or extremely expensive, even if the ideal defined medium could be developed it would cost more than medium containing serum. Depending on the product of interest and the cell line used for its manufacture, it is smometimes possible to adapt the cells to simple serum-fee or even protein free media without compromising product titres or quality. Researchers involved in such media development efforts must consider the fact that the cell line in use is probably undergoing both selection and adaptive change to adjust to the altered media, for example cells with high growth rates and low productivity can replace higher producing cells. The product resulting from adaptation to a given medium should be carefully compared to that produced by cells from the seeds bank using the original medium, to ensure that glycosylation patterns and functional activity have been altered during the adaptation process. COMPONETS OF CELL CULTURE MEDIA WATER The first and important ingredient in a cell culture medium is good quality water the importance of a highly purified and carefully monitored water supply can hardly be over emphasized. Mammaliancell cells are sensitive ti common imuritlies in water such as metal ions, bacterial endotoxins, and organic molecules. Water used for cell culture should be distilled at lleast once andpassed through successive ion exctiang and

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activated cabon filtered to remove ions and activated cabon filtered to remove ions and organic compounds. Once purified., the collected water should ideally be maintained at high temperature and circulated to prevent growth of microorganisms. Materials used for storage or circulation should not leach metal ions or plasticizers into the water. The filters should have a schedule for regular maintenance and replacement, and the water should be tested regularly for conductivity, presence of organics and presence of pyrogens from contaminating organisms. (2) BASAL MEDIA Most cell culture media contain a mixture of salts, vitamins, amino acids, coenzymes, and minerals generally celled basal media. The most common formulas have an osmolality near 300 MOSM and are based on animal cell nutritional studies. The common mediums are 199, DMEM, F12, and RPMI 1640, used for large scale cell culture. Variations of these formulas are numerous and can be expected to proliferate as media are adapted to particular cell lines are requirements. Most cell culture media use sodium bicarbonate as the pH buffering system. Animal cells have a metabolic requirement for a small amount of bicarbonate, however, the primary reason for using bicarbonate is buffering of the medium pH. In closed systems or large flasks without adequate potential for air exchange, organic buffers such as HEPES can be used to improve pH control at the expense of possible harmful effects on the cells. The energy and material requirements for cell growth and product formation and generally met by adding glucose and glutamine to the culture medium. The patterns to biochemical utilization vary among cell lines. Some cell lines have been observed to generate half of the ATP necessary for cellular metabolism through the conversion of glutamine even when glucose is available. Both glucose and glutamine can generate metabolic byproducts that inhibit further activity. Glucose and glutamine both undergo glycol tic processing with lactate as a product and the utilization of glutamine causes the evolution of ammonium ion. Cell and product yields can be enhanced by avoiding high concentrations of lactate and ammonia, through the use of nutrient control strategies, substitution of alternative carbon sources, or continuous perfusion of medium through the reactor vessel. The addition of antibiotics to cell culture media to prevent microbial contamination is a common practice, but one that should be discouraged in a manufacturing setting. If used, antibiotics must be completely removed by down stream purification before in vivo use of any cell culture product as many people are strongly allergic to antibiotics., antibiotics can also have the effect of suppressing but not eliminating microbial contamination: for this reason all seed stock should be preserved from cultures that have been maintained in Antibiotic free medium for several weeks.

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C.

Animal sera as Medium supplements

Animal serum is widely used in a animal cell culture at concentration up to 15 vol % the optimal concentration and type of serum are cell line dependent. For much cell work, fetal ovine serum (FbS) appears to provide the best overall combination of growth. Promoting activities and lack of contaminating proteins. Serum serves several purposes in cell culture media: aside from the obvious nutritional effect serum provides. 1) 2) 3) 4) Hormones and growth factors Carrier proteins for transport of ions Fatty acid, lipids and like substances Attachment factors to promote cell- surface interactions.

Serum also has “detoxifying” effect in that metal ions and even nutrients that could inhibits cellular functions at full strength are instead bound to albumin and other serum proteins. FBS prices and supplies vary by season and in response to economic pressures on the beef and dairy industries: one carit expect serum supplies to keep pace with the current expansion in cell culture activity. Close atension to serum quality is also necessary: concentrations of some hormones and factors can vary as much as 10 fold in individuals lots of serum. Some of this variability is unavoidable and influenced by factors such as the diet of the source animals. Serum can contain trace amounts of pesticides, antibiotics asd toxins ingested form plants and biological contamination of the culture. Although filter sterilization of bacterial contaminants is fairly reliable, but mycoplasma and viruses such as bovine diarrhea virus are sometime present in filtered serum.

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SELECTED COMPONENTS OF FETAL CALF SERUM S. No. 1. Component PROTEINS Albumin Transferrin Hemoglobin 2. LIPIDS AND FATTY ACIDS Triglycerides Phospholipids Cholesterol 3. HORMONES AND STEROIDS Insulin Growth hormone Progesterone Testosterone Corticosterone Deoxycorticosterone Dehydroepiondrosterone Aldosterone Cortisol Estrone Estriol 17B-Estradiol Thyroid stimulating Hormone Parathyroid hormone Follidestimulating Heutinizing hormone Platelet-derived growth factor 8 mg/L 42 Pg/1 121 ng/L 261 ng/L 67 ng/L 13 ng/L 270 ng/L 10 ng/L 6000 ng/L 23 ng/L 17 ng/L 11 ng/L 1790 ng/L 1929 ng/L 199 ng/L 900 ng/L 770 mg/L 210 mg/L 630 mg/L 320 mg/L 2300 mg/L 1790 mg/L 81 mg/L Quality

D.

Defined Media

Much of the early work with defined media was motivated by efforts to study the behaviors of cells in response to individual component such as hormones. HAM and Mckeehan (1979) Published a generalized

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protocol for development of an optimal defined medium to support the growth of any cell line. Following all the recommended steps would require years of patient work with each cell line. One can achieve satisfactory result much more quickly by recognizing that important effects of serum can be divided in to a few major categories. Most serum-free media developed by individual researchers or marketed commercially provide some means of addressing the functions of each group. 1. Carrier Proteins Fetal bovine serum contains high levels of bovine serum albumin (BSA) and transferring, BSA functions primarily as a carrier of fatty acids, lipids and other small molecules and peptides in serum, some of which are insoluble in native form. Albumin also may have the ability to detoxify some inhibitors of cell function such as hydrogen peroxide. BSA from commercial soureces is often bound to trace amounts of tatty acids and lipids. These impurities may actually provide better result than use of purified BSA. Transferring binds iron in solution and delivers it in to transportable form to the cell membrane, serum- free medium typically contain between 1 µ g/Ml and 50 µ g/Ml of added transferring. Transferring from different source of serum can differ markedly in their ability to promote growth. A recent study showed and ability of 9 given cells to bind the different form transferring. 2. Insulin Most serum – free media contain insulin at ferrins, insulin form different source may differ in their ability to support growth. Insulin influences many aspects of cell metabolism: some cell lines can be adapted to insulinfree media but lower yields and poor long term stability are usually observed. 3. Trace elements A wide variety of trace elements are known important to cell metabolism, yet the provision of these components in basal media is inconsistent. Water and basal media components often contribute sufficient trace metals to support growth. One element that may be insufficient, however, is selenium. Many investigators add amounts of the order of 10-8 Mol L-1 HAMILTON AND HAM F12 medium featuring the addition of a trace metal mixture to avoid any potential for deficiencies. Growth and attachment factors Numerous hormones and growth factors have been used for culturing cell lines in serum – free media. A partial list includes epidermal growth factor, growth factor, fibroblast growth factor, progesterone, testosterone, and hydrocortisone the requirement for these factors in a serum free medium is very much cell-line dependent. Highly differentiated cell lines such as cell form endocrine organ typically require the provision of several hormones or releasing factors to retain their natural functions, while highly transformed line such as hybridomas require minimal supplementation. Most of the published research in this field has described lab-scale investigations of cell biology and media requirements: cell culture for commercial purposes

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often occurs at sufficient scale that medium supplementation with purified hormones is inconvenient or completely impractical. 5. Miscellaneous Numerous other medium ingredients have been tated and may have value for a particular process. Some (such as lipids, fatty acids, amino acids and Nucleic acids derivatives) are standard ingredients in available basal media. Extra amount of these components may be added to meet special nutritional needs or compensate for degradation. B-mercaptoethanol is reported to enhance the stability of medium ingredients by preventing oxidation and ethanolamine has been described as an essential ingredient in serum – free medium for hybridoma growth. Other ingredients such as methyl cellulose and plutonic F68 have added to media to alleviate the effects of physical shear, and phenol red is sometimes used to provide visual indication of pH. E. Preparation and Handling of Media The proper handling of cell culture media is largely a matter of common sense and attention to details, with reorganization of the fact the mammalian cells are relatively sensitive to impurities and deteriorated components. Care should be taken to freeze or refrigerate heat labile components, and to avoid unnecessary exposure to light to open air. Sterilization of medium – component by heat has long been traditional in industrial fermentation processes, but the labile nature of many ingredients in cell culture media has limited the use of this technique to salt and stock solution used to make up complete media filtration has become the method of choice for most large – scale work. Filters with pore size sown 0.1µ m have been developed that are suitable for sterilization of any commonly used media component filters use of sterilization of cell culture media should be tested on a regular schedule, and should not leached any foreign materials such as detergents and binders into the medium. 1. Quality Control The quality of any ingredients used in cell culture media should be monitored closely, as trouble in this regard can cause costly delays and wasted effort. Animal sera can introduce contaminants. Blood products such as human transferring should be deactivated or tested for pathogenic human viruses. In cases where cell line is particularly sensitive to the quality of serum or other medium components, it may be necessary to pretest bulk lots with the cells of interest before purchasing. Many medium components should be used faitlly soon after thawing or formulation from powder: for example. Glutamine in solution at room temperature will decay spontaneously to ammonia and pyrodidoncarboxylic acid. 2. Sterility A low rate of contamination in an absolute requirement for a successful cell culture operation. Both mechanical and operational problems contributes to contamination. Mechanical problems can be reduced by the use of double instead of single seals in reactor ressels, regular maintenance and

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replacement schedules for critical parts such as sterilizing filter, and a solid system design that minimizes dead legs and build up of steam condensate. Operational problems can best development of clear and through documentation of operating procedures. Automations of critical processes such as sterilization can in some case help prevent operator errors and also reduce variability in the process. V. A. PROCESS DESIGN AND OPERATION Introduction

Cell culture is unusual among biotechnology processes in that a wide variety of reactor types have been used successfully for industrial manufacture of cell culture product. The search for and “optimum” reactor system must take into account the peculiarities of the cell line and product being considered as well as factors not directly related to the cell culture process it self, such a down stream purification requirements. The situation can be stream purification requirements. The situation can be generalized by stating tat the optimum reactor system is one that reliably produces acceptable product in the amounts required. While minimizing cost from labor, media, purification, and general operations. This is not necessarily the reactor system that must closely imitate the cells natural environment. Traditional fermentation processes have increased productivities enormously by creating very unnatural environment for producing organisms. In cell culture reaction future maximum cell densities varying from spiner flask (2 x 106 per mL) to near densities observed in solid tissues (greater then 1 x 108 per ml) achieved with several types of perfusion reactors. Low density technology is simple and robust. High ensity reactor offer much higher productivity for a given size of reactor but face a greater nutrient and mainly taining adequate nutrient and oxygen supply throughout the reactor volume. Process control requirements are also more stringent. A high – density reactor that loses its perfusion pump or pH control system will generate 9 total environment very quickly compared to a suspension culture reactor. Temperature control is rather easy to a achieve in either case due to low metabolic rate of mammalian cells. B. Suspension Culture Suspension culture of lymphocytes or hybridoma cells, for example, is possible in small-scale fermeners of up to 10 mc3 capacity, in which cells are magnetically stirred gently. Alternatively, small scale air lift fomenter may be employed. Very cell suspension culture has very limited application, however, and has been surpassed in biotechnological application by the development of micro carriers, which allows for large-scale suspension culture of anchorage – dependent cell lines is a common and practical technology for manufacture of biological. Low – density cell culture can be a practical approach for large-scale manufacture of products from animal’s cells. Control system for suspension culture are rather simple. Sterilizable pH and oxygen probes penetrating the reactor wall can provide signals for automate feedback control of these variables. Maximum cell

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densities achievable by un supplemented batch mode approach S x 106 cells ML. Suspension culture can be conducted in either Semi = -continuous or batch mode. Semi continuous mode has also been called “semi batch”, “fedbatch” and “continuous” culture. Agitation can be provided either by traditional Rushton impellers, marine impellers, or an air lifted configuration, stirred suspension culture of mammalian cells has been described at 8000-liter scale and airlift suspension culture at the 1000-liter scale. Air lift cell culture used air bubbles both for agitation and aeration for the culture. Stirred culture generally provide oxygen for the culture through the use of sparged air: oxygen. Supplementation can be used if necessary. Silicone tubing is also used inside the reactor for aeration. One can improve call and product yields in suspension culture through the use of nutrient feeds. A feed strategy can be developed by analyzing medium samples from different stages of a production culture. The continuous “Cytostat” operational mode may be helpful in maintaining the cells in a particularly productive status. C. High – density suspension culture The productivity of suspension cultures in stirred reactors can be improved by avoiding the wash out of cells two good methods are: 1) 2) Separation of the effluent, with recycle of a cell-enriched stream back to the reactor The spin filters technique.

