TAMILNADU VETERINARY AND ANIMAL SCIENCES UNIVERSITY

Training Manual OnBACTERIOLOGICAL TECHNIQUES USED IN ANIMAL

DISEASE DIAGNOSIS

(Training to the Laboratory technicians from Animal Husbandry department, Government of Bhutan)

Dr. Parimal RoyDr. S.SureshkannanDr. G. Balakrishnan

CENTRAL UNIVERSITY LABORATORYCENTRE FOR ANIMAL HEALTH STUDIES

MADHAVARAM MILK COLONY, CHENNAI - 600 051.Telephone: 044-25551581; Fax: 044-25551577;

Email: culcahs@rediffmail.com

INDEX

1

01. Sterilization

The explicit identification of etiological agent depends highly on the use of sterile

laboratory accessories and procedures. To avoid contaminants, one must remove or kill all

microorganisms from equipment and media used for microbiological work to eliminate or

reduce the possibility of unwarranted contaminants. All the equipments such as glassware,

scalpels, needles, forceps, etc., and the media for culturing microorganisms must be sterilized

thoroughly using principles of aseptic technique.

Sterilization: Means complete destruction of all-forms of microbial life. A sterile object, in

the microbiological sense, is free of all living microorganisms. There is no such thing as a

"practically sterile" or "nearly sterile"; it is either sterile or it is not sterile.

Methods of Sterilization

S.No Title Page No.1. Sterilization

1.1 Physical Method 1-41.2 Chemical method of Sterilization 41.3 Gaseous method 4-51.4 Filters 5-6

2. Culture and identification of bacteria2.1 Bacteriological media and different media to be used 7 -122.2 Sample processing and culture flow chart 132.3 Bacterial genus identification flow chart 142.4 Streaking methods 15-162.5 Bacterial colony morphology 17-182.6 Maintenance and preservation of pure culture 19-21

3. Staining of bacteria3.1 Preparation of Clean slide 213.2 Grams Staining 22-233.3 Alternative test for Grams Staining 233.4 Ziehl - Neelsen (ZN) Staining 23-243.5 Staining Technique for Anthrax bacilli 24

4. Secondary level identification of Bacteria by biochemical tests4.1 Biochemical tests procedures and observations 26-334.2 Gram negative bacteria Biochemical tests 344.3 Gram Positive bacteria Biochemical tests 35-36

5. Biochemical tests 376. Antimicrobial Susceptibility testing

6.1 General guidelines 38-396.2 Interpretation 39-416.3 Antimicrobial sensitivity chart 41-42

7. Coliform count in water samples 438. Laboratory test for fungus 44-469. Diseases and Materials to be collected from Livestock and poultry 47-52

References 53

2

There are many methods of sterilization but most of them fall under the broad classification

of physical, chemical and gaseous ones.

1.1 Physical Methods of Sterilization

A. Dry Heat

(a) Flaming : Small objects, e.g., inoculating loops and needles that are not easily damaged

by heat, can be sterilized by thrusting them into the flame dull-red heating to a temperature

high enough to destroy any organisms present upon the surface.

(b) Hot-air sterilization : In this method the glass wares are first dried properly, wrapped in

brown paper and then exposed to hot-air in electric or gas oven to a temperature of 160°C for

twohours. This treatment is sufficient for complete sterilization because at this temperature

destruction of all living cells and viable spores take place due to destructive oxidation of the

cell contents. Uniform heating depends upon proper loading in the oven. A further rise in

temperature may char the paper or cotton plug.

Hot-air oven is operated by electric power and used for sterilizing glasswares, e.g.,

Petri dishes, flasks, tubes, pipettes and other glass wares in microbiological laboratories. The

walls of the sterilizer are made of stainless steel or aluminium and are so devised that the

heat conduction from inside to outside is completely prevented.

The oven consists of a big chamber into which the materials to be sterilized are kept.

A fan is set at the bottom of the oven which forces steam of hot dry air circulating through

the chamber resulting in rise in chamber-temperature to sterilize the materials. A

thermometer is fitted for recording the temperature of the oven. Temperature at 160°C

sterilizes the glassware’s in a period of two hours.

HOT AIR OVEN1.Exhaust 2. Diffusion Wall 3. Glass Wool Insulation

3

4. Air Flow Sampler 5. Turbo Blower 6. Motor

A schedule of time and temperature for sterilization with dry air is as follows:

B. Moist Heat

Moist heating is more efficient in sterilization than the dry heating because heat conduction is

less rapid, the process takes much longer time and the death rate is lower in dry heat as

compared to the moist heat. Culture media, aqueous solutions, cloths, rubber and other

materials that would be destroyed by dry heating are sterilized by moist heating. Following

are the commonly used methods of moist heat sterilization.

Tyndallization or

Intermittent sterilization

Certain media containing

gelatin, milk and sugars get

adversely affected by heating

at high temperature and it would, therefore, be prudent to use intermittent (also called

'fractional') sterilization with the help of earlier described Arnold Steam Sterilizer. This

method involves heating the material at 100°C for 30 minutes on three successive days with

incubation periods in between.

Resistant spores germinate during the incubation periods; the newly formed

vegetative cells get destroyed on subsequent exposure to heat. The disadvantage of this

process is that it is time consuming and non-nutrient solution can not be sterilized by this

method because resistant spores may not germinate but remain dormant in such solution.

(c) Steam Under Pressure : This method is useful for sterilization of media as well as

apparatus. The laboratory apparatus designed to use steam under regulated pressure is called

an autoclave. The autoclave is an essential unit of equipment in every microbiological

laboratory.

Principle

The steam is allowed to form in the inner cylinder of an autoclave by heating water.

The steam pressure inside the cylinder increases with the time of heating and the steam valve

Temperature (°C) Time (minutes)120 480140 180150 150160 120170 60180 20

4

is shut off. The pressure developing inside can be read out by a pressure gauge.

Generally an autoclave is operated at a pressure of approximately 15 lbs/in2 (121°C).

The time of operation to achieve sterility depends on the nature of the material being

sterilized, the type of container, and the volume. For example, test tubes of liquid media can

be sterilized in 10-15 minutes at 15 lbs/in2 (121°C) the same media in 10 litre quantities

would require an hour or more at the same temperature for complete sterilization. In general

sterilization is done at variable heat penetration time, constant sterilization and arbitory

safety margin time; when volume of the liquid is less than 50 ml taken in thin glass

containers, heat penetration time is 7 minutes, sterilization time is 5 minutes and safety

margin is 3 minutes.After the desired time of exposure to steam under pressure, the supply of

heat is cut off and steam pressure in the autoclave allowed to come down to zero before

opening the lid for removal of sterilized materials.

Horizontal Autoclave

(Source - http://www.microbiologyprocedure.com)

Sterilization of test tubes containing liquid media

Exposure periods required for aqueous solution or liquids in various containers affording a

reasonable factor of safety for sterilization by autoclaving

Container Size Minutes exposure at 250-254ºF (121-123ºC)Test tubes 18 x 150 mm

32 x 200 mm38 x 200 mm

13-1712-1415-20

Erlenmeyer flask 50 ml500 ml1000 ml2000 ml

12-1417-2220-2530-35

Fenwal flask 500 ml1000 ml2000 ml

24-2825-3040-45

5

Milk-dilution

bottle

200 ml 13-17

Serum bottle 9000 ml 50-55

1.2 Chemical Methods of Sterilization

A variety of non-volatile chemicals are generally used in the laboratory to sterilize

discarded glassware, desk, hand gloves, etc. The primary objective of the use of such

chemicals is to kill potentially dangerous microorganisms present on such articles, and also

to reduce the laboratory atmosphere from fungal spores. There are wide varieties of

disinfectants, some important examples are as follows:

Chemical group Name of the Chemical

Halogens Chlorine Chlorine and its compounds

Work as general disinfectant and sanitizer

Iodine and Iodophores AntisepticAlcohols Ethyl and Isopropyl Skin antisepticHeavy metals Mercuric chloride and

organomercurialsUseful disinfectant for surface sterilization of bench tops, inanimate objects; orgnanomercurials as antiseptics for skin

Phenolic compounds Lysol, Cresol etc. Germicidal agents, effective against wide range of microorganisms

Quarternary ammonium compounds

Alkyl-dimethyl-benzyl-ammonium chloride etc.

Skin antiseptic, disinfectant for utensils.

Nitrates Silver nitrate Disinfectant for the surface of test materialsAldehydes Formaldehyde Use as microbiostatic; it is less commonly used

because of its irritating vapour

1.3 Gaseous Methods of Sterilization

Ethylene oxide

By far, the most effective and useful gaseous disinfecting agent known is ethylene

oxide (C2H4O). Ethylene oxide is used only in special cases when sterilization is not possible

by any other easy method because the gas is explosive and toxic to man. The vapour of

ethylene oxide is used under pressure in a special equipment. This vapour is highly toxic to

viruses, bacteria and fungal cells and also to the heat resistant spores and endospores.

Ethylene oxide is now widely used in hospitals and in industries for sterilizing heat

sensitive materials. In hospitals, it is used for disinfecting chemical respirators, heart-lung

machines, opthalmoscopes, etc.

Polymer of formaldehyde

In recent years, a polymer of formaldehyde, namely, paraformaldehyde is being used

as bactericidal, sporicidal and virucidal agent. Its action is rapid at temperature of 54-59°C

and at relative humidity of 82-90%..

6

1.4 Filters:

There are different types of filters used in microbiology viz. 1.Seitz filter 2. Sintered glass

filter 3. Membrane filter

(i) Seitz Filter (Asbestos filter).

This filter consists of 2-6 mm compressed asbestos fibre filter sheet. A variety of

filter sheets containing different pore sizes are available in discs or squares ready for use and

work satisfactorily only for a few hours. The medium to be filtered (sterilized) is poured into

the funnel-like structure and drawn through filter sheet by vacuum.

When the filtration is complete the filter sheet is discarded and the filtrate is obtained.

A modified Seitz filter in which vacuum-drawn filtrate technique has been replaced by

centrifugal technique is also used now-a-days where the filter is mounted on a centrifuge

which forces the filtrate into the tube.

Seits filter (asbestos filter) Membrane Filter

(Source -

http://www.microbiologyprocedure.com)

Diagrammatic representation of

graded filtration principles

7

(Source - http://www.microbiologyprocedure.com)

(ii) Membrane or Molecular Filter

A new type of filter called “membrane" or 'molecular' filter has been developed in

recent years. Unlike bacteriological filters which retain only bacteria, membrane filter retains

all forms of microorganisms according to their pore size. Examples are syringe filters of

porosity 22 μm or 45 μm. Similar filters are used for media filtration.

These filters are made up of biologically inert cellulose esters, and are prepared as

circular membranes consisting of millions of pores of uniform and specifically predetermined

size. Membrane filters were originally manufactured by the Millipore Filter Corporation

(USA) and therefore they are also known as "Millipore' or 'Ultra filters'.

