handbook of media stains and reagents in...

HANDBOOK OF MEDIA STAINS AND REAGENTS IN MICROBIOLOGY

- A. M. DESHMUKH

Preface

For conducting microbiology practical’s there is always a problem of getting

composition of media, stains and reagents. We have to search one or other book for

reference. The purpose of "Handbook of Media, Stains and Reagents in Microbiology" is

to collect all necessary information in one book. Author hopes the book will save the

time and efforts of research workers, students; teachers, laboratory technicians,

pathologists, environmental scientists and others.

The author is indebted to Dr. R. K. Trivedy, Dr. P. K. Goel, Shri. S.G. Pathak, Dr.

S.S. Jagdale, Dr. S.C. Kale, Dr. M.B. Gandhi, Dr. S.S. Bajekal, Shri. N.R. Shaikh, and

Shri. T.B. Sawant for graciously offering their suggestions.

The author wishes to express his thanks to -Dr. S.G. Sabnis and Prof. S.H. Shinde

for constant encouragement for writing this book. The author is also thankful to Dr. G.R.

Tathade for reading the manuscript and providing suggestions for improvement.

The author acknowledges his thanks to many known and unknown persons

involved in invaluable help during completion of this work.

- A. M. Deshmukh

Contents

Preface

1. Media for Biochemical Test 4

2. Cultivation Media 35

3. Staining Methods 153

4. Reagents 192

5. The Standard Solutions 206

Appendices 228

References 233

Index 236

1

Media for Biochemical Test

Amino acid Decarboxylation Test

A. Moeller's medium

Composition: Peptone 5.0 g

Meat extracts 5.0 g

D --- Glucose 0.5 g

Pyridoxal 0.005 g

Bromcresol purple 5.0 ml

( 1 in 500 solution)

Cresol red 2.5 ml

( 1 in 500 solution)

Distilled water to 1000 ml

PH 6.0

Preparation: Dissolve all in gradients except indicators in water and adjust the pH. Add

indicators. Add 1 per cent amino acid (di or mono) Hydrochloride whose decarboxylation

is to be tested. (L-lysine, L-ornithine or. L-arginine hydrochloride). Distribute 1 ml in

each tube.

Add sterile liquid paraffin to provide a layer about 5 mm think above the medium.

Autoclave at 1210 C for 15 min.

Use: To test amino acid decarboxylation ability of bacteria. Inoculate the tubes and

incubate. After incubation positive test is indicated by violet colour of the medium. The

control remains yellow.

B. Falkow’s medium

Composition:

Peptone 5.0 g

Yeast extract 3.0 g

D - Glocose 1.0 g

0.2% bromocresol purple 10 ml

Aminoacid 5.0 g

(L-LysineiL-ornithinek-arginine)


PH 6.7

Preparation: Dissolve all ingredients in water except bromocresol purple. Adjust the pH.

Add the indicator. Autoclave at 12 1 O C for 15 min.

Use: To test aminoacid decarboxylation ability of bacteria. The medium first becomes

yellow due to acid production. Later violet colour of the medium appears due to

decarboxylation. The control remains yellow.

Amylase Production

Starch agar

Composition:

Starch (soluble) 20.0 g

Peptone 5.0 g

Beef extract 3.0 g

Agar 20.0 g


pH 7.0

Preparation: Dissolve all ingradients in distilled water. Adjust the pH. Add agar. Steam

sterilize the medium for one hour at 100°C.

Use: To study the starch hydrolysis by microgranisms. After incubation, flood the plate

with Gram's iodine. Amyl lytic colony will be surrounded by clear zone against purple

coloured background.

Casein Hydrolysis

Milk agar

Composition

Nutrient agar (sterile) 875.0 ml

Skimmed milk (sterile) 125.0 ml

Preparation: Sterilize the nutrient agar and milk separately at 121°C for 30 minutes.

Cool the agar at 50°C. Add cooled milk aseptically in the melted nutrient agar.

Use: To set casein hydrolyzing ability of microorganisms. Casein hydrolytic colonies

will develop clear zones around the colony.

Chitin Hydrolyse

Composition:

Chitin 4.0 g

K2HPO4 0.7 g

MgSO4.7H2O 0.5 g

KH2PO4 0.3 g

FeSO4.7H2G 0.01 g

MnCl2.4H2O 0.001 g

ZnSO4.7H2O 0.001 g

Agar 20.0 g

Distillen water to 1000 ml

pH 7.0

Preparation: Autoclave the medium 1210C for 20 minutes.

Use: For the isolation of chitin lytic bacteria.

Note: Adjust the pH 8.0 and use the medium for isolation of actinomycetes.

Citrate Utilization Test

A. Citrate broth (Koser's modified)

Composition:

NaCl 5.0 g

MgSO4.7H2O 0.2 g

NH4H2PO4 1.0 g

KH2PO4 1.0 g

Na3C6H5O7.2H2O 5.0 g

(Sodium citrate)

Distillen water to 1000 ml

pH 6.8

Preparation: Dissolve all ingredients in distilled water. Adjust the pH. Distribute in the

tubes and steam sterilize at 12 1 "C for 15 minutes.

Use: To test the ability of an organism to utilize citrate as the sole carbon and energy

source for growth. Positive test is indicated by the presence of turbidity in the broth after

incubation. Observe the turbidity by comparing uninoculated control tube.

B. Citrate agar

(Simmon's citrate medium)

Composition:

Koser's (modified) broth 1000 ml

Bromothymol blue (0.2%) 40.0 ml

Agar 20.0 g

Preparation: Add the indicator in the broth. Dissolve agar. Distribute in the tubes, steam

sterilize at 12 1°C for 15 min. and allow to set as slants.

Use: To test the ability of an organism to utilize citrate as the sole carbon and energy

source for growth. Positive test is indicated by the blue colour of medium after

incubation. Colour of original medium is green.

DNase test

DNase agar

Composition:

Casein (pancreatic digest) 15.0 g

NaCl 5.0 g

Soybean meal (papaic digest) 5.0 g

Deoxyribonucleic acid 2.0 g

Agar 20.0 g


PH 7.3

Preparation: Add all comporients in distilled water by gentle heating. Steam sterilize at

1180C for 15 min. Pour plates.

Use: Spot inoculate the medium heavily by suspension of test organism. Incubate for 24

hrs. flood the plate with IN HCI. A clear halo around the colony indicate positive test. 1

N HCI precipitates unchanged deoxyribonucleic acid.

Esculin Hydrolysis

Bile esculin medium

Composition :

Peptone 5.0 g

Beef extract 3.0 g

Bile (oxgall) 40.0 g

Esculin 1.0 g

Ferric citrate 15.0 g

Agar 15.0 g


PH 7.0

Preparation: Dissolve all ingredients in distilled water. Distribute into tubes, sterilize in

autoclave at 12 1 OC for 15 min. and prepare slants.

Use: To set esculine hydrolyzing ability of bacteria. Esculin hydrolysis is indicated by

causing the slant to blacken after incubation.

Note: This test is usually applied for indentifiecation of group D streptococci. Both

enterococcal and nonenterococcal species of group D are able to hydrolyse esculin.

Eijkman Test

MacConkey's broth

Composition: (See 9)

Use: This test in used for identification of Escherichia coli. Inaculate MacConkey's broth

with test organism. Incubate at 44* 0.2OC for 24 hrs. E. coli produce gas at this

temperature.

Gelatin hydrolysis (Gelatin liqueefaction test)

Gelation agar

Composition:

Gelatin 4.0 g

Glucose 0.05 g

KH2PO4 0.5 g

K2HPO4 1.5 g

Nutrient agar (melted) 1000 ml

Preparation: Dissolve the phosphates, gelatin and glucose in melted nutrient agar slowly

for even distribution ofthe gelatin. Steam sterilize in a flask at 1210C for 30 min. Prepare

plates.

Use: To test gelatin hydrolyzing ability of microorganisms. Flood the plate containing

growth of the microorganisms with Frazier's solution. Gelatin hydrolysis is indicated by

clear zone around colony against opaque background.

Gluconate Test

Compasition:

Peptone 1.5 g

Yeast extract 1.0 g

K2HPO4 1.0 g

Potassium gluconate 40.0 g


pH 7.0

Preparation: Dissolve all components in water. Adjust the pH. Distribute in 1 rnl

quantities in each tube and steam sterilize at 121°C for 15 min.

Use: used to test the ability of an organism to oxidize gluconates to the 2 keto-gluconate.

Inoculate the tube with bacterial suspension and incubate at 370C for 48 hours. Then add

1 ml of qualitative benedict's solution and heat the tube for 5-10 minutes. Observe the

change in colours. Benedict's reagents is blue in colour. Positive test is indicated by

development of green to orange coloured precipitate.

Homo and Heterofermentation - Differentiation

Tomato juice-gelatin medium

Composition:

Gelatin 120.0 g

Yeast hydrolysate 2.5 g

Tomato juice 100.0 g

Glucose 50.0 g


PH 7.0

Preparation: Sterilize glucose separately in 500 ml of water by filtration and remaining

medium at 121°C for 30 minutes. Mix both the solutions aseptically.

Use: For differentiating homofermentative and heterofer-mentative lactic acid bacteria.

Inoculate the tubes with culture. Mix it properly, chill the tubes in cold water to solidity

the gelatin. Then seal with about I inch of melted nutrient agar. Incubate for 2-5 days at

the temperature indicated and examine for gas production.

Hippurate Hydrolysis

Sodium hippurate broth

Composition:

Peptone 5.0 g

Beef extract 3.0 g

Sodium hippurate 10.0 g


Preparation: Dissolve the ingradients in distilled water. Distribute in tubes. Sterilize in

autoclave at 121°C for min.

Use: To detect hippurate hydrolysis. Incubated broth is centrifuged and 0.8 ml clear

supernate is taken into a kahn tube. 0.4 ml of ferric chloride solution [12g FeC136H2O

dissolved in 100 ml of dilute (5.4 ml concentrated HC1 to 94.6 ml distilled water) HCI] is

added in a kahn tube. Mixture is allowed to stand with occasional shaking for 10 minutes.

Positive test: A heavy cloudy, precipitate persisting after 10 minutes.

Negative test: Clearing of the initial precipitate within 10 minutes.

Hydrogen Sulfide Production

A. SIM agar (Sulfide indole motility agar)

Composition:

Peptone 30.0 g

Beef extract 3.0 g

Ferrous ammonium sulphate 0.2 g

Sodium thiosulphate 0.025 g

Agar 4.0 g


pH 7.2

Preparation: Dissolve all components indistilled water. Adjust the pH. Disribute in the

tubes. Sterilize the medium at 121°C for 20 min. in the autoclave.

Use: This medium is used for detection of H,S, indole and motility. H2O producing

organism cause blackening of the medium. H2S reacts with ferrous ammonium sulphate

resulting black insoluble compound. ferrous sulphide. Indole production can be tested by

the method prescribed in the illdole test. Motility is indicated by diffused growth

from the inoculation point.

B. Peptone water 1

Composition:

Peptone 20.0 g

Sodium chloride 5.0 g


PH 7.2

Preparation: Dissolve all components in warm water. Adjust the pH. Autoclave at 121°C

for 15 min.

Use: Used to test hydrogen sulphide production ability of bacteria. Inoculate the tube of

peptone water by given organism. Dip the filter. paper in saturated solution of lead

acetate. Dry the filter paper, sterilize and keep hanging in the inoculated tube of peptone

water with the help of cotton plug. Incubate the tube.

Positive test: Filter paper turns black.

Negative test: No change in colour of filter paper.

C. Kligler's iron agar I

Composition:

Meat extract 3.0 g

Yeast extract 3.0 g

Peptone 20.0 g

Lactose 10.0 g

Glucose 1.0 g

Sodium chloride 5.0.g

Ferrio citrate 0.3 g


Phenol red 0.05 g

Agar 12.0 g


pH 7.4

Preparation: Dissolve the ingredients in water. Dispense in about 5 min amounts in test

tubes and sterilize them at 121°C for 15 minutes. Prepare slants with a butt.

Use: To detect H,S production and fermentation of lactose and glucose. Interpret the

result like TSI agar.

D. Lysine iron agar

Composition: Yeast extract 3.0 g

Peptone 5.0 g

L-lysine 10.0 g

Dextrose 1.0 g

Sodium thisulphate 0.04 g

Ferric ammonium citrate 0.5 g

Bromocresol purple 0.02 g

Agar 20.0 g


Preparation: Dissolve the ingredients by boiling in water. Dispense into tubes. Sterilize

at 121°C for 20 minutes. Prepare slants with deep butts.

Use: To test production of H2O and lysine decarboxylase by Proteus.

(a) The production is detected by blackening of the medium.

(b) The lysine decarboxylase production is detected by development of red colour around

the colony.

Indole Test

Durham's peptonewater (Fermentation basal medium)

Composition: (See peptone water in hydrogen sulphide production test)

Use: 1. This medium is used chiefly as the basic for carbohydrate fermentation media.

2. It is also used fortesting the indole formation ability. Inoculate the tube with

given bacteria. After incubation add few drops of xylene. Shake the tube

vigourously. Add 2 to 3 drops of kovac's reagent along tlie side of the tube.

Note: 1. Use of tryptone water instead of peptone gives good results in indole formation.

2. All commercial peptones do not contain sufficient tryptophan for good indole

production. Therefore the suitable brand of peptone must be selected for this

purpose.

Lysozyme Sensitivity Test

Lysozyme broth

Composition:

A. Glycerol broth

Peptone 5.0 g

Beef extract 3.0 g

Glycerol 70.0 ml

Distilled hater to 1000 ml

Add all components in distilled water. Sterilize at 1210C for 20 minutes.

B. Lysozyme solution

Lysozyme 0.1 g

0.01 N HCI 100.0 ml

Add lysozyme to 0.01N HCI. Mix. Filter sterilize. This solution can be stored at

4°C for 7 days.

Preparation of complete medium

Lysozyme solution 0.5 ml

Glycerol broth 100 ml

Add aseptically and distribute in sterile tubes of 5 ml each.

Use: For differentiation of genera of actinomycetes based on sensitivity to lysozyme

sensitivity actinomycetes do not grou in lysozyme broth. Growth can be compared by

inoculating in glycerol broth.

e.g. Nocardia asteroids -- not sensitive to lysozyme Streptomyces griseus -- sensitive to Iysozyme.

Lactate Fermentation

Lactate agar

Composition:

Trypticase 20.0 g

Yeast extract 5.0 g

Sodium lactate 12.0 g

Agar 25.0 g


pH 6.8

Preparation: Dissolve all components in distilled water except agar. Adjust the pH. Add

Agar. Distribute in the tubes. Steam sterilize the medium at 1210C for 20 min.