Cell recycle can be accomplished through the use of cross flow membrane micro filtration. A membrane permits the flow of medium and product but can retain cells without harming viability. Problems with membrane recycle include clogging of pores with cell debris, and long term sterility and performance. High speed centrifugation can also be used for recovery of viable cells from spent medium. Spin filter suspension reactor feature a rotation filter that will passed medium but not cells, due tangential forces at solid liquid boundary layer. D. Entrapped – Cell Suspension Reactors Researchers have attempted to improve the performance of suspension culture through the use of immobilized or entrapped cells. The most common technique involves production of small polymer spheres or beads with mammalian cells either trapped inside during the polymerization step, or seeded the beeds by mixing in the reactor. Alginates, collagen and agarose have been used for entrapment of mammalian cells. The polymer particles have an open, sponge-like structure: ideally cells will populate the entire structure. Cell density up to 108 per mL beads have been reported. The beads can be suspended in reactors at high densities, resulting in net cell concentration up to S- 107 per mL of

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culture fluid. A fluidized bead reactor using weighted collagen beads has also been usefull. Tractors with entrapped cells have good potential for scal-up because the beads can be placed in well mixed reactors. The technique is also helpful in preventing shear damage when culturing sensitive cell types, and in providing a micro environment with elevated concentrations of cellproduced growth factors. The high density also introduces some problems. The cells are no longer in a homogenous environment. As a result the nutrient and oxygen conceration supplied to individual cells cannot be assessed by using probes in the culture medium. Mass transfer limitations with in beads can creates a central region without cell growth or necrosis of lightly packed cells once beads is fully populated. E. Micro carrier culture Micro carrier technology, in most common usage, involves attachment of cell to non – porous polymer beads with a diameter of 100 µ m. Because cells reside only on the surface of micro carriers, the cellular environment can be measured can controlled easily with pH and oxygen probes in reactor vessel. New cell densities between 2 x 106 and 1 x107 can be achieved, depending on the cell line used. The micro carrier should have a density between 1.02 and 1.10 g/ml to allow easy suspension in stirred reactor. Modified dextrins, polystyrene, s and other support materials have given. Large-scale micro carrier culture presents some unique challenges. One complication is the large amount of inoculums required. Seed culture must be prepared in roller bottles or removed from a smaller number of confluent micro carriers. Bead-bead and bead-impeller collisions and hydrodynamic shear forces, are reported to cause loss of viability, still, micro carrier culture provide one of the few technique for anchorage- dependent cell culture that dose not face obvious mass transfer limitations to scale up. F. High density perfusion

The cell culture reactors most similar to the native environment are the high density perfusion ractors. These configurations have proven to be very successful for small-scale research. Improvements is recent year have brought some of these type of reactors up to productivities needed for largescale manufacture of cell culture products. Many types of reactors have been used for maintaining a high cell density; most provide sufficient surface area for culture of anchorage dependent cells. All these reactors culture cells at densities high enough to inhibit or half growth. Ideally this will allow improved product yields when medium is perfused past the cells. Hollow fiber reactor has been used for high density cell culture. Most reactors use medium flowing through fiber lumens to support cells in the spaces between fibers. Typical designs utilize an altrafiltration membrane that retains the product outside of the fibers. This product concentration effect can

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be beneficial: hybridoma culture can typically produce several milligrams per millimeter of the product. Higher viable cell densities and better over all productivity can be achieved by using on open pore structure and collecting toward density product from the lumen side of the fibers. Hollow fiber reactor generates very high cell densities. This can crate nutrient limitations and subsequent cell sheath. Some techniques have been developed to correct this situation, e.g.; regularly reversing the flow direction, imposing cyclic pressure variation to force medium flow across the fibers, or providing for circulation of cells on the shell side of the fibers. A simple and effective technique for culturing anchorage dependent cells is the use of a ceramic support with interior channels. Cells attach to the surfaces of each channel and are perfused with medium from an external reservoir. This technique also faces engineering limitation to scale-up. Columns packed with glass beads have also been used for culture of anchorage dependent cells. This reactor configuration would appears to suffer from scale up difficulties as well. However, the technology has reportedly been used with success at scale up to 100 liters. Other successful designs have incorporated large membrane filter for perfusion of entrapped cells: A “static maintenance” reactor design used hollow fibers to aerate the interior of a high density reactors and flat porous membranes above and below the culture chamber that supply and removed medium but retain cells with in the reactors. VI. Downstream processing A. Introduction Most downstream operations for mammalian cell culture products are familiar from manufacture of other biological products depends greatly upon end use. For example, antibodies manufactured for in vitro diagnostic kits may follow cell removal with one step precipitation process. Cell culture products for human intravenous injection must undergo extensive purification and testing. B. Cell Removal Some type of cell culture reactors provide a cell free product perfusion reactors entrap most cells but debris and whole cells are still present in the effluents suspension culture broth contains between 106 and 107 cells per mL, and some reactors required recovery of product from broth with even higher cell densities. Centrifugation is a common and reliable technique for removing cells from the product containing medium. Two approaches are possible: low speed centrifugation and high speed centrifugation that attempts to pellets these suspended cell components. Cell removal can also be accomplished using microporous membranes. Cross flow filtration and improved membrane technology have established membrane filtration as a reliable large scale operation. C. Concentration Cell culture products can be concentrated by several techniques. Ammonium sulfate precipitation is a popular small scale technique, however, the process is difficult to control at large scale and the protein phase change sometime causes loss of activity.

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The most popular concentration technique is ultrafiltration. The cell free product stream is passed across a membrane that passes water and salts but retains large molecules. cross flow filtration units with molecular weight cut offs between 10000 and 10000 daltons are suitable for concentration of most product stream. One can prepared the product for uncoming chromatographic purification at this point by diafiltration with the buffer to be used in the next purification step. Adsorption and affinity columns can also be used for concentrating cell culture products. D. Purification Well established protein purification techniques are suitable for purification of cell culture products. Ion exchange chromatography is the dominant technique for large scale protein purification. Affinity purification HPLC and FPLC techniques are becoming increasingly important as new methods are developed and support materials improved. VII A. Regulatory aspects of products from animal cell culture Biohazards associated with cell culture The primary hazard to be considered in cell culture operations are the potential for viral contamination of working cell line, and the possible presence of harmful nucleic acids in the final product. One can reduced most of the concern of microbial and viral contamination through testing of the master seed stocks. The safety tissues and available procedures have been described HOPP. Once a clean cell line has been established, viral contamination is still there by several routes. Viruses can be introduced from the physical environment from the cell culture media or through expression of an endogenous viral sequence integrated in the host cell chromosome. B. Regulatory Environment The regulation of biological product manufacture using animal cells has for the most part been a function of regulation are based on earlier experiment with viral vaccines. In recent years agencies have responded to developers in biotechnology by issuing new regulation and guidelines. VIII. references 1. Animal cell Biotechnology vol. 2 2. Mommalian cell technology W.G. Thilly 3. Bashor, Methods in Enzymology – 58 4. Large scale mammalia cell culture by J. Feder and W.R. Talbert.

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COMPONENTS OF BASAL MEDIA Medium DMEM 199 RPMI`1940 AMINO ACIDS Arginine 4.0 E-4 3.3 E-4 1.1 E-3 Cysteine 6.3 E-7 Halfcysteine 4.0 E-4 6.8 E-4 4.2 E-4 Glutamine 4.0 E-3 1.0 E-4 2.1 E-3 Histidine 2.0 E-4 1.0 E-4 9.7 E-5 Isoleucine 2.0 E-4 3.8 E-4 3.0 E-4 (± ) Leucine 8.0 E-4 3.8 E-4 9.1 E-4 (± ) Lysine 8.0 E-4 3.8 E-4 2.2 E-4 Methionine 2.0 E-4 1.0 E-4 2.0 E-4 (± ) Phenylalanine 4.0 E-4 9.1 E-5 3.0 E-4 (± ) Threonine 8.0 E-4 1.7 E-4 5.0 E-4 (± ) Tryptophan 8.0 E-5 2.4 E-5 9.8 E-5 (± ) Tyrosine 4.0 E-4 1.1 E-4 2.2 E-4 (± ) Valine 8.0 E-4 1.7 E-4 4.3 E-4 (± ) Alanine 5.6 E-4 (± ) Asparagine 3.3 E-4 Aspartate 1.5 E-4 4.5 E-4 (± ) Glutamate 1.4 E-4 9.1 E-4 (± ) Glycinene 4.0 E-4 6.7 E-4 1.3 E-4 Proline 3.5 E-4 1.7 E-4 Serine 4.0 E-4 2.9 E-4 4.8 E-4 (± ) SUGARS, NUCLEIC ACIDS AND INTERMEDIATES Glucose 5.6 E-3 5.6 E-3 1.1 E-2 Pyruvate 1.0 E-3 Actate 6.1 E-4 Ribose 3.3 E-6 Deoxyribose 3.7 E-6 Adenine 4.9 E-5 AMP 5.8 E-7 ATP 1.8 E-6 Guanine 1.6 E-6 Hypoxanthine 2.2 E-6 Xanthine 2.0 E-6 Thymine 2.4 E-6 Thymidine Uracil 2.7 E-6 LIPIDS AND DERIVATIVES Cholesterol 5.2 E-7 Choline 2.9 E-5 3.6 E-6 2.1 E-5 Innosital 3.9 E-5 2.8 E-6 1.9 E-4 Linolic acid 3.0 E-7 VITAMINS AND CO-ENZYMES Ascorbic acid 2.8 E-7 Biotin 4.1 E-8 8.2 E-7

F12 1.0 E-3 2.0 E-4 1.0 E-3 1.0 E-4 3.0 E-5 1.0 E-4 2.0 E-4 3.0 E-5 3.0 E-5 1.0 E-4 1.0 E-5 3.0 E-5 1.0 E-4 1.0 E-4 1.0 E-4 1.0 E-4 1.0 E-4 1.0 E-4 3.0 E-4 1.0 E-4 1.0 E-2 1.0 E-3 3.0 E-5 3.0 E-6 1.0 E-4 1.0 E-4 3.0 E-8

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Folic acid 9.1 E-6 a Amino Benzoic acid a-lipoic acid Nicotinic acid Nicotinamide 3.3 E-5 Pentothenic 1.7 E-5 Acid Pyridoxine Pyridoxal 2.0 E-5 Riboflavin 1.1 E-6 Thymine 1.2 E-5 Vitamin B12 Vitamin D Vitamin K Vitamin E Vitamin A INORGANIC IONS (TOTAL) Calcium 1.8 E-3 Magnessium 8.1 E-4 Potassium 5.4 E-3 Sodium 1.6 E-1 Chloride 1.2 E-1 Phosphate 9.1 E-4 Sulphate 8.1 E-4 Nitrate 7.5 E-7 TRACE ELEMENTS Iron 2.5 E-7 Cobalt Copper Zinc -

2.3 E-8 3.6 E-7 2.0 E-7 2.0 E-7 4.2 E-8 1.2 E-7 1.2 E-7 2.7 E-8 3.0 E-8 2.5 E-7 5.8 E-8 2.0 E-8 3.5 E-7 1.8 E-3 8.1 E-4 5.4 E-3 1.5 E-1 1.3 E-1 1.0 E-3 8.6 E-4 1.2 E-6 4.1 E-7 -

2.3 E-6 7.3 E-6 8.2 E-6 1.0 E-6 4.9 E-6 5.3 E-7 3.0 E-6 3.7 E-9 4.2 E-4 4.1 E-4 5.4 E-3 1.4 E-1 1.1 E-1 5.6 E-3 4.2 E-4 8.4 E-4 3.7 E-7 -

