5 Microbiology Manual

7/30/2019 5 Microbiology Manual

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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 carefulwhile 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.


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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, orscarves that could catch fire orknock over reagents.

Report all accidents orspills toyourteacher, no matter how minor it may be. Such accidents

will not count againstyourgrade.

Do not put yourpencils inyourmouth orears. Do not scratch yourbody while working inthe lab.


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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 freefrom 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 forheating 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 45Cfor 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).


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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 oranaerobic 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 suspendingparticles; 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


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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. Thisenergy 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).


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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 into100 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 isused 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 afront 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 Conductivity meter Demineralizer

1. Steam outlet I-Centrifuge

2. Palladium sachet II-Hot air oven

3. Safety valve III-Water bath

4. Shallow round depression IV-Anaerobic jar

5. Perforated sieve V-Seitz filter

6. Seitz filter VI-Cavity slide

7. Tube holders VII-Autoclave


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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 amicrobiologist. There are different methods to sterilize the sampling bottles, culture media, glass-

wares, 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

Cellulose membrane filter

Sintered glass filter

Asbestos pad filter-Seitz filter

Ultrasonics

Radiation Freeze drying (alternate freezing and thawing)

B. CHEMICAL METHODS


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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.


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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 thatsimulate 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.


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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 musthave 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 thepressure 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.


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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 offilters 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.


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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, bufferingsolutions, 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 adisinfectant 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 mediumApply 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 +++


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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: autoclavingc: 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?


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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 culturemedia 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.

INDICATORSThese 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


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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-IIOmedium 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 110medium, 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.


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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) 90-95 m1

Fresh defibrinated sheep blood 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 ofstaphylococcus. 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. .


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5: SIMMON CITRATE AGAR

Composition

MgSO4 0.2g (NH4)H2PO4 LOg K2HPO4 1 g

Sodiu citrate 2 g

Sodium cWoride 5gAgar 20 g

Bromothymol blue 0.08 g

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


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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 citrate12m1

600ml

Dissolve by heating and sterlize by autodaving at 121C for 25 minutes. MALACHITE GREEN


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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 MEDIUMMineral 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) 7 g

Disodium hydrogen phosphate 4 g

Sodium pyruvate 12.5

Water 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


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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 acetateSodium 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 chlorideSodium 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


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horse serum 50 ml

Glucose 25% solution 40 ml

Bacitracin 500,000 units

per 250 ml (2000 units/ml) 12.5 ml

Polyxin 500,000/l00ml

(5000 units per ml) 1.2 ml

Cyclohexamide solution( 1 0 mg/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 LactoseSucrose

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


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Lactose

Sucrose

Glucose

Ferrous ammonium sulphate Sodium thiosulphate Phenol red

Agar

Distilled water

20 g

5g

10 g 10 g1 g 0.2 g 0.2 g

0.025 g 13 g 1000 ml18: MYCOPLASMA BROTH

COMPOSITION

PPLO broth without crystal violet

Glucose

Swine or horse serum

Fresh yeast extract

Cysteine hydrocWorideNicotinamide 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. 19: SABOURAUD,S

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


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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 MEDIAThese 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 2.0 gram

Beef extract 0.5 gram

Sodium chloride 0.5 gram

Agar 2.5 gram

Water upto 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.


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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 selective, differential,

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 bacteriummultiply 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.

1STREAK PLATE METHOD

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


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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 countingcolonies 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.

QUESTIONSWhat 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?


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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. Thejar 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.


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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 inoculationwith 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 inthe 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.


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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: ELEVATIONFlat

Raised, Convex,

Pulvinate,

D: SURFACE

Umbonate


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Smooth glistening,

E: COLOUR

Green, Yellow,

Rough, Wrinkled,

Black, Pink, Red,

Dry / PowderyWhite

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 variousmorphological 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


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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 thetop.

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.


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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

tain 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.

% solution of safianin in 95% alcohol. Satranin (2.5% solution) Distilled water

10.0 rnl 1 00 rnl

IfPROCEDURE

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

m positive bacteria: violet colour

Examples: Species of Bacillus, Clostridium, Corynebacterium,

Streptococcus, Staphylococcus, Mycobacteria etc.

m 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 andadd 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


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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.

D 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 m1100 ml

Examine under oil immersion objectiveRESULTS

Acid fat positive bacteria: red colour

Examples: Mycobacteria, Nocardia, Actinomycetes etc.

Acid fast negative bacteria: blue colourExamples: All other bacterial species expect

those depicted above.

Note: All gram positive bacteria are not acid fast positive, but all acid fast positivebacteria 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 b: Welch's method

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


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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 bedecolourized 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: Wash with water and counter stain with safranine for 30 seconds.

4: Wash with water and blot dry, observe under oil immersion objective.

RESULT

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


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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 : Apply vaseline to the edge of cavity of the slide.

2: 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: Turn the slide to bring the side of the coverslip upward.

5: 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 Acid fast positve Acid fast negative

bacteria bateria

(Carbol fuchsin +steaming) Acid alcohol Methylene blue

What is the significance of capsule formation in bacteria?


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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?


36/59

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 skillin 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


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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 Magnification

Objective(X) Ocular(X) (X)

Low power 10 10 100

Dry High power 45 10 450

Oil imersion 100 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 0n = refractive index of the medium between slide and objective0 = 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


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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 specimenpass 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 ofocular micrometer covering the full length of bacteria.

Suppose 3 divisions are fully covering the length of bacteria. It mean total length of bacteria is3xO.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


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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 systembelow 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?


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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?


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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


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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 dryingof 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?


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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 manyareas 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 Yersinia enterocolitica

Treponema pallidum Bovine cardiolipin

Intestinal Microflora Human blood group antigens

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:

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


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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

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