2. CULTURE AND IDENTIFIATION OF BACTERIA

The role of suitable quality culture media for cultivation of microorganisms cannot be

over emphasized. The success in isolation of aetiological agents depends on selective

inoculation in specific media. Only in exceptional cases, an organism could be identified on

the basis of its morphological characteristics alone. But in most cases other biochemical tests

and diagnostic aids are necessary to be performed to identify an etiological agent.

2.1 BACTERIOLOGICAL MEDIA

Types of media

Bacteriological media can be broadly sub-divided into four categories.

1. Generalised culture media

Generalised culture media are routinely employed in a laboratory for primary isolation of an

organism. This media will support the growth of many type of bacterium in general. e.g.

nutrient broth, nutrient agar, Brain Heart Infusion (BHI) broth, Tryptose Phosphate broth

etc..

2. Enriched media

8

Certain organisms do not grow on ordinary nutrient media. They require growth- promoting

ingredients such as blood, glucose, serum, egg, etc. The media containing ingredients which

enhance their growth-promoting qualities are enriched media e.g. blood agar, chocolate agar

and loeffler medium.

3. Enrichment media

Enrichment media are liquid media containing chemical constituents which inhibit some

normal flora and allow pathogens which may be present in very small number in the

specimen, to grow unhampered and thus enriching them. Isolated colonies of these organisms

may be obtained by subculturing onto solid media. e.g. Selenite broth, tetra thionate broth for

the enrichment of Salmonella and listeria enrichment broth for Listeria spp.

4. Selective and differential:

Selective and differential media are used to isolate or identify particular organisms.

Selective media allow certain types of organisms to grow, and inhibit the growth of other

organisms. The selectivity is accomplished in several ways. For example, organisms that can

utilize a given sugar are easily screened by making that sugar the only carbon source in the

medium. On the other hand, selective inhibition of some types of microorganisms can be

achieved by adding dyes, antibiotics, salts or specific inhibitors which affect the metabolism

or enzyme systems of the organisms. For example, media containing potassium tellurite,

sodium azide or thallium acetate (at concentrations of 0.1 - 0.5 g/l) will inhibit the growth of

gram-negative bacteria. Media supplemented with penicillin (5-50 units/ml) or crystal violet

(2 mg/l) will inhibit the growth of gram-positive bacteria. Therefore, tellurite agar is used for

selective isolation of gram-positive organisms, and nutrient agar supplemented with

penicillin can be used to select gram negative organisms.

Differential media are used to differentiate closely related organisms or groups of organisms.

Owing to the presence of certain dyes or chemicals in the media, the organisms will produce

characteristic changes or growth patterns that are used for identification or differentiation. A

variety of selective and differential media are used in medical, diagnostic and water pollution

laboratories, and in food and dairy laboratories. Here are few commonly used selective and

differential media described below

1. Blood agar (Enriched media) – It enhance growth and also differentiates haemolytic

with non haemolytic organism.

9

2. Mac Conkey agar–(Differential media) - Differentiates lactose fermenting and non

lactose fermenting bacteria).

3. Xylose Lysine Deoxycholate agar (XLD) – (Differential media) – Differentiates

various enteric organisms.

4. Sabourauds Dextrose agar (SDA) – Selective isolation of fungus and yeasts

5. Manitol salt agar (MSA) – Selective isolation of Staphylococcus spp.

6. Edwards medium - Selective isolation of Streptococcus

But in diagnostic bacteriology it is better we could streak the specimen in one generalized

medium along with blood agar and two differential ,medium of suspected pathogen e.g. for

enteric pathogen we should streak on Mac Conkey and XLD agar.

Blood Agar:

A non-selective medium enriched medium for the isolation and cultivation of many

pathogenic and non-pathogenic microorganisms like Staphylococcus, Streptococci etc. The

medium is often used to observe different forms of haemolysis from pathogenic

microorganisms.

Composition:

Ingredients Grams/Litre

Meat extract - 10.0

Peptone - 10.0

Sodium chloride - 5.0

Agar - 15.0

Final pH 6.8+/-0.2 at 37°C

Suspend 40 g of dehydrated medium in 1 litre of distilled water and boil it to dissolve the

suspended agar completely. Sterilize by autoclaving at 121°C for 15 minutes. For the

addition of defibrinated blood, cool the autoclaved medium to 45-50°C and add aseptically

6% (5-10%) of sterile defibrinated blood. Check for sterility by keeping the plates overnight

in incubator. Store the plates below 8°C, protected from direct light. Store dehydrated

powder, in a dry place, in tightly-sealed containers at 2-25°C.

Principle and Interpretation:

Meat extract and Peptone provide nitrogenous compounds, vitamins, carbon, sulphur

and amino acids in Blood Agar Base. The medium contains sodium chloride for the osmotic

balance. Blood Agar Bases are relatively free of reducing sugars, which have been reported

to adversely influence the hemolytic reactions of beta-hemolytic streptococci. Sheep blood

gives best results for Group A Streptococci. When horse blood is used, Haemophilus

10

haemolyticus colonies produce haemolysis and mimic Streptococcus. Haemolytic patterns

may vary with the source of animal blood or type of base medium used. Slight acidic pH (6.8

± 0.2) favours distinct hemolytic reaction and is advantageous for cultivation of Streptococci

and Pneumococci. The low pH helps in stabilization of red blood corpuscles and favours the

formation of clear haemolysis zone.

Chocolate agar :

Preparation of chocolate agar is same as in blood agar, But after the addition of blood, the

media is kept in water bath for about 15 minutes (until haemolysis occurs) at 72°C .

Chocolate agar containing 10% sterile defibrinated blood is suited for the isolation of

Haemophilus and Neisseria species. Normally X factor (Haemin) and V factor (Nicotinamine

Adenine Dinucleotide) is required for the growth of Haemophilus organisms. While the

media is kept in water bath at 72°C, RBCs breaking down takes place and release the

required factors in to the medium. For the selective isolation of tubercle bacilli, addition of 1

% glycerol and 25 % human blood is recommended.

MacConkey’s Agar

MacConkey’s agar is a differential plating medium used in the detection and isolation

of all types of enteric bacterium. It is generally used for differentiating strains of Salmonella

from the members of the coliform group which ferment lactose sugar; however, the medium

supports the growth of all Salmonella and Shigella strains and gives good differentiation

between these enteric pathogens and the coliform group. When grown on MacConkey’s

medium, colonies of coliform bacteria are pink in color and are surrounded by a zone of

precipitated bile; Klebsiella organism produce partial pink coloured mucoid adjoined

coalescent colonies; Salmonella, Shigella and Proteus organisms produce colour less

colonies. These reactions are due to the acid produced by the fermentation of lactose. The

acid end-products act on bile salts, and neutral red is absorbed by the precipitated salts.

Colonies of these organisms are noncolored and transparent. The growth of Grampositive

organisms is inhibited because of the crystal violet and bile salts in the medium.

Eosin Methylene Blue Agar (EMB)

Eosin methylene blue agar is a differential medium used for the detection and

isolation of Gram-negative intestinal pathogens. A combination of eosin and methylene blue

is used as an indicator and allows differentiation between organisms that ferment lactose and

those that do not. Saccharose is also included in the medium because certain members of the

Enterobacteria or coliform group ferment saccharose more readily than they ferment lactose.

In addition, methylene blue acts as an inhibitor to Gram-positive organisms. Colonies of E.

11

coli normally have a dark center and a greenish metallic sheen, whereas the pinkish colonies

of Enterobacter aerogenes are usually mucoid and much larger than colonies of E. coli.

Mannitol Salt Agar (MSA)

Mannitol salt agar is a selective medium used for the isolation of pathogenic

staphylococci. The medium contains mannitol, a phenol red indicator, and 7.5% sodium

chloride. The high salt concentration inhibits the growth of most bacteria other than

staphylococci. On MSA, pathogenic Staphylococcus aureus produces small colonies

surrounded by yellow zones. Mannitol fermentation is the reason why S. aureus produce

yellow coloured colonies due to acid production, which changes the indicator from red to

yellow.

Microorganisms of Veterinary importance and some specific media used

S.No. Name of the organisms and

disease

Media used with the Media Catalogue

No. of Himedia and Difco

Staining

1. E.coli

Colibacillosis

MacConkey agar (M082A-50G),

EMB agar (M317-100G)

Gram negative rods

2. Salmonella spp.

(Salmonellosis)

MacConkey agar (M082A-50G),

Brilliant green agar (M021D500G),

XLD (MM 016-100G),

SS agar (M108 100G),

Hektoen agar (M467-100G),

Gram negative rods

12

3. Campylobacters

(Campylobacterisis)

Thiol medium (M 852-500G),

Skirrow agar (FD 158 Selective

supplement to Campylobacter agar),

MacConkey agar (M082A-50G),

Butzler medium (FD007

supplement)

Gram negative rods

4. Pasteurella multocida

(Fowl cholera)

Blood agar (M027-100G),

BHI agar (M211-100G),

DSA agar (M183-500G)

Bipolar

Gram

negative rod 5. Mannhermia haemolytica

(Haemorrhagic Septicaemia)

Blood agar (M027-100G),

MacConkey agar (M082A-500G)

Bipolar

Gram

negative rod6. Haemophilus paragallinarum

(Infectious Coryza)

Chocolate Agar ( Modified from

Blood agar),

Levinthol medium (M472-500G),

Blood agar (M027-100G)

Gram

negative rod

7. Brucella spp

(Brucellosis)

Albimi medium (M074 500G),

Columbia Agar (MU144 500G),

Dextrose starch agar (M183-500G)

Modified acid fast

organisms

8. Actinobacillus lignieresi

(Wooden tongue)

Blood agar (M027-100G)

MacConkey agar (M082A-50G)

Gram

negative rod 9. Mycoplasmas spp.

(Mycoplasmosis)

PPLO Agar (Difco-BD 255420),

Ox heart infusion agar (Prepared at

Laboratory)

Gram negative

pleomorphic

10. Leptospira serovars

(Leptospirosis)

EMJH medium,

Korthof medium (M457-100G),

Fletcher’s medium (M239-100G)

Gram negative

spirochaete

11. Staphylococcus spp.

Mastitis, Bumbled foot)

Mannitol salt agar (MU118-100G),

Baird-Parker medium (ME 043-

100G), Purple agar (Maltose agar)

Blood agar (M027-100G)

Gram

positive cocci in

bunches

13

Note: Catalogue No. starts with M are Himedia products and others are Difco products.