3.0 E-6 1.0 E-6 3.0 E-7 1.0 E-7 3.0 E-6 1.0 E-7 1.0 E-6 1.0 E-6 3.0 E-4 6.0 E-4 3.0 E-3 1.5 E-1 1.4 E-1 1.0 E-3 1.0 E-6 3.0 E-6 1.0 E-6 1.0 E-8 3.0 E-6

*e.g. 2.5 E-7 means (2.5 × 10-7 moles)

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ANIMAL CELL AND TISSUE CULTURE The foundation of animal cell and tissue culture was laid at the beginning of the present century when cells could be shown to survive and divide in vitro. Ross Harrison (1907) later Alexis Carrel (1912) used tissue and embryo extract as culture media. They successfully demonstrated that animal cells can be grown indefinitely in vitro. The following terms are used for animal culture techniques. *Cell culture *Tissue culture *Organ culture ORGANOTYPIC CULTURE If the explants maintain its structure and function in culture it is described as organotypic culture. HISTOTYPIC CULTURE If cells is culture reassociate to create a 3-D structure, irrespective of the tissue from which it was derived, it is described as a histotypic culture. Different areas of research in biotechnology depend largely on tissue culture. These areas are : 1. Production of antiviral vaccines 2. Cancer research 3. Cell fusion techniques 4. Genetic manipulation 5. Monoclonal antibodies 6. Production of pharmaceutical drugs 7. Chromosome analysis of cells derived from womb 8. Study of the effect of toxins and pollutants 9. Use of artificial skin 10. Study of the function of nerve cell. ADVANTAGES AND DISADVANTAGES OF THE USE OF ANIMAL TISSUE CULTURE ADVANTAGES 1. Controlled physiochemical environment (pH, temp. osmotic pressure, O2, CO2 etc.) 2. Controlled and defined physiological conditions (constitution of medium etc.)] 3. Homogencity of cell types (achieved through serial passaging) 4. Economical, since smaller quantities of reagents are needed than in vitro. 5. Legal, moral and ethical question of nimal expt. Ion are avoided. DISADVANTAGES 1. Expertise is needed, so that behaviour of cell in culture can be interepreted and regulated.

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2. Ten times more expensive for same quantity of animal tissue therefore reason for its use should be compelling. 3. Unstable aneuploid chromosome constitution. TISSUE CULTURE FACILITIES Minimum requirement (essential) 1. Sterile area 2. Preparation area 3. Storage area a. Liquids ambient, 40-200C b. Glass ware c. Plastics d. Small items e. Specialized equipment f. Chemcials Desirable features (beneficial) 1. Filtered air (air conditioning) 2. Hot room with temperture recorder 3. Microscope room 4. Dark room 5. Serivice bench adjacent to culture area 6. Separate sterilizing room 7. Separate sterilizng room 8. Cylinder store Useful additions 1. Piped CO2 and comapred air 2. Store room for bulk plastic 3. Containment room for biohazard work 4. Liquid N2 sotrage tank (-2001). TISSUE CULTURE EQUIPMENTS Minimum requirement (essential) 1. Incuabtor 2. Sterilizer (Autoclave pressure cooker) 3. Referigertor 4. Freezer (fr -200C) 5. Inverted microscope 6. Soaking bathc or sink 7. Pipette cylinder 8. Pipete washer 9. Water purifier 10. Bench centrifuge 11. Liquid N2 freezer (1,500-3000 ampules) 12. Liquid N2 sotrage flask 1. 2. 3. Desirable features (beneficial Laminar flow hood Cell counter Vaccum pump

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4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.

CO2 incubator Coarse and fine balances pH meter Osmometer Phase contrast and fluorescence microscope Ortable temperature recorder Roller racks for roller bottle culture Magnetic stirrer ipette dirve Pipette plugger Auto pipette. Useful additions -700C Glasswater washing machine Closed circuit TV for inverted microscope Colony counter High capacity centrifuge Cell sizer Time lapse cinemicrographic equipment Interference contrast microscope Polythene bag scaler (for packing of long term storage) controllled rate cooler Filling for freezer records and catalogues Centrifugal elutriator centrifuge and rotor Fluorescence activated cell sorter Densitometer Density meter THE SUBCULTURE ON WHICH CELLS GROWN Cells shown to require attachement for growth are said to be anchorage dependent. However, cell which have under goen transformation frequently become anchorage independent and can grow in suspension. Substrate may be adhesive (glass, plastic palladium etc. or a metallic surface) or nonadhesive (agar, agarose, methocel). Class as a substrate Glass is the most commonly used substrate in the form of slides, converslips, flasks and test tubes. Disposable plastic as asubstrate Poly styvene is often used as the most common substrate. Others are poly vinyl chloride (PVC), polycarbonate, polytetrafluorethylene or feflon (PTFE), thermanox (TPX). Palladium and metallic surface as substrate For 90% of animal cell and tissue cultures, glass or plastic is employed as a substrate. However, other substrate have been usedfor specialized applications e.g. Palladium diposited on agarose (using electron microscopy shadowing equipment) was used as a substrate for growth of fibroblast and glia stainles steel discs and other metallic surfaces were also used.

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CULTURE MEDIA FOR CELLS AND TISSUES Culture medium is one single most important factor for culturing cells and tissues. It provides. (i) The optimum conditions of factors like pH 0 p etc. (ii) The chemical constituents, which the cells or tissuesare incapable of synthesizing. Animal cell need either a completely natural medium or an artifical medium supplemented with some natural products. 1. a) b) c) NATURAL MEDIA Plasma dots Biological fluids e.g. scrun Tissue extracts

2. DEFINED MEDIA Synthetic media have been designed for a variety of tissues and purposes like Media for immediate survival Media for prolonged survival Media for definite growth Media for specilized functions 1) RPMT-1640 (Moore et al., 1950) 2) DMEM (Delbecco’s) 3) MEM 4) F12 5) TCM (Ham, 1965) 6) IMDM 7) CMRL 1066 (Parker et al., 1957) These media are serum media Serum free media The use of serum in culture media has several disadvatnages, which led to the developments of many serum free media. Some of the disadvantage include the following: (i) Serum varies from batch to batch and determiorates within one year (ii) Changing serum batch requires fresh testing. (iii) If more then one cell types one used, each may require different serum batch. (iv) When cell culture is used for down stream processing to recover cell products, the presence of srum is an obstacle to purification. (v) Serum increases the cost of the medium, since its cost is 10 times the cost of its constituents if used as a substitute. (vi) Serum may stimualte undesirable groth and may even inhibit growth in some cases. The major advantage of serum free media is the ability to make a medium selective for a particular cell type. (i) (ii) (iii) (iv)

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Example of serum free media 1. MC DB 110 2. MC DB 202 3. MC DB 402 4. MC DB 153 5. Iscove’s 6. LHC Selection of a suitable medium Cells or cell line 1. Hemopoietic cells 2. Human diploid fibroblast 3. Human leukemia 4. Human tumours 5. Mouse leukemia 6. Mouse erythroleukemia 7. Mouse myloma 8. Hela cells 9. Glial cells 10. Continuous cell lines Medium RPMI, 1640 Eagle’s mem RPMI 1640 RPMI 1640, DME Fischer’s RPMI SF12, RPMI 1640 RPMI, DME Eagle’s meme MEM Eagle’s Meme

STERILIZATION OF EQUIPMENT AND APPARATUS Item Sterilization Disposable tips for micropipettes Autoclave Filter Millipore Autoclave Glass ware Dry heat Magnetic sterrer bar Autoclave Pasteur pipette Dry heat Agar Autoclave Amino acid Filter Antibodies Filter DMSO Self sterilizing EDTA Autoclave Glucose-20% Autoclave HELPES Autoclave HCI in Filter Glucose 1 – 2% Filter NaHCO3 Filter NaOH In Filter Phenol red Autoclave Serum Filter Trypsin Filter Vitamins Filter Water Autoclave

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Experiment No. Objective To isolate single cell suspension from spleen and liver rat to culture than in DNEM Requirement Medium, NaHCO3, bacterial filter, automatic pipette, Laminar flow, inverted microscope petridishes, Milk bottle, culture plate etc. Principle Single cell culture can be isolated by either mechanical dispersion or by enzymatic method. In mechanical disruption recovery is low and possibility to damage. Cells may also be damaged by enzymatic activity. Procedure (i) Media preparation We prepare DMEM medium by bacterial filter. After filtration we add 1% NaHCO3 and antibiotic. NAHCO3 is acts as a buffering agent. ii) Kill the experimental animal (Mice) and dip it into 70% alcohol iii) Dissect the animal from ventral side and remove the liver and spleen iv) Mechanical disaggregation: In this method tissue is carefully sliced by operation blade and the cells which spell out are collected or cells may be repeatedly pipetted. v) Enzymatic disaggregation: Crude trypsin is the most common enzyme used for disaggregation for the following reasons. It is tolerated by a variety of cells It is effective for various tissues Expose the cells to the warm enzyme for a minimum period; dissociated cells are collected every half an hour and the trypsin is removed by centrifugation vi) Incubate the culture in CO2 incubator

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Experiment No. Objective Counting of total cell viability by “Trypam Exclusion Test” Requirement Floemoaytometer, cell culture, trypem blue Microscope (Polyvar), Pasteur pipette. Principle Trypam blue is the stain which can stain only dead cells the viable cells can not be stain by this due. So it is very useful stain to distinguish viable and dead cells in a population. By using this stain we can determine the half life of a cell line. Procedure i) Take 2 ml of culture and add 1 ml of trypen blue in it ii) Keep the stain and culture mixture for 2 minute iii) Charge the sample to moemocytometer iv) Observe the cells under polyvar microscope Observation The viable cells looks colourless and dead cells appear in blue color Discussion Trypan blue a blue coloured stain capable of entering into dead cells and dead cells appear blue in color. Not permit the entry of trypen blue. So that they appear colourless under microscope. Hepatocyte - Emzymatic means Total no of cells = 20 x 16 x 5 x 2 x 10 = 320 x 102 =32 x 104 No of dead cells = 4 x 16 x 5 x 2 x 10 = 64 x 102 = 32 x 104 2.54 104 Viability = ___________ x 100 3.2 104 Mechanical means = 80% Total no of all = 30 x 16 x 5 x 2 x 10 = 4.8 x 104 0.8 4 No of dead cell = 4 x 10 , Via =________ x 104 x 100 4.8 = 16.6%

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Experiment No. Objective To culture MACROPHAGES in RPMT medium Requirement RPMI medium, NaHCO3 bacterial filter, automatic pipetter, laminar flow, inveted microscope petridishes, Milk plate, culture plate etc. Procedure i) Media preparation We prepare RPMT medium and filter by bacterial filter. After filteration we add 1% NaHCO3 and antibiotic. NaHCO3 is acts a buffering agent. ii) Macrophage separation We inject 3 ml of sodium thioglycolate (1%) in the peritoneum cavity of white rat. After the injection, macrophages of blood get excited and accumulated in the peritoneal cavity. After 2 days we take out the macrophages the cavity after disscting the mouse from ventral side wash the cell with buffer. iii) Macrophage culturing We transfer the macto phages in the RPMI medium and them into well culture plate. Incubate it into CO2 incubator. Observation See under inverted microscope. macrophages are part of body defence mechanims they act as phagocytes or antigen presenting cells. They usually gets accumulated at the site of injection we can induce macrophages production artificially by infecting any foreign particle. Intraperitonial injection of sodium thioglycolate will induce the macrophages.