Use: To test lactate fermentation ability of bacteria. Stab inoculates the tubes. Incubate.

Lactate ferernentation is detected by gas production.

Lecithinase Production

A. Tween phosphate buffered substrate medium

Composition:

Phosphate buffer pH 7.0 40.0 ml

Tween 80

(Polyoxyethylene sorbitain

monooleate) 0.2 ml

Neutral red (1 g/litre) 0.8 ml

pH 6.8 - 7.2

Preparation: Add neutral red solution to phosphate buffer and mix. Add tween 80 and

mix gently. Distribute in 5 ml amounts in tubes and sterilize at 1210C for 20 minutes.

Use: It is used in tween80

hydrolysis test to detect lecithinase production.

B. Egg yolk agar

Composition:

1. Melted nutrient agar (sterile) 85 ml

2. Egg yolk suspension l5 ml

Preparation of egg yolk suspension:

Take fresh egg (less than four days old). Wash with water by brush and a plain alkaline

soap. Rinse in running water for 10 min. Dry the egg by sprinkling methylated spirit and

burning it off. Crack the, tapered end of egg with sterile knife. Separate the egg white

from egg yolk. Collect the egg yolk in sterile container aseptically.

Preparation of complete medium:

Melt tlie agar. cool to 55°C and add tlie egg yolk. Pour plates.

Use: To test lecithinase activity of bacteria. Lecithinase production is shown by wide

zones of opalescence around colonies more intense and larger than the zones caused by

lipolysis.

C. TweenTM

80 hydrolysis

Composition :

Peptone 10.0 g

NaCl 5.0 g

CaCI2 0.1 g

TweenTM

80 10.0 ml

Agar 20.0 g


pH 7.2

Preparation: Autoclave at 1210C for 20 min.

Use: For testing the microorganisms for hydrolyzing TweenTM

80 Colonies hydroluzing

TweenTM

80 are surrounded by an opaque zone.

Lipolysis Test

A. Fat agar

Composition:

Sterile melted nutrient agar 100 ml

Sterile melted butter (unsalted) 10 ml

Preparation: Mix thoroughly and pour plates.

Use: To study lipolytic activity of bacteria. Inoculate the centre of the solidified fat agar

plate with bacterial suspension. Incubate for 5-7 days at given temperature. Flood the

plates with a saturated aqueous solution of' CuSO4. After 20 minutes pour off the excess

solution. Bright greenish blue insoluble copper soap is formed with the fatty acids around

the colony.

B. Egg yolk agar

composition:

(See lecithinase production)

Use: Used to test lipolytic activity of bacteria. Flood the date with copper sulphate

solution. After 20 minutes pour off the excess solution. Bright greenish blue insoluble

copper soap is formed with the fatty acids around the colony.

C. Tributyrin hydrolysis method

Composition:

Peptone 5.0 g

Yeast extract 3.0 g

Tributyrin 10.0 g

(Glyceryl tributyrate)

Agar 20.0 g


pH 7.5

Preparation: The medium is prepared such that the tributyrin forms A stable emulsion in

the nutrient agar and adjust the pH

Use: To test hydrolysis of tributyrin. An emulsion of microdroplets of the fat, tributyrin,

in a solid medium makes it opaque. Lipolytic organisms remove the opacity by

converting the fat to water soluble butyric acid.

Litmus Milk Test

Litmus milk medium

Composition:

Preparation of litmus solution

Litmus granules 80.0 g

Ethanol, 40% aq. 300 ml

Grind up the granules, add to a flash containing 150 ml aqueous ethanol. Boil for 1

min. Decant the solution in other flask. Add remainder aqueous ethanol and boil for I

min. Decant and combine both solutions, make the volume 30 ml with 40% aq. ethanol.

Add HCI (IN) drop by drop, shaking continuously until the solution becomes purple.


Skimmed milk 250 ml

Litmus solution 6.25 ml

Mix both the solutions. Distribute in tubes 5 ml in each. Steam for 20 min. on three

successive days. Colour of medium is blue or purple.

Use: Litmus milk test indicates both saccharolytic and proteolytic properties of bacteria.

Lactose fermenter in litmus milk form acid cause it to become pink or red. Proteolytic

bacteria decompose milk protein and change to a clear dark purple solution.

Melanine Production

Tyrosine agar

Composition:

Glycerol 15.0 g

L-Tyrosine 0.5 g

L-Aspargine 1.0 g

K2HPO4 0.5 g

MgS04.7H2O 0.5 g

NaCl 0.5 g

FeSO4.7H4O 0.01 g

Agar 20.0 g


Preparation: Steam sterilize the medium at 12I0C for 20 min.

Use: For testing melanine production by actinomycetes.

Malonate Utilization Test.

Composition:

Yeast extract 1.0 g

(NH4)2SO4 2.0 g

K2HPO4 0.6 g

KH2PO4 0.4 g

NaCl 2.0 g

Na – malonate 3.0 g

Bromothymol blue 0.025 g

Agar 20.0 g


pH 7.4

Preparation: Dissolve all components except indicator in water. Adjust the pH. Add the

indicator. Distribute in the tubes. Steam sterilize at 121°C for 15 min.

Use: For testing the ability to utilize malonate by bacteria. Positive results are indicated

by the change in colour of the indicator from green to blue due to the rise in pH (due to

utilization of sodium malonate).

Malonate Utilization and Phenylalanine

Deaminase test

Composition:

(NH4)SO4 2.0 g

K2HPO4 0.6 g

KH2PO4 0.4 g

NaCl 2.0 g

Sodium malonate 3.0 g

DL-Phenylalanine 2.0 g

Yeast extract 1.0 g


PH 7.4

Preparation: Dissolve all components in distilled water. Steam for 5 minutes and filter

through paper. Add 5ml of a 0.5% solution of bromothymol blue in absolute ethanol.

Distribute Iml in each tube, steam sterilize at 121°C for 20 min.

Use: For following two tests:

1. Malanate utilization test:

Positive test, green to blue colour of medium after inoculation and incubation.

2. Phenylalanine deaminase test:

Add few drops of 0. IN HCI until the colour of the medium becomes yellow. Add few

drops of a 10% aqueous solutions of FeCI3, shake and observe the colour for

phenylalanine deaminase test.

Dark green = Positive test

Yellow buff = Negative test.

Methyl Red Test

MR-VP broth (Glucose phosphate broth)

Composition:

Peptone 5.0 g

K2HPO4 5.0 g


Glucose 10% solution 50 ml

PH 7.6

Preparation: Dissolve the peptone and phosphate. Adjust the pH Filler. Distribute in 5

ml amounts and steam sterilize and at 121°C for 15 minutes. Sterilize the glucose

solution by filtration and add 0.25 ml to each tube. (final concentration 0.5%).

Use: To perform methyl red and Yoges Proskauer test. Inoculate the tube with given

organism. Incubate. Add about 5 drops of methyl red reagent after incubation. Mix and

observe the colour.

Positive test: Development of bright red colour.

Negative test: No development of bright red colour. Colour remains yellow.

Motility Test

Motility agar

Composition:

Peptone 1.0 g


Agar 4.0 g


PH 7.2

Preparation: Dissolve all the components in water by heating. Adjust the pH. Distribute

in tubes. Steam sterilize at 1210C for 15 minutes.

Use: Motile bacteria forms spreading colony on soft motility agar.

Motility and Nitrate Reduction Test

Motility nitrate medium

Composition:

Beef extract 3.0 g

Peptone 5.0 g

KNO3 1.0 g

Agar 3.0 g


PH 7.2

Preparation: Dissolve all the ingradients in distilled water by heating. Ajust the pH.

Distribute in tubes. Steam sterilize at 12I0C for 15 min.

Use: For detection of motility and nitrate reduction. Diffuse growth along the line of

inoculation indicates motility. For nitrite detection add few drops of sulphanilic acid

followed by equal volume of alphanaphthylamine. The rapid development of a pink

colour indicates the presence of nitrite.

Nitrification Kest

A. Ammonium medium

Composition: (NH4)2S04 1.0 g

K2HPO4 1.0 g

NaCl 2.0 g

MgSO4.7H2O 0.5 g

FeSO4 trace


Preparation: Excess of the sterilized CaCO3 is added to the medium. Sterilisation of the

medium is not necessary if the medium is used freshly.

Use: To test ammonia to nitrite (nitrification) conversion ability of bacteria. This medium

is also used to isolate Nitrosomonas from, soil. Pesence of nitrite can be tested by using

an appropriate colorimetric reagent. (See page 245)

B. Nitrite medium

Composition:

NaNO2 1.0 g

MgSO4.7H2O 0.5 g

FeSO4.7H2O 0.03 g

NaCl 0.3 g

Na2CO3 1.0 g

K2HPO4 1.0 g


pH 7.3

Preparation: Sterilisation is not necessary if the inoculations are made in the freshly

prepared medium.

Use: To test nitrite to nitrate (nitrification) conversion ability of bacteria. This medium is

also used to isolate Nitrobacter from soil. Fresence of nitrate can be tested by using an

appropriate colorimetric reagent. (See .page 245)

Preparation: If sterilization is desirable for storage, adjust the pH aseptically after

sterilization with sterile 0.1 N HCI.

Nitrate Reduction I

A. Tryptone nitrate broth

Composition:

Trytone 20.0 g

Potassium nitrate 1.0 g

Dissodium phosphate 2.0 g

Dextrose 0.1 g

Agar 1.0 g


pH 7.2

Preparation: Dissolve the ingredients in water. Adjust the pH. Distribute in the tubes.

Steam sterilize the medium at 1210C for 20 min.

Use: To test nitrate reduction ability of bacteria. Presence of nitrite can be tested by an

appropriate colorimetric reagent. (See page 245)

B. Nitrate m'edium

Composition:

KNO3 0.2 g

Peptone 5.0 g


pH 7.2

Preparation: Dissolve the ingredients in water. Adjust the pH. Distribute in the

tubesfflasks. Steam sterilize the medium at 1210C for 20 min.

Use: To test nitrate reduction ability of bacteria. (Presence of the enzyme nitrate

reductase which causes the reduction of nitrate to nitrite.) Presence of nitrite can be tested

by an appropriate colorimetric reagent. (See page 245)

ONPG Test

ONPG broth

Composition:

Preparation of ONPG solution: I

ONPG (0-nitrophenyl-β -D Galactopyranoside) 1.5 g

Sodium phosphate buffer to (0.01 M, pH 7.5) 250 ml

Preparation of complete medium: I

ONPG solution 250

Peptone water 750 ml

pH 7.2 : 7.4

Filter sterilize ONPG solution. Sterilize peptone water saperately. Adjust the pH of

peptone water 7.2 : 7.4 before sterilization. Sterilize peptone water at 1150C for 20 min.

Aseptically combine the sterile ONPG solution with cooled sterile peptone water. Mix

thoroughly. Distribute in tubes.

Use: For differentiation of late lactose fermenting (having β galactosidase and lacking

permease) and non-lactose fermenting bacteria. (without β galactosidase).

Inoculate ONPG broth and incubate for 24 hrs. If galactosidase is present a yellow

colour (due to o-nitrophenol) is formed.

Oxidation Fermentation Test

Hugh and Leifson medium (of glucose medium)

Composition:

Peptone 2.0 g

NaCl 5.0 g

K2HPO4 0.3 g

Bromothymol blue 3 (1% aq. solution) 3 ml

Glucose (1 0% solution) 100 ml

Agar 3.0 g


pH 7.1

Preparation: Dissolve all the ingredients except the indicator and sugar solution in water.

Adjust the pH. Add the indicator and steam sterilize the medium is a flask at 1210C for

30 min. The sugar solution is sterilized separately by filtration and then added as given in

the composition. The medium is then distributed in the sterile tubes to depth of about 4

cm in aseptic condition.

Use: Used to distinguish between aerobic and anaerobic break down of a sugar.

Duplicate tubes of medium are inoculated by stabbing, 1 tube is promptly covered with a

layer of sterile melted petroleum jelly to a depth of 5-10 mm and tubes are incubated.

After incubation, if acid is produced only at surface of the medium, where conditions are

aerobic, the attack on the sugar is oxidative. If acid is found throughout the tube,

including the lower layers where conditions are anaerobic the break down is fermentative

Pectin Hydrolysis

Hankin's medium

Composition:

Mineral salt solution 500 ml

Yeast extract 1.0 g

Agar 15.0 g

Pectin (citrus or apple) 5.0 g


pH 7.2

Preparation: Dissolve agar power in 400 ml of water with heat. Dissolve pectin and

yeast extract in another 100 ml of water and mix Both solutions. Add mineral salt

solutions. Sterilize by autoclaving at 121°C for 20 min.

Composition of mineral salt solution:

(NH4)2SO4 2.0 g

KH2PO4 4.0 g

Na2HPO4 6.0 g

FeSO4.7H2O 0.2 g

CaCl2 1 mg

H3BO3 10 µg

MnSO4 10 µg

ZnSO4 70 µg


Use: To test degradation of pectin by bacteria. If the plates are flooded with 1 percent

aqueous solution of hexadecyltrirnethyl ammonium bromide, penctinolytic organisms

produce clear zone around colonies. Pectin precipitates in presence of hexadecyltrimethyl

ammonium bromide.

Note: Adjust the pH 5 if fungi are to be tested.

Penicillin Assay

A. Antibiotic assay medium (Muller-Hinton agar)

Composition:

Infusion from beef 300.0 g

Acid hydrolysate of casein 17.5 g

Starch 1.5 g

Agar 20.0 g


pH 7.2 – 7.4

Preparation: Sterilize the medium at 1210C for 15 minutes.

Use: It is used as medium for antibiotic susceptibility testing by Kirby Bauer disc

sensitivity method.

B. Antibiotic assay medium (Trypticase soy broth)

Composition:

Trypticase peptone 17.0 g

Phytone peptone 3.0 g


Dipotassium hydrogen phosphate 2.5 g

Dextrose 2.5 g


Preparation: Sterilize the medium at 121°C for 15 minutes.

Use: It is used for testing antibiotic sensitivity (Kirby Bauer method.)

C. Penassay seed agar

Composition:

Beef extract 1.5 g

Yeast extract 3.0 g

Casein digest 4.0 g

Peptone 6.0 g

Glucose 1.0 g

Agar 20.0 g


pH 7.0

Preparation: Steam sterilize the medium at 12 1 "C for 20 min.

Use: Used to test penicillin sensitivity of bacteria.

D. Penassay seed agar

Composition:

Gelatin 6.0 g

Yeast extract 3.0 g

Beef extract 1.5 g

Agar 20.0 g


pH 7.0

Preparation: Steam sterilize the medium at 121 O C for 20 min.

