EXPERIMENT NO 1 SUGGESTIONS AND REGULATIONS

GENERAL INSTRUCTIONS Many of the dyes used in microbiological laboratory can stain your clothing permanently. It is therefore advisable to wear a laboratory coat during practical work. Every laboratory meeting will start with a short instruction period. Do not hesitate to ask questions, if the procedures in laboratory manual or instruction of your teacher, are not clear to you. Listen to the instructions given by the teacher about the exercise to be done and plan your work carefully before each laboratory work. Keep a record in black and white of your observations. Final examination will cover all the information provided to you about the practical and your own observations. Highlight or answer all questions on your practical manual after every exercise as home assignment. Mirco-organisms in the laboratory may be pathogenic for animals and man, so be careful while handling them.

LABORATORY REGULATIONS It is important for a student to develop a positive and responsible attitude towards his work with laboratory microorganisms. Minor mistake may contaminate the bodies and clothes of laboratory workers and those who come in contact with them may have bacterial infection. Never trust the students who worked at your laboratory bench before you but always respect the students who come after you. Begin every laboratory period by swabing your bench with a piece of cotton dipped in alcohol or spirit methylited and clean it again before leaving the laboratory.

 Keep your bench free of non-essential materials. Never pipette materials by mouth, use the pipetting devices provided in the laboratory. Do not eat, smoke, apply cosmetics in the laboratory. Wash your hands after every laboratory period. Place all contaminated glass-ware in recycle glass container and plastic-ware and papers in the provided containers so as to dispose them off after autoclaving. Never lay contaminated material on your bench and remember to sterilize platinum loop before and after use. Remove labels from the reusable glass-wares before you place them in recycling bin. Do not wear loose hanging clothes, or scarves that could catch fire or knock over reagents. Report all accidents or spills to your teacher, no matter how minor it may be. Such accidents will not count against your grade. Do not put your pencils in your mouth or ears. Do not scratch your body while working in the lab.

EXPERIMENT NO-2

INTRODUCTION TO LABORATORY EQUIPMENTS

1. PLATINUM LOOP It consists of a handle and a platinum wire forming loop at one end. The platinum wire has the property to get red hot rapidly in the flame and cool down quickly away trom the flame. This wire neither burn nor rusts. It is used for preparation of bacterial smears on the glass slides, for transferring bacterial samples to the culture medium and streaking for isolation of pure culture (plate-I). It is sterilised in the blue part of the flame. 2-PETRIDISH It is made of a clear transparent quality glass or plastic. It consists of a base and a cover. The medium is poured in the base. The glass-dishes are sterilized by autoclaving. The petridishes are used for pouring solid medium (Plate-2). The petridishe is usually used for studying colonial morphology of bacteria. 3. BUNSEN BURNER It may be gas or kerosene oil burner. It provides a well lit flame. Some area around the flame is free from microbes. ( 6 " around gas burner). This area is used for inoculation of microbial samples on culture medium (Plate-3). The flame is used for sterilization of loop and for heating the slides. . 4. WATER BATH It is a rectangular shaped meatlic utensil which is electrically heated and usually has a temperature controling device (thermostate). It may be equipped with stirring device or a shaking apparatus. Its temperature range is trom ambient to 100e. This is used to keep solutions or culture medium at a specified temperature. Serum is kept at 56OC for 30 minutes to inactivate the complement: basic agar medium in a molten state may be kept in it at 45C for adding blood to prepare blood agar). 5. HOT AIR OVEN It is an electrically operated and thermostatically controlled double jacketed metalic instrument. Oven may be conventional or equiped with forced air circulation. Temperature range of ambient to 250OC may be obtained in it. It is used for sterilization of glass-ware, heat stable powders, oily preparations and metallic but not sharp instruments (plate-5).

6. AUTOCLAVE It consists of cylinder, heating element, water container, pressure guage, a safety valve and steam outlet. It is available in various shapes (vertical/horizontal) and sizes. In some instruments, steam of required temperature is generated at the base of autoclave by a heating element. In autoclave, pure steam of 121C temperature at 15 lb pressure for 15 minutes is lethal for all microbes including spore forming bacteria. This is used for sterilization of heat stable solutions, media etc, (plate-6). It works on the principle that boiling point of water is directly proportion to the atmospheric pressure and steam at higher temperature is more penetrating and lethal to the bacteria. 7. ANAEROBIC JAR It consists of a cylinder and a lid. The lid have valves for introducing gases and a sachet containing catalyst- palladium salt that enhances the reaction of oxygen and hydrogen to form water. Absence of oxygen is indicated by an indicator present in neoprene tube at the side of the jar. Aerobic or anaerobic environment in this jar can be maintained for growth of bacteria (plate7). 8. INCUBATOR It is an electrically operated and thermostatically controlled rectangular shaped instrument. Incubators are of four basic types: I. Low temperature incubator-psychrophilic bacteria II. Normal incubator for aerobic and mesophilic bacteria, III. Carbon dioxide incubator for micro-aerophilic bacteria, IV. Anaerobic incubator for anaerobic bacteria Temperature within the incubator may range from ambient to 100C. Incubator is used according type of bacteria under investigation. 9. CENTRIFUGE MACHINE It consists of an electrically operated and thermostatically regulated motor and a head with tube holders. The head may be horizontal, vertical or angular. The machines can be classified as low speed, medium speed and ultracentrifuge machines according to the speed. Motor revolves the tube holder and generates a centrifugal force which increases gravitational pull on the suspending particles; the suspending particles settle down at he bottom of the tube from the suspension. With implementation of density medium, the components of different densities are

8separated.

For example, by the use of density medium, lymphocytes can be separated from monocytes (plate-9). 10: FREEZE DRYER It consists of three parts such as vacuum pump, tube holders and condenser. According to law of heat, the boiling point of a liquid can be decreased by reducing pressure and can be increased by increasing pressure. In this instrument a vacuum (very low pressure) is created by the vacuum pump in air tight container where liquid containing microbes, is solidified and then water is evaporated below the freezing point of water. The gaseous water produced is condensed to a liquid form on a hygroscopic salts or to ice form in condenser. In this way, microbes are freezed and dehydrated (dried)(plate-I 0). 11: AMPOULE CONSTRICTOR It is a small electrically operated instrument that revolves the ampoule on the flame. It is used to prepare constricted ampoule (plate-II). 12. COLONY COUNTER It consists of electrically enlightened base and a magnifying glass lens. This facilitate the counting. of colonies on the solid culture medium. Some counters are equipped with electric and ink operated marker and telecounter. The marker and telecounter help to highlight the counted colonies and rule out any error of colony counting (plate-I2). 13. ULTRASONIC It is an instrument that is used to disintegrate the microorganisms. This is composed of sound generator, transducer, and titanium probe. illtrasound of high frequency (60,000/second) is converted to mechanical energy that drive the probe with same speed up and downward in the solution. This movement induces cavitation in the solution. These bubbles release energy on disappearance. This energy is inversely proportional to the surface area of the bubble. This energy is sufficient to break the ionic and covalent bonds of the cell wall and cell membranes. This method is extensively used to prepare 1) antigens to coat SRBC , 2) antigens required for agar gel precipitation (AGPT) (plate-B). 14: TISSUE GRINDER This is available in various shapes and forms. It consists of a motor and metallic rod with blades at the distal end. The blades at end of a metallic rod are revolved at high speed which disintegrate the tissues (plate-I4). 15. SEITZ FILTER It consists of upper and lower holding parts that hold an asbestos pad filter of varying porosity (from 0.02 to 2 micron). The filter disc is supported by perforated metallic disc on the lower metallic holder. The seitz filter works under positive or negative pressure. It is used to filter the heat labile solutions, i.e., serum, sodium bicarbonate solution, glucose solutions etc. The filters are available in various porosities and forms i.e., syringe filter-where filter is of nitrocellulose membrane, sintered glass filters, earthen-ware filters etc. (plate-I 5).

16. BALANCE

These are used to weigh the chemicals. These are available in various forms and various ranges. Single pan balance of high precision are commonly used in clinical laboratories, i.e., Sartorius balance (plate-I 6). 17. MICROMETER This comprises of two parts such as a stage micrometer-it is a microscopic glass slide bearing an engraved line of 1 mm length and divided into 100 divisions (each division is of 10 micron length) and an ocular micrometer. It is a round glass disc bearing a line of unknown length but divided into 100 divisions and is fitted in the eye piece of the microscope during micrometry (plate-I 7). 18. CAVITY SLIDE

Cavity slide is a simple glass slide (3 II XI") with one round shallow depression in the centre. It is
used for demonstration of bacterial motility by hanging drop method (plate-I 8). 19. CULTURE HOOD It is a cabinet which provides closed and relatively sterile environment with still body of air. It has a front glass with a small window at the base. Some culture hoods have UV light fitting on the ceiling which can be switched on if required to sterilize the environment. This provides a sterile environment for pouring culture media, transferring aseptic cultures, making dilutions of ,the sterile solutions etc (plate-I9). 20. DEMINERALIZERS It consists of one plastic column containing resins, and conductivity meter. When water passes through this column, the positive or negatively charged resins adsorb ions of opposite charge and ions free water is collected from its upper exit. The conductivity meter indicates the salt removal efficiency in terms of resistance. This instrument is meant for demineralizing the water (plate-20).

QUESTIONS
1: Identify the instruments/or parts of the instruments? 2: Write down the function of instruments or highlighted parts of the instruments? 3: Match suitable parts in column A with respective instruments in column B? Example
1. Steam outlet 2. Palladium sachet 3. Safety valve 4. Shallow round depression 5. Perforated sieve 6. Seitz filter 7. Tube holders

Conductivity meter

Demineralizer
I-Centrifuge II-Hot air oven III-Water bath IV-Anaerobic jar V-Seitz filter VI-Cavity slide VII-Autoclave

EXPERIMENT-3 STERILIZATION AND DISINFECTANTS Sterilization means freeing of an article from all types of life. In routine work, microorganisms are isolated from the clinical samples. Manipulation of the samples to obtain pure culture of causal organisms is an art and to keep it tree from contaminating organisms requires the expertise of a microbiologist. There are different methods to sterilize the sampling bottles, culture media, glasswares, and other required objects. This can be achieved by physical and chemical methods. A. PHYSICAL METHODS Heat o Dry heat Flamming Hot air oven Red hot method Incineration o Moist heat Above 100 C Autoclaving At 100 C Tyndalization Boiling Syringe sterilizer Below 100 C Pasteurization Filtration o Earthen ware filter Ultrasonics Radiation Freeze drying (alternate freezing and thawing) Cellulose membrane filter Sintered glass filter Asbestos pad filter-Seitz filter

B. CHEMICAL METHODS

A. PHYSICAL METHODS A.I. HEAT Heat is the most widely applicable, economical and easily controlled method for sterilization. Heat kills the microbes by denaturing the microbial enzymes. Degree of heat resistance by bacteria can be expressed through the concept of thermal death point, thermal death time, and decimal reduction time. Heat used in sterilization may be in the form of dry heat, or moist heat. A.1.1. DRY HEAT Dry heat is believed to kill microbes by destructive oxidation of essential constituents. A.1.1.1. RED HOT l\1ETHOD Material to be sterilized is held in the flame until it becomes red hot. It is a highly effective method for sterilizing of platinum loop, points of forceps, and sharing spatulas. It is used for instruments which do not get oxidized by flame. A.1.1.2. FLAMING Various articles such as mouth of test tubes/flasks, syringe needle, scalpels etc. are kept in the flame for few seconds to get rid of environmental contamination during their operation, Moreover, eggs or trays are swabed with methylated spirit and ignited to flame. A.1.1.3. HOT AIR OVEN Items to be sterilized are paced in an oven, generally at temperature range up to 160C for one hour to ensure the sterilization. All type of oils with boiling point more than 250C, all heat stable powders, salts, glass-ware etc are sterilized in the oven. Procedure Clean, dry and wrap the glassware. Put these items in the oven. Close the door tightly, switch on the oven, and adjust the required temperature with the help of thermostat. Let the oven attain the required temperature before counting down the time. Record the time of 1/2 to I hour of instrument at temperature of 160C or 180C. The temperature can be noted from the thermometer fitted in the oven Switch off the oven and open the door carefully and take out the sterilized articles. Precautions Do not pack the oven completely but let 1/3 of its internal space remain free. The glass-ware must be properly cleaned to decrease the bacterial load.