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Experiment No. Objective To form animal cell lines of a) Cymphocyte cells from peripheral blood/ spleen b) Splenocyte from spleen Experimental out line Sacrifice mice Remove spleen Sieve spleen cells through wire mesh wash cells Viable cell count with trypan blue Cell count with hematocytometer Materials Balanced salt solution Acetic acid counting solution Trypan blue vital dye 70% ethanol Squeeze bottle Small/ Large surgical scissors Curved forceps Rubber policemen Stainless steel wire mesh Glass petridesh Hoemocytometer Pasteur pipettes Pre lab preparation 1. Balance salt solution (BSS) Stock # 1 10x Dextore - 10gm K2HPO4 - 0.6 gm 0.5% phenol red - 20ml Dissolve in 100ml distilled water Stock # 2 CaCl2 . H2O 1.86 gm KCl 4.00gm NaCl 80.00 gm MgCl2 1.04 gm MgSO4.7H2O 2.0gm Dissolve in 1000 ml distilled water Mix 10 ml of stock 1 and 10 ml of stock 2 bring upto 100ml with distilled water. The pH would be 7.2 – 7.4 Method 1. Sacrifice the mice 2. Make a cut through the loose skin by pulling gently upwards and suign the blunt scissors on the skin flap. This will expose the peritoneal wall. 3. Pull gently in opposite direction on the two sides of the skin incision to expose a wider area of the peritoneal wall 4. With the small surgical scissors make a cut over the spleen to expose it through the puritoreen

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5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

Group the spleen with the forcep and pull it gently group a folded corner of the stainless steel wire mesh with the hemostat. Insert the mesh into the petridish and add about 10 ml of BSS Put the removed spleen on the wire mesh with a rubber policemen. Rub the spleen back and forth orier of mesh continue until all the psleen dissociates With a sterile pasture pipette bring the cell suspension up and down several times to dissociate large clumps pipette the cell suspension into the glass centrifuge tube. Allow the cells to settle for 5 minutes Centrifuge at 1000 rpm resuspend the cell pellet Add 0.1 ml of tryptan blue soln to 1.8 ml of acetic acid solution. Add 0.1 ml of the cell suspension and mix thoroughly Fill hemo cytometer chamber with a drop of mixture Dilution factor = 20 volume factor = 10,000 Record the grid count

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Experiment No. Objective Stain the viable cells (lymphocyte) and observation requirement Cell suspension stains, spirit lamp, micro scope etc. Procedure 1. Wright’s Stain Smear 2 drop wright’s stain (2.3 minute) Add distilled water (8-10 minute) Wash with running water Air dry and observe 2. Giemsa stain Smear Fix in methyl alcohol (3-5 minute) Dry in air Dilute giemsa stain (9 dw:1 GS) 30 minutes Wash with distilled water Air dry and observe Result Lymphocyte Round/oval nuclei covering almost all parts of the cell nucleus deep purple blue with 1-2 nucleoli Cytoplasm is very small and stain sky blue color Monocyte Largest cell of blood Kidney/ bean shaped cytoplasm relatively than lympholyte Plae cells with pale staining round nucleus with chromatin being reticulum

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Experiment No. Objective Formation of gradient by diffusion method Requirement Sucrose (12%, 25%, hypodurmic syringe etc.) Procedure This is the simplest method of forming gradients, layers of medium solution at different densities are manually layered on top of each other and either used in this form (discontinuous) or allowed to stand for a period of time for diffusion to occur produce a continous gadient. Syringe fitted with a large bere needle is usually used to introduce known volumes of solution of medium. The least dense solution is added first followed by more dense solution which when added, slowly displace the less dense solution. This is repeated to produce a discontinuous gradient

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Experiment No. Objectives Discontinuous isopycnic separation of blood cell. Requirement Blood, Isotonic pereal solution tube, centrifuge etc. Principle and procedure Granulocyte can be purified from human blood by a 2 step discontinuous percoll gradient 4 ml of isotonic pereall with a density of 1.10 g.ml is overlayered with 4 ml of a 1-077 g/ml percoll solution creating a 2 step gradient. 4 ml of calf blood anotr layeed on the gradient and centrifuged for 20 minutes at 2000-3000 rmp. Lymphocytes and monocytes bond at the interface between the blood and the 1.077 g/ml percoll the granulocyte band between the two percall solution and the red cells pellet to the bottom of the tube. Experiment No. 18 Objectives Immobilization of yeast cell by formation of poly acrylamide beads. Requirement Aerylamide, N.N.- medhylae bis acrylamide, M./M. tetra ethylene methol diamine (TEMD) potassium persulfate, ammonium persulfate, amlyl alcohol. Procedure 10ml of washed yeast cell suspension added to 12.75 ml of eq solution of acrylamide, N2 gas passed to derive out oxygen. Since O2 is an inhibitor of polymerization. Add a layer of amyl alcohol. Add catalyze system i.e. 2 ml of 2.5 potassium/ammonium per sulfate then add 2.25 ml of 5% TEMED with constant stirring. Mixture is poured rapidly into a solid verticle glass chamber 0.2mm thickness keep the chamber for cooling at 10oC water bath kep it for 1 hr. Remove the 2mm thick sheet and cut it into 4 x 5mm and finally washed the blocks with phosphate buffer. Air dry it to room temperature.

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Experiment No. Objective Immobilization of yeast cell by formation of agar beads. Requirement Agar (2.5%), growing yeast cell culture, phosphate buffer, toluene, chloroform, hypodermic syringe. Principle Living cells use energy for its survival as well as multiplication. If cell multiplication is stopped, all the available energy will be used for its survival and the substrate will be converted to product. If the cell is incorporated on a solid support it growth will stop and the substrate will be converted to product. Procedure Take 25ml of 2.5% agar solution, and autoclaved it. Take 10ml of the yeast suspension and washout with phosphate buffer. After washing melt agar at 45-50oC and the yeast cell to the agar under aseptical condition take toluene and chloroform in a beaker in the ratio of 3:1 in cold, pour the agar drop by drop with the help of syringe in the liquid. Small beads are formed. Take out the beater and wash it with phosphate buffer and dry it keep at 37oC for 24 hrs. Observation Place the yeast is glucose solution. After some days, glucose is converted into ethanol. Test the glucose by Benedict’s reagent.

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Experiment No. Objective: Immobilization of yeast cell by formation of sodium alginate beeds. Requirement 3% sodium alginate, phosphate buffer, 0.025M, anhyd CaCl 2. hypodermic solution. Procedure The procedure of sodium alginate bead formation is exactly some as agar bead. Sodium alginate is taken it 40 ml phosphate buffer and melted in flask and then autoclaved. After autoclaving. Mix yeast cell suspension (previous washed.) Mixed well. pour into 2.5% calcium chloride (saturated) by using hypodermic sofringe drop by drop. Take out the beads from the solution and washed with buffer solution air dry at 37oC. Observation Place the beads in glucose solution after some days. Yeast converts the glucose into alcohol.

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INDEX S.No. 1. 2. 3. 4. 4.1 4.2 5. 5.1 6. 6.4 6.5 6.6 Content Introduction Tissue culture: Application General methodology 3.1.3 Preparation of stock solution Special techniques Fusion of protoplasts Products of plantlets from suspension culture Agrobacterium mediated transformation in plants Agrobacterium mediated transformation in Page

tobacco/pigenonpea Embryogenesis, organogenesis Somatic embryogenesis organogenesis Plant regeneration directly from cultured explants

Reference

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PLANT TISSUE CULTURE : AN OVERVIEW I. Introduction Agriculture, being the mainstay of our economy, received so much impetus from the immemorial. By and large and domestication of wild plants depends on the genuine effort of human being and we are now a logway away from the traditional farming practices. Land being the limiting factor in the cultivation, combined efforts of agromonists, plant breeders, plant pathologists and entomologists etc. have helped a lot to increase the grain yield. Of late, it was felt by the scientific community that, traditional breeding practices can no larger increase the grain yield. So they began to look upon the new found wisdom of biotechnology or for this stagnation in grain yield. No plant biotechnology programme will be complete without tissue culture as it is the only method for propagating these genetically engineered propagules which might have failed otherwise. This aspect has been reported by Levy (1981) who stated the micropropagation through in vitro techniques the advantage of satisfactorily propagating genetically superior phenotypes. Tissuec culture: Applications

II. 2.1

Production of true to type plantlets Tissue culture may be best described as clonal propagation as the plantlets produced are clones to each other. This aspect has been exploited in seed anther or v\ovules and later subjecting them to chromosome doubling by the application of colchicines for the production of either homozygous of heterozygous diploid. This isolation and culturing of haploid lines is normally possible only through tissue culture 2.4 Embryo rescue technique The production of intergeneric or interspecific hydrids is practically impossible due to the defective chromosome pairing which results in lose of viability so in these cases, as soon as the fertilization is over, embryo is separated and allowed to grow in artificial media which would have been otherwise repelled by the motherplant. 2.5 Somaclonal variation In single cell culture, when we are dealing with a very large population sometimes it may happen that one plantlet out 10 4 or 105 may deviate from parental lines and show novel phenotypes. If these novel phenotypes are agronomically desirable traits then they may be propagated further or else discarded. But sometimes, these variants may revert back to parental lines. 2.6 Mutation breeding In single cell culture or tissues by the application of suitable mutagens, evolved lines can be developed. This techniue, for plant breeders, is a very handy tool as they can be used for the isolation of herbicide resistant and other stress tolerant varieties. 2.7 Protoplast fusion Instead of dealing genome, scientists now deal with protoplast which is a cell without cell wall. By suitable means cell wall as dissolved and protoplasts of two different in suitable media and allowed regenerate into

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planlets. Even though the method sounds simple it is very tedious. As it was often found, it final product resulted is of an undesirable variety (eg. pomato, obtained by the fusion of potato and tomato). 2.8 Propagation of transgenics Transgenic crops which are the fruits of biotechnology are propagated only by tissue culture because most of them had lost their viability. III General Methodology 3.1 Media The success of any tissue culture practise depends on the careful selection of media. By the scrupulous work of available. This include MS (Muradhige and Skoog, 1962), Woody Plant Medicines, WPM (Loyed and Mcoum, 1980) ambroge’s these media and manipulate them by varying the salt concentration and growth regulation. 3.1.1 Chemical composition of media Compound Amount Mg e-1 MS NH4NO3 1650.00 H2NO3 6.20 CaCl2.2H2O 440.00 CaCl2.6H2O 0.025 CaNO3 0.00 FeSO4.7H2O 27.80 MnSO4.7H2O 22.30 MgSO4.7H2O 370.00 Na2EDTA 37.30 KCl 1990.00 KNO3 0.00 KH2PO4 170.00 NaMoO4.7H2O 0.25 ZnSO4.7H2O 10.60 Vitamins Glycine 2.00 Nicotinic acid 0.50 Pyridoxine –HCl 0.50 Thiamine – HCl 0.10 Other Inositol 100.00 Sucrose (in % w/v) 3.00 Agar (“) 0.80 WPM 400.00 6.20 96.00 0.00 0.00 27.80 22.30 370.00 37.30 0.00 990.00 170.00 0.25 8.60 2.00 0.50 0.50 0.10 100.00 2.00 0.80

3.1.2 Composition of stock solution for ms medium Stock No. Chemical I NH4NO3 KNO3 KH2PO4 MgSO4.7H2O II CaCl2.2H2O Stock Conc. 50x Quantity 82.5 (ge-1) 95.0 8.5 18.5 22.0

50x

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III IV

V`

Na2EDTA FeSO4.7H2O MnSO4.7H2O ZnSO4.7H2O H3BO3 KI Na2MoO= COCl2.6H2O CuSO4 Vitamins Glycine Nicotinic acid Pycidoxine-HCl Thiamine HCl

100x 100x

3.7 2.8 22.3 1.06 0.62 0.83 0.83 0.025 200mge-1 50.00 50.00 50.00 10.00

0.025

3.1.3 Preparation of stock solution Each stock should be prepared separately. For this each of the chemicals mentioned in the above description to be weighed accurately and dissolve in 200ml of distilled water taken in a beaker. The contents were then made upto one litre by adding distilled water. The stock solutions should be properly labeled indicating the stock number found date of preparation and stored in chamber coloured bottles under refrigerated conditions. Stock solution of the salts have to be prepared once in three months and that of vitamins once every month. The stocks should be abandomed whenever contamination is noticed. The iron stock (stock number III) precipitases readily. To avoid this Na2 EDTA and FeSO4. 7H2O were dissolved in separate beakers with approximately 200ml distilled water. Both beakers were placed on hot plates and bring them to the point of boiling. Then FeSO4.7H2O solution should be added slowly to Na2EDTA (never vice versa) over a 15 minute period with constant stirring. The mixture should be allowed to cool to room temperature in dark and then to be refrigerated. If the stock is cloudy, it is to be discarded. 3.2 Preparation of media Quantity of stock solution required for preparation of media 1 liter MS Stock No Quantity (in ml) I 20 II 20 III 10 IV 10 V 10 The specified quantities of stock solution should be pipetted into a 1000ml beaker in which about 1/3rd distilled water is taken. The required quantity of sucrose and myoinositol is to be added and dissolved. Make up the volume to 1 liter and adjust the pH to 5.7 – 5.8 weighed quantity of agar should be dissolved by boiling. About 20ml of the culture media should be taken in precleaned culture vials and plug tightly with non-absorbent cotton. The media is to be sterilized by autoclaving at 15 psi pressure for 15-20 minutes

