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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?
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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