12. Streptococcus spp.(Mastitis,

Strongyles in Equine )

Edward’s medium (M748-100G),

Blood agar (M027-100G)

Gram positive cocci in

chains 13. Bacillus anthracis

Anthrax

PLET agar (M269 -500G),

Blood agar (M027-100G)

Gram positive rods

14. Clostridium perfringens

(Enterotoxiemia)

Cooked meat medium (M149-

100G),

Blood agar (M027-100G)

Gram

positive rod

15. Clostridium chauvoei

(Black quarter)

Cooked meat medium (M149-500G)

Blood agar (M027-100G)

Gram

positive rod16. Clostridium tetani

(Tetanus)

Gram

positive rod Drumstick

bacilli 17. Listeria monocytogenes

(Listeriosis)

Blood agar (M027-100G) Gram

positive rod 18. Erysipelothrix

rhusiopathiae

(Swine erysipelas)

Serum agar (Prepared at Lab),

Blood agar (M027-100G)

Gram

positive rod

19. Actinomyces bovis

(Locked/Lumpy jaw)

Dorset egg medium (SL 025),

Loeffler’s serum (M1189-100G),

BHI agar (M210 -500G),

Glucose broth (M070),

Thioglycollate broth (M010-100G)

Gram

positive rod

20. Mycobacterium spp

(Tuberculosis)

Dorset egg medium (SL 025),

Lowenstein-Jensen’s medium

(M198, FD019-Hi-Media),

Stonebrinks medium

Acid fast orgnisms

21. Mycobacterium

Paratuberculosis

(Johne’s disease)

Herrold’s egg-yolk medium Acid fast orgnisms

14

2.2 SAMPLE PROCESSING AND CULTURE FOR ISOLATION OF BACTERIA

Sample(processed without delay and inoculated into appropriate media)

Brain heart infusion / nutrient broth4 to 6 hours incubation 37ºC

Inoculation into solid media (overnight growth)Colonies in different culture media

Blood agar MacConkey agarBrilliant Green

agar

Mannitol salt agar / Edwards

mediumNutrient agar

Sabourauds Dextrose agar

Beta haemolytic (Partial haemolysis) in Staphylococcus; Hot cold Phenomenon on refrigeration

Pink flat colonies (Lactose fermenting) with bile precipitation-Escherchia coli Pink Convex colinies without bile precipitation – Enterobactor sp.Partial pink mucoid colonies- Klebsiella sp.

Green colonies –Escherchia coli ;Pink colonies with pink background- Salmonella; Bright yellow colonies - Proteus; Yellow mucoid colonies- Klebsiella pneumoniae

Yellow colonies with yellow background – Staphylococcus aureus White colonies-Staphylococcus intermedius Staphylococcus epidermis

Medusa head colonies – Bacillus sp. Bluish green colour change – Pseudomonas sp.

Rapidly growing velvety granular / bluish green growth – Aspergillus fumigatusBlack and granular growth- Aspergillus niger Yellowish green with fluffy texture – Aspergillus flavus

Non-haemolytic minute pin point colonies in Pasteurella multocida

Colourless colonies- Salmonella, Shigella, Proteus No growth- Pasteurella sp. (except P.trehalosi)

No growth- Pasteurella Sp.

Only salt tolerant organism will grow(Halophilic)

This will support many type of organisms (The colony growth from this agar should be used for catalase and oxidase test)

This will inhibit the growth of most of the bacteria in addition Chloramphenicol and Cycloheximide increase selectivity of pathogenic fungi.

15

Grams staining

Gram +Gram --_

CampylobacterHelicobacterVibrio

Catalase

Catalase Positive

Catalase negative

MicrococcusStaphylococcusBacillusListeriaCorynebacteriumActinomyces*Rhodococcus

Streptococcus (α β γ)Enterococcus (α)Erysipelothrix (α)A.pyogenes (β)Nocardia*

If rods Motile

If β Staph Coagulase & MSA, Maltose agar

BacillusListeria

If α Strep, Bile SaltEsculin hydrolysis

If β Strep, Patho- Dx Latex Test kit

Oxidase +Oxidase --

Growth on MAC Growth on MAC

Growth + Growth --

Actinobacillus (pink)Aeromonas (PLF)Bordetella (Clear)Pseudomonas (Clear)Acinetobacter

BrucellaHaemophilusMoraxellaPasteurella

Growth + Growth --

Citrobacter YersiniaEnterobacter ProteusEscherchia SalmonellaKlebsiella Serratia

Brucella ovis

Lactose +/--

Fecal sample for Salmonella

Set up Selenite Broth, Brilliant Green Agar (BGA), MAC, Xylose Lysine Deoxycholate, HEA

Black colonies on Hektoen enteric Agar (HEA) ,Non Lactose fermenting (NLF) on Mac Conkey

No Black colonies on HEA

Selenite broth subculture on BGA

Pink colonies

Tiple Sugar Iron Agar & Citrate TSI (Alk/H

2S

Grouping by Salmonella antiserum

2.3 Bacterial genus identification flow chart

16

2.4 Streaking methods and isolation of pure culture

Common Methods of isolation of pure culture

Pure culture of microorganisms that form discrete colonies on solid media, e.g.,

yeasts, most bacteria, many other fungi, and unicelluar microalgae, may be most commonly

obtained by plating methods such as Streak plate method, Pour plate method and Spread

plate method.

But, the microbes that have not yet been successfully cultivated on solid media and

are cultivable only in liquid media are generally isolated by serial dilution method.

Streak Plate Method

This method is used most commonly to isolate pure cultures of bacteria. A small

amount of mixed culture is placed on the tip of an inoculation loop/needle and is streaked

across the surface of the agar medium. The successive streaks "thin out" the inoculum

sufficiently and the microorganisms are separated from each other. It is usually advisable to

streak out a second plate by the same loop/needle without re-inoculation.

These plates are incubated to allow the growth of colonies. The key principle of this

method is that, by streaking, a dilution gradient is established across the face of the Petri

plate as bacterial cells are deposited on the agar surface. Because of this dilution gradient,

confluent growth does not take place on that part of the medium where few bacterial cells are

deposited

Various methods of streaking

(Source - http://www.microbiologyprocedure.com)

Presumably, each colony is the progeny of a single microbial cell thus representing a clone of

pure culture. Such isolated colonies are picked up separately using sterile inoculating loop/

needle and re-streaked onto fresh media to ensure purity.

17

Pour Plate Method

This method involves plating of diluted samples mixed with molten agar medium.

The main principle is to dilute (1/10) the inoculum by transferring 1ml of specimen culture to

9 ml of liquid media, after proper mixing, 1ml will be transferred to the subsequent tube) so

as to permit a thorough distribution of bacterial cells within the medium. Here, the mixed

culture of bacteria is diluted directly in tubes containing molten agar medium maintained in

the liquid state at a temperature of 42-45°C (agar solidifies below 42°C). The contents of

each tube are poured into separate petri plates, allowed to solidify, and then incubated. When

bacterial colonies develop, one finds that isolated colonies develop both within the agar

medium (subsurface colonies) and on the medium (surface colonies). These isolated colonies

are then picked up by inoculation loop and streaked onto another Petri plate to insure purity.

Pour plate method has certain disadvantages as follows: (i) the picking up of subsurface

colonies needs digging them out of the agar medium thus interfering with other colonies, and

(ii the microbes being isolated must be able to withstand temporary exposure to the 42-45°

temperature of the liquid agar medium; therefore this technique proves unsuitable for the

isolation of psychrophilic microorganisms.

However, the pour plate method, in addition to its use in isolating pure cultures, is

also used for determining the number of viable bacterial cells present in a culture.

Diagrammatic representation of Pour Plate Method

A. Media/dilution B. Pouring of the

plate; and

C. Colony development after incubation. Control

consists of the sterilized plating medium alone

(Source - http://www.microbiologyprocedure.com)

18

The isolated colonies are picked up and transferred onto fresh medium to ensure purity. In

contrast to pour plate method, only surface colonies develop in this method and the

microorganisms are not required to withstand the temperature of the molten agar medium.

2.5 COLONY MORPHOLOGY

MacConkey agar: LF/NLF colony Haemolysis on blood agar

Lecitheniase activity on egg yolk agar Coli form count agar –Pink coli form

Pseudomonas aerogenosa mucoid colonies E.coli Metallic sheen on EMB agar

Staph sp. on Mannital salt agar Medusa head - Bacillus colony

19

Listeria on PALCAM agarPseudomonas : chromogenic colony

Salmonella on XLD Proteus-Swarming on blood agar

Vibrio colonies on TCBS agar Staph.on Baird parker agar Source : http://www.microbelibrary.org/ASMOnly/details.asp

Spread plate method

(Source - http://www.microbiologyprocedure.com)

20

The inoculum is subjected to serial dilution in a sterile liquid medium, and a large

number of tubes of sterile liquid medium are inoculated with aliquots of each successive

dilution and spread over the plate. Plates are incubated and observed for bacterial growth.

2.6 Maintenance and Preservation of Pure Cultures

Once a microorganism has been isolated and grown in pure culture, it becomes

necessary to maintain the viability and purity of the microorganism by keeping the pure

cultures free from contamination. Normally in laboratories, the pure cultures are transferred

periodically onto or into a fresh medium (sub-culturing) to allow continuous growth and

viability of microorganisms in slant. The transfer is always subject to aseptic conditions to

avoid contamination.

Agar slant Preparation

Agar slants are prepared to inoculate microbial culture. To prepare agar slant a

required number of culture tubes are taken and about 5 –10 ml of liquefied agar medium is

poured in each of them.

(Source - http://www.microbiologyprocedure.com)

The tubes are cotton-plugged and

sterilized in an autoclave. After

sterilization the tubes are taken out and are

placed in slanting (sloping) position for

sometimes; the tubes get cooled and the

medium in them is solidified resulting in a

slopy surface.

21

Inoculation Procedure

1. Immediately after deplugging and sterilizing the mouth of the tubes, the loop/needle is also

flame-sterilized and is inserted within the agar surface of the inoculum containing tube to

quench the heat, and a small bit of inoculum is taken on the loop/needle tip.

2. The inoculum containing loop/needle is taken out and brought in within the agar slant

containing tube where the inoculum is just rubbed on the surface of the agar slant.

3. All the steps starting from plug removal from the mouth of the tubes to the rubbing of the

inoculum on the surface of the agar slant should be done quickly to avoid contamination.

4 When inoculation is complete, the open mouths of tubes and the cotton plugs are sterilized

by flame and the cotton plugs are replaced.

5. The inoculated tube is incubated under suitable temperature to favour rapid growth of

microorganisms.

Since repeated sub culturing is time consuming, it becomes difficult to maintain a

large number of pure cultures successfully for a long time. In addition, there is a risk of

genetic changes as well as contamination. Therefore, it is now being replaced by some

modern methods that do not need frequent subculturing. These methods include refrigeration,

paraffin method, cryopreservation, and lyophilization (freeze drying).

Refrigeration

Pure cultures can be successfully stored at 0-4°C either in refrigerators or in cold-

rooms. This method is applied for short duration (2-3 weeks for bacteria and 3-4 months for

fungi) because the metabolic activities of the microorganisms are greatly slowed down but

not stopped. Thus their growth continues slowly, nutrients are utilized and waste products

released in medium. This results in, finally, the death of the microbes after sometime.