Use: Used as the antibiotic assay medium.

Phenylalanine Deaminase Test

Composition:

Yeast extract 3.0 g

DL phenylalanine (or L phenylalanine 1g) 2.0 g

Na2HPO4 1.0 g

NaCl 5.0 g

Agar 20.0 g


pH 7.4

Preparation: Dissolve all the components in water except agar. Adjust the pH. Dissolve

the agar by heating. Distribute in tubes and sterilize by autoclaving at 121°C for 15 min.

Allow to solidify in tubes as slope.

Use: To test phenylalanine deamination ability of an organism. After incubation add few

drops of 10% solution of ferric chloride over the growth on the slant.

Positive test: Development of green colour.

Negative test: No development of green colour.

Phosphatase Production

Phenolphthalein phosphate agar

Composition:

Melted nutrient agar (pH 7.2) 98 ml

Sodium phenolphthalein phosphate solution (0.5%) 2ml

Preparation: A stock solution of sodium phenolphthalein phosphate is prepared,

sterilized by filtration and stored at 40C. Mix the melted sterilized nutrient agar with

sodium phenolphthalein phosphate solution.

Use: To isolate phosphatase producing organism, specifically staphycococci.

Phosphatase liberate free phenolphthalein, colonies become pink when held over an open

bottle of ammonia.

Phosphate Solubilization

A. Katznelson and Bose medium

Composition:

Glucose 1.0 g

K2HPO4 (1 0% solution) 5.0 ml

CaCl2 (1 0% solution) 10.0 ml

Agar 2.0 g

Soil extract 100.0 ml

pH 7.0.

Preparation: Sterilize the soil extract containing glucose and agar at 121°C for 20 min in

autoclave. Sterilize the K2HPO4 and CaCI2 solutions separately. Mix. Adjust the pH with

sterile 0.1N sodium hydroxide. Pour the plates immediately and allow to solidify.

Use: To test phosphate solubilizing activity of micro-organisms. The clearing around the

colonies indicate phosphate solubilization.

B. Pikovskaya's medium

(Modified by Sundara Rao and Sinha)

Composition:

Glucose 10.0 g

Tricalcium phosphate 5.0 g

CaCl2 (10% solution) 0.5 g

KCl 0.2 g

MgS04.7H20 0.1 g

MnSO4 trace

FeSO4 trace

Yeast extract 0.5 g

Agar 20.0 g


pH 7.2

Preparation: Dissolve all the components except tricalcium phosphate in 700 ml water.

Adjust the pH. Sterilize tricalcium phosphate separately in 300 ml water. Sterilize at

121°C for 20 min in autoclave. Cool at about 50°C and then mix both the solutions.

Use: To test phosphate solubilizing activity of microoganisms. Clearing around the

colony indicates phosphate solubilization.

Potassium Cyanide Test

Composition:

A. Basal medium

Peptone 3.0 g

NaCl 5.0 g

KH2PO4 0.23 g


pH 7.4

Steam sterilize the medium at 12 1 OC for 30 min.

B. Cyanide solution

KCN 0.5 g

Distilled water to (sterile) 100 ml


Basal medium 1000 ml

Cyanide solution 15 ml

Add the cyanide solution to the cold medium. Distribute insterile tubes, 1 ml in each

tube.

Use: To test the ability of an organism to grow in the presence of cyanide.

Note: The medium can be stored for 4 weeks at 40°C.

Sodium Chloride Tolerance Test

Sodium chloride tolerance broth

Composition:

Heart infusion broth 25.0 g


Indicator (1.6g Bromcresol purple in 100 ml 95% ethanol) 1.0 ml

Dextrose 1.0 g


pH 7.2

Preparation: Add all ingredients except. indicator in 1000 ml water. Adjust the pH. Then

add indicator. Distribute in tubes and sterilize in an autoclave for 15 min at 1210C.

Use: This test is used for differentiating the two types of group D streptococci. All

enterococci of group D produce heavy growth in this broth. None of the nonenterococci,

group D grow in this medium. A positive result is indicated by turbidity within 72 hrs. A

colour change of purple to yellow may also be present.

Note: Heart infusion broth (obtainable in dehydrated form) already contain 0.5% sodium

chloride.

Sugar Fermentation

A. Sugar fermentation medium

Composition:

Peptone water 100 ml

Test sugar 0.5 to 1 g

Andrade's indicator (1 %) 0.25 ml

or

Neutral red indicator (1 %) 0.25 ml

or

Phenol red indicator (1 %) 0.18 ml

or

Bromothymol blue (1 %) 0.25 ml

Preparation: Sugar is added in peptone water. Adjust the pH. Distribute in tubes.

Sterilize in autoclave at 121°C for 20 minutes. (steam sterilize at 100°C for 1 hr if the

sugar is thermolabile, like diassaccharide lactose.)

Use: Used to test sugar fermentation ability of bacteria. Durham fermentation tube in

inverted position in the broth is used to detect the gas production. Acid production is

indicated by change in colour I according to type indicator used, e.g., yellow to red when

andrade's indicator is used.

B. Sugar fermentation medium

Composition:

Peptone 5.0 g

Meat extract 3.0 g



Test sugar 1%

pH 7.2

Preparation: Dissolve the ingredients in water. Adjust the pH. Add indicator solution as

given in above medium. Distribute in tubes. Place a Durham's tube in an inverted

manner. Sterilize the tubes at 121°C for 15 min.

Use: To test sugar fermentation ability ~f bacteria, if a more nutritious base is needed.

C. Sugar fermentation (for actinomy'cetes)

Composition:

(NH4)2HPO4 1.0 g

MgS04.7H2O 0.2 g

KCl 0.02 g

Carbohydrate 100.0 ml

solution (10%)

Bromocresol purple 20.0 ml

Agar 20.0 g


pH 7.0

Bromocresol purple solution


Ethanol 50.0 ml

Distilled water to 100.0 ml

Preparation: Add all components except carbohydrate solution, bromocresol purple

solution and agar in distilled water. Adjust the pH. Add bromocreasol purple solution and

agar. Dissolve. Steam sterilize at 12 1 "C for 20 minutes. Sterilize carbohydrate solution

by filtration. Mix aseptically.

Use: For identification of actinomycetes to perform carbohydrate fermentation test.

Actinomycetes produce acid from carbohydrate turn the medium yellow.

D. Serum agar fermentation medium

Composition:

Basal medium

Peptone 20.0 g


Digest broth pH 7.6 100 ml

Agar 25.0 g

Phenol red solution (0.2 %) 20 ml


pH 7.6

Preparation: Dissolve the peptone and salt in the water by steaming. Adjust to pH 8.4 by

making just alkaline to phenolphtlialein. Steam for 30 min. Filter through a coarse filter

paper. Adjust the pH to 7.6. Add the broth and agar and steam at 100°C for one hr. Bottle

in 100 ml amounts and add 2ml phenol red to each bottle. Autoclave at 100°C for 1 h and

then 110°C for 5 min.

Complete medium

Basal medium 100.0 ml

Sterile serum (guinea pig or rabbit) 5.0 ml

Test sugar (10 % solution, sterile) 10.0 ml

Preparation: Melt the basal medium, cool to 55°C. Add other ingredients with sterile

precautions and distribute in tubes. Prepare slants.

Use: This medium is used for organisms such as meningococci and qnococci that grow

poorly in liquid media. Acid production is indicated by change in colour of the medium

from red to yellow.

E. Sugar fermentation

Serum sugar fermentation medium (broth)

Composition:

Peptone water (PH 7.4) 80.0 ml

Serum 20.0 ml

Phenol red (0.2 %) 5.0 ml

Test sugar 1.0 g

Preparation: Serum medium is sterilized usually by filtration. It may be steamed for 30

min for three successive days.

Use: This medium is suitable for more exacting organisms such as Corynebacterium

diphtheria to test sugar fermentation.

Precaution: Some sample of horse serum may contain saccharolytic enzymes and give

false results.

Sulfatase Test

wayne's sulfatase medium

Composition:

Na2HP04 2.5 g

L-Asparagine 1.0 g

KH2PO4 1.0 g

K2HPO4 1.0 g

Trisodium phenolphthalein 0.65 g

sulfate

Casein (pancreatic digest) 0.5 g

Ferric ammonium citrite 0.05 g

MGS04-7H2O 0.01 g

CaCl2.2H2O 0.5 mg

ZnS04.7H2O 0.1 mg

CuSo4 0.1 mg

Glycerol 10.0 ml

Agar 20.0 g


pH 7.0

Preparation: First add glycerol 700 ml water. Mix thoroughly. Add other components

and make the volume 1000 ml with distilled water. Gently heat. Distribute in tubes.

Autoclave at 121°C for 20 min.

Use: This test is commonly use for identification of Mycobacterium tuberculosis.

Inoculated tubes with the test organism for 3 to 14 days. Add few drops of amino

solution or .5 to 1 ml of 2N Na2CO3 in the tube. Pink colour indicate positive test.

Mycobacterium

ruberculosis gives negative test.

Snyder Test

Snyder test agar

Composition:

Trytone 20.0 g


Dextrose 20.0 g

Bromocresol green 0.02 g

Agar 20.0 g


pH 4.8

Preparation: Steam sterilize at 1 180C for 30 min.

Use: It is used to measure the rate of acid production from the metabolism of the glucose

by the lactobacilli. Lactobacilli grow on the snyder test agar. They utilize glucose of the

medium converting it to organic acids and these by lowering the pH to 4.4 or lower. At

this pH bromocersol green becomes yellow. Lactobacilli present in mouth (saliva)

responsible for dental caries are tested by referring following table. Caries process begins

at pH 4.4.

Standardized table to determine dental caries susceptibility .Caries

susceptibility

Hours incubation

24 48 72

Marked Positive --- ---

Moderate Negative Positive ---

Slight Negative Negative Positive

Negative Negative Negative Negative

Thiocyanate Utilization Test

Composition:

Basal solution 100 ml

Solution A 0.5 ml

Solution B 2.0 ml

Solution C 20.0 ml

Basal solution

Na2HPO4 0.48 g

KH2PO4 0.44 g

MgS04.7H2O 0.05 g


Steam sterilize at 121°C for 20 min.

Solution A

FeC12.6H2O 1.0 g

CaCl2 0.1 g


Sterilize by filtration.

Solution B

D-Glucose 10.0 g


Sterilize by filtration.

Solution C

Sodium thiocynate 0.5 g

Distilled water to 100 rnl

Sterilize by filtration

Use: Used for testing thiocyanate utilization ability of microoganisms. Thiocyanate is

utilized as a source of nitrogen and sulphur.

TSI Agar Test

Triple sugar-iron agar

Composition:

Peptone 20.0 g

Tryptone 10.0 g

Beef extract 3.0 g

Yeast extract 3.0 g

Lactose 10.0 g

Sucrose 10.0 g

Dextrose 1.0 g


Ferrous sulphate 0.2 g


Phenol red 0.024 g

Agar 15.0 g


pH 7.4

Preparation: Dissolve all components in distilled water except phenol red and agar.

Adjust the pH. Add phenol red and agar. Distribute in tubes. Steam sterilize at 1180C for

15 minutes.

Use: To test fermentation of these three sugars and H,S production in one medium.

Key reaction for TSI agar

Reaction in

slants

Reaction in

butts Gas H2S Interpreation

Orange red

Red

(Alkaline)

Yellow

(Acidic)

Red

(Alkaline)

Orange red

Yellow

(Acidic)

Yellow

(Acidic)

Yellow

Black

-

-

+

+

-

Slight

-

+

Control

Dextrose fermentation

H2S production.

Dextrose and lactose

/sucrose fermentation.

Dextrose fermentation

And excess H2S

Production.

+ = Positive - = Negative

Culture characteristics ofmembers of enterobacteriaceae on TSI agar after 18-24

hours at 37°C are as follows.

Organism Slant Butt Gas H2S

Escherichia Sp.

Shigella Sp.

S. Typhimurium

S. typhi

Arizona Sp.

Citrobacter freundil

Klebsiella Sp.

Entrobacter Sp.

Serratia Sp.

P. vulgaris

P. mirabilis

P. morganii

P. rettgeri

Providencia Sp.

Pasteurella Sp.

Y

R

R

R

R

R

Y

R

Y/R

Y/R

R/Y

R

R

R

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

(48 hrs)

+

-

-

+

+

+

+

-

-

+

+

-

-

+/-

-

-

-

+

+

+

+

+

-

-

+

+

-

-

-

+

Y = Yellow colour + = Positive

R = Red (pink) colour - + Negative

Urea Hydrolysis

Urea agar (Christensen's medium)

Composition:

Peptone 1.0 g

NaCl 5.0 g

K2HP04 2.0 g

Phenol red (1:500 in aq. sol.) 6.0 ml

Agar 20.0 g


Glucose 10% sol.

Urea 20% sol.

pH 6.8 – 7.0

Preparation: Sterilize the glucose and urea solutions by filteration. Prepare the basal

medium without glucose or urea. Adjust the pH and sterilize by autoclaving in a flask at

121°C for 15 min. Cool to above 50°C add the glucose and urea and tube the medium as

slant.

Use: The medium used to detect urease production by the bacteria.Red colour after

incubation indicate urease production.

Note: The medium may be used as broth by omitting the agar.

Voges Proskauer Test

Glucose phosphate broth

Composition: (See methyl red test).

Use: Used to test carbohydrate fermentation ability of bacteria with the production of

acetylmethyl carbinol or 2-3 butylene glycol. This test is usually done in conjuction with

the methyl red test since the production of acety1 methyl carbinol or butylenes glycol

usually results in insuff~cient acid accumulation, giving methyl red test negative.

Method: After incubation add 1 ml of 40 per cent potassium hydroxide and 3 ml of an a-

naphthol solution. A positive reaction is indicated by the development of pink colour in

2-5 min.

Precaution: a-naphthol is carcinogenic. Avoid body contact.

Xanthine Decomposition

Xanthine agar

Composition:

Medium A

Pancreatic digest of gelatin 5.0 g

Beef extract 3.0 g

Agar 20.0 g


Add components to water gently heat to dissolve the components.

Medium B

Xanthine 4.0 g

Distilled water to Gently heat to dissolve. 100 ml

Complete medium

Medium A 900 ml

Medium B 100 ml

Combine medium at medium by mix thoroughly. Steam sterilize at 121°C for 20

minutes. Poor plates.

Use: Used for testing xanthine decomposition ability of bacteria. Examine the plates

under a low power microscope for disappearance of xanthine crustals. (clearing) around

the bacterial growth.