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3.3

Collection and preparation of explants Explants should be collected with care since they will affect the quality of plantlets regenerated. The motherplants should be so taken that, middle aged plants that too without defects, free of diseases and pests should be selected for explants. Explants collected were surface sterilized using mercuric chloride, chlorine water, calcium hypochlorite etc. at varying durations depending on the tissue selected. They were later washed with sterilized distilled water, dried and cultured under aseptic conditions in a klenzoids laminar air flow cabinet and put for incubation at 25±2oC and light intensity varying with species and tissue used. Special techniques Isolation and culture of protoplasts brassica /nicotiana Introduction Plant protoplasts provide the starting point for many techniques aimed at the genetic modification of plant cells and whole plants. The isolated protoplasts, a wall less cell can be induced to fuse with protoplasts of different species thus providing the opportunity for the creation of noval plant hybrids (somatic hybridization) 1) The protoplast is also an ideal recipient of foreign DNA, often in the form of plasmids and providing incorporation and expression ensure, such transformation can lead to the production of genitically modified plants. 2) Protoplasts can also incorporate larger partials (organelles) thus facilitating the exchange of extra techniques can not by definition, be described in isolation and are deliberately linked therefore, to subsequently recover the whole plants Back ground One of the most significant developments in the field of plant tissue culture during recent years has been the isolation culture and fusion of protoplasts the techniques are especially important because of their teaching implications in studies of plant improvement by cell modification and somatic hybridization. Isolated protoplasts also offer the means of tackling various fund omental research problems in experimental plant biology. This is realized because of the totipotent nature of plant cells. The literature accumulated so far has revealed that protoplasts is culture can be induced to undergo intra and inter specific fusion to form a somatic hybrid and also to take-up foreign organelles and genetic materials. Protoplasts have also been used to investigate various problems in plant physiology. This fascinating field of research, though still informative stages as for as the technology is converted has awakened the interests role in opening up new vistas and has awakened the interests of plant physiologists, pathologists, molecular biologists and cytogeneticists. It is envisaged that in future years protoplasts will be one of the most frequently used research tools for tissue culture studies. Plant and cell requirements for protoplast isolation 1. Growth of plants The genetic and physiological status of the green house grown plant can influence the ultimate yield and viability of protoplasts. An example of green house conditions suitable for petunia is given below:

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a. Germinate seeds of petunia in the light on soilless compost a t o 20-30 C b. Grow the seedling in the green house at 20-25oC wit an illumination of 10,000 lux and 0-16 hours day length. c. Transfer the seedlings to seed trays and grow as futher for 5-6 weeks under the same conditions plants 8-10 cm high, with 7-10 fully expanded leaves are used as a source of protoplasts. N.B. Plants in bud are never used. Pest control Two of the main causal agent of bacterial contamination through leaf damage once white fly and red spoiler.These can be controlled by a sequential treatment withvarious fumigants including DdT/ lindane. Growth of cell suspensions Cellsuspensions to be used as a source to protoplasts should be their exponential phase of growth.At thin and cellulosic in nature thus requiring minmum incubation with enzyme mixtures. Growth of Seeding (1) Surface sterilize seeding in 0.1% mercuric chloride for 10 minutes. (2) Wash thououghly in sterile tap water (3) Transfer the seeds aseptically to petri dishes cohtaining MS mediu without phytohormones but including 0.6%agar and 0.2 % sucrose. (4) Germinate the seeds in the dark or light (2000 lux) at 250C. Isolation and cultur3e of mesophyll protoplasts from tobacco shoot culture Plant species Nicotiano glauca x N. congsdoffi Origin of protoplasts Mesophyll cells Enzyme mixture Macerozyme (0.4% + cellulase onazyka (4%) in 0.6 M sucrose. Culture medium NAGATA and TAKEBE + NAA (3 mg/l)+6-BAP (1 mg/l) Growth response Somatic hybrids Steps 1. Take two to four fully expanded leaves of a 6 week old shoot culture under sterile conditions and place them in a 9 cm plastic culture dish wet the leaves thoroughly with enzyme solution and remove the mid ribs. 2. Cut the leaves into pieces and transfer leaf pieces bottom side down in to a 9 cm dish containing 12 ml enzyme solution. Seal the dish with peafilm and incubate over night at 25oC in the dark wihtout shaking. 3. entlyagitate the digest release of protoplasts from leaf debris inaided by smoothly pumping once the suspension up and down a 10 ml pipette with a wide opening takeup through a 100 µ m stainless steel mess seave on a 100ml glass beaker. Add 6-8 ml of Ku medium to thedish and disrupt remaining tissue by carfully pumping it up and down the

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pipette. Sieve this suspension to collect both fractions in the glas beaker. 4. Mix the protoplast suspension gently an distribute in to two 14 ml sterile plastic centrifulge tubes. Carefully overlay thesuspension with 1 ml w5 solution. Spin for 7-10 protoplasts at the interphase w3iht a 2 ml pipette, taking as little as possible of the loer phase transfer the protoplasts to one new 14 ml centrifuge tube combining theprotoplasts from twointerphases. 5. Remove the protoplasts at the interphase with a 2 ml pipette,taking as little as possible of the lower phase transfer th protoplasts to one new 14 ml centrifuge tube combining theprotoplastw from two interphases. 6. Gradually add 8-10 ml W5 solution, close the tube and resuspend th4 protoplats by gently titting thecapped tube. Pellet the protoplasts. Remove the supernatant repeat this step. 7. Resuspend protoplast pellet in 5-10 ml. w5 solution. To determine protoplastyield with a 1 ml pipette take up 100µ l aliquot of the protoplasts suspension, add 900 µ w5 solutionto it and count the protoplasts in a hemocytometer. 8. Pellet the protoplats. Remove the supernatant and resuspend protoplast pellet inmedium K3 to yield a suspension with a cell density of 1x 106 protoplast/ml . 9. Place 0.5 ml of the protoplat suspension in a 6 cm falcon petridish. Add 4.5 ml prewarmed 1: 1 mixture of media K3 and H containing 0.6% sea plaque agarose. Mix gently and allow to set. 10. when midium is solidifide seal thedishes with parafilm and culture the protoplasts fro 24 hr in the dark at 24oC, followed by 6 days incontinuous dim light where first and mutliple cell divisionoccur. 11. cut theagarose containing the dividing Protoplasts quadrants and place these in 50 µ l of medium A, ina 9 cm plastic culture vessel. Incubate at 24oC in continuous dim light on a rotary shaker at 80 rpm. 12. After 102 weeks a loan of protoplast- derived colonies is formed within th agarose sectors floating in liquid medium A in bead type culture under continuous shaking and the same culture conditions. Solutions 1. Enqyme solutin: Dissolve 1.2% (w/v) cellulase” onoquka” R10 and 0.6%(w/v) macerozyme R10 in K4 medium ( K3 with 0.4 instead of 0.3 m sucrose). Spin down to pellet starchcontaminating the enzyme preparations. Adjust to pH 5.6 withkOH and filters- sterilize store at 4oC for no longer than 3-4 weeks. 2. Washing Solution: W5 : Dissove 15.4µ M NaCL 125 mm CaCl2, 5 mM KCL and 5 mM glucose in distilled water, adjust to pH 5.8 with KOH and autoclave store at room temperature. 3. Media: The compotion of the media used at final concentrations of their individual ingradients is medium A, Medium H, MediumK3 Medium Ms morpho. 4. Stock Solutions for preparation of media: for all couture media wsed, prepare macro elements as 10 fold concentrated stocks. Micro elements, vitamins, and organic acid stock solutions are 100 fold

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concentated in all cases.For preparation of 1000 ml microelement stock, distilled water each. Mix dissolved solutions and heat at 60 o C. After cooling down, add remaining stock ingradients dissoved in distilled water and complete to stock solution of organic acids for medium H only stock solutions of carbohydrates are 10 fold concentrated and wihtout glucose for medium H. 1000 fold concentrated but fold concentrated but without sucrose for medium A. 5. Medium Preparation: Prepare the couture media using stock solutions and dilute them glass distilled autocluave. Keep sterile liquid media A, N, and K3 in autoclaed glass bottles at 4oC . medium K4 is prepared as medium K3 but contains 0.4 M sucrose final concentration. Prepare 1:1 mixture of media K 3 and H solidifide with 0.6% (w/v) low getting tempeerature sea plaque agarose. Autoclave dry agarose first, add sterile K3 medium, melt in a microwave oven, and after cooling to 45 oC, add filter steilized midium. Adjust to pH 5.8 with KOH for medium MS add 0.8% (w/v) agar and autoclave.Pour 100 ml fractions of melted meium in autoclaved couture glass jars and store at room temperature. To prepare medium MS morpho solidified with 0.6 (w/v) agarose, first autoclave the agarose in three-fourths of required volume of distilled water separately diluted corresponding stock solutions and other medium in gradients to complete remaining fourth of final volume with distilled water and after filter-sterilization, and it to the autoclaved, melted agarose to yield and final medium pour 80 to 100 ml fractions of melted agarose to yield the final medium in 9 cm sterile plastic culture vessels and is autoclaved culture glass jars and store at room temperature. Compositions of the Media used Media A Macro elements (mg/litre final concentration) KNO3 1010 NH4NO3 800 CaCl2.2H2O 440 MgSO4.7H2O 740 KH2PO4 136 (NH4) succinate 50 Micro elements (mg/litre final concentration) Na2EDTA 37.3 FeSO4.7H2O 27.8 H3BO3 3.0 KI 0.75 MnSO4.7H2O 10.0 ZnSO4.7H2O 2.0 CuSO4.5H2O 0.025 Na2NoO4 0.25 CoCl2.2H2O 0.025 Carbohydrates (g/litre final concentration)

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Sucrose 30 Mannitol 50 Myo inositol 0.1 Hormones (mg/litre final concentration) NAA 0.1 BAP 1.0 Vitamins (mg/litre final concentration) Pyridoxin HCl 1.0 Thiamine HCl 10.0 Nicotinic acid 1.0 Media H Macro elements (mg/litre final concentration) KNO3 NH4NO3 CaCl2.2H2O MgSO4.7H2O KH2PO4 1910 600 300 300 170

Micro elements (mg/litre final concentration) Na2EDTA FeSO4.7H2O H3BO3 KI MnSO4.7H2O ZnSO4.7H2O CuSO4.5H2O Na2NoO4.2H2O CaCl2.2H2O 37.3 27.8 3.0 0.75 10.0 2.0 0.025 0.25 0.025

Hormones; mg/lt final concentration) 2, 4-D NAA BAP 0.1 1.0 0.2

Carbohydrates (g/litre final concentration) Sucrose Glucose Mannitol Sorbitol Cellobiose Fructose Mannose Rhammose 0.125 68.40 0.125 0.125 0.125 0.125 0.125 0.125

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Ribose Xylose Myo inosital

0.125 0.125 0.1

Vitamins (mg/litre final concentration) Pyridoxin HCl 1.0 Thiamine HCl 10.0 Nicotinic acid 1.0 Folic acid 0.2 Ca Pautothenate 0.5 P-amino benzoic acid 0.1 Calcium chloride 0.5 Riboflavin 0.1 Ascorbic acid 1.0 Vitamin A 0.005 Vitamin D 0.005 Vitamin B12 0.01 B-Biotin 0.005 Organic acids (mg/litre final concentration) Sodium pyruvate 5 Citric acid 10 Malic acid 10 Fumari acid 10 Other organics Casein hydrolysate 250 Media K3 Macro elements (mg/litre) KNO3 NH4NO3 CaCL2.2H2O 900 MgSO4.7H2O 250 (NH4)2SO4 NaH2PO4H2O 150 CaH PO4 Micro elements (mg/litre) Na2EDTA FeSO4.7H2O H3BO3 KI MnSO4.7H2O ZnSO4.7H2O CuSO4.2H2O Na2NoO4.2H2o CoCl2.2H2O 37.3 27.8 3.0 0.75 10.0 2.0 0.025 0.25 0.025

250 250 250 50

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Carbohydrates (g/litre) Sucrose Xylose Myo inositol Hormones (mg/litre) 2,4-D NAA BAP Vitamin (mg/litre) Pyridoxin HCl Thiamine HCl Nicotinic acid MS media Macro elements (mg/litre) KNO3 NH4NO3 CaCl2.2H2O MgSO4.7H2O 370 KH2PO4 Micro elements (mg/litre) Na2EDTA FeSO4.7H2O H3BO3 KI MnSO4.7H2O ZnSO4.7H2O CuSO4.5H2O Na2NoO4 CoCl2.2H2O

102.69 0.25 0.1

0.1 1.0 2.0

1.0 10.0 1.0

1900 1650 440 170

37.3 27.8 6.2 0.83 16.9 8.6 0.025 0.25 0.025

Carbohdyrates (g/litre) Sucrose 10 Myo inositol 0.1 Vitamins (mg/litre final concentratino) Pyridoxin HCl 0.5 Thiamine HCl 0.1 Nictonic acid 0.5 Other organic (mg/litre) Glycine 2.0 MS Morpho media