Paraffin Method

This is a simple and most economical method of maintaining pure cultures of bacteria

and fungi. In this method, sterile liquid paraffin in poured over the slant (slope) of culture

and stored upright at room temperature. The layer of paraffin ensures anaerobic conditions

and prevents dehydration of the medium. This condition helps microorganisms or pure

culture to remain in a dormant state and, therefore, the culture is preserved for several years.

22

Glycerol Preservation:

Glycerol prepared in 10% v/v with autoclaved BHI broth could be used as

preservative and maintenance medium for bacterial culture. After inoculation of culture the

medium is incubated for 4hours and then kept in -20°C for as long as 2years. At the time of

requirement the glycerol culture is revived by sub culturing in a suitable medium.

Cryopreservation

Cryopreservation (i.e., freezing in liquid nitrogen at -196°C) helps survival of pure

cultures for long storage times. In this method, the microorganisms of culture are rapidly

frozen in liquid nitrogen at -196°C in the presence of stabilizing agents such as glycerol that

prevent the formation of ice crystals and promote cell survival.

Lyophilization (Freeze-Drying)

In this method, the culture is rapidly frozen at a very low temperature (-70°C) and

then dehydrated by vacuum. Under these conditions, the microbial cells are dehydrated and

their metabolic activities are stopped; as a result, the microbes go into dormant state and

retain viability for years. Lyophilized or freeze-dried pure cultures and then sealed and stored

in the dark at 4°C in refrigerators. Freeze-drying method is the most frequently used

technique by culture collection centres.

3. STAINING BACTERIA

3.1 Preparation of Clean slide:

It is essential to use clean, dry, dust-free slides: remember that grease and residual

detergent are equally liable to spoil a blood film and interfere with staining.

New slides:

Boxes of clean grease-free slides may be used. If not, available proceed as follows.

Leave the slide overnight in a detergent solution. Then wash thoroughly in running tap-water,

rinse in distilled water if available and wipe dry with a clean linen cloth. Before use, wipe the

surface with methylated spirits (95% ethanol) or methanol and dry with a clean cloth; then

keep covered to avoid having dust settle on the surface.

Used slides:

Discard in detergent solution, heat to about 60oC for 20 minutes. Then wash in

running tap-water, rinse in distilled water if available and treat as for new slides as described

above.

23

3.2 Gram Stain:

Grams staining reaction classify bacteria broadly in to Gram positive and Gram

negative. Bacteria that decolorize easily are called Gram-negative (Pink/red) and those that

retain the primary stain are called Gram-positive (purple). Bacteria stain differently because

of differences in their cell walls. Gram-positive cell walls consist of many layers of

peptidoglycan. The crystal violet-iodine complex is larger than either the crystal violet or

iodine molecules that entered the cell and the complex cannot pass through this thick cell

wall. Gram-negative bacteria have a thin layer of peptidoglycan and an outer

lipopolysaccharide layer. The alcohol dissolves the lipopolysaccharides so that the crystal

violet-iodine complex can be washed out of the cell.

Before bacteria can be stained, a smear of bacteria must be made on a slide and heat

fixed. A smear is made by spreading a bacterial suspension on a clean slide and letting it air

dry. The dry smear is heated on a hot plate or passed through a flame several times to heat fix

it.

Prepare a bacterial smear.

• Place 10 μl of sterile water in the center of a clean glass slide.

• Mix bacteria with water drop on slide. Spread the water-bacteria mixture over an area

of about 1 inch square.

• Allow the smear to air dry.

24

• Hold the slides with forceps and heat fix the smears by heating on the hot plate for

several minutes.

Staining Procedure:

• Cover the smear with a few drops of crystal violet and leave for 1 minute.

• Turn the slide over so the smear is facing down. Wash the slide carefully over the top

with distilled water from a wash bottle over the sink until no large amounts of color

wash off. Do not squirt water directly onto the smear.

• Cover the smear with Gram’s iodine and leave for 1 minute.

• Without washing, decolorize with 95% ethyl alcohol (Decolorizer

• Wash the slide carefully over the top with distilled water

• Cover the smear with safranin and leave for 1 minute.

• Gently rinse with distilled water.

• Blot dry with paper towel or absorbent paper. Do not rub slide, as the bacterial smear

can be rubbed off.

• Observe under microscope.

• Record the observations from the microscope.

3.3 Alternative test for Grams reaction - 3 % Potassium Hydroxide

a. Add heavy inoculum of pure culture of bacteria grown on solid medium to a drop of 3

% potassium hydroxide (KOH) solution (3 grams KOH per 100 mL distilled water)

on a clean glass slide

b. Stir for about one minute, occasionally lifting the loop to look for thickening and

“stringing” of the slurry

Observation:

1. Gram positive bacteria will not appear to change the viscosity of the KOH solution

2. Gram negative bacteria will cause the KOH solution to become stringy or mucoid in

appearance and consistency.

3.4 Ziehl-Neelsen (ZN) staining :

The high mycolic acid content of certain bacterial cell walls, like those of Mycobacteria,

is responsible for the staining pattern of poor absorption followed by high retention. The

most common staining technique used to identify acid-fast bacteria is the Ziehl-Neelsen

stain, in

25

which the acid fast bacilli are stained bright red and stand out clearly against a blue

background Eg : All Mycobacteria - M. tuberculosis, M. leprae, M. smegmatis and

atypical Mycobacterium , Nocardia

Procedure :

• Cover the smear with carbol-fuchsin solution and heat under the slide with spirit lamp

to make the carbolfuchsin pierce the cell wall mycolic acid components.( Slide should

not get dried)

• Allow the slide to stand in hot solution for 5 minutes.

• Wash with running tap water.

• Decolorize with 1% Acid alcohol until light pink color appear.

• Wash in running tap water for 5 minutes.

• Cover the smear with methylene blue for 30 seconds.

• Rinse in water.

• Air dry the slide and observe under microscope

3.5 Staining technique for Anthrax bacilli

Objective

Microscopic examination of clinical material for anthrax bacilli to establish a ‘suspect’

diagnosis of anthrax and of environmental materials for the presence of anthrax spores.

Preparation of smears:

• Make two thin smears of clinical/animal material by rolling over the swabs or

spreading a small drop on a microscope slide

• Air dry and fix by dipping the slide in absolute alcohol for 30–60 seconds.

• Slide should not be heat dried to avoid distortion of morphology of the capsule.

Observe the typical morphology of the bacillus: In clinical material B. anthracis are Gram

positive thick, short, straight bacilli with square or truncated ends with parallel sides found

usually single, in pairs or chains of three or four bacilli. The chain of bacilli with truncated

and swollen ends gives a characteristic "bamboo stick" appearance.

Polychrome methylene blue stain for capsule (M’Fadyean reaction)

• This is the ideal method for demonstration of the capsule.

• Put a large drop of polychrome methylene blue on the smear to cover it completely.

• Leave for 30–60 seconds and air dry.

• Wash off (into hypochlorite solution, 10,000 ppm). A wash bottle is better than a tap.

• Examine under oil immersion.

26

• The capsule is seen clearly as pink amorphous material surrounding the blue-black

bacilli.

Other staining techniques:

Leishman and Giemsa stains are recommended for staining Cerebrospinal fluid

(CSF), other body fluids and blood, as the organisms are clearly visualized from the cellular

background.

Leishman stain

• Pour 8–12 drops of Leishman stain on the smear.

• Wait for two minutes.

• Dilute with double the volume of buffered distilled water (pH adjusted to 7.2).

• Mix by rocking the slide or with a Pasteur pipette.

• Keep for 7–10 minutes.

• Run tap water on to the slide to float off the stain.

• Drain and dry vertically in air.

• Examine under oil immersion.

The bacilli stain purple with a clear space around indicating capsule

Giemsa stain

This is used especially for staining blood smears.

• Prepare fresh Giemsa stain by diluting stock stain 1:10 in buffered distilled water (pH

adjusted to 7.2).

• Air dry films and fix in methanol for one minute• Wash off with wash bottle into hypochlorite solution.

• Flood the slide with Giemsa stain and stain for 25–30 minutes.

• Run tap water on to the slide to float off the stain and to prevent deposition of

precipitate on to the film.

• Drain, dry vertically and examine under oil immersion.

The bacilli stain purple with red capsule.

Leishman Giemsa staining

Preparation of Leishman Giemsa stain-stock solution

1. 250 ml of glycerine is heated to 60°C in a light proof container (1litre flask covered with black or brown paper).

2. 3.8g of Giemsa powder and 1 gm of Leishman powder is added to it and held at 60°C for 1 hour under constant stirring.

27

3. 250 ml of acetone free methyl alcohol is added after bringing the above solution to room temperature.

4. The solution is then filtered using Whatman No.1filter paper. The filterate is then collected in an amber coloured bottle and used as stock solution.

Staining procedure

The smear is fixed by immersing the slide in methanol for one minute.

1. One drop of the stock solution is added to one ml of tapwater. Smear is then

flooded with the diluted stain. It is allowed to act for 45 min.

2. The slide is then washed in tap water.

3. The slide is air dried and examined under oil immersion of microscope.

4. SECONDARY LEVEL IDENTIFICATION OF BACTERIA BY

BIOCHEMICAL TESTS

Most bacteria are identified and classified largely on the basis of their reactions in a

series of biochemical tests. Some tests are used routinely for many groups of bacteria (eg.

oxidase, nitrate reduction); others are restricted to a single family, genus, or species (eg.

coagulase test for staphylococci).

4.1 Biochemical Procedures and observation

1. Catalase Test

Catalase test is used to detect the production of catalase enzyme by the bacterium.

Catalase enzyme is produced by most bacteria. The enzyme break hydrogen peroxide (H2O2)

and release free Oxygen. Catalase is found in most aerobic and facultative anaerobic bacteria.

The main exception is Streptococcus spp. That’s why the test is used to differentiate between

Staphylococcus and Streptococcus.

• Dip a capillary tube into 3% H2O2 and touch a bacterial colony

• Observe the tube for bubble production

• Bubbling indicates a positive result.

2. Oxidase

The oxidase test identifies certain organisms that produce the enzyme cytochrome

oxidase. Cytochrome oxidase participates in the electron transport chain by transferring

electrons from a donor molecule to oxygen. The oxidase reagent contains a compound that

changes color when it becomes oxidized. If the test organism produces cytochrome oxidase,

the colorless reagent used in the test will detect the presence of the enzyme oxidase and,

reacting with oxygen, turn violet to purple. The oxidase test is a key test to differentiate

28

between the families of Pseudomonadaceae (oxidase positive) and Enterobacteriaceae

(oxidase negative).

• The test reagent is smeared on a filter paper or readymade reagent discs

• The target single colony is picked up in a non reacting material like glass rod or

plastic stick and then streaked on the disc

• Development of Violet colour within 15 -30seconds is considered as positive

3. Glucose Fermentation (OF Basal with 1% Glucose) –

Bacteria metabolize carbohydrates by oxidative and/or fermentative pathways.