The temperature of the oven must be below 50C before removal of glassware to avoid their breakage. Oily preparations and powders/salts must be in the glass ware which must be properly wrapped. Temperature above 180C may cause charing of cotton plugs and papers. The fumes of such burning are toxic for growth of certain fastidious bacteria. All the items should be placed with sufficient distance between them for hot air circulation. A.1.1.4. INCINERATION It is an effective method to sterilize and dispose off contaminated material like papers, bags, dressing and infected carcasses. A.1.2. MOIST HEAT Moist heat inactivates the microbes by coagulating the microbial components, i.e., enzymes, other proteins. It can be applied in many forms. A.1.2.1. TEMPERATURE BELOW 100C A.1.2.1.1. PASTEURIZATION This method is applied to milk to inactivate pathogenic bacteria. The bacteria causing diseases such as tuberculosis, brucellosis, scarlet fever and Q-fever, are killed by this method. This method will not deteriorate the nutritional contents of the milk. Pasteurization means exposing the milk to temperature of 63 C for 30 minutes, or 72 C for 15 seconds. High temperature for slightly longer time is common with an aim to lower the bacterial load. The milk may be free from milk-borne pathogens and also mitigate less coliform count, but never free the milk from microbes. Effectiveness of pasteurization is determined by phosphatase test. Presence of the active phosphatase in the milk after heating indicate faulty pasteurization. Biological materials such as serum, egg medium, body fluids, and stock solutions of sugars, are sterilized by a method that simulate tyndalization but in this case the temperature used for sterilization is between 70C to 80C for 1-4 hours. A.1.2.2. AT 100 C BOILING Boiling at 100C kills the vegetative forms of bacterial pathogens, some endospores, many virus and fungi within 10 minutes while some viruses and endospores are resistant. Glass syringes, surgical instruments, and suturing instruments are usually sterilized by this method.
A.1.2.2.1. TYNDALIZATION (FRACTIONAL STERILIZATION)

Material to be sterilized is exposed to steam at 1000C for 45 minutes for 3 consecutive days. The material is kept at room temperature or at 370C in between the heat treatments. During first heat treatment, vegetative cells are killed while spores remain alive which germinate during incubation before second heat treatment. The second and third heat exposure will kill the vegetative cells arising from endospores, rendering the material germ free.

A.1.2.3. ABOVE 100 C A.1.2.3.1 AUTOCLAVING Autoclaving is the most effective method of sterilization in which steam under pressure is used to kill the microbes. This equipment works on the principle of law of heat. Water under pressure of 15Ib per square inch will boil at 121C and will kill all the vegetative and sporulated bacteria. Procedure Check the quantity of water and place the properly wrapped/plugged material in the autoclave. Make it airtight, open the steam outlet and switch on the instrument. When pure steam starts coming out, close the steam outlet, and record the rising pressure on the pressure gauge of the autoclave. The pressure guage needle (which indicate the temperature, and pressure in side the apparatus) reaches to a required pressure (15 Ib/in). The apparatus will automatically maintain the required pressure and temperature. Let the autoclave work at this pressure for 10-2O minutes. Switch off the instrument, open the steam outlet slowly to avoid spoilage of the media, or leakage of the flasks. Unlock the autoclave when pressure reaches to zero on the pressure gauge. Let the instrument cool down and take out sterilized materials.
Precaution

Ample quantity of water must be present in the autoclave. An autoclave of 18 inches height must have 3+ 1/2" water. Autoclave should be airtight and safety valve be in working condition. There must be complete elimination of air and some steam before closing the steam outlet. Steam outlet must be opened slowly after complete sterilization. Never attempt to open the autoclave during operation or just after it, when the pressure inside the chamber is higher than atmospheric pressure. A-2: RADIATION The ability of sunlight to kill microbes is mainly due to V.v. light. The UV light ranging 2400 to

32000 A has bactericidal activity. The effective radiation is generated by mercury vapor
lamps for sterilization of closed environments such as culture hood or culture rooms.

1. It is equally effective against gram positive and gram negative bacteria while sporulated bacteria and viruses are somewhat resistant. Bactericidal U V rays have low energy and unable to penetrate more than a few mm of a liquid. All plastic materials, clean surfaces, and contaminated air of closed vessels can be easily sterilized. U V. exposure must be accompanied with darkness to avoid regeneration of bacterial damages. (X-rays and gamma radiation can kill bacteria by disrupting their DNA sequence. The techniques are too dangerous to be used in any microbiological laboratory). A-3: FILTRATION It is a mechanical method for sterilizing heat labile solutions (serum, vitamins, sugars, antibiotics, toxins etc.) and purification of soluble products ITom particulate matter. There are four type of filters depending upon material used in their preparation. These filters work on sieve principle. Earthen-ware filters The filter candle is made of diatomacious earth. The material is forced through the candle either by negative or positive pressure. Candles are sterilised by dry heat. A large quantity of the material may be adsorbed by the filter. Examples are Berkfeld filters. chamberland filters Sintered glass filters A small disc prepared from finely ground glass is supported in holding apparatus. It can be sterilized by hot air oven or autoclaving, It works under positive or negative pressure. Cellulose membrane filters Filter is composed of cellulose nitrate, or cellulose acetate. The filters are very thin and resemble to cell membrane. These are available in various porosities, 200 millimicron to 400 millimicron. Special devices are used for holding these filters during operation. These are available in various forms such as syringe or membrane filters. These can be sterilised by autoclaving and can be stored for indefinite period. Membrane filters are less absorptive and rate of filtration is much greater. Bacteria retained on the filter surface can be grown by placing on the culture media. Asbestos filter It consists of a filtering disc made of asbestos and a holding apparatus. Filtering pad is fixed in holding apparatus by placing on the sieve to avoid mechanical damage. Procedure: Sterilize the holding apparatus and filtering pad with the tube of lower metallic part which must be plugged and wrapped.

Connect the sterilized flask or test tube to the lower part of holding apparatus aseptically (in culture hood) to avoid environmental contamination. Pour the solution to be sterilized in the upper part of apparatus and apply positive or negative pressure with the vacuum / pressure pump until filtration is completed. Avoid bubbling. Disconnect the flask/test tube having filtered solution. Sterilize the apparatus in autoclave and discard the filtering pad.

DISADVANTAGES

Small quantity of solution is difficult to sterilize due to its absorbing property. Air can pass through filtering pore after complete filtration of solution. It may contaminate the filtered solution. Large quantity of micro and macro molecules present in the solution are adsorbed by the filtering pad; at the same time, some ions tram the pad may pass into the filterate. NOTE: It is commonly used for sterilization of heat labile solutions such as tissue culture media, buffering solutions, toxins, serum, antibiotics and sugar solutions. It is rapid as compared to tyndalization. B: CHEMICAL METHODS There are variety of chemicals extensively used as disinfectants which kill microbes on contact. The chemicals that are used on living tissues are known as antiseptics/antibiotics. Germicidal, bactericidal, fungicidal, virucidal, sporicidal etc, and bacteriostats, coccidiostats, coccidicidals etc

are routinely used terms.

Following are major disinfectants: The efficacy of a disinfectant is determined by phenol co-efficient. The standard time for disinfectant is 10 minutes. The efficacy of an antibiotic is determined by sensitivity test and that of a disinfectant is by Phenol Co-efficient. Pheno-Co-efficient: The highest dilution of the disinfectant that kills the test organism in 10 minutes but not in 5 minutes is divided by the greatest dilution of phenol showing the same result. The number obtained in this way is called the Phenol Co-efficient of the test chemical. ANTIBIOGRAM ASSAY/ DISC METHOD Prepare the culture medium in a petridish. (For sulpha drugs sensitivity, a medium tree trom folic acid is used). Swab the sample on the culture medium Apply sensitivity discs containing antibiotics on the medium at equi-distance Incubate the culture plate for 24-48 hours Observe the size of zone of growth inhibition and record the results as follows: Penicillin= Streptomycin --Gentamycin +++ T etracyclin +++ Ampicillin +++

QUESTIONS What is the theory upon which intermittent sterilization is based? Is moist heat more efficient sterilising method than dry heat, why? What temperature in degree F is 170C? What should be the pore size of bacterial filters? Fill in blanks with suitable correct word/number? a: Sterilizing temperature and time for following 1 : autoclave ( temperature C), ( time in minutes) 2:hot air oven (temperatureoC, (time in minutes) 3 :pasteurization tempoC, (time in minutes) 4:Tyndalization tempoC, (time in minute) for three consecutive days. Write down the items which can be sterilized by a: hot air oven b: autoclaving c: tyndalization d: filtration e: Pasteurization f: red hot method g: Ultraviolet light (distilled water, serum, petridishes, platinum loop, plastic rods, glucose MacConkey's agar, Milk, overcoats, phosphate buffered saline, table surface) How do dry heat, moist heat, and UV light kill the microbes? What are precautions to operate autoclave?

EXPERIMENT NO-4 PREPARATION OF CULTURE MEDIUM It is a substrate that supports the growth of microbes. It is available in liquid (nutrient broth) or in solid form (nutrient agar). Medium is singular and media is plural. Important ingredients of culture media are as follows: WATER It dissolves the ingredients (solutes) and enhances their absorption and bacterial metabolism (chemical reactions). It facilitates the absorption and excretion of water soluble products from the cytoplasm. Complete removal of water from the bacterial surrounding can cause complete cessation of bacterial growth (dessication). AGAR. It is a carbohydrate complex obtained from sea-weed (geladium). It is available in powder form or in brand form. It is used as solidifying agent for medium. It is not used as nutrients for growth of bacteria. It is a least inhibitory substance used for solidifying of medium. It melts at 9SoC and solidifies into uniform gel at 43C. This wide range of temperature facilitate the addition of enrichment agents or bacterial samples in the medium for viable count. It remains in solid state at 37C which is helpful for streaking, growth and purification of the cultures. When pH of the agar is low or high, it may loose its solidifying property if heated at high temperature for long time - Sabouraud's agar PEPTONE It is a product resulting from the digestion of protein substances i.e., meat, casein etc. It is a principle source of organic nitrogen, may also contain some vitamins and carbohydrates depending upon the type of material being digested. BEEF EXTRACT An aqueous extract of lean beef tissues which has been concentrated to a paste. It contains water soluble substances of animal tissues which include carbohydrate, organic nitrogen, water soluble vitamins and salts.
INDICATORS

These are chemical substances e.g., phenol red that change their colour according to pH of the medium. These are useful in culture media to detect acidic or alkaline products of microbial metabolism. They are also used to adjust the pH of the media.
Name of pH indicator: Phenol red, Neutral red Bromocresol purple Bromothymol blue Methyl red

Phenolphthalein

SELECTIVE AGENTS These are special type of chemicals added in the media to selectively inhibit the growth of some type of microbes and allowing the growth of others i.e., Bile salt in MacConkey's agar inhibit species of Bacillus genus, Pasteurella multocida etc. (MacConkey,s agar is a selective medium for intestinal bacteria) ENRICHMENT AGENTS Addition of some ingredients in the medium facilitate some fastidious type of organisms i.e., blood in the medium support the growth of streptococci (Enrichment media-blood agar). KINDS OF MEDIA I: Basal media The medium commonly used for growth of ordinary environmental bacteria and can als,o be used as a base for preparation of selective or enrichment media. Examples: Nutrient agar, Nutrient broth, Peptone water etc. 2: Enrichment media The medium that supports the growth of fastidious type bacteria or support the growth of those bacteria that do not grow on ordinary laboratory media Examples: Blood agar, Chocolate agar, Serum dextrose agar etc. 3: Selective media The medium that allows the growth of selective organisms and inhibits the growth of others. Examples: MacConkey's agar that selectivly allow the growth of members of intestinal bacteria i.e., Salmonella and Shigella agar allows the growth of Salmonella and Shigella species, Staph-IIO medium supports the growth of Staphylococcus species. 4: Differential media The medium that are used to differentiate the growth of two types of bacteria. Example: MacConkey,s agar can differentiate lactose fermenting from lactose non fermenting bacteria. NOMENCLATURE The medium is either named after the type of bacteria grown on such as Staphylococcus 110 medium, Salmonella and Shigella agar, Brucella selective agar, Mycoplasma agar etc, or in the memmory of the inventor of the medium such as MacConkey's agar, Stuart,s medium, Albimi agar, Stonebrink's medium, Lowenstein-Jenson's medium etc.