Catalase Test

Solution required: 3% H,02 solution -1 ml approx.

Procedure: Place a drop of three percent H20, on colonies of nutrient agar.

Result: Prompt effervescence indicates catalase production.

Note: Culture media containing blood are unsuitable for the test asblood contain catalase.

Or

Procedure: Take colony growth on nichrome wire. Insert wire loop in to the H2O 2

solution.

Result: Prompt effervescence indicates catalase production.

Oxidase Negative

Regent Required

a. Oxidase reagent: One percent solution or tetra methyl-p-phenylene diamine

hydrochloride.

Procedure:

1 . Put a drop of freshly prepared one percent solution of oxidase

reagent on a piece of filter paper.

2. With a sterile loop pick a test colony and rub it on the paper in the area

impregnated with oxidase reagent.

Result: 1f organikm is oxidase positive, paper will become deep purple blue in colour in

a few seconds.

Procedure:

Pour oxidase reagent over the colonies of the test organism on the culture plate.

Result: The colonies of oxidase positive organisms rapidly develop a deep purple blue

colour.

Bile Solubility Test

Solution required: 10% sodium deoxycholate solution.

Procedure:

1. Add one drop of 10% sodium deoxycholate solution to microbial suspension.

2. Incubate at 37°C.

3: Examine for clearing at 15 min., 20 min. and 1 h.

Result: Clearing should occur within 30 min.

Coagulase Test

1. Slide method

Solution required: Citrated plasma.

Procedure:

1. Place three separate drops of saline on a clean slide.

2. Suspend a loopful of test colony in two of these and a loopful of positive

control organism (Staphylococcus aureus) in the third.

3. With a sterile loop, add a drop of citrated plasma to one test and the positive

suspension.

Result: Occurrence of clumping in test and positive control within. 10 s indicates a

positive result. The saline control should remain evenly suspended. (Negative control)

Note: This test detects the presence of “clumping factor” and is not a true coagulase test.

a. Tube method

Procedure:

1. Prepare 1/10 dilution of plasma in 0.85% saline.

2. Emulsify a few colonies of positive control (Staphylococcus aureus) and the

test isolate into two separate tubes containing 1 a 1/10 dilution of plasma in

0.85% saline.

3. Incubate at 37OC.

4. Examine for coagulation at 1, 3 and 6 h.

Result: Conversion of the plasma into a soft 'or stiff gel, seen on tilling the tube to a

horizontal position indicates a positive, result.

Negler Test I

This test indicates the production of alpha-toxin, an exotoxin by Clostridium perfringens.

Requirement:

Egg yolk agar: Composition-See Lecithinase production.

Use: Divide an egg yolk agar plate into two equal sections. Spread a loopful of

Clostridium perfiingens antitoxin over half the plate and allow to dry. Inoculate the plate

with a loopful of the test culture covering both halves by single streak. Incubate at 37OC

under anaerobic conditions.

Result: Production of an opaque zone of precipitation around the area of growth and

absence of this precipitation in the area with specific alpha-antitoxin indicates positive

test.

Optochin Test

Requirement: Blood agar: See page 78

Use:

1. Divide a blood agar plate into three equal sections.

2. Inocula one with a known Streptococcuspneumoniae, another I with a

Streptococcus viridans and the third with the test isolate.

3. Keep the cultures separately.

4. Place a 5 microgram Optochin disc (ethylene hydrocupreine hydrochloride) in

the centre of the plate.

5. Incubate at 37OC overnight and observe the zone of inhibition.

Hydrolysis of Fats

This test detects the ability of an organism to decompose fats. This causes rancidity in

foods due to the formation of fatty acids.

Use: Prepare nutrient agar plates containing 1% fat (oil/butter/ghee) and neutral red

indicator. Streak the test organism in these plates. Incubate at 37'C for 2 days. Observer

plates for the presence of red globules under the colonies.

Result: Acid production is indicated by change in the colour of indicator from yellow to

red. Maintain a control with uninoculated.

Dehydrogenase Activity

Prepare six tubes containing 1.0 ml. of inoculum. 1.0 of M/10 phosphate buffer of pH 7

and 0.5 ml of resazurin solution.

Add 1.0 ml of water to tube number 1.

Add 1.0 ml of M/10 glucose to tube number 2.

Add l .0 ml of M/10 lactose to tube number 3.

Add 1.0 ml of M/10 sodium lactate to tube number4.

Add 1.0 ml of M/10 sodium succinate to tube number 5.

Add 1.0 ml of M/10 sodium fumarate to tube number 6.

Mix the contents thoroughly.

Incubate at room temperature and observe for colour change from blue to pink every 15

min for one hour. Record the time of colour change.

Use: This test detects the ability of an organism to produce dehydrogenases. This causes

removal of hydrogen from the substrate. The indicator dye reacts with this hydrogen and

changes colour.

2

Cultivation Media

1. Bacteria

Actinomycetes medium

(Bennet’s agar)

Composition:

Glucose 10.0 g

Casein 2.0 g

Yeast extract 1.0 g

Beef extract 1.0 g

Agar 20.0 g

Distilled Water to 1000 ml

pH 7.3

Preparation: Autoclave the medium at 1210C for 20 min.

Use: Used for the cultivation of actinomycetes, specially Streptomyces species. This

medium enhances sporulation.

Actinomycetes medium

(Dextrose tryptone agar)

Composition:

Glucose 10.0 g

Tryptone 5.0 g

K2HPO4 0.5 g

NaCl 0.5 g

FeSO4.7H2O 0.1 g

Agar 20.0 g


pH 7.2

Preparation: Steam sterilize the medium at 121°C for 20 min.

Use: Used for isolation of actinomycetes.

Actinomycetes Medium

(Glycerol yeast extract agar)

Composition:

Glycerol 10.0 g

K2HPO4 1.0 g

L-aspargine 1.0 g

Agar 20.0 g

Trace salt solution(see oatmeal agar) 1.0 ml


pH 7.4

Preparation: Steam sterilize the medium at 12 1°C for 20 min.



(Oatmeal agar)

Composition:

Oatmeal 20.0 g

Agar 20.0 g

Trace salt solution 1.0 ml


pH 7.2

Pridham and Gottlieb trace salt solution

CuSO4.5H2O 0.64 g

FeSO4.7H2O 0.11 g

MnCL2.4H2O 0.79

ZnSO4.7H2O 0.15 g


Preparation: Cook the oatmenl in 1 lit. water for 20 min. Filter through cloth. Restore

volume of filtrate to 1 litre. Add I ml of trace salt solution. Adjust the pH. Add agar.

Steam sterilize the medium at 121°C for 20 min.



(Potato-carrot agar)

Composition:

Diced potato 150.0 g

Diced carrot 30.0 g

Tap water to 1000 ml

pH 6.5

Preparation: Cook the potato and carrot in 1 litre of boiling tap Mater for 30 min. Filter

through muslin and adjust the volume to 1 Litre. Adjust the pH and add agar. Steam

sterilize the medium at 121°C for 20 min.



(Carbon utilization medium-Shirling and Gottlieb)

(NH4)2SO4 2.64 g

KH2PO4 2.38 g

K,HP04 5.65 g

MgS04.7H2O 1.0

Trace salt solution (See page No. ....) 1.00 ml

Agar 20.0 g

Carbon source(Sterilize separately by filtration) 10.0 g


pH 7.2

Preparation: Steam sterilize the medium at 1210C for 20 min. Add sterile carbon source

solution after sterilization in the medium.

Use: Used to test carbon utilization ability of actinomycetes, important in identification.


(Synthetic agar)

Composition:

K2KPO4 1.0 g

MgSO4.7H2O 0.5 g

KCl 0.5 g

FeSO4.7H2O 0.01 g

NaNO3 2.0 g

Glycerol 30.0 g

Agar 20.0 g


pH 7.2



Actnomycetes Medium

(Yeast glucose agar)

Composition:

Yeast extract 10.0 g

Glucose 10.0 g

Agar 20.0 g

Distilled water.to 1000 ml

pH 7.2

Preparation: Steam sterilize the medium at 121°C for 20 minutes.



(Yeast extract-malt extract agar)

Composition:

Yeast extract 4.0 g

Malt extract 10.0 g

Glucose 4.0 g

Agar 20.0 g


pH 7.3

Preparation: Steam sterilize the medium at 1210C for 20 minutes.



Composition:

Glycero l5.0 g

Sodium propionate 4.0 g

Sodium caseinate 2.0 g

K2HPO4 0.5 g

Asparagine 0.1 g

MgS04.7H2O 0.1 g

FeS04.7H2O 0.001 g



Use: Used for cultivation of Actinomyces species.

Acetobactor Medium

Composition:


Glucose 3.0 g

CaCO3 10.0 g

Agar 20.0 g

pH 7.1

Preparation: Autoclave at 1210C for 20 min. Mix CaCO3 b: shaking the medium before

pouring.

Use: Used for isolation of Acetobacter and Gluconobacter.

Acetobacter Medium

(Hoyer's medium)

Composition:

(NH4)2SO4 0.1 g

K2HPO4 0.01 g

KH2PO4 0.09 g

MgS04.7H2O 0.025 g

FeCl3.6H2O 0.002 g

Ethanol (15% v/v) 20 ml

Distilled water 80 ml

Preparation: Dissolve the salts in 80 ml of distilled water. Dispense as 4 ml aliquots in to

test tubes and autoclave at 1210C. Ethanol may then be added as sterile filtered solution

at the rate of 1 ml per tube.

Use: Used to cultivate Acetobacter. Acetobacter grow slowly, may take up to 14 days to

develop.

Acetobacter Medium

(Acetobacter broth)

Compostion:

Peptone 3.0 g

Glucose 18.0 g

(NH4)2SO4 1.0 g

Yeast extract 2.0 g

Solution A 5 ml

Solution B 5 ml


Solution A

K2HP04 50.0 g

KH2PO4 50.0 g


Solution B

MgSO4.7H2O 20.0 g

NaCl 1.0 g

FeSO4 1.0 g

MnSO4 1.0 g

Conc. HCL 1.0 ml


Preparation: Steam sterilize at 1210C for 20 minutes.

Use: Used for cultivation of Acetobacter.

Acholeplasma Medium

Composition:

Papaic digest of soyabean meal 10.0 g

PPLO broth without crystal violet 900.0 ml

Fresh yeast extract solution 100.0 ml

(25.0 g Baker's yeast/100 ml water)

Agar 3.0 g

pH 7.8

PPLO broth without crystal violet

Beef heart, infusion from 225.0 g

Peptone 9.0 g

NaCl 4.5 g

Preparation: Autoclave at 1210C for 20 min.

Use: Used for cultivation of Acholeplasma.

Alcaligenes Medium

Composition:

K2HPO4 7.32 g

CaCl2.2H2O 0.014 g

MgSO4.7H2O 0.002 g

FeSO4.7H2O 0.04 g

Ammonium tartarate 4.6 g

KH2PO4 1.09 g


pH 7.5

Preparation: Autoclave the medium at 1210C for 20 min.

Use: Used for cultivation of Achromobacter and Alcaligenes speck.

Agrobacterium Isolation Medium

Composition:

Mannitol 10.0 g

Sodium nitrate 4.0 g

Magnesium chloride 2.0 g

Calcium propionate 1.2 g

Magnesium phosphate. 0.2 g

Magnesium sulphate 0.1 g

Sodium bicarbonate 0.075 g

Magnesium carbonate 0.075 g

Agar 20.0 g


pH 7.2

Preparation: Autoclave the constituents at 115°C for 20 min. Allow to cool to 50-550C

and aseptically add the following:

Berberine 275 ppm

Sodium selenite 100 ppm

Penicillin G 60 ppm

Streptomycin sulphate 30 ppm

Cyclohexamide (85-100% active) 250 ppm

Tryothicin 1 ppm

Bacitracin (65 units/mg) 100 ppm

Use: Highly specific for the isolation of Agrobacerium tumefaciens,

A. radiobacter group from soil.

Ammonification Medium

Composition:

KH2PO4 3.00 g

KCl 0.20 g

MgSO4.7H2O 0.20 g

NaCl 0.20 g

CaSO4 0.10 g

FeSO4 0.01 g

Peptone 10.00 g



Use: Used to isolate ammonifying bacteria. Bacteria are inoculated into tubes containing

5 ml of the above medium. After 10 days of incubation production of ammonias is testd

by means of Nessler’s reagent.

Anaerobic Bacteria Medium

Cooked meat broth (Robertson)

Composition:

Cooked meat filtrate

Fresh bullocks heart 500.0 g

Water 500 ml

Sodium hydroxide (1 N NaOH) 1.5 ml

Mince the heart. Add in alkaline water Simmer for 20 min. Filter through muslin cloth.

Dry minced meat, on filter paper partially.

Complete broth

Cooked meat filtrate 500 ml

Peptone 2.5 g

NaCl 1.25 g

pH 7.8

Preparation: Steam at 100°C for 20 min. Add 1 ml pure HCL and filter. Adjust the pH

8.2. Steam again at 100°C for 30 min. Adjust the reaction to pH 7.8.

Complete medium Place the meat pieces in tubes to a depth of about 2.5 cm. Cover these pieces with about

10 ml broth. Autoclave at 1210C for 20 min.

Use: Used for isolation of anaerobic bacteria.

Note: Before inoculation boil the tube vigorously in water bath to drive away oxygen.

Cool and inoculate. Put a layer of liquid paraffin if essential.

Anaerobic Broth

Composition:

Casein 20.0 g

Glucose 10.0 g

NaCl 5.0 g

Sodium thioglycollate 2.0 g

Sodium formaldehyde

Sulfoxylate 1.0 g

Methylene blue 2.0 mg


pH 7.2

Preparation: Distribute the broth in to tubes. Autoclave the medium at 1210C for 20 min.

Use: For cultivation of anaerobic microorganisms especially Clostridium.

Anaerobic Medium

(Thioglycollate medium)

Composition:

Yeast extract (water solube) 5.0 g

Casein hydrolysate (Pancreatic digest) 15.0 g

Glucose 5.5 g

L-cysteine 0.5 g

Agar 0.75 g



Methylene blue 1.0 ml


pH 7.1

Preparation: Dissolve the ingredients except thioglycollate and indicator in water by

heating. Add thioglycollate and adjust the pH. Filter. Add indicator solution. Sterilize at

121°C for 15 min. Cool and use.

Use: Used for isolation of anaerobic bacteria.

Note:

1. If the upper third is blue in colour, anaerobic condition should be stored by

boiling again for few minutes and cooling rapidly.