Oxidation occurs in the presence of atmospheric oxygen (aerobic), whereas

fermentation takes place in an anaerobic environment. Metabolism of the

carbohydrate dextrose by either an aerobic or anaerobic pathway results in acid

production. The resulting acidic environment causes the Bromo Thymol Blue pH

indicator in the medium to turn from green to yellow. The presence of bubbles in

the tube indicates gas production (aerogenic). If no reaction occurs, the medium can

remain unchanged or become alkaline (blue at the surface).

• A deep butt tube (~7 mL in 16 × 125mm) is used for this test.

• With a sterile needle take a small inoculums from an isolated colony and stab to the

bottom of the tube.

• Incubate at 20-24°C for 24-48 hours. Check tubes at 24 hr for acid and/or gas

production.

RESULTS: A = acid(yellow); AG = acid + gas, N = no change or alkaline

Reaction Top of Tube Bottom of TubeOxidative A NFermentative AG or A AG or ANon-reactive N N

4. Triple Sugar Iron (TSI)

This medium can determine the ability of an organism to utilize specific carbohydrates

incorporated in a basal growth medium, with or without the production of gas, along with the

determination of hydrogen sulfide (H2S) production. TSI agar contains the three sugars in

varying concentrations: glucose (1X), lactose (10X), and sucrose (10X). It also contains the

pH indicator phenol red. If sugar fermentation occurs, glucose will be initially used and the

butt of the tube will be acidic (yellow). After glucose utilization the organism may continue

to ferment the remaining sugars. If this occurs the entire tube will become acidic. Certain

bacteria are unable to utilize any sugars and will breakdown the peptone present. Peptone

29

utilization causes an alkaline (red) shift in the medium that causes a color change from

orange to red. Blackened medium is caused by hydrogen sulfide production, which changes

ferrous sulfate to ferrous sulfide. In addition, splitting of the medium or presence of bubbles

in the butt of the tube can determine gas production.

• With a sterile needle inoculate the TSI slant by stabbing to the bottom of the tube and

then streaking the surface of the slant as the needle is drawn out of the tube. Incubate

with loosened cap.

• Read the results after 18-24 hours.

RESULTS:

A = Acid; K = Alkaline; H2S = Hydrogen sulfide produced; N =No change

Slant/Butt Color/Reaction InterpretationK/N or K/A Red/ Orange (Oxidative) or red /yellow

(fermentative)

Only peptone utilized or

only glucose-fermented A/A Yellow/Yellow and /or sucrose –fermented Glucose, plus lactose Gas Splitting or bubbles Gas production H2S Black butt Hydrogen sulfide produced 5. Carbohydrate Utilization

The following carbohydrates are normally used in laboratory to aid in bacterial species

identification: Arabinose, Rhamnose, Mannitol, Salicin, Sorbitol, and Sucrose (saccharose).

The procedures to be followed for each of these media are identical.

• Inoculate carbohydrate tube with a loop full of 18 to 24 hour pure culture.

• Incubate with loosened cap for 18 to 24 hours at 37oC. A prolonged incubation of

up to

four days may be necessary for some negative results.

RESULTS

a) Positive - Acid is produced from fermentation, which turns media yellow.

b) Negative - No fermentation of carbohydrate, media remains same.

c) Aerogenic - Gas bubbles are present within the media.

6. O/129 Discs

This test determines the sensitivity of a bacterial organism to the vibriostatic agent

2,4-diamino-6,7 di-isopropylpteridine (O/129).

• Suspend bacteria in sterile saline or PBS

• Streak suspension on plate in three planes with a cotton swab.

• Aseptically place sensitivity disc in the center of inoculum.

30

• Incubate at 20-24°C for 24 hours.

• This test can be done on the same plate as the antibiotic sensitivity test.

RESULTS: Sensitive: Zone of inhibition around disc

Resistant: Growth adjacent to disc

7. IMViC Tests

The IMViC series includes four tests:

A. Indole production B. Methyl red test C. Voges-Proskauer test D.Citrate utilization

Except for the lowercase “i”, which is added for ease of pronunciation, each of the letters in

“IMViC” stands for one of these tests. “I” is for indole; “M” is for methyl red; “V” is for

Voges-Proskauer, and “C” is for citrate. IMViC tests are particularly useful for

differentiating Escherichia coli and Klebsiella pneumoniae

A. Indole production

The ability to degrade amino acids to identifiable end products is often used to

differentiate among bacteria. Some bacteria can hydrolyze tryptophan using an enzyme

called tryptophanase. Large amounts of energy can be obtained by this hydrolysis. A

byproduct of the hydrolysis is indole, which is excreted by the organism. Indole can be

detected by reaction with Kovac's reagent (para-dimethylaminobenzaldehyde in alcohol)

which produce a red color. A red layer indicates a positive result. It is used mainly in the

differentiation of genera and species. For example; E. coli gives a positive result while

Proteus mirabilis and Klebsiella give a negative one.

Indole production is dependent upon the presence of tryptophan in the culture medium.

• A 1% solution of tryptone is generally used in tests for indole production because it

is rich in tryptophan. Bacteria to be tested are incubated for 48-72 hours.

• 5 drops of Kovac's reagent are added to the culture broth.

• Indole reacts with Kovac's reagent to give a red product in the alcoholic layer. A

yellow color indicates a negative result.

B. Methyl Red and C. Voges-Proskauer Tests (MR-VP)

The fermentation of glucose by bacteria results in end products that vary from species

to species depending on metabolic pathways that are available to them under the culture

conditions. A number of genera of Gram negative bacteria ferment glucose to produce lactic,

acetic, succinic, and formic acids (mixed acid fermentation). They also typically produce

large amounts of CO2, H2, and ethanol. Acid accumulation can reduce the pH to 5 or lower.

Other organisms ferment glucose to form only one fermentation product, usually acetic acid

31

which is converted to acetoin. MR test (methyl red) is used to determine if glucose can be

converted to acidic products like lactate, acetate, and formate, acid products that will cause

the pH to drop below 4. Methyl red, an indicator, turns red below 4.4 and will thus turn red if

acid products are present, otherwise it will remain yellow. VP (Voges-Proskauer) test is used

to determine if glucose can be converted to acetoin. Barritt’s Reagent A and B are added. The

reagents will react with acetoin, and a positive reaction will show a red color.

Procedure:

Methyl red test:

1. Inoculate the organism and incubate it for 48 hrs at 37ºC.

2. Add a few drops of methyl red to the original culture tube and mix the contents.

Results :A red color indicates a positive result. A yellow color indicates a negative result.

C. Voges-Proskauer test:

1. Inoculate the organism and incubate it for 48 hrs at 37ºC.

2. Add of Barritt's reagent A to the tube and mix.

3. Add 15 drops of Barritt's reagent B. and mix.

4. The culture should be kept on table for about 20 minutes for color development to

occur.

5. A red color indicates a positive result. A yellow color indicates a negative result.

D. Citrate Utilization

Some bacteria may be able to use organic compounds other than sugars as their sole

source of carbon. The citrate test determines if a bacterium can grow utilizing citrate as its

sole carbon and energy source. These bacteria are able to cleave citrate to oxaloacetate and

acetate via the citritase enzyme.

Simmons/Koser's citrate medium

This medium is used to test for citrate utilization. Koser Citrate Medium is prepared

so that no sources of carbon (other than sodium citrate) or nitrogen (other than ammonium

salts) are present. Bacteria that are able to use citrate as their carbon source will grow in the

medium and change the green coloured medium to blue colour change which indicates a

positive reaction.

Procedure:

1. Transfer growth from a single colony or a loopful of liquid suspension and

inoculate the Cintrated slant medium.

2. Incubate at 37º C for 18-24 hours.

3. The development of blue colour indicates a positive result while a clear green colour is

32

considered as negative.

The results of the IMViC Test can be used to differentiate between E.coli and

Klebsiella. E.coli gives ++-- results on the IMViC tests, while Klebsiella give the reverse: --

++ .

8. Coagulase Test

The test is used to detect the production of the enzyme coagulase which converts

fibrinogen to fibrin. In the laboratory, it is used to distinguish between different types of

Staphylococcus isolates (Coagulase Positive staphylococcus (CPS) or Coagulase Negative

Staphylococcus (CNS). Coagulase reacts with prothrombin in the blood. The resulting

complex causes blood to clot by converting fibrinogen to fibrin. The test is perfomed as slide

Coagulase test (for bound Coagulase) or tube Coagulase test (for free Coagulase)

A suspension of Staphylococcus culture is mixed with rabbit plasma either on a glass slide or

in small tube.

• Formation of clump in slide or gelling and clot formation of rabbit plasma in tube is

positive.

• In tube test time is noted as Coagulase positive in 2 hrs, 4hrs, 6hrs etc.

• Slide test is presumptive and tube test is confirmative coagulase test.

9. Urease

Urease is an enzyme that catalyzes the hydrolysis of urea into carbon dioxide and

ammonia. The reaction occurs as follows:

(NH2)2CO + H2O → CO2 + 2NH3

Urease broth is a differential medium that tests the ability of an organism to produce urease.

The ready made urea broth contains two pH buffers, urea, a very small amount of nutrients

for the bacteria, and phenol red as pH indicator. Urea broth should not be autoclaved but

filter sterilization is required before inoculation. Members of the genus Proteus and

Bordetella can degrade urea rapidly. The test is used to differentiate urease-positive Proteus

species from others members of the Enterobacteriaceae. Some strains of Klebsiella species

are also urease positive.

Results :

Phenol red turns yellow in an acidic environment and fuchsia in an alkaline environment. If

the urea in the broth is degraded and ammonia is produced, an alkaline environment is

created, and the media turns pink.

33

10. Nitrate reduction test

The identification of some bacteria is aided by determining if the organism can

reduce nitrate (NO3) to nitrite (NO2) or another nitrogenous compound such as ammonia

(NH3) or nitrogen gas (N2). This reaction is expressed as:

NO3 ----> NO2 ----> NH3 or N2

In order to determine if a bacteria can reduce nitrate, the test organism is inoculated

into nitrate reduction broth. Alpha-napthylamine and sulfanilic acid are added and if a red

color develops then the bacteria is capable of reducing nitrate to nitrite. If the bacteria

converts nitrate to nitrogen gas this can be detected using a Durham’s tube which will be

elevated if gas is evolved. Nitrate positive bacteria include E. coli, Salmonella, Klebsiella

pneumoniae. Enterococcus faecalis, Bacillus subtilis.

11. Decarboxylase Test (Lysine and Ornithine)

A determination of bacterial enzymatic capability to decarboxylate an amino acid to

form an amine with resultant alkalinity.

• For each isolate to be tested, it is necessary to inoculate a decarboxylase control tube

and lysine or ornithine test tube. Use very small amount of inoculum from 18 to 24

hour pure culture.

• Add 1 to 2 mL oil overlay to each tube.

• Incubate at 37°C for 24 hours

• Some time a prolonged incubation of up to four days may be necessary.