COMMON MEDIA USED IN VETERNARY SCIENCES l:NUTRIENT AGAR COMPOSITION Peptone Beef extract 1-1.5% 0.5-1.5%
Sodium cWo ride

0.5-1% Agar agar 2-3% Note: Simple addition of peptone in the water is known as peptone water, while addition of beef extract and sodium cWoride in the peptone water is known as nutrient broth, addition of agar in the nutrient broth is known as nutrient agar."
2:BLOOD AGAR

Composition Sterilised moltem nutrient agar( 45C) Fresh defibrinated sheep blood 90-95 m1 5-10 m1

The nutrient agar is cooled to 45C after autodaving and admixed with the required amount of defibrinated blood. After mixing, the medium is poured in the sterilised petri dishes. 3: CHOCOLATE AGAR During preparation of this medium, the blood is mixed in sterilized moltem nutirent agar at temperature of 80C so as to have lysis of RBC. This lysis of the RBC will impart the medium chocolate colour. This will also provide X and V factors for growth of some of the pathogens such as Haemophillus, Campylobacter species etc. 4: STAPHYLOCOCCUS 110 MEDIUM Composition Sodium cWoride Agar PhenolphtWeine Ditilled water 109 5g 19 75g 20g 19 1000ml Peptone Beef extract Mannitol Addition of sodium cWo ride upto 7.5% make this medium selective for the growth of species of staphylococcus. The pathogenic staphylococci can ferment mannitol and produce acid metabolites which will change the pH of the medium. The colour of the phenolphtWein will change to pink. .

5: SIMMON CITRATE AGAR Composition MgSO4 0.2g (NH4)H2PO4 LOg Sodiu citrate Sodium cWoride Agar Bromothymol blue K2HPO4 2g 5g 20 g 0.08 g 1g

After preparation, the medium can be dispensed into test tubes and is required to be placed in slanted position. The bacteria use sodium citrate as source of carbon and ammonium salts as source of nitrogen and can grow on such inorganic salts. Some of the bacteria growing on this medium, can ferment sodium citrate in such a way that sodium hydroxide is produced, which change the colour of bromo thymol blue to green colour. The growth of the bacteria/development of green colour on this medium is indication that bacteria can use sodium citrtate as source of carbon.
6: MACCONKEY,S AGAR Composition Peptone Beef extract Neutral red 2%in ethanol

2% 0.5% Bile salt 0.35% 0.5% Agar Agar 1% 2-3% Lactose The bile salt makes this medium selective for growth of bile resistant bacteria while lactose and neutral red enable the differentiation of iactose fermenting (Escheria, Enterobacter, Citrobacter, Klebsiella etc ) from lactose non-fermenting bacteria ( Salmonella, Shigella, Proteous, Provedentia ). 7: TETRATHIONATE BROTH The tetrathionate inhibits the coliform growth while permitting the salmonella group to grow freely. This medium is selective for growth of Salmonella species. THlOSULPHATE SOLUTION Sodium thiosulphate Sterile water 24.8 g 100 ml

IODINE SOLUTION Potassium iodide Iodine Sterile water 20.0 g 12.7 g 100 m1 COMPLETE MEDIUM Calcium carbonate Nutrient broth Thiosulphate solution Iodine solution
2.5 g 78 m1 15m1 4m1

Phenol red 0.02% in 20 % ethanol 3 ml Add calcium carbonate in nutrient broth and sterilize by autoclaving then add iodine solution, thioglycolate, phenol red solution aseptically. Distribute 10 ml in screw capped bottles. This medium can be stored for only 2 weeks. 8:SELENITE BROTH The tetrathionate inhibits the growth of coliform while permitting the growth of Salmonella freely. This medium is selective for pure growth of Salmonella species.
COMPOSITION

Lactose Disodium hydrogen phosphate Sodium dihydrogen phosphate Sterile water 4g 5g 4g 9.5g 0.5 g 1000 ml Sodium acid selenite Peptone Dissolve the ingredients and sterilise by steaming at 100C for 30 minutes. 9:

LOWENSTEIN-JENSEN MEDIUM Melachite green inhibits the growth of organisms other than mycobacteria. Glycerol potentiates the growth of human type of Mycobacteria for which this medium is recommended. This medium can differentiate the human and bovine type of mycobacteria. COMPOSITION: MINERAL SALT SOLUTION Asparagine Glycerol Water 2.4 0.24 0.6 3.6 Pottassium hydrogen phosphatee anhydrous) Magnesium sulphate Magnesium citrate 12m1 600ml Dissolve by heating and sterlize by autodaving at 121C for 25 minutes. MALACHITE GREEN

SOLUTION Prepare 2% solution of malachite green in sterile water and dissolve the reagent in incubator (37 C) for 1-2 hours. This can be stored indefinitly. PREP ARATION OF COPMLETE MEDIUM Mineral salt solution Malachite green Beaten eggs(20-22 eggs) 600 ml 20 ml 1000 m1 Mix the medium and distribute 5 ml amounts in sterile McCartney's bottles (60 ml capacity). Screw the caps and lay horizantlly in the oven at 80C for 1 hour. Since the medium has been prepared with sterile precautions. This heating will coagulate the egg content and hence solidify the medium. 10: STONEBRINK'S MEDIUM This medium is recommended for bovine type of Mycobacteria. It does not contain glycerol which has no effect on bovine type of mycobacteria. The addition of sodium pyruvate supports the growth of bovine mycobacteria. MINERAL SALT SOLUTION Potassium dihydrogen phosphate(anhydrous) Disodium hydrogen phosphate Sodium pyruvate Water 7g 4g 12.5 1000 ml

Dissolve by heating, autoclave at 121C for 15 minutes, keep it indefinitely. MALACHITE GREEN SOLUTION Prepare 2% solution of malachite green in sterile water and dissolve the reagent in incubator (37C) for 1-2 hours. This can be stored indefinitely. PREP ARATION OF COMPLETE MEDIUM Mineral salt solution Malachite green solution Beaten eggs Prepare the beaten eggs, mix and dispense the medium with sterile precautions and. inspissate at 80C for one hour. 1000 ml 40ml 2 000 ml l1:CLOSTRIDIUM SELECTIVE AGAR

This medium contains reducing agents such as glucose, ascarbic hydrochloride and antibiotics to make it selective for clostridium species. Composition: Peptone Yeast extract Meat extract Glucose Sodium acetate Sodium chloride L-Cystine hydrochloride Soluble starch Agar Distilled water acid and cystine 10.0 g 3.0 g 10.0 g 5.0 g 3.0 g 5.0 g 0.5 g 1.0 g 10.0 g 1000 ml Steam the ingredients in a flask, and filter when dissolved. Adjust pH to 7.4, add agar and autoclave 121C. 12:THIOGLYCOLATE BROTH This medium contains methylene blue or resazurine as an oxidation-reduction potential indicator, glucose, cystine, thioglycolate as reducing agent. Composition Yeast extract: Caseien hydrolysate glucose L-cystine Agar Sodium chloride Sodium thioglycolate Resazurine soultion (1 : 1000) Water 5.0 g 15 g 5.5 g 0.5 g 7.50 g 2.5 g 0.5 g 1.0 ml 1000 ml Dissolve the reagents other than thioglycolate and resazurine, by steaming at lOOoC. Add thioglycolate and adjust pH 7.2. It can be filtered to remove precipitate if any. Add resazurine solution and autoclave. Store at cool place and use within one week. 13:BRUCELLA SELECTIVE AGAR Composition: Agar Peptone Sodium cWoride Meat extract Water Sterile heat inactivated

horse serum Glucose 25% solution Bacitracin per 250 ml (2000 units/ml) Polyxin 500,000/l00ml (5000 units per ml) Cyclohexamide solution( 1 0 mg/ml)

50 ml 40 ml 500,000 units 12.5 ml 1.2 ml 10 ml

(The cycloheximide powder is dissolved in 20 ml acetone and dilute 400 ml with sterile water). 15 g 10 g 5g 5g 1000 ml 14:DINGER,S MODIFICATION OF NOGUCHI MEDIUM This semisolid medium is slower to evaporate than liquid medium especially in tropics. Subcultures are made less frequently so as to maintain the virulance.
COMPOSITION

Nutrient agar Distilled water Inactivated serum 6 ml 100 ml 10 ml Mix the agar and water, autoclave, cool and admix with serum aseptically. Distribute in test tubes and test for sterility. 16: EOSIN METHYLENE BLUE AGAR COMPOSITION Peptone Lactose Sucrose Dipotassium phosphate Agar Eosin Y Methyylene blue Distilled water 10 5 5 2g 13.5 0.4 gram 0.065g 1,000 ml This medium is used for pure isolation of coliform of the cultures. 17: TRIPLE SUGAR IRON AGAR COMPOSITION Peptone Sodium chloride

Lactose Sucrose Glucose Ferrous ammonium sulphate Sodium thiosulphate Phenol red Agar Distilled water 20 g 5g 10 g 10 g 1 g 0.2 g 0.2 g 0.025 g 13 g 1000 ml
18: MYCOPLASMA BROTH

COMPOSITION PPLO broth without crystal violet Glucose Swine or horse serum Fresh yeast extract Cysteine hydrocWoride Nicotinamide adenine dinucleotide(NAD) Phenol red (1% solution) Thallium acetate (10%) Penicillin G potassium Distilled water 14.7g 109 150 ml 100ml 0.1 g 0.1 g 2.5 ml 2.5-5ml 1000,000 units 1000 ml Adjust pH 7.8 with 20% NaOH solution, distribute and filter sterilise. AGAR COMPOSITION; Peptone Beef extract Glucose Agar (pH to 5.6) 1-1.5 % 0.5-1 % 4% 1.5-2% Addition of glucose at rate of 4% and low pH 5.6, make this medium selective for growth of mold and yeast. The components are dissolved im water and boiled for 10 minutes. This heat treatment will inactivate the bacterial and fungal conataminents and dissolve the agar. 19: CORNMEAL AGAR COMPOSITION 19: SABOURAUD,S

Yellow corn/cornmeal Agar Tween-80 Distilled water 40 g 20 g 10 ml 1,000 ml Mix the cornmeal in 500 ml distilled water. Heat at 56C for one hour. Filter through gauze, then filter paper until clear. Adjust to pH 6.8. Add agar in remaining 500 ml water and dissolve by heating. Mix both the solutions and add tween-80. Autoclave at 121C for 15 minutes. This medium is for isolation of candida species.
COMPOSITION 20: RICE GRAIN AGAR White rice Water 8g 25 ml

This medium is used for isolation of mold. METHOD FOR PREPARATION OF CULTURE MEDIA These basic four steps are essential for preparation of all type of culture medium. Weigh individual ingredients of medium, mix properly and dissolve by heating if necessary. Composition of Nutrient agar Peptone Beef extract Sodium chloride Agar Water upto 2.0 gram 0.5 gram 0.5 gram 2.5 gram 1000 ml

Adjust Ph of the medium according to nature of organism to be grown using 4% HCI or 4N NaOH. Normal pH for pathogenic bacteria is 7.2-7.4 Sterilize the medium by autoclaving. Occasionally components other than components of nutrient agar are sterilised by different methods and admixed together by aseptic method. Nutrient agar--autoclaving Blood agar Nutrient agar autoclaved and cooled down to 45C and mixed with defibrinated blood from healthy animals. Pouring of culture medium in a: petri-plates-> used for isolations of pure culture, b: slants->used for storage of bacteria, c: stabs -> used for studying biochemical properties of bacteria The petriplates are incubated overnight at 37C to determine the sterility of the media. The plates in the incubator must be in the upside down otherwise evaporating water will accumulate on the medium surface. This water will disturb normal streaking of culture on such media.