2. Resazurin sodium solution 1 in 1000, may be used replacing methylene blue.


(Lapage et al., 1970)

Composition:

K2HOP4 0.5 g

NH4CL 1.0 g

Na2SO4 1.0 g

MgSl4.7H2O 2.0 g

CaCl2.2H2O 0.1 g

Yeast extract 1.0 g

Ferrous sulphate (see below) 10.0 ml

Reducing agent (see below) 10.0 ml

Sodium lactate (70%) 3.5 g

Resazuri n (see below) 1.0 ml


pH 7.4

Gas N2 : C02 80 : 20

Ferrous sulphate

FeSO4, 7H2O 0.5 g


Reducing agent

Ascorbic acid 1.0 g



Redox indicator (Resazurin)

Resazurin 0.10 g


Use: Used for the cultivation of Desulfomonas, Desulfobacter Desulfotomaculum and

Desuvovibrio.


(Bagnra et at. 1985)

Composition:

K2HPO4 2.21 g

KH2PO4 1.5 g

(NH4)2SO4 1.3 g

MgCl2 0.1 g

CaCl2 0.02 g

FeS04.7H2O 0.001 g

Yeast extract 5.0 g

NaHCO3 0.8 g

Cellulose 10.0 g

Cysteine hydrochloride 0.5 g

Resazurin (See page 51) 1.0 ml


pH 7.4

Gas Argon or Nitrogen

Use: Used for cultivation of cellulomonas species.


(Hungate, 1950)

Composition:

K2HPO4 0.5 g

KH2PO4 0.2 g

(NH4)2SO4 0.5 g

CaCl, 0.05 g

NaCl 1.0 g

MgSO4 0.05 g

Rumen fluid (see below) 150.0 ml

NaHCO2 5.0 g

Resazurin (see page 51) 1.0 ml


Cellulose 10.0 g


pH 7.5 7.5

Gas N2 : CO2 80 : 20

Rumen fluid

Take 2000 ml fresh rumen fluid. Allow to settle down coarse particles. Decant supenatant

to 1000 ml. Add 4.0 g yeast extract to it, pass nitrogen and incubate at 370C for

overnight. Then centrifuge at 8000 rpm for 10 min at 6 to 8OC. Collect the supernatant in

screw – capped bottle, pass nitrogen and store in refrigerator or -20°C. Pass nitrogen

on every use. Fermenting slurry of anaerobic digester may be processed in

similar manner and used in place of rumen fluid.

Use: Used for cultivation of clostridiurn, Bacteroides Butyrivibrio

and Bacterium species.


(Sijpesteijn, 1951)

Composition:

K2HPO4 0.6 g

KH2PO4 0.4 g

(NH4)2SO4 0.4 g

NaCl 1.2 g

MgSO4 0.2 g

CaCI2 anhydrous 0.2 g


NaHCO3 5.0 g

Cellulox 10.0 g




pH 7.5

Gas N2 : CO2 80 : 20

Use: Used for cultivation of Ruminococcus species.


(Sowers et al., 1984)

Composition:

K2HPO4 0.06 g

NH4Cl 0.05 g

NaCl 2.34 g

KCI 0.08 g

MgSO4.7H2O 0.63 g

CaCl2.2H2O 0.014 g

Yeast extraat 0.10 g

Trace elements (see p.54) 1.0 ml

Trace vitamins (see p.54) 1.0 ml

Sodium acetate 4.1 g

Resazurin (see below) 1.0 ml

Reducing agent (see below) 12.5 ml


pH 6.8

Gas N2 : CO2 80 : 20

Trace elements

Nitrilotriacetic acid 4.5 g

FeC12.4H2O 0.4 g

MnC12.4H2O 0.1 g

CaCl2 0.02 g

CoC12.6H2O 0.17 g

ZnCl2 0.1 g

H3BO3 0.019 g

Sodium molybdate 0.01 g


Trace vitamins

Biotin 2.0 mg

Folic acid 2.0 mg

Vitamin B12 0.1 mg

Pyridoxine HCl 10.0 mg

Thiamine 5.0 mg

Riboflavin 5.0 mg

Nicotinic acid 5.0 mg

DL-Calcium pantothenate 5.0 mg

p-aminobenzoic acid 5.0 mg

Lipoic acid 5.0 mg


Reducing agent


Adjust pH 9.0 g

Sodium sulphide 9H2O 2.0 g


Use: Used for cultivation of Methanosarcina, Methanococcoides, Methanlobus and

Methanococcus.


(Corder et al., 1983)

Composition:

K2HPO4 0.3 g

KH2PO4 0.3 g

NH4Cl 2.7 g

(NH4)2SO4 0.3 g

NaCl 0.61 g

MgSO4.7H2O 0.13 g

MgCI2.6H2O 0.053 g

FeSO4.7H2O 0.01 g

Cocl2 0.001 g

Na2MoO4.2H2O 0.0002 g

NiCl2.6H2O 0.0002 g


Trace vitamins (see page 54) 1.0 ml


Resazurin (see page 5 1) 1.0 ml

Reducing agent (see page 54) 12.5 ml


pH 6.8

Gas H2 : CO2 80 : 20

Use: Used for enrichment and cultivation of methanogenic bacteria specifically

Methanogenium.


(Balch et al., 1979)

Composition:

K2HPO4 0.14 g

NH4Cl 0.25 g

NaCl 18.0 g

KCl 0.35 g

MgSO4.7H2O 3.45 g

MgC12.6H2O 2.75 g

CaCl2.2H2O 0.14 g

Fe(NH4)2(SO4)2 0.002 g

Yeast extract 2.0 g

Trypticase 2.0 g

Trace elements (see below) 1.0 ml

Trace elements (Balch et al.)


MgSO4.7H2O 3.0 g

MnSO4.2H2O 0.5 g

NaCl 1.0 g

CoCl2 0.1 g

FeSO4.7H2O 0.1 g

CaCL2.2H2O 0.1 g

ZnSO4 0.1 g

CuSO4.5H2O 0.01 g

AIK(SO4)2 0.01 g

H3BO3 0.01 g

Na2MoO4.2H2O 0.01 g


Dissolve nitrilotriacetic acid with KOH to pH 6.5 and then add other salts.



Resazurin (see page 5 1) 1.0 ml

Reducing agent (see page 5 1) 12.5 ml


pH 6.8

Gas H2 : CO2 80 : 20

Use: Used for enrichment and cultivation of Methanobrevibacter spp, Methanococcus,

Methanomicrobium, Methanogenium and Methanoplanus.


(Balch et d, 1979)

Composition:

K2HPO4 0.3 g

KH2PO4 0.3 g

(NH4)2SO4 0.3 g

NaCl 0.6 g

MgSO4.7H2O 0.13 g

CaCl2.2H2O 0.008 g

FeSO4 0.002 g

Yeast extract 2.0 g

Trypticase 2.0 g

Trace elements (see page 54) 1.0 ml



Sodium formate 2.5 g


Reducing agents (see page 54) 12.5 ml


pH 6.8

Gas N2 : CO2 or H2 : CO2 80 : 20

Use: Used for enrichment of methanogenic bacteria, specifically Methanobacterium,

Methanobrevibacter, Methanogenium and Methanospirillum.

Azotobacter Medium

(Nitrogen free glucose broth)

Composition:

K2HPO4 1.0 g

MgSO4.7H2O 0.2 g

FeSO4.7H2O 0.05 g

CaCl2.2H2O 0.1 g

Na2MoO4.2H2O 0.001 g

Glucose 10.0 g


Preparation: If this medium is to be used immediately, sterilization is not necessary.

When it must be stored for long time, it should be sterilized.

If it is to be sterilized, the glucose should be sterilized separately in 100 ml of water at

1150C for 15 min. The remainder of the medium is steam sterilized at 121°C for 20 min.

After sterilization, the two solutions are mixed aseptically and dispensed in to

sterilized bottles.

Use: Used for isolation of Azotobacter.

Nitrogen free glucose agar

Add 2.5% agar in above broth. Prepare the medium in similar manner.

Azotobacter Medium

(Ashby's nitrogen free medium-modified)

Composition:

Mannitol 15.0 g

MgSO4.7H2O 0.2 g

K2HPO4 0.2 g

Ferric chloride (10% aqueous solution) 0.5 ml

Molybdenum trioxide (10% aqueous solution) 1 drop

Agar 15.0 g


pH 7.2

Prepamtion: Steam sterilize at 1210C for 20 minutes.

Use: Used for the isolation of Azotobacter. Colonies appear flat, soft, milky and mucoid.

Azotobacter Medium

(Ashby's mannitol agar)

Composition:

Mannitol 20.0 g

K2HPO4 0.2 g

MgSO4.7H2O 0.2 g

NaCl 0.2 g

K2CO4 0.1 g

CaCO3 5.0 g

Agar 20.0 g


pH 7.2

Preparation: Sterile CaCO3 should be added after steam sterilization of remainder

medium at 1210C for 20 minutes.


Azotobacter Medium

(Burk's nitrogen free medium)

Composition:

Burk's salt 1.3 g

Fe Mo mixture 1.0 ml

Sucrose 20.0 g


pH 7.0

Burk's salt

MgSO4 20.0 g

K2HPO4 80.0 g

KH2PO4 20.0 g

CaSO4 13.0 g

Fe Mo mixture

FeCl3 1.45 g

Na2NoO4 0.253 g


Prepnration: Steam sterilize the medium at 11 5°C for 20 minutes.

Use: Used for isolation of Azotobactor.

Azotobacter Medium

(Jensen's medium)

Composition:

Sucrose 20.0 g

K2HPO4 1.0 g

MgSO4.7H2O 0.5 g

NaCl 0.5 g

FeSO4 0.1 g

Na2MoO4 0.005 g

CaCO3 2.0 g

Agar 20.0 g


pH 7.0

Preparation: Add all components except CaCO3 and agar. Adjust the pH. Add agar.

Sterilize at 115°C for 30 min. (not sterilize if freshly used). Add sterile CaCO3 in melted

sterile agar medium.


Azospirillum Semisolid Medium

Composition:

K2HPO4 0.1 g

KH2PO4 0.4 g

MgSO4 0.2 g

NaCl 0.1 g

CaCl2 0.02 g

FeCl2 0.01 g

Na2MoO4 0.002 g

Sodium malate 5.0 g

Bromothymol blue (0.05% Ehanol) 5.0 ml

Agar 2.25 g


pH 6.8

Preparation: Steam sterilize the medium in autoclave at 121°C for 20 min.

Use: Used for cultivation of Azospirillum.

Azospirillum Medium

Composition:

K2HPO4 0.50 g

MgSO4.7H2O 0.10 g

NaCl 0.02 g

MnSO4.H2O 0.01 g

KOH 4.00 g

FeSO4.7H2O 0.05 g

Na2MoO4 0.002 g

CaCl2 0.01 g

Malic acid 5.0 g


pH 6.0 : 7.0

Preparation: Steam sterilize the medium in autoclave at 121°C for 20 min.

Use: Used for cultivation of Azospirillum.

Bacteroides Medium

(VL medium)

Composition:

Sodium azide

Sterile beef bile

Tryptone

NaCl

Meat extract

Yeast extract

Cysteine hydrochloride

Glucose

Agar

Distilled water to

pH

Preparation: Adjust the pH. Sterilize by autoclaving at 1150C for 20 minutes. Use: Used

for cultivation of Bacterioides fragilis from fecal matter. After anaerobic incubation for

48 hrs. The colonies appear round (1mm in diameter) translucent and are found at the

highest dilutions. Colonies are surrounded by a whitish precipitate of bile salts.

Bdellovibrio Medium

(Cation - supplemented yeast extract, peptone, Na acetate and cysteine agar1YPSC agar)

Composition:

Bacto yeast extract 0.1 g

Bacto peptone 0.1 g

Sodium acetate trihydrate 0.5 g

L-Cysteine hydrochloride 5.0 mg

CaCl2 0.002 .M

MgSO4 0.003 M


pH 7.6

Preparation: Autoclave at 1210C for 15 minutes.

Use: It is a medium of choice for plaque counts in most combinations of Bdellovibrio

strains and associate bacterial strains.

Beijerinckia Medium

(Becking's medium)

Composition:

Socrose 20.0 g

KH2PO4 0.8 g

K2HPO4 0.2 g

MgSO4.7H2O 0.5 g

FeCl3 0.1 g

Na2MoO4 0.005 g

Agar 20.0 g


pH 6.5

Reparation: Steam sterilize at 1210C for 20 minutes.

Use: Used for isolation of Beijerinckia.

Bioluminescent Bacteria Medium

Composition:

Basal medium

(Tris) Hydrozyrnethyl amino methane 12.1 g

NH4Cl 1.0 g

K2HPO4 0.075 g

FeSO4.7H2O 0.028 g

Artificial sea water 1000 ml

(NaCl -23.4 g, KCl 1.5 g, MgSO4.7H2O -24.6 g, CaCl2 -2.9 g, D.W. -1 litre)

pH 7.5

Biochemicals should be prepared in the above basal medium as follows

Amino acids 0.5%

Sugars 1%

Preparation: Dissolve Tris. Adjust pH. Then dissolve remaining ingredients. Steam

sterilize at 12 1 "C for 15 minutes.

Use: Basal medium is used for cultivation of bioluminescent bacteria.

Note: By adding 2% agar, prepare a solid medium.

Bordetella Medium

(Bordet Gengou medium)

Composition:

Horse blood (defibrinated) 50.0 ml

Potato slices 125.0 g


Glycerol 10.0 ml

Proteose peptone 10.0 g

Agar 22.5 g


pH 7.0

Preparation: Boil the slices in 250 ml water. Dissolve agar in 750 ml water and add to

potato decoction, glycerol and peptone. Adjust the pH. Sterilize in autoclave at 115°C for

10 minutes. Add sterile defibrinated horse blood to the cooled agar. Mix and pour into

sterile

Use: It is used for isolation of Bordetella.

Bordetella Medium

(Chacol blood agar)

Composition:

Charcol agar base

Beef extract 10.0 g

Strach 10.0 g

Peptone 10.0 g

Charcoal (Bacteriological grade) 4.0 g

Yeast extract 3.5 g

Agar 15.0 g


pH 7.4

Adjust the pH before addition of charcoal. Steam sterilize the medium 121°C for 20 min.

Complete medium

Charcoal agar base 100.0 ml

Horse blood (sterile, defibrinated) 10.0 ml

Penicillin (100 i.u./ml) 0.3 ml

Cool the charcoal agar base at 50°C. Add other ingredients aseptically. Pour plates.

Use: It is a selective medium of Bordetella pertussis.

Note: Some peptone brands are inhibitory to Bordetella, hence may be omitted from the

medium.