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Macro elements (mg/litre) KNO3 NH4NO3 CaCl2.7H2O MgSO4.7H2O 370 KH2PO4 Micro elements (mg/litre) Na2EDTA FeSO4.7H2O H3BO3 KI MnSO4.7H2O ZnSO4.7H2O CuSO4.5H2O Na2NoO4.2H2O CoCl3.2H2O

1900 1650 370 170

37.3 27.8 6.2 0.83 16.9 8.6 0.025 0.25 0.025

Carbohydrates (g/litre) Sucrose Myo inositol 30 0.1

Vitamins (mg/litre final concentration) Pyridoxin HCl 0.1 Thiamine HCl 0.1 Nicotinic acid 0.1 Ca-Pantothenate 1.0 Hypocotyle, stem and petiole protoplasts (e.g. Brassica napus) (i) For the isolation of hypocotyl protoplasts, germinate sterilized seeds and cut the hypocotyl into 0.5-1.0 mm transverse sections. Cut the stem of petiole tissue in a similar manner. (ii) Transfer to CPW 13 M for 1 hr and then to the enzyme solution incuabte for 16-18 at 220C in the dark with slow shaking. (iii) Pass the incuabtion mixture through a 50 µ m nylon sieve, washing the digested tissue pieces with small volumes of CPW 13M medium. Spin at 100 g for 10 min in order to sediment the prtoplasts. (iv) Remove the supernatant resuspend the protoplasts in CPW 215 medium and spin. Protoplasts will collect at the surface. (v) Remove the protoplasts, transfer to the appropriate culture medium and count. 4.1 Fusion of Proroplasts: Protoplats fusionof plants involves four discrete atages. (a) Isolation of protoplasts (b) Fusion

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(c) Selection and plant regeneration (d) The analysis of regenerated plant material Somatic hybridization provides a method wherby sexual incompatibility can be bypassed.Since, therre are no barriers to protoplast fusion incompatibility and therfore protoplast level. Followning fusion, heterokaryons containing nuclei of two species in common cytoplasm regenerate a new cell, enter division and nuclear fusion results in the formation of somatic cell hybrid cell. Like the normal cell, the sometic hybrid cell is totipotent dand theerfore capable of regeneration embryogenesis or organogenesis into whole hybrid plants.It is terefore, an approach of immense value iin the general area of plant breeding and plant improvement. Many methods have been described for the induced fusion of protoplassts, these differ mainly in relation to the necessary to visualize by mrkers i.e. green mesophyll of other parental species. Heterokaryons are identifided as those protoplat5s prossesssing chloroplasts ( from the mesophy11 parent) in a richly cytoplasmic background ( from the nonmesoplyll partner). As an example, a protocol is described below for the fusion of leaf protoplasts of Ptunia parodii with those of a cell suspension of albino Petunia hybrida. Alternative fusion treatments are also described. Stages invovled - Isolation of protoplasts - Fusion - Selection and plant regeneration - Analysis of regenerated plant material. 4.1.1 Poly Ethylene Glycol Method (PEG) a. Adjust the denstities of protoplasts 2 × 105 ml-1 in MS medium b. Mark each centrifuge tube at 4 and 8 ml level c. Spin all tubes except viability cntrol and allow the protoplasts to settle. d. Remove the medium so as to leave the protoplats suspended in a maximum of 0.5 ml of medium. e. Add 2 ml of PEG per tube and left for 10 min. PEG- 30%(W/V) Sucrose- 4%” 0.01M – CaCl2.2H2O f. Dilution of PEG at 5 minute intervals by adding 0.5, 2.0,3.0 and 4ml ofmS P1 DM medium per tubes. Resuspend the protoplasts after ech dilution. g. Spin ( 100 gm ,10 min) soas to sediment the protplasts and remive the supernatant. h. Wash theprotplats in M/S P1 2M medium. i. Suspend the fusion treated protoplasts iin 1 ml of media. j. Prepare twelve 2 cm petidishes containing 8 ml of M/S P1 2 M agar. k. Adjust thevolume of viability controls. l. Dispension of protoplast.

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m. Mix together the remaining 8 mleach of the two treatments and dispense into two dishes. n. Seal all dishes with film and iincubate. Conditions Vaies with species and variety Temp :25o -30o C Light: - 1000 lux. 4.1.2. Plant regeneration After 24-36 hours division starts beginning. A well established colony will occur within 21 days. From 4th week onwards colonies are suficiently large for transfer, using the tip of a scalpel to an agar solidified regenrationmediu,. In this stage colonies are maintained in petridishes and may sometimes require subculturing roots are also induced. PRECAUTIONS 1. peerfoem ail manipulationsunder sterilize conditions. 2. pipette very smoothly and avoid stromg sucking of the tube. 3. in centrifugation steps, speed should be increased only gradually. 4. Density should be optimum. 4.2. Productio of Plantlets from Suspension Culture: INTRODUCTION Plant cell suspension celtures provide the biochemist with a relativelhhomogenous populatio of cells. Readilyaccssible to exogenously applied chemicals and growingunder defined aseptic conditions. Cell suspension cultures are widely used as model systems for studying pathways of secondarymetabolism, enzyme induction and gene material for enzyme purificatio the lack of chlorophyll and carotenoid pigments in most plant cell suspension cultures is of great benefit fro work involving isolationof enzumes of secondary products. However, susensions can’t be maintaned for long periods of time,and are usually prepared fresh fo9r each experiment. Steps Involved 1. initiation of suspension cultures from callus. 2. Subculture and meaurement of growth. 3. Regeneration into free living plants. 1. Initiation of suspension cultures from Callus: Most suspension cultures are obtained by tansfeer of friable callus lumps of agitated medium of the same composition as that used for calus growth. A relativelh large initial inoculum is advantageous as this will ensure that sufficient single cells and / or small clumps are released into themedium to provide a sutably high cell density for subsequent

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growth.Agrtation rates on orbital shakers should b in the range of 30-150 rpm with an orbital motion stroke of 2-4 c,. At the first subculture into fresh medium, remive large clumps of initial incoulum either by transferring the material with a pipette or syringe of suitable orifice diameter to exclude large cell aggregates. 1. Aseptically transfer a volume of cell cultrue, which is udeergoing rapid increase in cell density to a dialysis tube connected to the end of a glass tube of equal diameter. 2. Place the dialysis tube in a conical flask containing fresh couture medium such that the bottom of the glass tube is level with the surface submergd. The top of the glass tube may be held in the neck of the conical flask with a cotton wool plug. 3. Incubation in an orbital shaker, the extent of conditioning of the fresh medium will depend upon a) The density of actively growing suspension b) The relative volumes of suspension and fresh medium. c) The time of incubation. Subculture and Measurement of Growht Cell suspension cell cultures usually require a regular subculture at more frequent intervals than the callus cultures transfer of a suitable size inoculum to fresh meidum using either a ) Pipettes b) autoclaves metal syringes or c) transfer by simply tipping culture into the new vessel upto a graduation mark to ensure approximately constant inoculum size. The growht of cell suspensioncultures may be monitored by measurement of one of more of the following parameters. 1. packed cell volume 2. Cell number 3. Wet and dry weights 4. Protein or DNA content 5. medium conductivity 6. Cell viability Protocol Production of free living Plants 1. Subculture 2-4 g of embryonic callus into a 250 ml Erlenmeyer flask containing 25 ml of mS Liquid medium supplemented with 0.45 µ M 2.4D place theflask on an orbital shaker and agitate at 50 rpm. 2. Inclubate under 1000 lux illumination using a 16h photoperiod at 25-29oC . 3. initially, subculture using a 1:1 dilution ratio by pipettin off onehalf of the medium and replaceing ith fresh medium after 2 weeks. 4. Subculture using a 1:4 dilution ratio using large month pipettes, transfer a 5 ml of suspension to 20 ml of fresh medium every 10-18 days. Generally repidly proliferatin cultures are subcultured every 10 days. Cultures can be maintained in tis manner for several years. 5. Asepticallyaspirate off 2,4-D containing medium and transfer the suspension callus to Ms liquid medium to uniform embryo pooulation

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can be produced by sieving the callus suspension through 43-200 µ m stainless steel mesh and culturing onMS liquid medium. 6. Initiate the plantlet formation via embryogenesis by subculturing 0.5-1 cm2 pieces of callus to the surface of mS agar medium. 7. Incubate thecultures under 16h photoperiod conditions ( 1000 lux) at 25-29oC. Within 2 weeks culturesw will exhibit theformation of visible embryos and green plantlets. 8. Agter 4-8 weeks, individual plantlets can be treased a part from the other callus- embryo-plantlet mass an recultured separately or halfstrength MS agar medium. 9. Incubate the cultures under 5000 lux ilumination using 16h photoperiod conditions at 25-29oC . Within 4-8weeks cultures will enlarge considerably and resemble seedling carrots 10. otinue reculturing every 4-8 weeks until plantlets reachsufficient size to be trasplanted to soil (~ 10-15 cm inlength), The selectionof large well developed plantlets with functional shoot and root systems is criticla fro success in obtaining free living plants. 11. Transfer platntlets intact to distilled water for 10-15 min to avoid dehydration and remove excess adhering medium Rinse plantlets three times with distilled water spray, with 0.5%(W/V) benolate fungicide and transfer to 7.6 cm2 diameter platic pots or fifty pent pots containing sterile peatmiss and vermiculets in 1:1 (V/V) ratio.Enclose plantlet and container with two interlocking cloear plastic polystyrens tubmblers to maintain high humidityconditions. 12. Administer weeklyapplicantions of 0.5% benolate sprays on foliage to minimise fungal growth water the pots every other day with distiled water and once a week with one forth strenght Hoagland’s solution during the first 1 or with 8000 lux light intensity under a 16h photoperiod at Gradually acclimatise the plantlets to green house covering.After 2 months, covers maybe removed and the plants treated as normal seedlings. Pit Falls Suspension culture regenerated plantlets are similar to callus regeneerated plantlets byt, regeneration thio suspension culture is a tedious process. 4. AGROBACTERIUM MEDIATED TRANSFORMATIONIN PLANTS Agrobacterium tumefarients causes crowngalls in plants. Crown galls are tumors of plants that arise at the site of infectin.The tumour inducing agent in Agrobacterium chromosome.Ti plasmids are large double- stranded circular NA molecules of about 200 kb and 1 key exist as independent replicating unit. Ti plsmids are maintained in Agrobacterium, because a part of the plasmid DNA called T-DNA , carries the genes coding for the synthesis of unusual amino acids called opines. The infected plant cell is indued to synthesixe these opines. Selective advantage for Agrobacterium,. A second set of genes in 7-DNA causes the unregulated growth of the plant cells.

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Production of auxins. The third gene codes for an enzyme that hormones cause t5he infected plant cell to divide and thus induces gall formation. T-DNA, the part of the Ti-plasmid, which get integrated into the plant chromosome can be used ofr genetic manipulationt. T-DNA is first cloned into the standard E. coli.vector, and thepalnt gene subsequentlycloned into a second cloning site carried in the vector. This intermediate vector4 is then introduced into Agrobacterium containing intact Ti Plasmids. Re-Combination occurs between the homologous plasmid, and on infectin of a plant with Agrobacerium, the recombinant plasmid is trasferred to the plant cells. The E. Plasmid because it becomes part of the ti plasmid. Recombinant DNA technology has modifided agriculutre sector completely virus resistant plants are prodced by introducing real coat proteins into plants. Similarly the transfer of toxic gene from Bacillus thuringiensis produced insect tolerant plant. Very recently scientists at IARI. New Delhi developed transgenic bringal with BT gene. 5.1 Agrobacterium mediated Transformationof tobacco/pigeon pea: protocol 1. In Nicotiania tonacum a) Prepare leaf protoplasts b) culture the protoplasts in3.0 ml of liquid M/S p, 9M medium over 12 ml agar-solidified M/S P, 9 M medium in 9 cm petridishes. Incubats them at 25oC with an illuminatin of 700 lux. c) Agter 7 days transger the regenerating cells to M/S P , 7M medium and inoculate with 0.4 ml of a 24 h culture of a. tumefaciencs. For effective transformations the resulting mixture should contain 5x 107 bacteria/ml and 0.8 x 102 bacteria per regeneratin tobacco cell. Incubate for 36 h at 25oC in the dark. Selection for Transformed colonies 1. Transfer the cells to fresh M/S P, 7 M medium to which has been added 1.0 mg ml-1 carbenicillin. 2. By subsequent transfer rreduce the 0.P to 3.5% mannitol and finallyafter 4 weeks to 6% mannitol or in M/S P1, OM medium but still in presence of carbencillin. 3. Place the cells on solidified M/S P1, OM medium with antibiotic.After 4-6weeks , using a scalpel, transfer individual colonies to agar solidified.M/50 medium and subculture after every 4 weeks. Surviving colonies are trasform events. 5.1 Confirmatin of Transformationand Tumour Geneticity