QUESTIONS What are properties of agar? What is composition of nutrient agar? Define selective medium, enrichment medium, differential medium, and indicator medium? medium, basal How will you proceed for preparation of following media: a: Blood agar, b: Sabouraud's agar, c: MacConkey's agar What is colour of following Ph indicators in acidic and basic solutions? Phenol red, Bromothymol blue, Methyl red, Neutral red, Bromocresol purple. What are ingredients of the folowing media for enrichment growth of microbes. MacConkey's agar, L-J medium, Sabouraud's agar, Staph 110 medium EXPERIM:ENT NO-5 ISOLATION OF PURE CULTURES-STREAKING METHODS AND CULTURE CHARACTERISTICS When a bacterium is placed on the surface of a medium. It multiplys by binary fission such as 1->2>4->8-> 16->32 so on. A single bacterium is not visible to the naked eye but when such bacterium multiply through many generations on the surface of solid medium, the bacterial mass becomes visible and is called colony. Colonies of each organism differ trom other in shape, size, colour, and texture, therefore colony appearance is a valuable clue to identify a culture and to confirm its purity. Using following exercise, pure culture may be isolated by streak plate method and pour plate method. STREAK PLATE METHOD selective, differential,

Plate the bacterial culture on the surface of solid medium and spread with a loop or bent needle. This is called streaking. The objective of streaking is to produce well separated colonies of bacteria from a concentrated bacterial culture. The cells are closely packed at the start of a streak and form a dense mass of cells running together but at the end of streak, the bacterial cells distribute singly and hence form well separated colonies. There are many methods used for obtaining pure growth of bacteria but one most commonly used is exercised here. Apply a drop of culture near one edge of the petri-plate

Flame the loop, cool it by touching on the sterile surface of the agar, hold the petri-plate in your left hand with in 6 inch diameter of the burner. From the surface of culture on the edge away trom you, streak the bacteria on one half of the plate in parallel lines. Flame the loop, rotate the plate at 45 and streak the remaining one fourth of the plate from the last line. Flame the loop and rotate the plate at 45, streak the remaining of the plate trom the last line. Incubate the plate at 37C and record the morphology of colonies such as colour, elevation, size, texture, edge, and surface of the growing colonies. If the colonies show a different growth characters, the original culture is a mixture of many bacterial species. in such case, each colony is further streak_d on the tresh solid culture medium until all colonies of the same characters are obtained. If all colonies are similar characters, the isolated culture will be pure. POUR PLATE METHOD In this method, the sample is diluted to 10 fold in normal saline and one ml from each dilution is admixed in 15 ml melted agar (45C) in the sterilized petri-dish. These dishes are incubated at 37C for 24-48 hours. The dish showing 30-300 colonies is selected for counting colonies and isolation of pure-culture. Suppose, there are 40 colonies in the dish which was having one ml of sample dilution 10-5. Total count of viable organism/ml of the sample will be 40x 1 0, 0000=4, 000, 000 bacteria. The bacteria growing in such culture will be inside the agar and will appear as spindle shaped. However, discrete colony can be streaked on the surface of fresh petri-plate. After incubation, if all colonies show similar characters then the culture will be pure.

QUESTIONS What are different methods to get pure culture? Write down five colony characters? Make.different model sketch for streaking? Write down procedure of streaking buffalo pus sample for isolation of pure culture? What are the objectives of streaking? What are advantages and disadvantages of pour plate method? How will you proceed for making ten fold dilution of sample of Urine of infected guinea pig?

EXPERIMENT -6

CULTIVATION OF BACTERIA The culture medium streaked with microbes are provided with suitable temperature, oxygen tension, light and suitable time for bacterial growth. The time required for growth (incubation period) depends on the available physical and chemical factors and generation time of bacteria. It varies from few minutes to months. TEMPERATURE Depending on the type of bacteria, the streaked plates are incubated at suitable temperature Psychrophiles 0-25OC (opt.20oC). Mesophilic 35-42C (opt.37C). Thermophiles 40-90oC (opt. 55C). OXYGEN REQUIREMENT On the basis of oxygen requirements, bacteria are typed as: 1) Aerobic The bacteria that grow in the normal atmospheric oxygen, i.e., Salmonella, Mycobacteria, Listeria etc,. 2: Anaerobic The bacteria that grow in the absence of oxygen, i.e. Clostridium, Spherophorus, Bacteroides etc,. 3: Microaerophilic The bacteria that grow in reduced oxygen tension, i.e. Campylobacter species. CULTIVATION OF AEROBIC BACTERIA The plates are placed in ordinary incubator and the required temperature can be adjusted accordingly. CUL TIV A TION OF MICROAEROPHILIC BACTERIA I: The plates streaked with microaerophilic bacteria such as Campylobacter species are kept in the anaerobic jar along with burning candle. The jar is sealed airtight and the candle will go off when 90-95% atmospheric free oxygen has been consumed providing microaerophilic environment. The jar is then incubated incubated. The liquid media may be boiled for 10 minutes to remove dissolved oxygen. It is cooled to temperature of 44-450C and is inoculated heavily with microaerophilic bacteria. The agar media containing reducing agents, after heat treatment, is solidified in test tubes by placing in cold water. The tubes are incubated at 37C.

ANAEROBIC CULTIVATION Anaerobic bacteria can be grown by eliminating ftee oxygen ftom the environment or by establishing a low O-R potential by providing sufficient reducing agents in the growth medium. The anaerobic environment can be achieved in several ways: Free oxygen from the medium is eliminated by boiling and then prevented its reintroduction by adding a relatively solid barrier such as a layer of mineral oil. The media is boiled for 10 minutes, cooled rapidly to 45C and inoculated with heavy culture (a drop or severalloopful) with out agitation. The top of the media is sealed immediately after inoculation with one centimeter deep layer of liquid parafin. This is useful for those media used for fermentation tests. The media containing various reducing agents such as thioglycolate, creatinine, ascorbic acid, sugar, which react with oxygen and keeps the enzymes in the reduced form. These are not sealed. Anaerobic jar: The jar contains a special catalyst (palladium) and can be sealed air tight. The streaked tubes or plates along anaerobi kit with water are placed in the jar which is then immediately sealed. The anaerobic kit in prsence of water releases hydrogen and carbon dioxide in the jar. The pladium catalyses the reaction between ftee oxygen and the hydrogen. This reaction results in formation of water. In this way anaerobic environment is induced. The streaked plates or tubes can be placed in the anaerobic jar which is then sealed as air tight. The atmospheric air of the jar can be replaced with hydrogen. This hydrogen will react with residual oxygen in presence of the catalyst. In this way an anaerobic environment in the jar is obtained. The anaerobic incubator is sealed unit in which air is replaced with a particular gas mixture, that provides the proper atmosphere for the survival and growth of anaerobic bacteria. The sealed in rubber gloves are used to manipulate the cultures. The box is fitted with internal fixtures to provide gas, electricity, and ultraviolet gas.

QUESTIONS Write the examples of bacteria of veterinary importance growing in following environments: a: Anaerobic bacteria b: Microaerophilic bacteria c: Aerotolerant bacteria d: Aerobic bacteria What are the chemicals present in gas pack and also explain the biochemical reaction when exposed to water. Why does the oxygen that dissolves in thioglycolate broth fail to inhibit the growth of anaerobes. Make diagrammatic sketch of displaying the relationship of oxygen with bacterial growth in a solid medium in test tube.

Why Clostridial bacteria species can not grow in the aerobic environment.
EXPERIMENT NO.6

ISOLATION OF PURE CULTURE INTRODUCTION The samples collected ITom air exposed areas such as skin, ear canal, foot pad, vagina, intestines, nares etc, usually contain more than one bacterial species. Therefore to isolate the pathogenic bacteria or required bacteria, either selective media are used such as MacConkey,s agar for E.Coli, or selenite broth for Salmonella species, or samples are streaked on the enrichment media to spread the bacteria at distance apart. Each bacterium multiply for a certain incubation period to display visible growth that is known as colony. The colony characters of one bacterial species are different ITom those of other bacteria species. From the colony characters, the number of bacteria in one sample can be determined. The required bacteria are transferred to broth medium for its pure growth. Different criteria for describing the colony characters are size, shape, edge, surface, colour, consistancy, elevation etc. A:WHOLE COLONY Punctiform Circular Rhizoid Irregular, Filamentous B: EDGE Entire, Undulate, C: Lobate, Filamentous, Curled C: ELEVATION Flat
Raised, Convex,

Pulvinate, D: SURFACE Umbonate

Smooth glistening, E: COLOUR Green, Yellow, Rough, Wrinkled, Black, Pink, Red, Dry / Powdery White EXPERIMENT NO.7 STAINS AND STAINING OF MICROBES Bacterial morphology can be examined by two ways: 1) Living un-stained organisms, 2) Dead cells stained with dyes. Living bacteria are almost colourless and lack sufficient contrast (with the water in which they are suspended) to be clearly visible. Staining the organism increases their contrast with their surrounding so that they are visible. Certain stains can identifY internal structures of cells that would otherwise be unseen. Further, the use of the oil immersion objective of the microscope to obtain the greatest magnification is more convenient with stain preparations than with wet mounts. Although bacteria do not look greatly different ITom their surrounding but they differ chemically. Stain or dye reacts chemically with the bacterial cell but not with the back ground, enabling us to distinguish the bacteria. Thus, the main advantages of staining are that it provide contrast between microorganism and their background which enables differentiation among various morphological types and enables study of internal structures of the bacterial cells such as cell wall, vacuole and spores. A wide range of dyes are available and is used in various modifications of basic staining techniques. Simple stains Differential stains Acidic dyes, Basic dyes Gram's stain and acid fast stain Special stains Capsule stain, spore stain and flagella stain

Stain/dye is a salt of which one of the ion is coloured. A salt is a compound composed of a positively charged ion and a negatively charged ion. The simple dye methylene blue is actually the salt methylene chloride which dissociate as follows:

Methylene blue chloride

>Methylene blue + + chloride

THE SIMPLE STAIN Procedure: Place a drop of sterile water in the centre of the slide and transfer a loopful culture from a bacterial colony or a drop of broth culture on the slide. Spread the suspension of the culture on the slide and allow it air dry. Fix the smear by gently passing the slide over the flame for 2 to 3 times with smear surface on the top. Flood the smear Methylene blue or Carbol fuchscin, and allow the stain to remain on the slide for one minutes, wash it in slow running water. Dry the smear in air or pressing it against a filter paper. Place a drop of immersion oil on the smear and observe under the oil immersion objective. Depending upon stain, the bacterial film will either stain red or blue. DIFFERENTIAL STAINING Bacteria differ chemically and physically trom one an other and thus react differentially to staining procedure. This is the basic principle of differential staining. There are two commonly used staining procedures. GRAM'S STAIN a: Basic dye: Crystal violet or methyle violet is used as principle or basic dye. Gentian violet Crystal violet (85%dye content) Ethyl alcohol(95%) Dissolve the crystal violet in the alcohol. Ammonium oxalate Distilled water 4.0 grams 20 rnl 0.8 grams 80 ml Dissolve the ammonium oxalate in the water and dilute the concentrated crystal violet solution in 1: 10 with distilled water, mix one part of the diluted crystal violet solution with four parts of the ammonium oxalate solution. b: Mordant: It is a substance that increase the affinity between stain and cell. Examples are acids, bases, metallic salts, iodine Iodine Solution Iodine Potassium iodide Distilled water
1. 0 gram 2.0 grams 300 rnl