RESULTS

Test Result Lysine or Ornithine Tube Control Tube

Positive Turbid to faded purple (glucose fermented, decarboxylase produced)

Yellow (glucose fermented)

Negative Yellow (glucose fermented, decarboxylase not produced)

Yellow (glucose fermented)

Negative Purple (glucose not fermented, decarboxylase not produced)

Purple (glucose not fermented)

12. Esculin Test

To determine the ability of an organism to hydrolyze the glycoside esculin (aesculin) to

esculetin (aesculetin) and glucose in the presence of bile (10 to 40%).

• Inoculate the surface of the bile esculin slant with inoculum from an 18 to 24 hour old

pure culture.

34

• Incubate at 37°C for 24 to 48 hours.

RESULTS

a) Positive - Presence of a black to dark brown color on the slant.

b) Negative - No blackening of the medium.

PRECAUTIONS

False positives may occur with hydrogen sulfide producing organisms. Neither of the

target organisms for these protocols will, however, produce hydrogen sulfide.

13. Gelatinase

A test to determine bacterial production of gelatinase enzymes that liquefy gelatin.

• Inoculate by stabbing ½ to 1 inch deep into the nutrient gelatin media with a heavy

inoculum from an 18 to 24 hour pure culture.

• Incubate 18 to 24 hours at 20-24°C.

RESULTS

a) Positive – Media is liquefied. Weak results can be visualized by rapping the tube

against the palm of the hand to dislodge droplets of liquid from the media. Any drops

seen are considered positive.

b) Negative – No liquefaction occurs in media.

14. Motility

This test determines if a bacterial isolate is motile by means of flagella.

• Place a drop of distilled water or sterile PBS onto the center of a clean microscope cover

glass. Place an additional tiny drop in one corner of the cover glass (to adhere the cover

glass to the depression slide when it is inverted).

• Inoculate the center drop from a pure culture that is 24-48 hours old using a sterile loop,

however 6-8hrs culture will show actively motile bacterium. Carefully invert the cover

glass and place over the concave portion of a hanging drop slide. Observe for motility

using 400× magnification on a compound microscope. Care should be taken to not

interpret “drift” or “Brownian motion” as motility. Record results as motile or non-

motile.

If this method fails to show motility then:

a) Inoculate a nutrient broth with the isolate and incubate at room temperature until

growth is obtained, usually 24 hours. After incubation use a sterile loop or sterile

dropper and place a drop on a clean cover glass. Place a tiny drop of distilled water

in one corner of the same cover glass. Continue as above.

35

b) Semi-solid motility test medium can also be used. Stab the medium with a small

amount of inoculum. Incubate overnight at room temperature. If the bacterial

species is motile, the medium will become turbid with growth that radiates from the

line of inoculum. If the bacterial species is non-motile, only the stab line will have

visible bacterial growth.

4.2 MAJOR GRAM NEGATIVE BACTERIA OF VETERINARY IMPORTANCE AND ITS BIOCHEMICAL REACTIONS

ORGANISMS TSI H2S INDOLE UREA OXIDASE CITRATE MOTILITY Escherichia A/A - + - - - +Klebsiella A/A - - + - + -Enterobacter ALK/A - - - - + +Citrobacter ALK/A + + + - + +Salmonella ALK/A + - - - + +Shigella ALK/A - + - - - -Proteus ALK/A + + - - + +++Aeromonas A/A - + - + + +Providencia ALK/A - + - - + +Edwardsiella ALK/A + + - - - +Serratia ALK/A - - - - + +Pseudomonas ALK/NC - - - + + +Acinetobacter ALK/NC/ALK - - - + + -Pasteurella hemolytic

A/A - - - + - -

Pasteurella multocida

A/A - + - + - -

Pasteurella multocida (Poultry)

A/A - - - + - -

Pasteurella Pneumontropica

A/A - + + + - -

Actinobacillus A/A - - + + - -Yersina ALK/A - - + - - +Bordetella ALK/NC - - +++ + + +Moraxella ALK/NC - - - + + -Brucella ALK/NC + - ++ + - -

36

4.3 MAJOR GRAM POSITIVE BACTERIA OF VETERINARY IMPORTANCE AND ITS BIOCHEMICAL DIFFERENTIATIING FACTORS

ORGANISMS

Nit

rate

Lac

tose

Coa

gula

se

DN

AS

E/ A

lkal

ine

phos

ph

atas

e

hae

mol

ysis

Man

nit

al

Suga

r fe

rmen

tati

on

Ure

ase

Mal

tose

/ P

urp

le a

gar

Aes

culin

hyd

roly

sis

Gel

atin

CA

MP

Lab

ani

mal

Mot

ilit

y

Lec

ith

inas

e

Staph. aureus + + +

HC

+ VP, G, M, MAN, L, S

D + + +

Staph. Intermedius + + +

HC

D + +

Staph. epidermidis - D D - + +Streptococcus pyogenes

- + - + V Sorbital, trehalose, Lactose

-

Strep. agalactia - + + - +Strep.dysagalactia - + + Bluish

grey

-

Strep.Uberis - + - +

Dark

-

Coryne.Pseudotuberculosis

V + D + - +

Coryne. Renale. - + Casein, G ++ -Coryne. ovis + G,L,M +Rhodococcus equi + + + +Mycobacteriumtuberculosis

+ - + GP die

Mycobacterium bovis

- + R, GP die

Mycobacterium avium

- - R,GP survive

37

Listeria monocytogene

- + - L-R,

G, M

+ + GP die, R-antons + test

+

Erysipelothrix rhusiopathiae

NG +

H2S

Dn, L - Bottle brush

Pigeon -

Bacillus anthracis + - or

wea

k

G,M,S,

Trehalose,Dn,VP

Inverted furtree

- weak

Anthracoides + +A.ctinomyces bovis - -

Actinomyces pyogenes

+ +

Clostridium tetani

--

- furtree + -C. Blutinium G,M,Sor + + VC. chauvoei G,M,L,Salicin - + -C. septicum G,M,L,Salicin - + -C.novyi G, M + + +C. novyi type D G - + +C.perfringens G, L, S, M + - +

D- delayed; V – varied ; + Positive ; - Negative ; GP- Guinea pig die; R – Rabbit die ; G- Glucose ; VP- Voges proskauer M- Maltose ; Man- Mannital ;S- Sucrose ;Sor –Sorbital ; L-Lactose ; L-R- L Rhamnose ; Dn-Dextron

38

4.4 BIOCHEMICAL TESTS

Carbohydrate utilization test Catalase test

Oxidase testTSI Slant

Urease test Gelatin Liquifaction

Indole testMethyl red test

Citrate utilization testSource :http://users.stlcc.edu/kkiser/biochem.html

39

6. ANTIMICROBIAL SUSCEPTIBILITY TESTING

Introduction

Antimicrobial susceptibility tests measure the ability of an antibiotic or other

antimicrobial agent to inhibit bacterial growth in vitro. This ability may be estimated by

either the dilution method or the diffusion method. The recommended method for

intermediate and peripheral laboratories is the modified Kirby-Bauer method, the

methodology of which is given below:

6.1 General guidelines for routine susceptibility testing

♦ Susceptibility tests should be carried out only on pure cultures of organisms considered to

be causing the infectious process

♦ The selection of antibiotic discs depends on the clinical consideration including the drugs

that are available and in general use by the veterinarian.

♦ A tetracycline disc will predict the results against all other tetracyclines.

♦ Sulphisoxazole is a suitable representative for all the sulphonamides

♦ Aminoglycosides and quinolones should be tested separately

♦ Pencillins, Streptococci should be tested against either pencillin G or ampicillin. Testing

♦ against both is not necessary.

♦ Erythromycin will predict the result of all other macrolides

♦ A clindamycin disc will predict the results for lincomycin.

♦ Chloramphenicol, vancomycin, nitrofurantoin and trimethoprim/sulphamethoxazole are

tested separately as required.

♦ A cephalothin disc will indicate the results for other first-generation cephalosporin’s

(cefalexin, cefradine, cefaloridine, cefazolin, cefapirin). Cefatozime represents

ceftazidime, ceftizoxime and ceftriaxone.

Modified Kirby-Bauer method

Materials required

♦ Tryptic soy broth or sterile saline broth

♦ Mueller-Hinton agar

♦ Antibiotic discs

o Any commercially available discs can be used and the stocks of antibiotic

discs preferably should be kept at -20ºC.

40

o A small amount of discs can be kept in the refrigerator for up to one month

before starting the work discs should be left at room temperature for about one

hour to allow the temperature to equilibrate.

♦ Sterile swabs

♦ Disc dispenser or forceps

Procedure

♦ If the sample provided is milk, then the sample could be directly smeared on agar

plate with a sterile swab.

♦ Incase of Pus swab, heart Blood swab or any organ, the sample should be cultured

on standard nutrient or BHI agar medium. After standard incubation period, pick

3-5 colonies of similar appearance and transfer to BHI broth or tryptic soy broth.

♦ Incubate the broth cultures at 35 + 2 oC for 2-4 hours for development of light to

moderate turbidity or adjust the turbidity with 0.5 McFarland standards.

♦ Inoculate the plates by dipping a sterile swab into the inoculum. Remove excess

inoculum by pressing and rotating the swabs firmly against the side of the tube

above the level of the liquid.

♦ Streak the swab all over the surface of the medium three times, rotating the plate

through an angle of 60o after each application. Finally, pass the swab round the

edge of the agar surface. Leave the inoculum to dry for a few minutes at room

temperature with the lid closed.

♦ Apply the antibiotic discs to the inoculated plates using an antibiotic disc

dispenser or sterile forceps.

♦ The plates should be placed in an incubator at 35 - 37oC within 30 minutes of

preparation and incubated for 16-18 hours (24 hours for Staphylococci).

Temperatures above 35oC invalidate the results for oxacillin/ methicillin.

♦ Do not incubate in an atmosphere of carbon dioxide.

6.2 Interpretation

After overnight incubation, the diameter of each zone (including the diameter of the

disc) should be measured and recorded in mm. The results should then be interpreted

according to the critical diameters by comparing with standard tables as susceptible,

intermediate or resistant to the antimicrobial agents used.

41

♦ The end-point of inhibition is judged by the naked eye but the extent of zone is

complicated in the following situations.

♦ Large colonies growing within an otherwise clear zone of inhibition should be

subcultured, identified and retested. Faint growth of tiny colonies at the edge of the zone

can be ignored.

♦ With sulfonamides and co-trimoxazole, slight growth occurs within the inhibition zone;

such growth should be ignored.

♦ When b-lactamase producing staphylococci are tested against penicillin, zones of

inhibition are produced with a heaped-up, clearly defined edge; these are readily

recognizable when compared with the sensitive control and regardless of the size of

the zone of inhibition, they should be reported as resistant.

♦ Certain Proteus spp. may swarm into the area of inhibition around some antibiotics, but

the zone of inhibition is usually clearly outlined and the thin layer of swarming growth

should be ignored.