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Transformation events arising from protoplasts and regenerated cells require a detailed biochemical analysis of the opine aminoacides. The presence of these opines coupled Agrobacteriun and /or theTi-plasmid.a simple test confirming the tumourous state, from the habituatee state involves grafting of selected cultures. Confirmationby grafting sterilize. Young shoots of greenhouse grown plants and surface

Take 5 cm lengths of shoots and insert some of the test tissue into a 0.5 cm slit on each stem explant. tape. Seal the graft with a 3.0 cm length of sterile micropore

EMBRYOGENESTS, ORGANOGENESIS AND PLANT REGENERATION 6.1 Mode of plant regeneration The term tissue culture has generated into an all encompassing, convenient term to describe all types of steril plant culture, cells, tissues, organs, embryos and plantlets. Plant regeneration through tissue culure can be accomplished using one of three methods: embryo culture, somatic culture of a zygotic embryo. Somatic or asexual embryogenesis is the production of embryo-like structures from somatic cells. Thesomatic embryo is an independent bipolar structure and is not physically attached to the tissue or origin. Plant development through organigenesis is the formation and outgrowth of shoots from callus or initiation and outgrowth of axillary trends generated from culture tips and their subsequent adventious rooting. 6.2 Explant factors The term explant is used to describe the initial piece of the plant introduded in vitro. For optimum success, explant must be obtained from healthy vigorous plants. Pre-culture plant conditions may greatly influence the subsequent growth of explants in culture. Practically any part of the plant can be successfully cultured in vitro and can regenerate plantlets provide the explant is obtained at the proper physiological stage of develoment. Meristematic tissue should be given preference to others. Following explant selection, the distinfection procedure are adopted. Fungal and bacterial contamination in cultures is usually detectable in 1-14 days. Surface sterilization is adopted to combat with this. Sterilant NaOCl (0.2%) Plant Part Flowers, stem Duration 10-20 minutes

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NaOCl (0.25%) NaOCl (2.6%) NaOCl (0.25-2.6) Iodine (3%) Hg Br2 (5%)

Leaves Roots Tap root Seeds Seeds

20 minutes 30 minutes 30 minutes 10 minutes 10 minutes

6.3 Nutrient medium factors A multitude of plant tissue culture media are suitable for plant regeneration. The formulation derived by Murashige and Skoog (Table ) is the most common medium employed in the plant tisseu culture field. Adverse chemical discharge into the media like polyphenols which causes the browning of media can be thwarted through frequent subculturing. 6.3.1 Media requirement for selected plant species caable of whole plant regeneration in vitro Species Anthurium Phoenix Daetylifera Brassica campestris Chrysanthemu m norifolium Cucurbita pepo Oryza sativa Triticum aestivum Zea mays Prium sativum Explant Type of Growthing conc : UM) regeneratio Callus Shootin Rooting n g Leaf 0-C 0.42,43.2 PBA 0.5 NAA D 3.2 PNA Axial bud SE-C 135 2,4- 0.54 D NAA 13.9 Zip Node AS 4.4 BA 4.4 BA 0.44 BA 5.4 NAA Shoot tip 0-C 5.4 NAA 0.11 NAA 0.054 NAA Cotyledon SE-C 4.5 0.5 2,4-D 2, 4-D Root 0-C 102, A- 0.054 D NAA Seed 0-C 4.5-9 5.7 IAA 0.054 2,4-D NAA Mesocotyl 0-C 67.8 0.054 2,4-D NAA Epicotyl 0-C 10.7 22.2 BA NAA 1.11 IAA 4.7 BA

6.4 Somatic embryogenesis The direct mode of somatic embryogenesis invovles the formation of an asexual embryo from a single cell or group of cells on a part of the explant tissue without an intervening callus phase.

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PROTOCOL CARROT a)

FOR

PRODUCTION

OF

EMBRYOGENIC

CALLUS

IN

Procurement of carrot seeds surface sterilization using 2.6%

b) 15 minutes sodiumhypochlorite solution c) d) Drying the seeds

Place 1-3 seeds on the surface of MS agar

e) Incuabte the seeds in a culture room at 25-290C under a 16 b photoperiod until seeds germinate. f) Transfer germinating plant to a sterile petridish and cut off the petiles. Place 1.3 pieces of petiole on the surface of MS medium containing 0.45 µ M 2,4-D. g) Incuabte the cultures in compete darkness at 25-290C for 4-8 weeks. h) Maintain the callus on agar medium by sub-culturing 0.5-1 cm2 pieces of callus to fresh medium every 3 weeks. 6.5 Organogenesis The production of adventitious shoot in vitro is more common and easier to control than the development of somatic embryos from cultured explants. Plant production through organogenesis can be achieved through one of three modes.  Production of adventitious organs from a callus derived from the explant.  Emergence of adventitious organs directly from the explant without an intervening callus phase.  Production of plantlets from outgrowth of axillary buds. Protocol for the production of shoot from callus a) Plants are grown in the greenhouse and leaf expalnts are used prior to flowering. Leaves are taken either 1-2 h after sunrise or from dark pretreated plants. b) Wash leaves with soap and water, rinse with 40% ethanol and then rinse with distilled water. c) Surface sterilize leaves in 1% NaOCl – 10 minutes.

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d) Cut leaves using scalpels and transfer into MS containing 4.5 µ M 2,4D and incuabte. e) Subculture pieces of callus obtained to MS containing 1 µ M 6-BAP to initiate shoots. f) Incubate at 25-290C under 16 h photoperiod at 1000 lux. Shoots will be produced is 305 weeks. g) Incubate cultures (shoots) in rooting media. h) Transfer rooted plantlets. 6.6 Plant Regeneration Directly from cultured explants The method of plant regenration is suited to herbaceous species using leaves, corns, bulbs, stems, Rhizomes and tubers. Here explant is established on a nutrient medium containing moderate levels of auxins and cytokinins (to avoid callus production) and subsequently initiates shoot organs. Callus production) and subsequently initiates shoot organs. Multiplication is achieved through subdividuon of shooting clump and planting out in separate vessels. This method should not be used to clone variegated plant since the resultant plants will either show modified pattern of lose the variegation completely. 6.6.1 Protocol for Regeneratino of plants via Direct Organogensis for Begonia a) Cut newly formed fully expanded leaves and accompanying petioles from Begonia plants grown in the green house. b) Disinfect leaves by treating with 5.25% NaOCl containing 0.5% tween20 for 10 min. Rise thrice with sterilized distiled water. c) Section leaves into 5 mm lengths and place, with the proximal side down, on the surface of MS, agar containing Nitsh & Nitsch vitamins, 1.8 µ M BA and 0.54 µ M NAA. d) Incuabte 1000 lux using a 16 h photoperiod at 25-290C. e) Subdivide each culture and transfer to the same medium after 4 weeks. f) Root shoots by transferring MS with 0.5 µ M NAA. g) Establish in soil. CONCLUSION Plant tissue culture is an inevitable method for propagation of plants. It forms a very conversant tool in the hands of plant breeders, plant biotechnologists etc. A detailed account of the general aspects of tissue

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culture with merits and demerits is described. Apart from that various types of plant regeneration techniques like callus culture, somatic embryogenesis etc. has been dealt in detail. Culture media compositions, preparation of media etc. had also been mentioned. Experimental protocol for protoplast fusion, Agrobacterium mediated transformation had also found a palce in the present discussion. REFERENCES Levy, L.W. 1981. A large scale application of tissue culture the mass propagation of pyrethrum clones in Ecuador. Enus. Exp.Bot.21L: 273-395. Lloyd, G and McCown, B. 1980. Commercially feasible microproparation of mountain laurel (Kalmia datifolia) by use of shoot-tip culture.Proc. Int. Plant. Prop. Soc. 30: 421-427. Murashige, T. and Skoog, F.1962. A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol. Pant 15 : 437-479. Rao, M.V.S., Rao, Y.V., Rao,Y.S. and Manga, V. 1988. Induction and growth of callus in Aza diraehta indica. Crop Improvement 15(2) : 203-205. Roy, S. K., Rahman, S.L. and Majumdar, R. 1990. In vitro propagation of jack fruit (Artocarpus heterophyllus). J. Hort. Sci. 3(65): 355-358. Watson, D.J., Gilman, M., Witkowshi, J., Zoller, M. 1992. Recombinant DNA Iied., W.H. Freeman and Co., Newyork : 273-290.

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I. II.

Objective: The generation of plantlets from callus materials required : Callus, media, flasks etc. 1. Subculture 2-4 g embryonic callus into a 250 ml. Ebermeyer flask containing 25 ml of MS liquid medium supplemented with 0.45 µ M 2,4D. 2. Incubate under 1000 lux illumination for a 16 hour photoperiod at 25280C. 3. Subculture using a 1 : 1 dilution ratio. 4. Transfer ml of suspension to fresh media every 10-15 days. Cultures can be maintained in this manner for several years. 5. Tranfer the callus into MS liquid media for inducing further stages of asexual embryogenesis. 6. Initiate the plantlet formation via embryogenesis by subculturing small callus pieces on MS agar medium. 7. When individual plantlets arise, separate them and culture on halfstrength MS medium. 8. Continue subculturing for a period of 4-8 weeks till the plantlets are ready for planting out.

Procedure

III. 1. M. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

Objective: To fuse protoplasts by PEG method Adjust the densities of protoplasts in culture to 2 ×10 5 /ml in M/SP1 9 Mark each centrifuge tube at 4 ml and 8 ml level. Spin all tubes, except the vaibility control and pelletize the protoplasts.

Procedure

Remove the medium so as to leave the protoplasts suspended in a maximum of 0.5 ml of medium. Add 2 ml of PEG per tube. Dilute the PEG at 5 minute intervals by adding M/S i1 PM medium standards. Spin (100 g, 10 min) so as to sediment the protoplasts and remove the supernatant. Wash the protoplasts and resuspend it. Prepare petridishes with M/S P1 9 IX agar medium Dispense the protoplasts into the petridishes. Mix together the remaining 8 ml each of the two selfed treatments and dispense into two dishes 8 ml/dishes.

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12.

Seal all the dishes with parafilm and maintain at 250C with continuous ilumination of 700 lux. IV. Objective: tobacco/pigeon pea Agrobacterium mediated transformation of

Procedure 1. Prepare leaf protoplasts. 2. Culture the protoplasts in 3.0 ml of liquid M/S P 1 9 M medium over agar at 250C. 3. After 7 days, transfer regenerating cells to M/s P1 7 M medium and inoculate with 0.4 ml of a 24 h cultue of agrobacterium tumefaciens. Selection of Transformed Colonies: 1. Transfer thecells to fresh M/s P1 7 M medium to which has been added carbencilin. 2. By subsequent transfer reduce the osmotic pressure to 3.5% mannitol and finally after 4 weeks to 0% mannitol as in M/s P1 9M medium. 3. Plate the cellls on solidified M/s P1 O M medium with antibiotiec. After 4-6 weeks transfer individual colonies to agar medium. Confirmation of Transformation by Grafting 1. Take 2-3 week old seedlings grown in green house. 2. Take 0.5 cm of the test tissue and insect the scion. 3. Seal the graft with a sealed microtape. 4. Insect the stem base into agar-solidified MS medium with 0.1 mg 1 -1 Mas in 6 OZ power round jars. Transformed tissues will proliferate while non transformed tissues will not.

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V. Objective

PLANT TISSUE CULTURE

To germinate the Brassica seeds in MS medium under aseptic conditions. Requirement Reagents, Atuclave, milk bottle, beakers, flasks, measuring cylinder, Culture bottle, etc. Principle In tissue culture maintanance of aspectic condition and aspetic handling has prime importance. Any contamination during handling will destory all material and our objective will be failed. The Brassica seeds can be sterilized by using usrface sterilizing agents such as 0.1% HgCl2, for 2 minutes 1% sodium hypochloride or 70% alcohol. Procedure (1) Media preparation MURASHIGE AND SKOOG MEDIUM Constituents Soluntion 1 1. 2. NH4NO3 KNO3 Solution 2 1. CaCl2.2H2O Solution 3 1. 2. MgSO4.7H2O KH2PO4 Solution 4 1. 2. Na2EDTA FeSO4.7H2O Solution 5 1. 2. H3BO3 MnSO4.H2O 100 X 1650 mg/lt 1900 mg/lt 100 X 440 mg/lt 100 X 370 mg/lt 170 mg/lt 100 x 370 mg/lt 27.8 mg/lt 1000 x 6.25 mg/lt 22.5 ,g/lt = = 0.3125 g/50 ml 1.15 g/50 ml = = 0.1865 g/50 ml 0.139 g/50 ml = = 1.85 g/50 0.55 g/50 ml = 2.2/50 ml = = 8.25 g/50 ml 9.5/50 ml

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3. 4. 5. 6. 7.