This solution should be prepared tresh every 2 to 3 weeks.

c: Decolourizer It is a substance that removes the dye trom the stained cell. Etyle alcohol or equal parts of the 95%alcohol and acetone

nterstain It is a basic dye of contrasting colour trom basic dye. The purpose of the counter stain is to colour the decolorised bacterial cell,i.e., neutral dye, basic fuchsin, satranine etc.

e 2.5% solution of safianin in 95% alcohol. Satranin (2.5% solution) Distilled water 10.0 rnl 1 00 rnl If
PROCEDURE

Prepare a smear with culture of the given organism and fix the smear Flood the slide with basic stain-crystal violet for one minute and wash with water Apply smear with Gram,s iodine solution (Mordant) for one minute and wash with water. Decolorise with alcohol (decoloriser) for 15 seconds and wash with water. Apply safTanine (counterstain) for 30 minutes and wash with water Blot or air dry and examine under the immersion objective. RESULTS Gram positive bacteria: violet colour Examples: Species of Bacillus, Clostridium, Corynebacterium, Streptococcus, Staphylococcus, Mycobacteria etc. Gram negative bacteria: red colour Examples: Species of Escherichia, Salmonella, Shigella, Proteus, Pseudomonas, Pasteurella, Haemophillus, Actinobacillus etc. THE ACID FAST STAIN The acid fast stain is a differential stain that measure the resistance of stained cells to acids. The property of acid fastness is correlated with the presence of high content of lipid in the cell wall of mycobacteria and actinomycetes. In this process, carbol fuchsin is principal dye, heat. (steaming) is mordant, acid alcohol or diluted mineral acids is decolourizer, and methylene blue is counter stain. PROCEDURE: Prepare the slide fTom the sputum and flood the smear with excessive quantity of carbolfuchsin and bring the slide to steam by heating over the small flame with out boiling or drying for 5 minutes and add more stain if it appears drying. Wash with tape water. Carbolfuchsin Basic fuchsin Ethanol95% Phenol crystal Distilled water 0.3 g lOml 5 ml 95 ml

Alcoholic basic fuchsin (saturated soultion) is prepared by dissolving 3 grams of basic fuchsin in 100 ml of 95% ethyle alcohol. Five percent phenol is prepared by dissolving 5 grams of phenol crystal in 100 ml distilled water. Mix 10 ml of alcoholic basic fuchsin and 95 ml of 5% phenol. Incubate at room temperature (2SoC) for 24 hours and then filter. Decolorise with acid alcohol on the tilted smear drop wise, until no more stain is removed from the slide. Wash to remove acid alcohol. ACID ALCOHOL Hydrochloric acid (concentrated) Ethanol 95% 3m1 97 ml Apply the counterstain methylene blue for 30 minutes, wash and dry the slide. COUNTERST AIN Saturated alcoholic solution of Methylene blue Potassium hydroxide (10% solution) Distilled water 30.0 ml 00.1 m1 100 ml Examine under oil immersion objective RESULTS Acid fat positive bacteria: red colour Examples: Mycobacteria, Nocardia, Actinomycetes etc. Acid fast negative bacteria: blue colour Examples: All other bacterial species expect those depicted above. Note: All gram positive bacteria are not acid fast positive, but all acid fast positive bacteria are gram positive.
.

CAPSULE STAIN IMany bacteria are enveloped in an additional slim layer of varying thickness called capsule. This layer stains very poorly. The capsule often appears as an unstained area around a stained cell. The best capsule stains are those which contrast the capsule with the cell and the environment. 1:a: Maneval's method Mix a drop of bacterial suspension with a drop of congo red solution on the slide. Dry the slide in air, and flood it with maneval's solution, keep for one minute and wash and then dry. Examine the preparation under oil immersion objective Prepare the bacterial smear and dry in air. Apply glacial acetic acid and keep for one minute Cover the film with carbol fuchsin for one minute after pouring off the excess acid. Wash off the stain with saline 5: place a cover slip over the wet smear and examine under oil b: Welch's method

immersion objective. c: Anthony's Method Prepare the smear and dry it in the air. Stain for 2 minutes with crystal violet (I %). Wash with 2% copper sulphate solution and then dry. 1: 2: 3: 4: Examine under oil immersion objective. OBSERVATIONS Bacteria appear deep purple and capsule colourless I-Diplococcus pneumoniae 2-Pasteurella multocida Examples: 3-P. haemolytica 5-Brucella abortus 4-Bacillus anthracis 6-Klebsiella pneumoniae ENDOSPORE STAINING Species of genera Bacillus and Clostridium produce a structure called endospore. Contrast to vegetative form, the endospore are higWy resistant to heat, adverse pH, depletion of nutrients, toxic chemicals and environmental factors. The endospore resist staining but once stained, can not be decolourized easily. PROCEDURE Prepare the smear, dry in air, and fix with heat. Cover the smear with filter paper, keep saturated with malachite green (5% aqueous solution) and continue heating for 5 minutes. 3: 4: Wash with water and counter stain with safranine for 30 seconds. Wash with water and blot dry, observe under oil immersion objective. The capsule is green and bacteria is red. Examples of spore forming bacteria Bacillus anthracis, B. subtilus, B. cerus, B. megaterium
Clostridium tetani, C1. oedematiens, C1. Chouvoei C1. welchii, C1. botulinum, C1. septicum

RESULT

EXAMINAION OF MOTILITY Some bacteria have whip like structures known as flagella. The flagellated bacteria are motile. Motility of bacteria is observed by hanging drop method. 1: 2: Apply vaseline to the edge of cavity of the slide. Place a drop of bacterial suspension in the centre of a cover slip. If the culture is on the solid medium, suspend the organism in a small drop of saline placed at the centre of cover slip. (Usually a 6 hours old broth culture is used for motility). Lift the cavity slide and carefully invert it over the cover slip, the drop on the cover slip should come in the centre of the cavity and should not touch the surface of the glass slide. 4: 5: Turn the slide to bring the side of the coverslip upward. Examine the slide under darkfield microscope and examine the motility of bacteria. Examples of motile bacteria: E. coli, Campylobacter, Proteus species, Clostridium oedimatiens etc. (All capsulated bacteria are non-motile, and all flagellated bacteria are motile). QUESTIONS What is the practical significance of staining? What is the function of gram's iodine? What is the mechanisms of gram's staining? What will be the colour after each staining process Staining G+ bacteria G- bacteria Crystal violet Gram's iodine Decolorizer Satfanine Name at least four species of bacteria of veterinary importance which are acid fast positive? Write a short note on the importance of acid fast staining? Why some bacteria are called acid fast. What accounts for this property? Fill in the blanks keeping in view the acid fast staining? Staining stage bacteria bateria Acid fast positve Acid fast negative

(Carbol fuchsin +steaming) Acid alcohol Methylene blue What is the significance of capsule formation in bacteria?

Name 5 organisms of veterinary importance that form the capsule? What is purpose of copper sulphate/india ink in capsule staining? Why the bacteria loose capsule forming property during in vitro culture? How can you proceed to acquire capsule forming property of the Pasteurella multocida? Draw a sketch of hanging drop method to demonstrate the set of bacterial suspension at time of microscopic examination? Why the vaseline is applied on the edge of slide? What is difference between flagella of gram positive and gram,s negative bacteria? Make a diagram of typical flagella of gram's + bacteria? What are different arrangements of flagella on bacteria? Name 5 species of spore forming bacteria which also cause disease in dairy animals? What is the role of spore in continuation of spore former-induced-diseases in the locality? What are the factors of spore due which they are higWy resistant to adverse environment? How the spore look like if stained with gram's stain, acid fast stain and simple stain? Is there any role of spore in identification of bacteria? What is maximum and minimum size of bacteria? Make diagrams of different morphological pattern of bacteria? How would you prepare a permanent slide of stained bacteria?

EXPERIMENT NO.8

MICROSCOPE AND MICROSCOPY Microbiology is a science concerned with the living organisms too small to be seen with the naked eye thus the advent of microbiology dates from the invention of microscope. A simple microscope is little more than a biconvex lens, but a compound microscope employs two separate lens systems thereby achieving greater magnification. Since the compound microscope is the primary tool in microbiology therefore, understanding of the basic principles of microscopy, a skill in the use and manipulation of this instrument are prerequisite to any study in this science. The microscope is basically an optical system for magnification and illumination for rendering the specimen properly visible. To comprehend the operation of optical and illumination system, the principles of the relationship of magnification, resolving power and illumination must be elucidated. Magnification Magnification in the compound microscope is obtained by a series of two lens system. The lens system nearest the specimen called the objective that magnifies the specimen and produces the real image. The ocular or eye piece lens system magnifies the real image yielding the virtual image that is seen by the eye. The total magnification is equal to the product of the ocular magnification and the objective magnification. The objective lens system is a combination of convex and concave lenses of different types of the glass that correct for various chromatic and spherical aberrations inherent in a simple convex lens. Microscope is equipped with three objective. Objective Low power objective Focal length Magnification 10 16mm 4mm High dry objective Oil immersion objective 40 100 1.8mm

The desired objective is rotated into place by means of revolving nosepiece. The shorter the focal length of the objective the shorter will be the working distance between the specimen and the objective. The objective lens for focuses the light rays passing through the specimen to form a real image with in the body tube. The real image is further magnified by the ocular lens system which is situated at the top of the draw tube. The ocular lens system has lower or field lens which place the real image in the focal plane of the upper or eye lens which serves. The eye lens is a simple magnifying lens enabling the eye to focus on the virtual image of the specimen. The total magnification obtained with a compound microscope is found by multiplying the initial magnification of the objective being employed by the magnification of the ocular. The initial

magnification of the objective is engraved on the objective mount. and the magnification of the ocular is usually marked on the top of the eye lens mount or on the side of the eye piece. The total magnification obtained with the objectives listed above is as follows: Name of objective Power of the lens Objective(X) Low power Dry High power Oil imersion 10 45 100 Magnification Ocular(X) (X) 10 100 10 450 10 1000