Brucella Medium

(Jones and Morgan medium)

Composition:

Basal medium

Peptone 10.0 g

Agar 20.0 g

NaCl 5.0 g

Meat extract 5.0 g


Steam sterilize the medium at 1210C for 20 min.

Complete medium

Basal medium 100.0 ml

Sterile inactivated

Horse serum (sterile, inactivated) 5.0 ml

Glucose solution 25% (sterile) 4.0 ml

Bacitracin solution (2000 units/ml in sterile water) 1.25 ml

Polyrnyxin B solution (5000 units/ml in sterile water) 0.12 ml

Cyclohexamides solution 10 mg/ml 1.0 ml

(Dissolve in acetone dilute with sterile water)

Preparation: Cool the sterilized basal medium at 550C. Add remaining solutions

aseptically. Pour plates.

Use: Used for cultivation of Brucella.

Brucella Medium

(Liver infusion broth-Huddleson)

Composition:

Minced beef liver 8 lbs


Steam for 2 hrs with frequent stirring. Strain through lint and autoclave for 30 min. Then

add

Peptone 10 g

Sodium chloride 5 g


Steam for 30 min, cool and adjust the pH to 7.0.

Use: Used for cultivation of Brucells abortus.

Brucella Medium

(Filde's eatract)

Composition:

Defibrinated sheep blood 50.0 ml

Hydrochloric acid 6.0 ml

Pepsin 8.0 g

Normal saline 100 ml

Preparation: Heat at 650C for 2 hrs. Add 20% NaOH, until a violet red colour develops

with cresol red indicator. Then add pure HCL drop by until a definite red tint develops

with phenol red.

Complete medium

Fildes extract 5.0 ml

Melted nutrient agar (Sterile) 100 ml

Use: Used for cultivation of Brucella.

Brucella selected medium

Composition:

Infusion from beef heart 500.0 g

Tryptone 10.0 g

NaCl 5.0 g

Glucose 2.5 g

Gelatin 1.0 g

Sheep blood 100.0 ml

Agar 15.0 g

Antibiotic solution 10.0 ml


pH 7.4

Antibiotic solution

cycloheximide 1.0 g

Bacitracin 250,000 u

Circulin 250,000 u

Polymyxin B 100,000 u


Filter sterilize the antibiotic solution.

Preparation: Add all components except blood and antibiotic solution in distilled water.

Steam sterilizer at 1210C for 20 minutes.

Use: Used for selective isolation Brucella species.

Campylobacter Medium

Composition:

Beef extract 10.0 g

Peptone 10.0 g

Charcoal 4.0 g

Casein hydrolysale 3.0 g

Sodium deoxycholate 1.0 g

FeSO4.H2O 0.25 g

Sodium pyruvate 0.25 g

Cefoperazone solution (0.32% in water) 10.0 ml

Agar 20.0 g


pH 7.4

Preparation: Add all components except cefoperazone solution in distilled water. Steam

sterilize at 1210C for 20 min. Sterilize the cefoperazone solution by filtration. Mix

aspectically.

Use: Used for the selective isolation of Compylobacter species.

Caulobacter Medium

Composition:

Peptone 0.5 g

Agar 20.0 g

Sea water (filered) 1000 ml

pH 7.0

Preparation: Steam sterilize the medium at 1210C for 20 min.

Use: Used for cultivation of Caulobacter.

Cellulolytic Bacteria Medium

(Omeliansky’s medium)

Composition:

(NH4)2SO4 1.0 g

K2HPO4 1.0 g

MgSO4.7H2O 0.5 g

CaCO3 2.0 g

Nacl trace


pH 7.0

Preparation: Add all components except CaCO3. Adjust the pH. Steam sterilize at

1210C for 20 min. (not necessary to sterilize if freshly used.) Add sterile CaCO3 in both.

Add

2-3 strips of filter paper to each tube, part of the paper remaining above the surface of the

medium. Cellulose decomposing organisms within 15-20 days.

Cellulytic Microorganism Medium

(Mandel and Reese medium)

Composition:

Ammonium sulphate 1.4 g

Potassium dihyydrogen 2.0 g

Phosphate

Magnesuim sulphate 0.3 g

Calcium Chloride 0.3 g


Manganese sulphate 0.0016 g

Proteose peptone 1.0 g

Urea 0.3 g

Zinc chloride 0.0017 g

Calcium chloride 0.002 g


pH 5.3

Preparation: Sterilize the medium at 1000C for 1 hrs.

Use: Used for assaying cellulase.

Clostridim Medium

(Lowbury and Lilly’s medium)

Composition:

(A) Agar base

Peptone water (see indole test) 1000 ml

Agar 50.0 g

Autoclave at 1210C for 30 min. This is used as is lower layer of the medium for the

upper layer.

(B) Human serum

Serum is prepared by treating plasma with 5% of sterile 10% calcium chloride at 370C.

Complete medium

Agar base 100 ml

Flide’s peptic 6.5 ml

Digest of sheep blood

Human serum (sterile) 40 ml

Neomycin sulphate 1.5 ml

(Sterile aqueos solution 10,000 µg/ml)

Heat fields digest at 550C for 30 min. Melt the agar base & cool it to 55

0C. Add the

remaining ingradients. Pour this as upper layer on the layer of agar base. Spread 150 I.U.

of CL. Perfringenes antitoxin over half of each surface.

Use: It’s both selective & differential medium.

1. Neomycin inhibits aerobic forming lecithinase producing organisms.

2. Swarming growth of Proteus is inhibited by excess agar.

3. Cl. Perfringenes produce lecithinase differentiating the colonies.

Clostridium Medium

(Sulphite polymyxin sulphadiazine agar)

Composition:

Basal Medium

Tryptone 15.0 g



Agar 20.0 g


pH 7.0

Preparation: Dissolve the ingredients by steaming and adjust the pH to 7.0. Sterilize by

autoclaving at 1210C for 15 min.

Complete Medium

Sodium sulphate (10% solution) 5.0 ml

Polymixin B (0.1% solution) 1.0 ml

Sodium sulphadiazine (1.2% solution) 1.0 ml

Basal medium 100 ml

Sterilize other solution by filtration separately and add to basal medium melted and

cooled to 450C. Mix well and pour plates.

Use: The medium is used for the enumeration of sulphite reducing clostridia, e.g.

Clostridium perfringens. Sulphate reducing bacteria produce black coloured colonies.

Clostridium Medium

(Sulfite agar)

Composition:

Tryptone 10.0 g

Sodium sulphite 1.0 g



pH 7.0

use: Cl. nigrificans, causeing sultlde spollage ofthe food, produce black coloured

colonies on the medium. (H2S producer).

Coliform Medium

(Briliant green lactose bile broth)

Composition:

Peptone 10.0 g

Ox bile 200 ml

Lactose 10.0 g

Brilliant green (0.1% aq. solution) 13 ml


pH 7.2

Preparation: Dissolve the peptone in approx. 500 ml water. Then ad the ox bile and

lactose, dissolve. Adjust the pH. Add the brilliant green solution. Make up the volume 1

litre by addition of water Distribute in tubes. Autoclave at 1150C for 15 min.

Use: This medium is used to determine coliforms in water. (in MPN test)

Significance: It eliminates false positive due to anaerobes.

Coliform Medium

(Glucose broth)

Composition:

Nutrient broth 100 ml

Glucose 1.0 g

Preparation: Sterilize at 1090C for 30 min.

Use: Glucose acts as reducing agent. The medium is useful for the growth of facultative

eanaerobes like coliforms.

Coliform Medium

(Endo's agar)

Composition:

Lactose 8.0 g

Basic fuchsin (10% solution in 95% alcohol) 0.3 ml

Sodium sulphite about

(2.5% solution) 5 to 10 ml


pH 7.2

Preparation: Sodiu, sulphite is added until the hot mixture turns pink in colour. Steam

sterilize the medium at 1000C for 1 hr.

Use: Selective medium for coliform. Coliform produce red coloured colonies on the

medium.

Coliform Medium

(Eosin methylene blue agar/EMB agar)

Composition: Peptone 10.0 g

Lactose 5.0 g

Sucrose 5.0 g

Dipotasium hydrogen phosphate 12.0 g

Eosin Y 0.4 g

Methylene blue 0.065 g

Agar 15.0 g


pH 7.2

Preparation: Steam sterilize at 1210C for 30 min.

Use: This medium is used for distinguishing lactose fermenting gramnegative bacteris

from non-lactose fermenting types by their appearance. Colonies of Escherichia coil have

a characteristic metallic sheen.

(Cystine tellurite blood agar)

Composition:

(A) Beef heart infusion agar

Beef heart infusion from 500.0 g

Tryptone 10.0 g



pH 6.8-7.3


(B) Cystine tellurite solution

Potassium tellurite(0.3%) (sterile) 15.0 ml

L-cystine 5.0 mg

Mix thoroughly 5.0 ml

C. Sheep blood (sterile)

Preparation: Melt 100 ml of 2% beef heart infusion agar and cool to 50°C. Add

aseptically cystine tellurite solution and sheep blood. Mix & pour into sterile petriplates.

Use: It is used for isolation Corynebacterium diphtheriae. Cystine allows detection of

H2S producing colonies of C. diptheriae from other Coiynebacterizrm species by their

characteristic brown halo around black colonies.

Corynebacterium Medium

(Hoyle's medium)

Composition:

Nutrient agar (sterile, pH 7.8) 200 ml

Horse blood 10.0 ml

Potassium tellurite solution (0.7 gm in 20 ml) 2.0 ml

Preparation: Cool nutrient agar at 550C. Add sterile blood laked by alternate freezing-

and thawing for 5 times. Then add potassium tellurite solution. Mix gently without

forthing and pour in plates.

Use: For isolation of Corynebacterium diphtheriae.


(Loeffler's medium)

Composition:

Ox, sheep or horse serum 30 ml

1 per cent glucose broth 80 ml

(see page 72)

Preparation: Mix and distribute. Sterilize by heating at 75°C for 20 min on 3 successive

days.

Use: This is used for the cultivation of Corynebacterium diphtheriae.


(Mueller-Hinton tellurite agar)

Composition:

Casein 5.0 g

Casamino acids 20.0 g

L-Tryptophan 0.05 g

Megnesium sulphate 0.1 g

Potassium dihydrogen phosphate 0.3 g

Agar 20.0 g

Tellurite serum(0.4% Pot. Tellurite in serum). 12.5 ml


pH 7.4

Preparation: Sterike the medium without tellurite serum in autoclave at 121°C for 20

min. Add tellurite serum to cooled sterile malted medium aseptically.

Use: Used for isolation Corynebacterium diphtheriae. C. diphtheria produce black

coloured colonies. Black colouration is due to diffusion of tellurite ions and later

reduction to tellurium metal that precipitates inside the bacterial cells.

Cytopltaga Medium

Composition:


Casein 0.5 g

Yeast extract 0.5 g

Beef extract 0.2 g

Agar 4.0 g


pH 7.2


Use: Used for the cultivation of Cytophaga species.

Cytophaga Medium

Composition:

Casein digest 0.05 g



pH 7.0

Preparation: Steam sterilize at I2 1 OC for 20 minutes.

Use: Used for the cultivation of Cytophaga species.

Derxia Medium

(Campelo and Dobereiner medium)

Composition:

Starch 20.0 g

K2HPO4 0.05 g

KH2PO4 0.15 g

MgSO4.7H2O 0.20 g

CaCl2.6H2O 0.02 g

FeCl3.6H2O 0.01 g

Na2MoO4.2H2O 0.002 g

Bromothyrnol blue (0.5% in absolute alcohol) 0.05 ml

NaHCO3 1.0 g

Agar 20.0 g


Preparation: Steam sterilize at 121°C for 20 minutes.

Use: Use for isolation of Derxia.

Denitrifying Baceria Medium

(Asparagine nitrate citrate solution)

Composition:

Solution A

KNO3 1.0 g

Asparagine 1.0 g


Solution B

Neutral sodium citrate 8.5 g

KH2PO4 1.0 g

MgSO4.7H2O 1.0 g

CaC12.6H2O 0.2 g

FeC13.6H2O trace


Solution C

Agar 20.0 g


Dissolve by heating.

Complete medium

Solution A 250 ml

Solution B 250 ml

Solution C 500 ml

Preparation: Steam sterilize solution A, B and C separately at 1150C for 20 minutes. Mix

aseptically.

Use: To test denitrifying activity of bacteria.

Enteric Bacteria Medium

(D.E.C. medium)

Composition:

Lab-Lemco 05.0 g

Peptone 0.5 g

Sodium taurocholate 0.85 g

Ferric chloride 0.3 g

Lactose 1.25 g

Agar 2.5 g

Neutral red (0.25 % aqueous solution) 1.5 ml


pH 7.4

Preparation: The medium is sterilized by boiling at 100°C for 1 hr.

Use: It is a selective medium for the isolation of intestinal bacteria.

Enteric Bacteria Medium

MacConkey's broth (Single strength)

Composition:

Peptone 20.0 g


Neutral red solution (2% in 50% ethanol) 3.5 ml

Lactose (10% aqu. solution) 100 ml


pH 7.5

MacConkey's broth (Double strength)

Prepare as above but use twice the quantity of all ingredients except the distilled water. It

is required to accommodate 10 ml water during MPN determination.

Preparation: Add all components except indicator. Dissolve. Adjust the pH. Add

indicator. Autoclave at 1 15OC for 20 min.

MacConkey's agar

Add 2.0 per cent agar to single strength MacConkey's broth prior to sterilization.

Use: The medium is selective for isolation of enteric bacteria. The bile salt inhibits the

growth of non-intestinal organisms. It is also a differential medium. The medium

differentiate coliform from noncoliforms. Coliforms colonies appear pink in colour as

they ferment lactose, producing acid. The noncoliform's colonies are colourless.

Erwinia Selective Medium

Composition:

(NH4)2SO4 5.0 g

K2HPO4 2.0 g

Eosin Y 0.4 g


Glycerol 10.0 ml

Antibiotic solution 10.0 ml

A gar 20.0 g


Antibiotic solution

Cycloheximide 2.5 g

Novobiocin 0.4 g

Neomycin sulfate 0.4 g


Prepamtion: Add all components in distilled water except antibiotic solution. Steam

sterilize the medium at 121°C for 1.5 minutes. Sterilize antibiotic solution by filtration.

Add aseptically. Pour plates.

Use: Used for selective isolation of Erwinia.

Flat Sour Bacteria Medium

(Dextrose tryptone bromocresol purple agar)

Composition:

Tryptone 2.5 g

Glucose 5.0 g

Agar 20.0 g



pH 7.2


Use: Used for the detection of flat sour bacteria. Acid producing coloured colonies are

detected on the medium.