♦ For bacteria grown on blood agar plates, the zone size for nafcillin, novobiocin, oxacillin

and methicillin will be 2-3mm smaller than the normal control limits.

General guidelines for routine susceptibility

testing

♦ Susceptibility tests should be carried out only on

pure cultures of organisms considered to be

causing the infectious process

♦ The selection of antibiotic discs depends on the

clinical consideration including the drugs that

are available and in general use by the

veterinarian.

♦ A tetracycline disc will predict the results against all other tetracyclines.

♦ Sulphisoxazole is a suitable representative for all the sulphonamides

♦ Aminoglycosides and quinolones should be tested separately

♦ Pencillins, Streptococci should be tested against either pencillin G or ampicillin. Testing

against both is not necessary.

♦ Erythromycin will predict the result of all other macrolides

♦ A clindamycin disc will predict the results for lincomycin.

42

♦ Chloramphenicol, vancomycin, nitrofurantoin and trimethoprim/sulphamethoxazole are

tested separately as required.

♦ A cephalothin disc will indicate the results for other first-generation cephalosporin’s

(cefalexin, cefradine, cefaloridine, cefazolin, cefapirin). Cefatozime represents

ceftazidime, ceftizoxime and ceftriaxone.

6.3 Antimicrobial sensitivity chart

Antimicrobial agent Disc contentZone diameter nearest whole mm

ResistantIntermediate

Susceptible

ß- LACTAMSAmpicillin 10 µg <13 14 -16 >17

Carbenicillin 100 µg <13 14-16 >16

Methicillin 5 µg <9 10-13 >14

Oxacillin 1 µg <10 11-12 >13

Penicillin 10 units <28 >29

Piperacillin 100 µg <17 >18

Ticarcillin 75 µg <14 >15

ß-LACTAM/ß-LACTAMASE INHIBITOR COMBINATIONSAmoxycillin/Clavulanic acid

20/10 µg <19 >20

Ampicillin/Sulbactam

10/10 µg <11 12-14 >15

Piperacillin/Tazobactam

100/10 µg <17 >18

Ticarcillin/Clavulanic acid

75/10 µg <14 >15

43

CEPHEMSCefazolin 30 µg <14 15-17 >18

Cefotaxime 30 µg <14 15-22 >23

Ceftizoxime 30 µg <14 15-19 >20

Ceftriaxone 30 µg <13 14-20 >21

Cephalothin 30 µg <14 15-17 >18

GLYCOPEPTIDESTelcoplanin 30 µg <10 11-13 >14

Vancomycin 30 µg <14 15-16 >17

AMINOGLYCOSIDESAmikacin 30 µg <14 15-16 >17

Gentamicin 10 µg <12 13-14 >15

Kanamycin 30 µg <13 14-17 >18

Streptomycin 10 µg <11 12-14 >15

Tobramycin 10 µg <12 13-14 >15

MACROLIDESAzithromycin 15 µg <13 14-17 >18

Erythromycin 15 µg <13 14-22 >23

TETRACYCLINESDoxycycline 30 µg <12 13-15 >16

Tetracycline 30 µg <14 15-18 >19

QUINOLONESCiprofloxacin 5 µg <15 16-20 >21

Nalidixic acid 30 µg <13 14-18 >19

Norfloxacin 10 µg <12 13-15 >16

Ofloxacin 5 µg <12 13-15 >16

OTHERSChloramphenicol 30 µg <12 13-17 >18

Clindamycin 2 µg <14 15-20 >21

Nitrofurantoin 300 µg <14 15-16 >17

Rifampin 5 µg <16 17-19 >20

Sulfonamides 250/300 µg <12 13-16 >17

Trimethoprim 5 µg <10 11-15 >16

Trimethoprim/sulfamethoxazole

1.25/ 23.75 µg

<10 11-15 >16

44

Note: Readymade antibiotic sensitivity discs and Mueller Hinton agar are available with Hi-medial Laboratory, India

45

7. COLIFORM COUNT IN WATER SAMPLES

Introduction

Total coliform bacteria are a collection of relatively harmless microorganisms that live in

large numbers in the intestines of man and warm and cold-blooded animals. They aid in the

digestion of food. A specific subgroup of this collection is the fecal coliform bacteria, the most

common member being Escherichia coli. These organisms may be separated from the total coliform

group by their ability to grow at elevated temperatures (40ºC) and are associated only with the fecal

material of warm-blooded animals.

Environmental Impact

The presence of fecal coliform bacteria in aquatic environments indicates that the water has

been contaminated with the fecal material of man or other animals. At the time this occurred, the

source water might have been contaminated by pathogens or disease producing bacteria or viruses

which can also exist in fecal material.

The presence of fecal contamination is an indicator that a potential health risk exists for

individuals exposed to this water. Fecal coliform bacteria may occur in ambient water as a result of

the overflow of domestic sewage or nonpoint sources of human and animal waste.

Escherichia coli

Method of measurement

Method

1. If the given water sample is apparently clean, the sample about 100µl should be spread plated

directly in Coliform agar/Mac Conkey/VRB Agar in duplicates and incubated at 37ºC for

18hrs.

2. Incase the water sample is turbid it should be serially diluted (101 , 102 , 103 ……109 ) by

adding 1ml of sample to 9ml of sterile peptone water and after proper mixing, 1ml from the

first tube should be transferred to the second tube containing 9ml of sterile peptone water.

Same way serial dilution should be done up to 109 .

3. 100µl of the diluted sample at tube no. 5 and 6 (105 and 106) should be spread plated in

Coliform agar/Mac Conkey/VRB agar in duplicates and incubated at 37ºC for 18hrs.

4. After incubation number of Pink colonies in Coliform agar or in Mac Conkey agar/Violet

colonies in case of VRB agar should be counted and the count is calculated according to the

dilution factor and expressed as cfu/ml.

46

Interpretation

1. Drinking water must have 0 colonies / 100 mL

2. Recreational bathing or swimming can’t be over 2000 colonies / 100 ml

3. For domestic animal water supply fewer than 2000 colonies / 100 mL

8. Laboratory tests for fungal infection

To establish or confirm the diagnosis of a fungal infection, skin, hair and nail tissue is

collected for microscopy and culture (mycology). Ultraviolet radiation (a Wood's light) can help

identify some fungal infections of hair because the infected hair fluoresces green

Specimen collection

• Specimens for fungal microscopy and culture may be:

• Scrapings of scale, best taken from the leading edge of the rash after the skin has been

cleaned with alcohol.

• Skin stripped off with adhesive tape, which is then stuck on a glass slide.

• Hair which has been pulled out from the roots.

• Brushings from an area of scaly scalp.

• Nail clippings.

• Skin biopsy.

• Moist swab from a mucosal surface (inside the mouth or vagina) in a special transport

medium.

• A swab should be taken from pustules in case of secondary bacterial infection.

• They are transported in a sterile container or a black paper envelope

Direct microscopy

The material is examined by microscopy by one or more of these methods:

• Potassium hydroxide (KOH) preparation, stained with blue or black ink

• Unstained wet-mount

• Stained dried smear

• Histopathology of biopsy with special stains.

• Microscopy can identify a dermatophyte by the presence of:

Fungal hyphae (branched filaments) making up a mycelium

47

• Arthrospores (broken-off spores)

• Arthroconidia (specialised external spores)

• Spores inside a hair (endothrix) or outside a hair (ectothrix).

• Fungal elements are sometimes difficult to find, especially if the tissue is highly inflamed, so

a negative result does not rule out fungal infection.

• A yeast infection can be identified by the presence of:

Yeast cells, which may be dividing by budding

• Pseudohyphae (branched filaments similar to those of a dermatophyte) forming a

pseudomycelium.

Culture

Culture identifies which organism is responsible for the infection:

• To find out the source of infection e.g. a particular animal

• To select the most suitable treatment.

• Growing the fungus in culture may take several weeks, incubated at 25-30ºC. The specimen

is inoculated into a medium such as Sabouraud's dextrose agar containing cycloheximide and

chloramphenicol. The cycloheximide is left out if a mould requires identification.

A negative culture may arise because:

• The condition is not due to fungal infection.

• The specimen was not collected properly.

• Antifungal treatment had been used prior to collection of the specimen.

• There was a delay before the specimen reached the laboratory.

• The laboratory procedures were incorrect.

• The organism grows very slowly.

Culture of yeasts and moulds may be due to harmless colonisation rather than infection. The

infection may be secondary to an underlying skin disesase such as psoriasis.

Blood tests are not useful for the diagnosis of superficial fungal infections. But in subcutaneous and

systemic infection, several tests may be useful.

• Culture

• Antibodies (histoplasmosis, coccidioidomycosis)

• Antigen (cryptococcosis, aspergillus, candidosis, histoplasmosis

48

49

9. DISEASES AND MATERIALS TO BE COLLECTED FROM LIVESTOCK AND POULTRY

S.NO. Name of the disease

Material to be Collected and Preservative if any

Methods of examination

1. Actinobacillus

(Actinobacillus lignieresi.)

• Pus from the abscesses. • Portion of tongue preserved in ice. Sterile Swabs from the lesions.

1.Microscopical: Pus Smear made by crushing between slides, Grams’s stain; Gram negative cell surrounded by gram negative filamentous sulphur granules,2.Straus’ reaction:Inoculation into male guinea pig produces orchitis.3.Cultural examination From affected tissues and lymph nodes cultured in serum or blood agar

2. Actinomycosis(Actinomyces bovis)

• Fresh portion of affected tissues or scrapings preserved in ice

1. Microscopical: Pus granules crushed between the slides, stained by Gram stain, Gram Positive filaments arranged like mycelia surrounded by gram negative filaments.2. Cultural examination Culture of affected portion in serum or blood agar.

3. Anthrax(Bacillus anthracis)

• Peripheral blood smear (Tip of ear / tail)

• Smear from swelling• Swabs from fluid

exudate/blood from natural orifices

• ear piece or muzzle in two or three pieces

• Blood stained soil

1. Microscopical:i) Blood smear stained by Leishman’s method will reveal characteristic rod shaped organisms with truncated ends,possessing a distinct capsule and arranged singly in very short chains. ii) Blood smear stained by Methylene Blue will evince Mc Fadyean reaction2. Cultural:

1. Cotton wool growth in broth clear supernatant

2. Medusa head colonies on agar3. Inverted fir tree growth in gelatin

agar.3. Biological:Guinea pigs and mice are used. On i/p inoculation animal dies in 48-72 hours showing characteristic lesions. Blood smear reveals characteristic organism.

50

4. Black disease(Clostridium novyi)

Pieces of liver in sterile container

1. Demonstration of anaerobic bacteria with sub-terminally located oval spores by gram staining 2. Anaerobic culture in Robertson cooked meat medium and identification

5. Black quarter (B.Q)(Clostrldium chauvoei) and (Clostridium septicum)

• Smears from the swelling

• Muscle impression smear.