Two adjustment wheels focus the lens system on the specimen. The course adjustment moves the body tube over a greater vertical distance and brings the specimen in approximate focus and the fine adjustment moves the body tube more slowly for precise final focusing. RESOLVING POWER Total magnification can not be indefinitely increased by use of additional lenses due to property of lenses called resolving power. The resolving power of a lens is its ability to show two closely adjacent point as distinct and separate entities. This character of a microscope is a function of the wavelength of the light used and a characteristic of the lens system known as its numerical aperture. Resolving power = 1 / d d= distance between two adjacent points to be resolved. d= wavelength! 2 x numerical aperture Numerical aperture = n x sin 0 n= refractive index of the medium between slide and objective 0= s half of the angle formed by two extreme rays passing through the specimen and entering into the objective. Thus the shorter the wavelength of light used the smaller the distance to be resolved and greater will be the resolution, i.e., blue light will give a greater resolution than with red light. However since spectrum of visible light is relatively narrow, increasing the resolution by decreasing wavelength of the light used is of limited value. The greatest increase in resolution of a light

microscope is brought about by increasing the numerical aperture. The numerical aperture is function of the effective diameter of the objective in relation to its focal length and the light bending power or reTactive index of the medium between the specimen and the objective. The optical factors limit the degree to which the objective may be altered to increase its numerical aperture. Because the refractive index of air is less than that of glass, light rays are refracted or bent as they pass Tom the microscope slide into the air (n=1.00). Thus many of the light rays reflected Tom the specimen are reTacted at so great angle that they completely miss the objective. By interposing immersion oil (n=1.52), which has essentially the same reTactive index as the glass (n=1.52) between the slide and the objective lens, and a greater percentage of the light rays from the specimen pass directly into the objective resulting in greater resolution and a clear image. MEASURING THE SIZE OF BACTERIA (MICROMETRY) Micrometer consists of an ocular micrometer and a stage micrometer. The ocular micrometer is a disc of glass on which equally spaced lines are engraved. When this disc is placed in the ocular the ruled lines are superimposed on the microscopic field. The scale of these lines is calibrated by superimposing them on to a stage micrometer. The stage micrometer is a glass slide on which 1 mm line is engraved into equal 100 divisions. Each division is 10 micron. It is determined that how many units of ocular division superimpose a known distance on stage micrometer. This will help to determine the exact length of each division of ocular micrometer. Once calibrated the ocular micrometer can be used to determine the sizes of various microscopic objects. When ever you change the objective or microscope, you are required to recalibrate the ocular micrometer. PROCEDURE I: Remove the ocular lens and insert the ocular micrometer in the eye piece. Replace the ocular lens and mount in the microscope and observe. Place the stage micrometer on the stage and centre the scale of stage micrometer with low power objective and observe the micrometer at oil immersion objective. Rotate the ocular until zero of ocular micrometer superimpose the zero of stage micrometer. Count the divisions of each micrometer to the point at which the lines of the micrometers coincide again. Suppose 75 ocular divisions are equal to 5 divisions of stage micrometer and one division on stage micrometer is equal to 10 micron. Five divisions are equal to 50 micron that are equal to 75 ocular divisions, therefore one division of ocular micrometer is equal to 50/75=0.67 micron. 2:Replace the stage micrometer with stained bacterial slide and measure the number of division of ocular micrometer covering the full length of bacteria. Suppose 3 divisions are fully covering the length of bacteria. It mean total length of bacteria is 3xO.67=2.01 micron. Measure the length of 10-20 bacteria in this way, and then take the average. DARKFIELD MICROSCOPE The effect produced by dark field microscope is a dark background against which objects are brightly illuminated. This is achieved by equipping the light microscope with a special type of

condenser that directs the light path trom the source of illumination in such a way (figures )that if specimen is completely transparent and homogeneous the light directed through the condenser does not enter the objective and the entire field of view is dark. If transparent medium contains objects that differ trom it in refractive index, there will be a scattering of light by reflection and refraction. The scattered light will enter the objective and thus the object will appear bright in the dark microscopic field. Dark field microscopy is important for the examination of unstained microorganisms suspended in fluid and hanging drop preparations. FLUORESCENT MICROSCOPE Fluorescence microscope is important in biology because a substantial level of specificity can be visualised by linking a fluorescent dye to an antibody that is raised against a particular antigen.. Fluorescent molecules are chemical substances that absorb the short-wave length of ultraviolet light and emit a light of longer wavelength. Thus a material may appear one colour by ordinary light and entirely different colour by ultraviolet light. Such materials are called as fluorescent and phenomenon is fluorescence. The fluorescent dyes are a: Flourescein Iso ThioCyanate (FITC), that absorbs blue light and emits green light, b:Rhodamine, absorb green light and emit red light, c: Auramine-O etc. This microscope is lamps (Mercury or tungsten halogen), source of ultraviolet light (shortwave length that increase the resolution power of the microscope) and two filters (exiter filter and barrier filter) that protects our eyes ITom the UV light rays. The exiter filter is placed between light source and specimen, allows only light of shortwavelength, while barrier filter, placed between eye piece and specimen, blocks the passage of light of short wave length and allows the light of longer wavelength. PRINCIPLE Antigen antibody reactions are specific. Fluorescent dyes can be coupled with protein molecules such as purified antibodies. Fluorescent dyes fluoresce on exposure to ultraviolet light. PHASE MICROSCOPE

This microscope is important for observing unstained living specimens such as

movements of Borrelia anserina- cause of spirochaetosis in poultry, spirochaete, leptospira etc.

Small difference in the thickness and chemical nature of various parts of the specimen retard the part of light (shift light in phase) as it passes through the specimen. Our eye can detect such phase shift, but phase microscope contains optical components that change phase differences into differences of brightness. To effect this change an annular phase ring is placed in the optical system below or between the element of condenser and a phase shifting element is placed in the real focal plan of each objective. QUESTIONS What are different microscopes and write down their working principle?

What are fundamental features for recording of stained bacterial slide while observing under light microscope? Define resolution, and magnification? and write down the formula for calculation of magnification and resolution of a light microscope? What is size of bacteria that you can see 1, 1.5, and 3 mm long rods in light microscope with oil immersion objective and with eye piece of lOX? What are unit of length for measurement of bacteria, and viruses? What are methods for observing unstained bacteria? Name three fluorescent dyes? Suppose 80 divisions of ocular micrometer are equal to 5 divisions of stage micrometer, and 4 divisions of ocular micrometer are covering the length of bacteria. Calculate the size of bacteria in micron?

EXPERIMENT NO.9 PRESERVATION OF LABORATORY CULTURES

INTRODUCTION It is a routine practice to preserve the isolate after its purification and characterization. Preservation will permit maximum survival of the population of pure culture with little or no genetic or physiological change. All the methods used in laboratory depends upon bacteriostasis, that is maintenance of cultures without growth or reproduction.
II

a: SHORT TERM PRESERVATION AEROBIC CULTURE The cultures are streaked on the agar slopes and incubated for 18 hours. After visible colonial growth, the culture slopes are stored at 4 Co. These cultures can survive depending upon their characteristics for different length of time, i.e., Actinobaccillus for 7 days, Staphylococcus for one month. FACULTATIVE ANAEROBES Some facultative anaerobes, for which oxygen is injurious, the cultures can be stored in refrigeraters as stab cultures. To avoid the oxygen exposure, and evaporation of the medium, a thin layer of mineral oil is applied on the top of the stab culture. OBLIGA TORY ANAEROBES Strict anaerobes are species of clostridium and spherophorus which are preserved in strongly reducing medium such as thioglycolate broth containing buffered salines. b: LONG TERM PRESERVATION Refrigerated culture remain viable for a few days to a month as remgeration is not suitable for long time preservation. The culture can be kept viable for years by subculture periodically, the bacteria in these conditions may mutate. This problem can be overcome by freezing the culture at low temperature (freeze drying or storage in liquid nitrogen). FREEZING

Most of bacterial cultures can survive for months if they are frozen in litmus milk or in 1 :20 dilution of glycerol and kept at -20C. LIQUID NITROGEN Bacterial cultures in a sterilized storage medium can be preserved indefinitly in liquid nitrogen. The bacterial cultures in vials can be sealed before storage. FREEZE DRYING (L YOPHILIZA TION) The microbial culture is suspended in sterile milk, serum, or equally protective medium. The culture is transfered into sterilized freezing ampoules (0.5 ml / ampoule). These ampoules are constricted at 1/2 below the top of the ampoules by ampoule constrictor. In this apparatus, the oxygen and natural gas are admixed together. The flame of two gasses produced is of very high temperature that melts the glass ampoule rotated at the same point. The ampoule with certain level of constriction is removed from the constrictor. The ampoules are fixed on the ampoule holder of the freeze dryer. The freeze dryer is switched on, the condensor will induce a -30oe in the product chamber and -60oe in condensor chamber. This will freeze the suspended cultures. Then the vacuum pump of the machine is switched on. This will create the vacuum upto -2xlO hg pressure, the frozen liquid of the medium will evaporate into steam. The gaseous vapours will be condensed into ice in the condenser chamber. This process will continue till all frozen water from the ampoules will be evaporated. The ampoules can be cut from point of constriction with oxyflame. Before and after cutting the ampoule the vacuum is checked with vacuum detector. The desiccated cultures can be stored for several years. A high percentage of microbial population survive lyophilization, and thus there is little or no selection for the more resistant genetic variants in the population. The high survival rate with this technique compared with other methods of preservation results from a combination of the protective effects of the stabilizer and freeze drying of the cultures.

QUESTIONS How can you preserve for short time the cultures of aerobic, facultative anaerobes, obligatory anaerobes in your laboratory? What is freeze drying? What is the composition of medium for preserving cultures in liquid medium, and freeze drying?

CHAPTER NO. 10 PREPARATION OF ANTIGENSN ACCINES Antigen is a chemically complex, degradable, large (minimum 1031 dalton), foreign molecule and can stimulate immunocytes of the host for antibody production. Complex particles such as bacteria, viruses, fungal spores, nucleated cells, erythrocytes are composed of mixture of proteins, polysaccharides, lipids, nucleic acids. Injection of such particles will stimulate an antibody response against each of the molecules of these particles. Moreover, a single protein may have many areas against which antibody response is directed and with which antibodies can bind. These areas are termed as epitopes. Epitopes on the protein molecules contain 4 to 6 amino acids and are found on the exposed surface of the molecule. Haptens Small compounds are not able to generate antibody response, and are not antigen, but can bind with specific antibodies. The purified form of carbohydrate moiety (C substance of streptococcus species) can not induce antibody response but can bind antibodies that are generated by the whole organism. Cross reactivity Identical antigenic determinants may be found on a number of different bacteria, cells, viruses and parasites. Antibodies directed against one antigen may be reactive with the antigen ITam an unrelated source. Cross-reactivity occur between following organisms: Brucella abortus Treponema pallidum Intestinal Microflora VACCINE Vaccines are mixture of complex molecules such as whole bacteria, virus, purified proteins, lipopolysaccharides, or glycoproteins from pathogens, and are injected in the host to induce an immune response or resistance against a pathogen. The vaccines are prepared in different ways in different conditions: 1: When the microorganism is pathogenic and can be cultivated in mass quantity. It can be inactivated with formalin or sonicated, adjuvanted and injected in the host for an immune response i.e., Pateurella multocida vaccine. When pathogenic organisms can be attenuated by different ways. In this way, the microbes become non-pathogenic but still are alive and immunogenic. i.e., Mycobacteria bovis, pathogenic for bovine was cultivated in bile containing medium for many times until became non-pathogenic for all animal species. Such live avirulent cultures are used as vaccines for human and animals (BCG vaccine). When avirulent strains of the pathogenic microbes are existing in the nature, which can be purified and cultivated in mass quantity and injected in the animal body to induce immune responses (Brucella abortus strain-19: lentogenic strains of ND virus). When the diseasing causing organism can not be cultivated in in vitro culture and avirulent strain of the organism is not known. In such case, the target organ can be trituated filtered through musclin Yersinia enterocolitica Bovine cardiolipin Human blood group antigens

cloth, and inactivated with formalin. This may be crude vaccine to immediately control the problem. In this experiment antigen! vaccine against Pasteurella multocida is prepared. PROCEDURE Prepare 100 ml of tryptone serum broth (pH 7.2) in a 250 ml flask according to the method described before. Sterilised tryptone broth Sterile bovine serum = 90 ml =lOml Transfer a loopful culture of 20 hours growth of Pasteurella multocida in the flask containing the broth. Incubate the culture at 37C for 24 hours. Count the number of bacteria in the culture by Miles and Misra technique. Dilute the culture to adjust 108 /ml of bacteria. This culture isformalinised formaldehyde in the culture: 0.12%: incubate 37C for 48 hours). Check the safety of the vaccine/antigen by injecting 1 ml intraperitonealy in susceptible mice. If mice do not die, the vaccine/antigen is safe to use. This is used as antigen for serum agglutination test or as vaccine to immunise animals against Pasteurella multocida (SAFETY TEST).