Frankia Medium

(BAP medium)

Composition:

K2HPO4 0.591 g

KH2PO4 0.952 g

NH4Cl 0.267 g

MgSO4.7H2O 0.095 g

CaCl2.2H2O 0.010 g

FeNa EDTA 0.010 g

Na propionate (filter sterilize) 0.480 g

Trace elements solution 1 ml

Vitamin solution 1 ml


pH 7.0

Trace elements solution

H3BO4 2.86 g

MnCL2.4H2O 2.27 g

ZnSO4.7H2O 0.22 g

CuSO4.5H2O 0.08 g

NaMoO4.2H2O 0.02 g

CoSO4.7H2O 0.001 g


Vitamin solution

Thiamine HCl 10 mg

Nicotinic acid 50 mg

Pyridoxine HCl 50 mg

Biotin 225 mg

Folic acid 10 mg

Ca Pantothenate 10 mg

Riboflavin 10 mg

Preparation: Steam sterilize at 121°C for 20 minutes. Sterilize the phosphate separately.

Mix.

Use: Used for isolation of Frankia.

Fusobacterium Medium

(Qmata and Disraely medium)

Composition:

Casitone 15.0 g

Yeast extract 5.0 g

Glucose 5.0 g

NaCl 5 g

L-cysteine 0.75 g

Crystal violet 0.01 g

Streptomycin 0.01 g

Agar 20.0 g


pH 7.2

Preparation: Sterilize the medium at 11 5°C in autoclave for 20 min.

Use: Used for cultivation of Fzlsobacteriunt.

General Medium

(Blood agar)

Composition:

Nutrient agar (PH 7.2) 500 ml

Defibrinated blood 25 ml

Preparation: Autoclave the nutrient agar at 12 10C for 15 minutes. Cool to 45-50°C and

add 25 ml of sterile blood aseptically. Rotate to mix thoroughly avoiding accumulation of

air bubbles and pour immediately into sterile tubes or plates, i.e., before solidification.

Use: It is differential medium for haemolytic organisms. Three patterns of hemolysis can

occur on blood agar plate.

a) Beta hemolysis (Complete hemolysis), where formation of a clear zone with a

clear edge around the colony takes place.

b) Alpha hemolysis (Incomplete hemolysis), where production of methemoglobin and

a green cloudy zone around the colony is formed.

c) Gamma hemolysis (No hemolysis), where no change in colour surrounding the

colony on blood agar takes place.

General Medium

(Chocolate agaraysed blood agar/Heated blood agar)

Nutrient agar (pH 7.2) 100 ml

Defibrinated blood 5 ml

Preparation: Sterilize agar at 121°C for 15 minutes. Cool to 50°C and aseptically add the

sterile blood and mix thoroughly. Gradually heat to 75OC in water bath by mixing the

blood and agar by gently agitation until the blood begins to coagulate and become

chocolate brown in colour.

Use: (1) Used for identification of Neisseria. Pour tetramethly-p-Phenylene diamine

hydrochloride over inoculated chocolate agar plate and observe for the pink to purple

colour to the colonies. Neisseria develop above colour.

General medium (for pathogens)

(Hiss serum water)

Composition:

It consists of 1 part of sterile ox serum and 3 parts sterile of water.

Use: It is used in biochemical media of Neisseria, Corynebacterium etc. requirig serum

for growth.

General Medium

A. Nutrient agar

Composition:

Peptone 1.0 g


Beef extract 0.3 g

Agar 2.0 g


pH 6.8

Preparation: Dissolve all components in distilled water except agar. Adjust the pH. Add

the agar. Sterilize in autoclave at 121°C for 20 min.

B. Nutrient broth

Preparation: Same as nutrient agar except agar is omitted.

Use: Used for cultivation of heterotrophic microorganisms.

General Medium

(Plate count agar)

Composition:

Tryptone 5.0 g

Yeast extract 2.5 g

D-Glucose 1.0 g

Agar 20.0 g


pH 7.0

Prepnration: Sterilize the medium in autoclave at 100°C for 1 hr.

Use: For counting the bacteria from soil samples.

General Medium

(Tryptone yeast glucose agar)

Composition:

Tryptone 5.0 g

Yeast extract 2.5 g

Glucose 1.0 g

Agar 20.0 g


pH 7.2

Preparation: Sterilize the medium in autoclave at 1150C for 20 minutes.

Use: This is a general media used for cultivation of microorganisms.

General Medium

(Tryptone agar)

Composition:

Tryptone 10.0 g

Agar 20.0 g


Preparation: Sterilize the medium at 1210C for 20 min.

Use: Used as a general media for isolation and cultivation of microorganisms.

General Medium

(Yeast extract agar)

Composition:

Yeast extract 3.0 g

Peptone 5.0 g

Agar 20.0 g


pH 7.2

Prepamtion: Dissolve the ingredients by heating. Steam sterilize at 1310C for 20 minutes.

(Yeast extract milk agar)

The medium is prepared in the same way as yeast extract agar but 10 ml of skim or whole

milk is added per litre of broth.

Use: This medium is used for making plate counts of viable bacteria in milk.

Note: Plates should be used fresh because its pH tends to fall on keeping.

Gram-negative Bacteria Medium

(Penicillin agar)

Composition:

Plate count agar fortified with 10-35 units penicillin per ml of agar nay be used in place

of EMB agar to detect gram-negative bacteria n given sample.

Gram-negative Broth

Composition:

Peptone 20.0 g

Dextrose 1.0 g

D-mannitol 2.0 g

Sodium citrate 5.0 g

Sodium desoxycholate 0.5 g

Dipotassium hydrogen Phosphate 4.0 g


Potassium hydrogen phosphate 1.5 g


pH 7.0


Use: It is an enrichment medium for Salmonella and Shigella.

Haemophilus Medium

(Levinthal's medium)

Composition:

Sterile nutrient agar 100 ml

Sterile rabbit or human blood 5 ml

Preparation: Melt the agar, add the blood and heat mixture in boiling water. Allow tlie

deposit to settle and distribute the clear supernateants in petriplates.

Use: Used for cultivation of Haemophilus.

Haemophilus Medium

(Bacitracin heated blood agar)

Composition:

Chocolate blood agar (see page 82) 100 ml

Add bacitracin to get the concentration 10 I.u./ml in the medium.

Use: Used for isolation and cultivation of Haemophilus species.

Halophiles Medium

(Abram and Gibbon's medium)

Composition:

Casmino-acid 0.5 g

Yeast extract 1.0 g

Propeose-peptone 0.5 g

Na citrate 0.3 g

KC1 0.2 g

MgSO4.7H2O 2.0 g

FeSO4.7H2O 0.005 g

NaCl 20-30 g


pH 7.0


Use: Used for cultivation of extreme halophiles.

Halophiles Medium

(Eimhjellen medium)

Composition:

Yeast extract 0.5 g

MgSO4.7H2O 2.0 g

CaC13.2H2O 0.5 g

NaCl 25.0 g


pH 7.2

Preparation: Steam and sterilize at 1210C for 20 minutes.

Halophiles Medium

(Milk medium of Dussault and Lachance)

Composition:

MgSO4.7H2O 5.0 g

Mg(NO3)2.6H2O 1.0 g

FeCl3.7H2O 0.025 g

Peptone 5.0 g

Glycerol 10.0 g

NaCl 200 g


Preparation: Dissolve the ingredients except salt in 500 ml of water. Add 30 g of agar.

Sterilize at 121°C for 20 min. Sterilize the slightly moist salt and 50 g of skimmed milk

powder dissolved in 500 ml water in separate vessels. Add the hot agar solution to the

salt and dissolve as much of the salt as possible. Add the milk. Mix to dissolve

the remaining salt.

Use: Used for isolation of halophiles.

Halophiles Medium

Composition:

MgSO4.7H2O 2.5 g

Casamino acids 1.0 g

Yeast extract 1.0 g

Peptone 0.5 g

Trisodium citrate 0.3 g

KCl 0.2 g

NaCl 25.0 g


pH 7.2


Use: Used for isolation of halophilic microorganisms.

Lactic Acid Bacteria Medium

(APT agar)

Composition:

Yeast extract 7.5 g

Tryptone 12.5 g

Glucose 10.0 g

Na citrate 5.0 g

NaCl 5.0 g

K2HPO3 5.0 g

MnCl2.H2O 0.14 g

MgSO4.7H2O 0.8 g

FeSO4 0.04 g

Na2CO3 1.25 g

Tween-80 0.2 g


pH 6.1

Preparation: Sterilize in autoclave at 1210C for 20 min.

Use: Used for cultivation of lactic acid bacteria.

Lactic Acid Bacteria Medium

(Neutral red chalk lactose agar/NRCLA)

Composition:

Peptone 3.0 g

Meat extract 3.0 g

Yeast extract 3.0 g

Lactose 10.0 g

CaCO3 15.0 g

Neutral red (1%) 5.0 ml

Agar 25.0 g


pH 6.8

Preparation: Steam sterilize at 109°C for 1 hr.

Use: Used for cultivation of lactic acid bacteria.

Lactobacilli Medium

(DeMan, Rogosa and Sharpe's medium)

Composition:

Peptane 10.0 g

Meat extract 10.0 g

Yeast extract 5.0 g

Glucose 20.0 g

Tween 80 1 ml

Dipotassium hydrogen Phosphate 2.0 g

Sodiuin acetate 5.0 g

Triammonium citrate 2.0 g

Magnesium sulphate 200 mg

Manganese sulphate 50 g

Agar 20.0 g


pH 6.0

Preparation: Dissolve the ingredients. Adjust the pH and autoclave at 1210C for 20 min.

Use: This is a selective medium supports good growth or Lactobacillus.

Lactobacilli Medium

(Tomato juice agar)

Composition:

Peptone 1.0 g

Casein hydrolysate 1.0 g

Tomato juice 40 ml

Agar 2.0 g


Preparation: Dissolve all components except agar in tomato juice. heat gently. Adjust to

pH 5.0 wit11 lactic acid. Dissolve the agar in the 60 ml water and mix both solutions

while they are hot. Filter through thin layer of cotton and autoclave at 1150C for 10 min.

Use: It is selective medium for the growth of Lactobacili.

Significance: Tomato juice provides growth factor and pH 5.0, makes the medium

selective.

Precaution: At pH 5.0, agar tends to be hydrolysed on heating and repeated melting

should be avoided.

Leuconostoc Medium

(Sucrose gelatin agar)

Composition:

Tryptone 10.0 g

Yeast extract 5.0 g

NaCl 5.0 g

K2HPO4 5.0 g

Glucose 1.0 g

Sucrose 50.0 g

Gelatin 50.0 g

Agar 20.0 g


pH 7.2

Preparation: Take 500 ml water. Dissolve all ingredients except geltain and agar. Ad-

just the pH. Add gelatin. Mix 4% agar solution (500 ml). Steam sterilize at 1210C for 20

min.

Note: Addition of 0.02% sodium azide makes the medium more selective.

Use: Sucrose inhances capsule or slime production. Used for cultivation of Leuconostoc

mesenteroides and better slime formation.

Leptospira Medium

(Dinger's modification of Noguchi's medium)

Composition:

(a) Semi solid medium

Nutrient agar, 3% 6 ml


Sterile inactivated serum 10 ml

Preparation: Mix the agar and water and sterilize by autoclaving at 1210C for 20 min.

Cool and add the serum with sterile precautions, distribute in tubes.

(b) Solid medium

Tryptose-phosphate broth (Dehydrated) 0.2 g

Agar 1.0 g


pH 7.5

Preparation: Sterilize mixture by autoclaving at 1210C for 15 min. After cooling add 10

ml sterile rabbit serum and I mi hemoglobin solution prepared by lysing washed and

packed sheep erythrocytes in 20 vol cold distilled water. Sterilize by filtration. Heat

mixture at 560C for 30 min and pour plates.

Use: Used for isolation of Leptospira

Leptospira Medium

(Korthof's medium-modified)

Composition:

(i) Peptone salt solution

Peptone 0.8 g

NaCl 1.4 g

NaHCO3 0.02 g

KCl 0.04 g

CaCl2 0.04 g

KH2PO4 0.24 g

Na2HPO4.2H2O 0.88 g


pH 7.2

Preparation: Steam the ingredients at 100°C for 20 min and filter through Whatman No. 1

filter paper. Adjust the pH. Bottle in 100 ml amounts and autoclave at 115OC for 15 min.

(ii) Blood serum

Rabbit's serum is inactivated by heating at 56°C for 30 min & sterilized by seitz

filtration.

(iii) Hemoglobin solution

Hemoglobin is prepared from blood clot after removal of serum & then adding equal

volumes of distilled water and then freezing & thawing repeatedly. It is sterilized by seitz

filtration.

Complete medium

Peptone salt solution 100 ml

Sterile blood serum 8ml

Sterile hemoglobin solution 0.8 ml

Use: Used for cultivation of Leptospira.

Leptospira Medium

(Modified Stuart's medium)

Composition:

(i) Stock solutions

(a) L-asparagine (dextro-rotatory) 1.3%

(b) NH4CI 0.54%

(c) MgC12.6H2O 2.03%

(d) Thiamine hydrochloride 0.1%

(e) Sodium chloride 0.58%

(f) Phenol red 0.02%

Preparation: Prepare the stock solutions with distilled water and sterilize by autoclaving

at 115°C for 15 min.

(ii) Phosphate buffer solutions of pH 7.6

Sterilize by autoclaving at 121°C for 30 min.

(iii) Blood serum

Serum is prepared as for Korthofs medium.

Complete medium

L-asparagine solution

Ammonium chloride solution

Magnesium chloride solution

Sodium chloride solution

Thiamine hydrochloride solution

Phenol red solution

Distilled water

Phosphate buffer, pH 7.6

Sterile inactivated rabbit s e m

Preparation: Mix all ingredients except the rabbit serum and autoclave at 11 5°C for 15

min. Add the serum with sterile precautions, distribute and use.

Use: Used for the cultivation of Leptospira.

Manganese bacteria medium

(Bromfield medium)

Composition:

KH2PO4 0.005 g

MgSO4 .7H2O 0.002 g

(NH4)2SO4 0.010 g

Ca3(PO4)2 0.010 g


MnSO4.4H2O 0.005 g


pH 6.0

Preparation: Medium is autoclaved at 11 5°C for 15 min. To solidify the medium add

2% agar.

Use: Used for isolation of (Manganese bacteria) bacteria capable of

converting (oxidizing) mn++

(mangnus) to mn+++

(mangnic) ions.