• Muscle piece-air dried

1. Microscopical:Muscle impression smear stained by Claudius method of staining will reveal rods with sub-terminal spores.2. Biological: Intramuscular injection into a guinea pig produces characteristic gangrenous necrosis and death. Extensive hemorrhagic edema and emphysema with s/c haemorrhage is seen. Injection of a drop of 5% calcium chloride before inoculation accelerates the reaction. Clostridium chauvoei and clostridium septicum can be differentiated by cultural characters and biological test by toxin neutralization test.

6. Botryomycosis(Staphylococcus aureus)

Pus smear

Pus swab

1. Demonstration of gram positive Cocci in clusters

2.Culture and identification on Mannitol salt agar, Maltose Agar and Blood agar –Beta hemolytic, Hot Cold Phenomenon3.Coagulation of plasma, Novobiosin sensitivity are the characteristic features of pathogenic organism.

7. Brucellosis (Brucella abortus- occurs commonly in cattle B. melitensis occurs commonly in goats and B. suis – occurs commonly in pigs.)

• Serum preserved in 5% chloroform

• Milk form affectd quarter

• Smears form vaginal exudates and foetal stomach contents

• Swabs from vaginal exudates or foetal stomach contents, foetal cotyledons and uterine discharge

1. Microscopical:Smears stained by Gram’s staining reveal Gram negative coccobacilli. Dark pink stained cocobacilli in blue backround with Modified Ziehl Neelsan staining. 2. Cultural: Brucella selective agar, Potato Dextrose agar, Tryptose agar, Serum dextrose agar or liver infusion agar can be used. 5 -10% increased tension of carbon dioxide favours the growth of B. abortus.3. Straus test. : Intraperitoneal inoculation into a male guinea pig produces orchitis – 4. Serological (a) Rapid plate test – for screening (b) Tube agglutination test – for confirmation (c) ABR test or MRT test –

51

to screen milk form affected animals.8. Bovine

Lymphangitis (Yersinia pseudotuberculosis type III)

• Pus smear/smear from gland Pus swab

1.Microscopical :Pus smear on Gram’s staining reveals Gram negative rods. 2.Isolation and identification of the organism in XLDor Brilliant Green Agar. 3.Biological: Straus test

9. Caseous Lymphadenitis,

Caseous Lymphangitis

(Corynebacterium pseudotuberculosis)

• Swabs from abscesses.

1.Microscopical :Smear from caseous lesions on Gram’s staining reveals Gram positive pleomorphic bacterium with metachromatic granules demonstrated by Alberts staining 2. Isolation and identification of the organism from Blood agar. Differentiation based on non growth of bacteria on Mac Conkey agar.

10 Calf Diphtheria(Fusobacterium necrophorum).

• Swab from the lesions.

Mere isolation and identification of this nonsporulating Gram negative, anaerobe do not confirm the etiological aspect. Biological test in rabbits is always necessary as this organism appears as secondary invader in most of the cases.

11. Calf Dysentry or white scour or Colibaciliosis.(Escherichia coli)

Rectal swabs Cultural: Isolation and identification of the organism using selective media and characterization.

12. Calf Pneumonia(Pasteurella multocida)Sheep pneumonia (Pasteurella haemiolytica)

• Swabs from pleura and lungs.

1. Microscopical: Demonstration of bipolar organism by giemsa/leishman staining.2. Isolation and identification of the organism in Blood agar ( tiny colonies) and non growth of bacteria on Mac Conkey agar and confirmation 3. Biological test in mice and rabbits.

13 Contagious Bovine pleuro pneumonia (CBPP) (M.mycoides subspecies mycoides (small colony types)

Contagious Caprine

Piece of lung in ice and Serum Piece of lung in ice

1. Culture and identification of the organism in PPLO medium supplemented with 20 % Horse serum, penicillin and Thalous acetate.

2. Plate agglutination test to demonstrate the antibodies from serum of ailing animals

52

pleuro pneumonia (CCPP)(M.capricolum subspecies Capri pneumonia (F38),

14. Chronic respiratory disease (Mycoplasma gallisepticum)

• Tracheal impression smear,

• Trachea in ice, Swabs from nasal secretion

• Serum from the infected birds

1. Demonstration of pleomorphic fungi like organism from the impression smears by Giemsa staining techniques 2.For culture and identification of the organism in PPLO medium supplemented with 20 % Horse serum, penicillin and Thalous acetate3.Plate agglutination test : To demonstrate the antibodies from serum of ailing birds

15. Enterotoxaemia.(Clostridium welchii Type D,)

• Intestinal contents or a loop of intestine tied at both ends and preserved in 0.5% Chloroform.

1. Biological test – in mice by caudal vein inoculation chloroform treated intestinal content after centrifugation . Death of mice with neurological symptoms in few minutes to two hours. 2. Toxin neutralization test :Actual toxin can be determined by neutralization with specific antitoxins.

16. Glanders(Burkholderia mallei)

• Smears from discharge

1. Mallein test in living animals. 2.Straus test positive.

17. Fowl spirochaetosis (Borrelia anserine)

• Blood buffycoat smears

• Piece of liver and spleen ice

Microscopical :The organisms can be demonstrated by Giemsa strain or silver impregnation staining techniques and could be viewed under DFM

18. Fowl cholera (P.multocida)

Blood smear • Blood swab in

charcoal mediumLong bone packed in table salt

1.Microscopical :Bipolar organisms could be detected by Giemsa staining

2.Culture and identification in Blood agar and Mac Conkey ( No growth of Bacteria)3. Biological test : Bone marrow from long bone will be injected in to mice. Death of mice occus in 24 to 48hrs

19. Haemorrhagic septicemia. (H.S.)(Pasteurella multocida)

• Auricular vein smear.Oedema fluid smear.

• Heart blood smear• Heart blood swabs.• Swabs from internal

Microscopial: Smears stained by Leishman’s stain reveal bipolar organisms.2.Cultural: Isolation and identification identification in Blood agar and Mac Conkey (No growth f bacteria)

53

organs• Long bone

preserved in charcoal in case of extensive putrification.

3. Biological Test: In mice and rabbits.Haemorrhagic tracheitis in rabbits.

20. Johne’s disease (Paratuberculosis)(Mycobacterium avium sub sp Paratuberculosis)

• Rectal pinch smear and Rectal washings.

1. Microscopial: Smear stained by Ziehl Neelsan’s method reveals acid fast bacteria. 2. Cultural examination in Dorset’s Egg or L.J. medium. 3. Biological test : Johnin or Avian tuberculin test in living animal.

21. Leptospirosis(Leptospira interohaemorrhagiae and L. canicola occur in dogs and produce jaundice. L.. pomona has been incriminated in bovines.)

Serum; Urine.

1. Demonstration of motile spiral organism by Dark field Microscope (DFM) and in silver impregnated staining technique.2.Microscopic Agglutination Test (MAT) in microtitre plates.

22. Listeriosis(Listeria monocytogenes).

• Brain, nasal swab and heart blood swab

• Heart blood of the foetus preserved over ice.

1. Isolation and identification of the organisms in Listeria selective agar, Blood agar incubated at 20ºC2. Microscopical :. Small pleomorphic Gram positive rod, motile, beta haemolytic and CAMP positive. ( Isolation is augmented by prior chilling in ice).

23. Mastitis(Str. agalactiae, Str. dysagalactiae, Str. uberis, Staph. aureus, Cory. Pyogenes)

• Smears from milk sediment.

• Milk from suspected cases of mastitis

1. Microscopical :. Demonstration of Gram positive cocci in chais and clusters and some pleomorphic organisms.2. Isolation and identification of the organisms by growing it in Edwards medium, mannitol salt Agar and blood agar.

24. Pullorum disease & fowl typhoid.(Salmonella pullorum and Salmonella gallinarum)

Cloacal swab. Ailing chicks. Serum from birds. • Piece of intestine tied

at both ends intestinal swab.

1. Isolation and identification of the organism in XLD, Brilliant Green agar and Bismuth sulphite agar.2. Agglutination test with Salmonella Pullorum Coloured Antigen (SPCA)

54

25. Salmonellosis • Depending upon the disease conditions swab should be collected.

1.Isolation and identification of the organism in XLD, Brilliant Green agar and Bismuth sulphite agar.

26. Strangles(Streptococcus equi)

Swabs from abscess. 1. Microscopic : Demonstration of chains of gram positive cocci in grams staining.2. The isolation and identification of Streptococcus equi in Edwards medum and in blood agar. CAMP positive

27. Swine Erysipelas(Erysipelothrix rhusiopathiae)

Affected organsSerum

1.Isolation and identification of the organism - Non hemolytic pin point colonies on blood agar.2.Coagulase positive organism grow as bottle brush like pattern in gelatin agar.

28. Tetanus(Clostridium tetani)

Smear from lesions

Serum from the affected animal

Necrotic wound tissues

1. Morphology :C.tetani organism (Drumstick appearance with terminal oval bulged spores)2. Biological :To demonstrate neurotoxin in mice3. Isolation and identification of the organism in Blood agar and in Roberson cooked meat medium.

29. Tuberculosis(Mycobacterium bovis; Mycobacterium tuberculosis;Mycobacterium avium).

• Smear from lesions. Swabs from lesions. Lesion preserved in ice.

1.Microscopical examination.: Demonstration of Acid fast organism by Ziehl neelsan staining.2.Cultural examination: Culture develops in 4 to 8 weeks in Dorset egg medium or Lowenstein Jensen medium. 3.Biological test: In guinea pigs, rabbits and fowl depending upon the type. Disease is reproduced in 4 to 8 weeks. 4. Tuberculin test :.Intradermal test in live animals (Development of inflammatory reaction at injection site)

30. Psittacosis (Chlamydia psittaci) affecting psittacine and domestic birds

• Impression smears from the organs

Conjunctival swab

Demonstration of Chlamydial elementary bodies by FA, Giemsa and MZN stains. Isolation in Yolk sac of fertile eggs.

55

REFERENCES

• OIE Terrestrial Manual, 2008 • Veterinary Clinical Pathology (Edn 1989) – G.A.Sastry

• Veterinary Microbiology and Microbial disease - P.J. Quinn, B.K. Markey, M.E. Cater, W.J. Donnelly and F.C. Leonard, Blackwell Sciences Ltd., Ed. 2002 • Identification of Bacterial Species - Kimberley Christopher and Elsa Bruno, Department of Biological Sciences, University of Alberta, Edmonton, Alberta, CANADA

• NWFHS –Laboratory Manual and Procedures ( From internet) – (Chapter-5) – Jason woodland, Pinetop, Arizona• Animal Microbiology Edn.(1978) –Buxton and Frasier• Web : Introduction to bacteria @ Science in the real world Microbes in action (1999)• Clinical Veterinary Microbiology (1994) - P.J. Quinn, M.E. Carter, B.K. Markey and G.R. Carter• Internet Source :http://users.stlcc.edu/kkiser/biochem.html

• Internet Source : http://www.microbelibrary.org/ASMOnly/details.asp

• Internet Source - http://www.microbiologyprocedure.com

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