Inoculate the loopful of the prepared culture on the nutrient agar plate and incubate for 48 .
hours. No growth on the agar indicate the culture is sterile (STERILITY). F or preparation of aluminised vaccine, the aluminium hydroxide is added in the culture at rate of 0.2% and pH is adjusted to 7.2 with 10% sodium hydroxide solution. Note: Antigens are also prepared on the basis of protein content of the inoculum. For one rabbit of one kilogram, 500 ug of protein of the antigen must be injected to induce good immune response. QUESTIONS What is antigen, hapten, immunogen, epitope and antigen determinant? What are physiochemical properties of an antigen? How you can prepare a vaccine? What type of immunity will be induced by vaccination and by injecting hyperimmune serum to cow calf?

EXPERIMENT NO. 12 OBSERVATION OF PRIMARY AND SECONDARY ANTffiODY RESPONSES OF CHICKENS TO NEWCASTLE DISEASE VACCINE INTRODUCTION Injection of vaccine/antigen in the host stimulate immunocytes and results in 2 types of response humoral immune response cellular immune response This response takes more than 7 days to reach a detecatable level and is called primary immune response. Second injection of the same antigen/vaccine on 21 days after first injection will induce boosted response (humoral/cellular response) and is called secondary immune response/amnesic response/boosting response. In the present experiment the humoral response will be recorded follows:

PROCEDURE
1: Inject 0.5 ml ofNDV vaccine (100 dose vial ofNDV is admixed in 50 ml normal saline) in 10 birds at age of 3 5 days. Collect serum from the 10 birds at weekly interval on 7,14,21 days post priming and store this serum at 20C till required. Give second injection of the same antigen sub cut (0.5 ml /bird) and collect serum samples on 7, 14, and 21 days post boosting and store at -20C until required. Determine haemagglutination titre of anti-NDV of all the sera and plot the titre graph. RESULTS Primary response is low while secondary response is high.

EXPERIMENT NO. 13 BASIC PRINCIPLES OF SERODIAGNOSTIC TESTS INTRODUCTION: Serodiagnostic tests are techniques commonly used in laboratories to identify the pathogens, specific antibodies, titre of microbes, or antibodies. Serum is a main source of antibodies. Antibody Antibodies are secreted by antigen stimulated B cells or plasma cells. These are proteins of immunoglobulins such as IgG, IgM, IgA, 19B, and IgD. IgG, 19B, IgD are bivalent monomers, IgA is in dimer form, IgM is pentamer form. Monomeric form of antibodies have two binding sites (see diagram). Antigens are mainly multivalent or has multiple sites for attachment to antibodies. AGGLUTINATION In agglutination, antigens are particulate matter i.e., whole cell, whole bacteria, fungal spore, or inert particles coated with soluble antigens. These antigens uniformly distribute in saline solutions. Antibodies of serum when mixed with antigen, form bridge between antigens. Such antigens form clumps or agglutinate. This test can be performed on slide/slabs-slide agglutination test (SAT)/ spot agglutination test, or in tubes- tube agglutination test. In most of microbial. diseases, SAT is performed to determine the antibody titre in carrier animalslbirds such as salmonellosis, mycoplasmosis, leptospirosis, brucellosis etc (Plate 10-1). 2: PRECIPITATION TEST .In precipitation test, the antigen is soluble and form solution in saline. In the antigenantibody interaction, insoluble complexes appear in the form of turbidity in the solution. Maximum turbidity appear when antigen antibody interact at equivalent proportion. i.e. 1: Ascoli test in Anthrax. 2: Lancefield classification of streptococci. COMPLEMENT FIXATION TEST (CFT) This is a highly sensitive test and can determine micrograms of antibodies. This test is based upon following principle: I: Antigen antibody interact specifically. 2: Antigen antibody complex can activate the complement components which bind with the complexes. Activated complements can cause lysis of sensitised (antibody coated cells) RBC. Subagglutinating level of antibody to sheep RBC can coat the cells without agglutination. Lysed and unlysed RBC can

be differentiated with naked eye. ENZYME LINKED IMMUNOSORBANT ASSAY (ELISA) This is also a highly sensitive test and can detect micrograms of antibodies in serum. It is commonly used in diagnostic laboratories. I: Antigen antibody interact specifically. 2: Antigen or antibody are coated on the bottom of wells of an immunoplates (microtitration plate). Enzymes are conjugated with proteins (antibodies /antigens). Enzymes are very specific to substrate and catalyse the reaction producing products. Colour intensity is determined by spectrophotometer. FLUORESCENT ANTffiODY TECHNIQUE (FAT) coloured This method is used for demonstration of microbes in tissues/secretions. It is based on

following principle:
1 : Antigen antibody interactions are very specific 2: Fluorescent agents are conjugated with proteins

'

3: Fluorescent agent has ability to enhance the wavelength of UV light. The UV light rays falls on the fluorescent material and the rays reflected from the material are of the wavelength of the range of visible spectrum. There are filters which will allow the passage of light rays of required wavelength (Plate 10-5). QUESTIONS Make model sketch to explian the CFT, ELISA, FAT, and Agglutination? What are basci principles of the following serological tests: Precipitation, agglutination, CFT, ELISA, FAT

CHAPTER-14 BIOCHEMICAL CHARACTERS OF BACTERIA The bacteria are unicellular procaryotic cells with nuclear material without nuclear membrane. The nuclear material is a circular DNA that controls the shape and physiology of the bacterial cell. The bacterial genes transcribe and translate into proteins/enzymes which catalyse some of the reactions. The enzyme production in bacteria is species specific, for example Salmonella species do not secrete enzymes for lactose fermentation, while E. coli can secrete the enzymes which ferment lactose. The bacterial genera or bacterial species are characterzed on the basis of their characters of enzyme production. 1: LECITHINASE OR PHOSPHOLIPID HYDROLYSIS TEST The phospholipids are major components of all cell membranes. They are glycerol esters of two fatty acid residues and phosphate that are esterified with non-fatty acid unit such as choline. As phospholipids are functional components of all cells, the ability of bacteria to breakdown the host cell phopholipids is an important factor in the spread of virulent .. rrucroorgarusms. Some of the bacteria secrete phospholipase enzyme that can hydrolyse phospholipid of the egg yolk. Cleavage of the phosphate ester bonds forms a water insoluble lipid. This enzymatic activity is detected by a zone of opalescence in the medium around the cell mass. PROCEDURE 1: Prepare egg yolk agar. Add 5-10% sterile egg yolk in the autoclaved molten nutrient agar asepticaly. Inoculate a drop of broth culture of the test organism in the centre of the agar. Incubate the plate at 37C for 24 hours and record the zone of opalescence. 2: 3: RESULT

_Appearance of zone of opalescence around the bacterial growth is indication about the ability of
bacteria to secrete phospholipase enzyme.

.2:
THE UREASE TEST

.The genus proteus can be distinguished trom some other gram negative rods by its ability
to produce large amounts of urease. The hydrolysis of urea releases ammonia as follows:

NH2 I C=O I NH2 2NH3 + CO2

If
Ammonia Carbon dioxide
The production of ammonia raises the pH of the medium, that change the colour of phenol

red (a pH indicator) to red.

PROCEDURE Inoculate the urea agar with the test organism. COMPOSITION Peptone Sodium chloride Dipotassium hydrogen phosphate Phenol red solution (one gram in 500 ml D.W) Agar Glucose sterile 10% soultion Urea 20% solution sterile (Note: Glucose and urea are heat labile solutions) 2: Incubate the agar at 37C for 1-4 days. RESULTS UREA AGAR 1 g 5g 2g 6 ml 20 grams lOml 100 ml Appearance of red colour is indication of urease production by the test bacteria.

CATALASE ACTIVITY:
This test is used to determine the catalase activity of aerobic bacteria. H2O2 Catalase H2O + O2

Cultures on blood agar should not be used for catalase tests because blood also contains the

enzyme. Old or poorly grown cultures may give false negative reactions. The enzyme catalase contain hemoporphyrin structure. This porphyrin structure is part of catalase, but also, with Mg++ in place of Fe, is part of the cytochromes (respiratory chain electron carrier) found in aerobic form of life, and in the chlorophils of all photosynthetic cells. Example: Staphylococccus aureus + MycoplasmaStreptococcus faecalisClostridium species. Lactobacillus PROCEDURE 1: 2: 3: Place a drop of 3 % hydrogen peroxide on a clean glass slide. With the help of sterile platinum loop, transfer a portion of a colony ITom an agar plate or slants to the drop. Observe for the production of bubbles for a positive reaction.

RESULTS: Oxygen forms bubbles in the water and test is regarded as positive.

4:0XIDASE TEST:
The NADP oxidase enzyme is secreted by many non-intestinal bacteria. This enzyme degrades tetramethyle-p-phenylene diamine dihydrochloride and produces a dark purple coloured compound. Examples: E.coli Salmonella Proteous Pseudomonas +

PROCEDURE 1: Grow the bacteria to be tested on the medium.

Prepare the oxidase reagent as follows: Add 1 ml of distilled water to bijou bottle containing few crystals of tetramethylepphenylenediamine dihydrochloride and mix thoroughly and pour it onto a filter paper inside a clean petridish. U sing sterile clean platinum loop, smear a portion of the colony onto wet filter paper. RESULTS NOTE: Development of a dark purple colouration within 10 seconds indicates a positive reaction. Nichrome loops may give false positive reactions. The reagent slowly become co loured due to nonbiological reactions so it should be used promptly. DIASTASE TEST Many pathogenic and non-pathogenic species of bacteria secrete exoenzymepolysacchradases that mediate hydrolysis of polysacchride (Starch). Iodine reacts with starch molecules and form blue black coloured product, while iodine can not react with hydrolysed starch products and retain its original iodine colour.

Bacillus subtilis E.Coli PROCEDURE: 1: Inoculate a starch agar plate lightly to give discrete colonines. 2: Flood the plate with lugol,s iodine solution. +RESULT: Clear iodine stained areas + (hydrolysis of starch) Blue back coloured areas - (no hydrolysis of starch)

CASEIN HYDROLYSIS TEST

Casein which exist in milk as colloidal suspension that gives milk its opaque whiteness. It is the principle protein in milk. Many bacteria produce enzymes that hydrolyze this protein into more soluble and transparent derivatives. Protein breakdown (some time is called peptonization) is useful in identifying microbial species. PROCEDURE Prepare skim milk agar (10% skimmed milk in peptone agar) and pour into 64 mm petridishes. Place a loopful culture of Rcoli in centre of the one plate and B. subtilis in the centre of the other plate. 3: Incubate at 37C for 48 hours and observe the results. RESULTS Colonies of proteolytic microbes (organism that digest casein) will be surrounded by clear zone.

GELATIN HYDROLYSIS TEST

The protein gelatin is obtained by hydrolysing collagen, a component of connective tissue and tendon of animals. Gelatin (12% aquoues solution) will soldify at 2SoC while liquify at 37C, but gelatin hydrolyzed by bacteria will not solidyfy even at low temperature. Gelatin is a good substrate for testing proteolytic activity of the microbes. PROCEDURE Prepare gelatin medium in five tubes as follows: Add 12% gelatin in nutrient broth and allow it to sland undisturbed overnight and dissolve the gelatin by heating upto 4SC. Steam at 11SoC for 10 minutes, cool to 4SoC and dissolve white of 2 eggs. Steam for 30 minutes and filter through hardened filter paper, bottle in 12 ml amounts in test tubes. Autoclave at 11SoC for 10 mintes, remove ITom the incubator quickly and keep at low temperature. Inoculate loopful culture of E.cloi, B. subtilis, S. faecalis, and P. vulgaris in tube number 1, 2, 3, and 4 respectively. Keep the fifth tube uninoculated control. Incubate the tubes at 37C for 48-96 hours.