ZnSO4.7H2O KI Na2MoO4 CuSO4.5H2O CoCl3.6H2O Solution 6

8.6 ,g/lt 0.83 mg/lt 0.025 mg/lt 0.025 mg/lt 0.025 mg/lt 1000 X 0.5 mg/lt 0.5 mg/lt 0.1 mg/lt 2.0 mg/lt

= = = = =

0.43 gm/50 ml 0.041 gm/50 ml 0.00125 gm/50 ml 0.00125 gm/50 ml 0.00125 gm/50 ml

1. 2. 3. 4.

Nictonic acid Pyridoxine HCl Thiamine HCl Glycine

= = = =

0.025 gm/50 ml 0.25 gm/50 ml 0.005 gm/50 ml 0.1 gm/50 ml

BAP = 2 ppm = 10 mg; Sucrose = 30 mg/lt NAA = 1 ppm = 5 mg Procedure 1. Prepare MS medium andautoclave it. 2. Soak the brassica seeds in 0.1% HgCl2 for 2 minutes, 3. Wash the seeds in sterile wate for 2 to 3 times. 4. Aseptically tranfer 3-5 seeds to culture bottle and 6-10 5. Aseptically transfer 3-5 seeds to culture bottle and 6-10 seeds using sterilized forceps. 6. Keeping the bottle in dark till radical emerges out. 7. Transfer the bottle to light after radical emergence. Observation The seeds took 2-3 days for germiantion and full sized plant (with 2 leaves) appeared on the medium after 5 days. Experiment No. 2 Objective Callus induciton from hypocotyl of Brassica seedling on callus inducing medium Requriment Forceps, scalpel, medium, bottle spirit lamp etc. Principle

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Callus is homogenous undifferentiated mass of cells. Any plant part or cell or tissue when inocualte into callus inducing media start multiplying and produce a gorup of cells which are homogenous. The callus inducing medium hasauxins and cytokinins in such as proportion that it is capabe of inducing callus. Procedure 1. Prepare the callus inducing medium and pour it into bottle. 2. Remove the seeldings from boiling tube in aspetic conditon and aseptically separate the hypocotyl region. 3. Cut the hypocotyl region into small pieces using sterilzied scalpel. 4. Aseptically transfer few pieces of hypocotyl into the bottle containing callus inducing medium. 5. Keep the flasks in dark. 6. Observe the callus produced after 6-7 days.

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IMMUNOLOGICAL TECHNIQUES Immunology is a rapidly developing field of study concerned with the discovery of the mechanisms of the immune system, the development of vaccines and the regulatory procedures for manipulating the immune response. Immune response may be cell mediated response (mediated by T. Lymphocyte) of Humoral response (mediated by B. lymphocyte). Antigen An antigen is any substance which when introduced into the vertebrate host stimulates the production of antiboides and reacts with preformed antibodies. Immunogenicity Capacity to stimulate an immune response. Specificity Their ability to react specifically with immune antibodies. Antibody Antibodies are also termed as immjnoglobulins immunoglobulins having 2 light and 2 heavy chains Ab are classified into 5 classes according to their size and heavy chain. Heavy chain IgG IgM IGA IgD IgE R µ α δ Σ Light chain K/ K/ K/ K/ K/ Mol. Wt. 15,000 900,000 160,000/370,000 180,000 185,000

Antigen Antibody interaction Epitopes of antigens interact with paratopes of antibody and form an immune complex. Ag + Ab – Ag. Ab [Ag Ab] K1 = ------------[Ag] [Ab] [Ag] [Ab] K2 = -------------[Ag Ab]

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K = Affinity constant If K1 is more = more affinity K2 is more = les affinity Antibody is the measure of strength of antibody and antigen interaction Cross reactivity Ab may react with many other molecules other, then the antigen agasint which it is raised. It is called cross reactivity and it will be raised due to presence of poly functional binding sites. Adjuvants Adjuvants which may or may not be antigenic, increae the level of circulating antibodies and thus improve immune responses. Antigenic adjuvants Gram –ve bacteria (Borderella-pertusis) endotoxin of ram –ve bacteria. Acid fast microorganisms, phytonemagglutinins and antibodies. Non-antigenic adjuvants It includes potassium aluminium tarterate, calcium phospahte, mineral oil, lanolin, surface active agents, calcium alginate, collodian, or acrylic particles. Freunds complete adjuvants It is such a mixture which contain killed mycobacteria, an oil and an emulsifier. Affinity The intrinsic binding power of a single antibody combining site with a single antigen bidning site, which may be expressed interms of a single binding constant. Avidity The net combining power of an antibody molecule with its antigen. Antigen combining site or paratope The site on the antibody that combinesspecifically with its corresponding antigenic determinant. It usually involves only a few amino acid residues antibody molecule but not usually directly linked to each other. Antigenic determinant or epitope A small site on an antigen to which a complementary antibody molecule may be specifically bound through its combining site. Antiserum A serum containing antibodies agasint a specific antigen. Avidin A protein that specifically binds biotin with very high affinity.

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Complement A group of serum proteins essential for antibody mediated immunolysis. Immunization is clonal expansion of immune cells. There are 2 types of immunization. Active immunization Here we use antigen. It is used as preventive or prophylactice measure. In active immunization vaccine may be comitve agent or toid or alttenuated vaccine. Factors afecting antibody production a) Dose of antigen b) Nature of antigen c) Mode of immunization d) Immunoization schedule e) Presence of immuno protentior Dose of antigen B.S.A. 500 µ g After immunization some non specific reactinos like enzyme activity may degrade the antigen. Molecules that can induce non-specific mechanisms of immune response is called as xenobiotics. So we have to protect the antigen from non-specific immune reactions. The protective agents are called immunopotentior or adjuvants. e.g. Fruends ion plete/ incomplete adjuvants, Alum, MgSO4 Mode of immunization 1. Intra pertionial (I.P.) 2. Intra dermal (I.D.) 3. Subcutaneous (S.C.) 4. Intra veinal (I.V.) 5. Foot Pad (F.P.) 6. Intramuscular (I.M.) Immunization schedule Ist IInd IIIrd IV V VI injection injection injection injection injection injection 0 day 7 daysSC./IP 14 days 21 days 28 days 35 days SC./IP SC/IP SC/IP I.V. SC. at multiple sties

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Mode of Bleeding 1. By ear (Marginal vein of pinnae. Dialate by xylene) 2. From eye (Retro orbital bleeding) 3. From heart (cardiac puncture method) 4. Juglar vein.

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Experiment No. Objective Observe agglutination reactions by blood grouping Requirement Blood, antiserum, mixing rod, slide etc. Principle Agglutination form a clump instead of using bacterial cells to demonstrate agglutination, whole human red blood cells can be employed. There are 2 types of antigen present on blood corpuscles. Blood Group 1. 2. 3. 4. A B AB O Antigen a b ab Ab Antibody b a Genotype IaIa/Ia/Ia IbIb/IbIb IaIb IoIo

Whatever Ag, a person has in his cell the corresponding Ab is lacking in the serum; this obviously must be so. Since if a person of groupo A had the antibodies agaisnt A in this serum his cells would be agglutinated when an Ag is not present, the corresponding Ab. B present. This a person of group A has no Aba as he has no Ag B but does have Ab : b. The blood groups are of great importance. Knowledge of their existance has removed the hazards which were associated with blood transfusion so that this valuable thereaputic procedure is commonly used. The information received from blood grouping in some times used in medico legal cases is case of disputed paternity or child mixing. Possible effects of transfusion of blood Donor’s blood group O A B AB Receipients Can be Agglutinates the blood given to of O A B AB O, A, B, AB + + A & AB + + B & AB + + + AB Can receive blood O O, A O, A O, A, AB Remark

Donor B, Univ. recipient

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Observation Table Name 1 . 2 . 3 . 4 . 5 . 6 . 7 . 8 . 9 . 1 0 . 1 1 . Anti A Anti B Blood Group

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1 2 .

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Experiment No. Objective To detect pregnancy by the presence of B HCG in urine by latex particles agglutinatino inhibition test. Requirement Sample, distt. H2O, black testing plate, Ab against GCG latex beads coated with HCG, Mixing rods. Principle HCG is human chorionic gonadotropic hormone. It is released from the placenta of a pregnant woman and can be collected from the urine of some. HCG is a dipeptide glycoprotein. Containing 2 subunit α and β . It is soluble in antibody. Hence the Ag Ab complex precipitation reaction is not so visible we have to anpigy it. This is done by the use of HCG coated latex beads, in the test reaction and positive test shows no agglutinatin, hence it is actually a precipitation inhibition test reaction. Procedure 1. Take immunological testing slide blakc in colour. 2. There were 2 circle outlines on the plate. On one add a drop of distilled water to the other place a drop of urine sample. 3. Place a drop of antigen specific antibody near the drop of sample. Mixed them with a mixing rod. 4. Now added a drop of the latex beeds coated with HCG to each circle. Mixed them by moving the slide gently. 5. Observed the plate after 2 minutes. Observation In the circle with control; agglutination was visible beginning from periphary of the urine sample there was no agglutination. Result The result implies the sample bears HCG and the donor of the sample is pregnant. Precuations (i) (ii) Do not use same end of the mixing rod for mixing control and sample. Only a drop of the constituent shall sufficient for the test.

Discussion The dark coloured plate is supplied in the kit enables use to visualize the agglutination reaction which as such may not be visible on normal slide. We can carry out the test even after 90 days of prenancy but is generally carried out in the mid term of pregnancy to yield visible agglutination reaction.

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HCG in sample acts as the antigen and react, pontanecously with antibody provided with antibody provided (excess) and form Ag-Ab complex. Now as we add HCG coated beads there out to be no agglutination as all the AB have already formed lattice of complex with antigen and hence no agglutination shows a the test for pregnancy. GEL DIFFUSION /OUCHTERLONY A major advantage of this type of gel diffusion assay is that more than one antigen antibody systme in a mixture can be detected. It is important to remember that whenantiboides one produced by injeecting an antigen into an animal, many different antibody molecules reacting with different part of the antigen will be found in the serum of the animal. In this serum referred to as antiserum C is usually used as a source of antibodies in experiment. A single antigen will combine with the antibody and it induces (its homologous antibody) to form a single precipitin live in a gel diffusion assay. When two Ags are present each behaves independently of the other. The position of the precipitin lines wil depend primarily on the sizes of the antigens and their rates of diffusion is the gel. Thus the number of precipitin lines indicates the number of Ag-Ab system present. Double diffusion, referring to the process described above in C both Ag-Ab molecule diffuse from a well, is a simple but very useful procedure invented by the Swedish scientist Auchterlony. It is used to compare antigens for the number of indentical or cross reacting determinants. If a solution of Ag in placed into 2 adjacent wells and the antibody is placed equidistant from the 2 antigen wells. The 2 precipitin lines that form will join at their closest ends and fuse. This is called reaction of identity. When unrelated Ag and placed inadjacent wells and the centre well in filled with Ab reacting with both Ag the precipitin lives will form independently of each other and will cross. This is called as a reaction of non-identity.

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EXPERIMENT NO. Objective Demonstration of gel diffusion/ouchterlony Requirement 1% purified Agar, phosphate buffer saline (pH 7.4), 4 microscopic slides. Sodium azide, 95% ethanol will cutter, B.S.A. antibody. Procedure Preparation of 1% agar 1. For phospahte buffer saline, dissolve the following in 150 ml distilled water 2.19 g NaCl 1.28 gm KH2PO4 2.06 gm Na2HPO4.7H2O Adjust pH 7.2, add distilled water to make 250 ml 2. Add 2.5 gm purified agar in 250 ml water and 0.25 gm sodium azide (bacteriostatic) heat to dissolve agar, and autoclaved 3. Cool the agar solution to 600C. Preparation of slides with sample wells Clean the slide by 95% alcohol and label it Using a 5 ml pipet, pipet 4 ml agar carefully and place agar on slide. Allow the agar to solidify. This will take approx. 10 minutes Cut the 5 wells with well cutter. Loading the sample Fill the center will with 3 drop of antiserum using a transfer pipet. Fill the outer wells with 3 drops of Ag.

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