Freeze all the tubes in ice-bath and observe the state of gelatin. RESULTS The control tube (the tube in which no hydrolysis taken place) will solidify while hydrolysed gelatin will remain liquid. PRECAUTION Do not agitate the tubes after incubation with bacterial cultures. If hydrolysis of gelatin is proceeding slowly from surface to downward as it often does, agitation can mix the hydrolysed with unhydrolysed gelatin and may give eroneous results. 8: DECARBOXYLATION AND AMINE PRODUCTION The decarboxylation of an amino acid is the splitting off of its caboxyl group to yield an amine and cabon-dioxide. The reaction can be expressed as follows: Decarboxylase R-CH (NH2)-COOH
R-CH2-NH2 + CO2

Bacterial decarboxylation can be demonstrated by showing the disappearance of the amino acid or by formation of amine and carbondioxide. The reaction results in accumulation of an amine which is basic, therefore, decarboxylation can be demonstrated by measuring rise in pH.
-------------------------------

Lysine Ornithine decarboxylation

-------------------------------

Proteus vulgaris Enterobacter aerogenes ++------------------------------PROCEDURE 1: Inoculate one tube of lysine decarboxylase broth with loopful culture of the test organism and keep the other as uninoculated control. COMPOSITION:

Peptone Meat extract Glucose Pyridoxal Bromocresol purple(1:500 ml water) crsol red(1 :500 ml water) Distilled water

5g5g 0.5 g 5 mg 5 ml 2.5 ml 1000 ml 1: Dissolve these components, adjust pH 6.0, distribute in four portions and dissove the amino acid under test at rate of 1 %. Distribute this medium into 1 ml amount and overlay with 5 mm thickeness of liquid paraffin and autoclave at 121C for 15 minutes. Incubate both the tubes for 24 hours at 37 C. 2: 3: 4: Test the rise in pH in both the tubes by pH indicator (bromocresol purple).
RESULTS

Appearance of purple colour of bromocresol in the tube indicate rise in pH or decarboxylation of the amino acid. 9: DEAMINATION Deamination of amino acids is the enzymatic splitting of the amino group to yield amonia (NH3) and usully the corresponding keto acid. Deaminase R-CO-COOH + NH3 H2O keto acid amonia
R-CH(NH2)-COOH

The bacteria that split amino acids and yield amonia and keto acids, can excrete deaminase. In deamination of phenylalanine, the phenyle pyruvic acid is produced which forms coloured complex with ferric ions. Phenylalanine deaminase Proteus vulgaris Enterobacter aerogenes

PROCEDURE
Inoculate one tube of phenylalanine agar with Proteus vulgaris and a second with Enterobacter aerogenes. COMPOSITION

.Yeast extract DL-phenyle alanine Disodium hydrogen phosphate Sodium chloride Agar Distilled water 3g 2g 1 g 5g 15 g 1000 ml

_2: 3:
Adjust the pH to 7.4, distribute and sterilize by autodaving at 121 C for 15 minutes and allow it to solidify in test tubes in slope forms. Incubate both the tube at 37C for 24 hours. Transfer 4 to 5 drops of a 10 % F eCh solution in both the tubes so that it flows on the surface of the slants. RESULTS Appearance of green colour indicate the positive test.

INDOLE PRODUCTION TEST .Indole is a nitrogen containing compound that can be formed from the degradation of amino acid -tryptophan- by certain bacteria. Tryptone is used as the substrate because it contains much tryptophan. The indole reacts with aldehyde compound of Kovac's reagent and forms red co loured compound that is more soluble in alcohol.
PROCEDURE 1: Inoculate the tryptone broth with the test organism Peptone Sodium chloride Distilled water Incubate at 37C for 24 hours. 20g 5g 1000 ml

2: 3: Add few drops of Kovac's reagent, mix properly and keep the tube in the stand undisturbed. Kovac's reagent Isoamyl alcohol 150 ml p- Dimethyl-aminobenzaldehyde 10g Con. hydrochloric acid 50 ml (dissolve aldehyde in alcohol and add acid slowly) RESULTS After few minutes, a thin layer of red colour will appear at top of the broth, that is indication of positive indole production test.

HYDROGEN SULPHIDE PRODUCTION TEST

Some bacteria act on sulfur containing amino acids to liberate H2S. The H2S production in the culture medium containing bismith or iron, is indicated by blackening the colony of sulfide producing bacteria. H2S react with iron or bismith and forms black coloured precipitate that blacken the colony.

.t_CH2-SH
I H2N-CH-COOH cysteine sulphide +2H CH3 I H2S + H2N-CH-COOH hydrogen alanine PROCEDURE 1: Prepare bismith sulphide agar. COMPOSITION Meat extract Peptone Sodium chloride Gelatin Ferrous chloride 10% aqueous solution 7.5 g 25.0 g 5.0 g 120.0 g 5.0 ml These components are dissolved except ferrous chloride and sterlize by autoclaving and dissolve the ferrous chloride solution at 55C and sterilize by filteration. Tube the medium and cork the tube with paraffin wax. 2: Inoculate the test bacteria on the agar plate. 3: Incubate the inoculated plate at 37C for 24 hours. RESULT

12: Growth of black colony is an indication ofHzS production by the test organism. COAGULASE
TEST: The pathogenic staphylococci produce coagulase that converts the plasma fibrinogen into fibrin which forms clot and entangle the bacterial cells. This reaction requires another factor present in plasma called coagulase reacting factor (C.R.F.). Some bacteria such as Klebsiella utilize citrate thus releasing Ca ++ and Mg ++ ions that ultimatly causes the clot formation. Some strains produce large amount of fibrinolysin which dissociate the clot. Coagulase positive bacteria Coagulase negative bacteria SLIDE METHOD Put 2 drops of the normal saline onto glass slide. EmulsifY into each drop a loopful culture from an agar plate to make a smooth suspension. Add a drop of undiluted plasma to one drop, mix gently and observe for clumping- a positive reaction. The second drop serve as control for the Staphylococcus pyogenese Staphylococcus epidermidis

granularity and degree of suspension. TUBE METHOD Dilute plasma 1/10 in saline solution. To 0.25 m1 of the plasma in a samll test tube, add 0.05 m1 of ovrnight broth culture of staphylococci. Proceed with the similar test with a known coagulase positive and negative bacteria as controls. Incubate the tubes in a water bath at 37C and examine after 1 hour interval until clot formation.

RESULT
Clot can be tested by laying the tubes on top of table. The clot will not flow while unclotted plasma will flow along the wall oftest tube.

13: NITRATE REDUCTION TEST

COMPOSITION Potassium nitrate Peptone Distilled watwr
0.2 g 5.0 g 1000 ml

Tube in 5 ml amounts and autoclave at 121C for 15 minutes. TEST REAGENT Solution A Dissolve 8.0 gram of sulphanilic acid in 1 litter of 5 N acetic acid Soltion B Disolve 5 grams of alpha naphthyl amine in 1000 ml of 5 N acetic acid. Immdiately before use, mix equal volumes of soluton A and solution B to give final test reagent. PROEDURE Add 0.1 ml of the test reagent in the culture. Development of red colour within a few minutes indicate the production of nitrite in the medium and hence the production of nitrate reductase.

ARYL SULPHATASE TEST

tThis test determine the sulphatase production ability of the bacteria. The enzyme splits the
phenolphthaleine sulphate into sulphate and phenolphthaleine. The free phenolphthaleine appears as pink or red-purple coloured compound in akaline pH. Mycobacterium avium Mycobacterium intracellulare Mycobacterium fortuitum negative positive positive

PROCEDURE Prepare the medium. Prpare 6.25 % solution of tripotassium phenolphthalein disulphate and sterilize by filteration. Add 1 ml of this reagent to 100 ml of sterilized Dubos and Davis Liquid Medium aseptically. Bovine albumin (fraction v Armour and Co) Water Mix and sterilize by filteration 4.5 ml 45.5 ml

COMPLETE MEDIUM Potassium dihydrogen phosphate(KH2PO4) Disodium hydrogen phosphate(Na2HPO4.12H20) Sodium citrate (C6H5Na307.7H20) Magnessium sulphate (MgSO4.7H20) Tween-80 (10% solution) Casein-hydrolysate 20% solution Distilled water Bovine albumin solution 1.00 g 6.25 g 1.50 g 0.60 g 5ml 10 ml 1145 ml 40 ml Adjust pH 7.2, aultoclave at 121C and add bovine albumin with sterile precautions. Inoculate 5 m1 ofthis broth with the drop of the bacterial culture. Incubate at 37C for 7 days. Add drop by drop a solution (1.0 M) of sodium carbonate to detect free phenolphthalein which is demonstrated by production of pink colour. RESULT 2) 3) 4) Positive Negative pink colour no colour

14: METHYL RED TEST

Some bacteria ferment glucose and produce sufficient amount of acid as end products. The acid metabolites are sufficient to reduce pH of the medium and to give red colour with methyle red. PROCEDURE Prepare glucose phosphate peptone water. Glucose 5 grams Peptone 5 grams Phosphate buffered saline---1 litre Transfer 5 m1 to each test tubes and sterlize by filteration. 2) Inoculate the medium with the pure culture of the test organism. 3) Incubate the tubes at 37C for 24-48 hours. 4) Add few drops of methyl red and mix gently.

RESULTS Red colour of the medium Yellow colour of the medium positive negative

15: VOGES PROSKAUR TEST

Some bacteria produce acetoin or acetyle methyl carbinol as an intermediate compound from glucose fermentation. It is detected by oxidation in alkali + air to diacetyl which give pink colour with creatine. PROCEDURE 1). Inoculate the test culture to the glucose phosphate peptone water, incubate at 37C for 24-48 hours. Add few drops of Omeara's reagent and read the results 2) RESULT Postive Negative pink colour no colour

16: CITRATE UTILIZATION TEST The ability to utilize citrate as a sole source of carbon and energy distinguishes certain gram negative rods. Simmon's citrate agar contains citrate as its only carbon and energy source. Growth on this medium is a positive test for citrate utilization. Certain organism that give a positive test increases the pH of the agar, changing the bromothymol blue indicator in the medium from green to blue. PROCEDURE 1) Inoculate a slant of Simmon's citrate agar (in culture media) with culture ofE. coli, E. aerogenes and P. aeruginosa.

POSITION MgSO4 (NH4)H2PO4 K2HPO4 Sodium citrate Sodium chloride Agar Bromothymol blue 0.2 g 1.0 g 1 g 2 g 5 g 20 g 0.08 g

After preparation, the medium can be dispensed into test tubes and is required to be placed in slanted

position. Incubate at 37C for 24-48 hours RESULT Growth during incubation is a positive test and appearance of blue colour is an indication of citrate utilization with the production of sodium hydroxide.

SUGAR FERMENTATION TEST

Bacteria have very complex genome that is different from one bacterial species to another even in the same genus, consequently they differ in the production of certain enzymes. For example E. coli produces a set of enzymes to ferment lactose while salmonella species lack of genes for production of these enzymes. During fermentation, there are either accumulation of acid/alkaline products with or without gas production. After growth of bacteria, acid/alkaline products are detected with addition of pH indicator while gas is detected with use of Durham's tube. The Durham's tube (1" long tube) is filled with the peptone water and added in inverted form in the test tube containing sugar peptone water. During fermentation of sugar by bacteria, the gas, if produced, will accumulate in the durm's tube. PROCEDURE Inoculate the test culture to peptone water containing test carbohydrate,and durhm's tube, incubate at 37C for 24-48 hours. 2) Add few drops of me thy Ie red and read the results RESULTS Acid + gas Acid + no gas No acid + No gas Red colour and gas in durhm's tube. :Red colour and no gas in durhm's tube : Yellow colour and no gas in durhm's tube