Minimal Mediuru

Composition:


Disodium hydrogen phosphate 6.0 g


Ammonium chloride 2.0 g


Glucose 8.0 g

Agar 20.0 g


pH 7.0

Preparation: Magnesium sulphate should be separately autoclaved and added to the rest

autoclaved constituents at 1210C for 20 minutes.

Use: It is a synthetic medium allow the growth of only prototrophs.

Minimal Medium

Composition:

Glucose 2.0 g

(NH4)2SO4 1.0 g

K2HPO4 7.0 g

MgSO4 0.5 g

Agar 20.0 g


pH 6.8

Preparation: Sterilize medium at 1210C for 20 minutes.

Use: It allows the growth of only prototroplls of Escherichia coli.

Minimal Medium

(Davis and Mingioli-synthetic medium)

Composition:

Glucose (sterile 10% soln) 20.0 ml

K2HPO4 7.0 g

KH2PO4 3.0 g


(Na3C6H2O7.2H2O) MgSO4.7H2O 0.1 g

(NH4)2SO4 1.0 g

Trace element solution 5.0 ml

Agar 20.0 g


PH 7.1

Trace element solution

Composition:

FeSO4.7H2O 0.5 g

ZnSO4.7H2O 0.5 g

MnSO4.3H2O 0.5 g

H2SO4 0.1 ml


Preparation: Glucose is added with sterile precaution, as a sterile solution after the

remainder ofthe medium has been autoclaved, other sugar may be substituted for the

glucose, particular amino acids or growth factors may be added. The citrate may be

omitted.

Use: Medium allows the growth of only protoroph.

Mutant Isolation Medium

(Streptomycin agar)

Composition:

To 1 litre of sterile liquidnutrient agar (50°C), aseptically add 100 mg of streptomycin

sulphate. Pour directly into sterile petri plates.

Use: To isolate streptomycin resistant mutants, only streptomycin resistant organism can

form colony on the medium.

Mycobactericrrn Medium

(Dorset's egg medium)

Composition:

Beaten egg (213 hen's eggs) 7.5 ml

Sterile nutrient broth 2.5 ml

Preparation: Prepare the beaten eggs. Mix and add in the nutrient broth with sterile

precaution. The medium is solidified in inspissator at 75°C.

Use: For the isolation of Mycobacteriuin tuberculosis.

Precaution: It is advised to add malachite green solution (2%), 1.25 ml per 100 rnl for

the isolation of Mvcobacterium tuberctrlosis.

Mycobacteriurn Medium

(Dubo's medium)

Composition:

Casein hydrolysate (20 % solution) 10 ml

NaH2PO4 6.25 g

M2PO4 1.00 g



Magnesium sulphate (7H2O) 0.6 g

Tween 80 (10% solution) 5.0 ml


pH 7.2

Preparation: Add all the components in water. Autoclave at 1150C for 20 min. Before

use add 0.1 ml of 9% solution of bovine albumin. Albumin solution is sterilized by

filtration.

Use: Used for cultivation of Mycobacterium tuberculosis.

Mycobacteriurn Medium

(Loewenstein and Jensen medium)

Composition:

(a) Mineral salt solution

KH2PO4 2.4 g

MgSO4 0.24 g

Magnesium citrate (. 14 H2O) 0.6 g

Asparagine 3.6 g

Glycerol 12.0 ml


Boil the solution for 2 hrs and cool overnight.

(b) Salt-starch solution

Mineral salt solution 600 ml

Starch 30 g

Mix. Heat in a water bath at 56°C with constant stirring for 15-20 min. Keep in

water bath for I hr. 1000 ml.

(c) Egg fluid

Obtained by beating 20-13 eggs aseptically. (see page 13)

Complete medium

Salt-starch solution 600 ml

Egg fluid 1000 ml

Mix. Strain through sterile gauze.

Then add Malachite green (sterile) (2 % aqueous solution) 20 ml

Preparation: Distribute in tubes. Sterilize by keeping in inspissator at 85°C for 30 min.

on first day and at 75OC for 30 min. on second day, without disturbing the tubes placed

in slanting position. The tubes are stored in the refrigerator and used within a months.

Use: Used in cultivation of Mycobacterium tuberculosis.

Note: According to Jensen 1955, starch may be omitted because (1) It makes the medium

more difficult to prepare. (2) It is unnecessary for the growth of A4ycohacteriunt

tuberculosis.

Myxobacteria Medium

(CT Agar)

Composition:


MgSO4.7H2O 2.0 g

Potassium phosphate 500 ml

buffer (0.02 M, pH 7.6)

Agar 20.0 g


pH 7.6

Preparation: Add casein digest and NgSO4.7H2O in distilled water. Adjust the pH.

Autoclave at 121°C for 20 minutes. Steam sterilize the phosphate broth at 121°C for 20

minutes, separately, mix both solutions aseptically. Pour plates.

Use: Used for cultivation of Myxobacteria.

Myxobacteria Medium

(Dowrkin's defined medium)

Composition:

Tryosine 0.6 g

Asparagine 0.5 g

Leucine 1.0 g

Isoleucine 0.5 g

Proline 0.5 g

Arginine 0.1 g

Histidine 0.05 g

GI ycine 0.05 g

Lysine 0.25 g

Methionine 0.05 g

Phenylalanine 0.15 g

Tryptophan 0.05 g

Serine 0.1 g

Threonine 0.1 g

Valine 0.1 g

Djenkolic acid 0.1 g

Alanine 0.05 g

Glycogen 3.0 g

MgSO4.7H2O 1.0 g


Make the medium 0.01 M in K2HP0, -KH,P02 buffer (pH 7.6).

Use: Used for the cultivation of Myxobacteria. Omission of Phenylalanine and

tryptophan initiate the fruiting stage.

Myxobacteria Medium

(McCurdy's medium)

Composition:

Raffinose 1.0 g

Sucrose 1.0 g

Galactose 1.0 g

Soluble starch 5.0 g

Casitone 2.5 g

MgSO4.7H2O 0.5 g

K2HPO4 0.25 g

Agar 20.0 g


pH 7.4

Use: Used for cultivation of Myxobacteria.

Myxobacteria Medium

(Noren's medium)

Compsotion:


Asparagines 2.5 g

K2HPO4 2.0 g

NaCl 1.2 g

MgSO4.7H2O 0.01 g

CaC12.2H2O 0.01 g

MnSO4.4H2O 0.001 g



pH 7.5

Use: Used for cultivation of fruiting Myxobacteria.

Myxobacteria Medium

(Stanier medium)

Composition:

KNO3 1.0 g

K2HPO4 0.2 g

MgSO4.7H2O 0.1 g

CaCl2.2H2O 0.1 g

FeC12.6H2O 0.02 g

Agar 10.0 g


Preparation: Sterilize at 1210C for 20 min. Pour plates. Place sterilize piece of filter

paper on the surface of agar.

Use: Used to isolate cellulolytic Myxobacteria.

Mycoplnsma Medium

(Specimen collection media/SCM)

Composition:

Trypticase soy broth 3.0 g

Bovine albumin powder 0.3 g

Phenol red 20% 0.5 ml


Penicillin 2000 µ/ml

Thallium acetate 1:2000

Preparation: Suspend the trypticase soy broth in water. Mix thoroughly and warm gently

till solution is complete, add phenol red and autoclave at 100°C for I5 min. Add the test

at temperature of 50°C or below. Pass through seitz filter, distribute in 2 to 3 ml

quantities in sterile capped vial. Store in the refrigerator.

Use: SCM for mycoplasmas.

Mycoplasma Broth

Composition:

Mycoplaslrza broth base 100 ml

Horse serum 10 ml

Fresh yeast extract 5.0 g

Dexyribonucleic acid (Calf thymus DNA) 0.02 g

Phenol red 0.002 g

Glucose 0.1 g

Arginine 0.1 g

Preparation: Mix all the constituents in mycoplasma broth base except serum and

autoclave at 121°C for 15 minutes. Allow the medium to cool to 45-50°C and add horse

serum aseptically.

PPLO agar: Add 2% agar.

Use: Used for isolation of mycoplasmas. They appear as round colonies 250-750 pm in

diameter with dense centre and less dense periphery giving the fried egg appearance.

My coplastnu broth

(Liquid medium)

Composition:

PPLO broth without crystal violet pH 7.8 70.0 ml

Yeast extract pH 7.0 10.0 ml

Horse serum (unheated) 20.0 ml

Glucose solution, 10 % w/v Sodium deoxyribonucleate 10.0 ml

(calf thymus) solution in 80 w/v 0.2 % 1.0 ml

Thanllous acetate solution in 80 w/ 1.0 ml

K2HP04. 1 M Pencillin solution 2.0 ml

50,000 units per ml 2.0 ml

Phenol red solution, 0.2 % w/v 1.0 ml

Yeast extract solution

Baker's yeast 1.0 kg

Dejonized water 1000 ml

HCL, Analar grade about 6.5 ml

Preparation: Add the yeast to 500ml water at 50°C. Mix well. Add the

remaining water. Warm to 80°C and add acid to adjust the pH to 4.5.

Mix well and heat at 80°C for 20 min. Allowthe cells to settle and filter

the supernatant through seitz filter. Adjust the pH 7.0 before use.

Sterilize the solutions by filtration and add them to the sterilize broth.

Use: This medium is used for primary isolation of Mycoplasmas.

Mycoplasma Medium

Composition:

PPLO broth or PPLO agar without violet pH 6.0 70.0 ml

Yeast extract pH 7.0 (see page 102) 10.0 ml


Urea solution, 20 % w/v 5.0 ml

Phenol red solution 0.2 % w/v 1.0 ml

Penicillin solution, 200,000 units per ml 0.25 ml

pH 6.0

Preparation: Sterilize the solutions by filtration and add to sterilize broth. Check the pH.

Use: Used for cultivation of T-strain mycoplasmas.

Mycoplasma Medium

(Sloppy agar medium)

Composition:

PPLO broth without crystal violet pH 7.8 70.0 ml

PPLO agar base without crystal violet pH 7.8 10.0 ml

Yeast extract pH 7.0 (see page 102) 10.0 ml


Sodium deoxyribonucleate (calf thymus) solution 0.2 % w/v 1.0 ml

Thallus acetate solution, 1 in 80 w/v 1.0 ml

Dipotassium hydrogen phosphate solution, 1M 2.0 ml

Penicillin solution, 50,000 unit per ml 0.2 ml

Preparation: Sterilize the solutions by filtration and add to the sterile broth. Add the 10

1n1 sterile molten PPLO agar base. Distribute in sterile tube. Prepare slopes.

Use: Used for cultivation of mycoplasmas.

Mycoplasmn Medium

(Transport medium - Staurt 1959)

Composition:

Anaerobic salt solution

Thioglycollic acid 2 ml

Distilled water (pass through anion exchange

resin column to remove chlorine, if present) 900 ml

IN NaOH 12-15 ml

Sodium glycerophosphate (20% aqueous) 100 ml

Calcium chloride (1 % aqueous) 20 ml

Agar solution

Agar 6.0 g

Distilled water to (Chlorine free) Dissolve by heating 1000 ml

Complete medium

Melt the agar solution and add the anaerobic salt solution. Adjust the pH to 7.2. Add 4 ml

methylene blue solution (0.1%). Mix. Distribute in tubes. Autoclave at 121°C for 15 min.

Use: Used as transport medium when delay for isolation of mycoplasmas/gonococci

from specimen is unavoidable.

Mycoplasma Medium

(Kelton medium)

Composition:

Heart infusion broth 1000 ml


Peptone 10.0 g

pH 7.9

Preparation: Autoclave at 1201C for 20 minutes and before use add 100 ml horse serum.

Use: For the storage of mycoplamas.

Mycoplasma Medium

[Chalquest and fabricant medium - modified)

Composition:

(1) Solid phase

PPLO agar, dehydrated 39.6 g


Trypticase 5.0 g

Thallium acetate Nicotinamide adenine 0.25 g

dinucleotide WAD) 0.1 g

Swine serum (inactivated) 100 ml

Penicillin 1,000,000 units


Preparation: Dissolve the agar by heating. Add trypticase, starch, thallium acetate and

phenol red. Autoclave at 121°C for 20 min. Cool to 50°C. Add swine serum, penicillin

and the NAD (prepare in 10 ml quantities). Sterilize by filtration.

( 2) Liquid phase

PPLO borth dehydrated, without crystal violet 18.9 g

Cysteine violet HCl 0.1 g

Phenol red 0.025 g

Thallium acetate 0.25 g

NAD 0.1 g

Swine serum (inactivated) 100 ml

Penicillin 1,000,000 units


Use: This medium is also very suitable for the propagation of M. pneumnoniae. And M.

synoviae.

Mycoplasma Broth

Composition:

Bacto tryptose 20.0 g

Dextrose 5.0 g

NaCl 5.0 g

Na2HPO3 2.5 g

Glycerol 5.0 g

Yeast extract (see page 102) 1.0 g


Pig serum (inactivated at 560C for 30 min) 100 ml

Penicillin 100 units/ml

Preparation: The complete medium is sterilized by seitz.

Use: This medium is used for all routine isolation, filtration and vaccine production of M.

mycoides var mycoides.

Mycoplasma Medium

(Hayflick medium)

Composition:

(a) Horse serum broth

Bacto PPLO broth (without crystal violet) 70 ml

Horse serum 20 ml

Yeast extract (see page 102) 10 ml

ThaIIous acetate (I % w/v) 2.5 ml

Penicillin 100,000 units per ml 0.2 ml

Preparation: Sterilize the PPLO broth at 1210C for 20 min Sterilize other solutions by

filtration. Add to broth aseptically. Distribute in sterile tubes.

(b) Horse serum agar

Use bacto PPLO agar instead of broth in above medium.

Use: Used for cultivation of niycoplasnias.

Mycoplasma Medium

(A2 medium)

Composition:

Trypticase soy broth powder 30.0 g


pH 7.0

Bottle in 76 ml amounts

Autoclave at 1210C for 15 min. Before use add

____

Unheated horse serum (Sterile, pH -6) 20 ml

Yeast extract (see page 102) 5 ml

Benzyl penicillin 1000 units/ml

Use: Used for cultivation of T-strain mycoplasmas.

Neisserin Transport Medium

(Amies transport medium)

Composition:

Potassium chloride 0.2 g

Calcium' chloride 0.1 g




Potassium phosphate 0.2 g


Charcoal 10.0 g

Agar 4.0 g


pH 7.3

Preparation: Dissolve the ingredients in water. Sterilize at 1210C for 20 minutes.

Use: It is used for transporting swab specimens especially suspected for N. gonorrhoeae.

Nitrosomonas Medium

(Ammonium medium)

Composition:

(NH4)2SO4 2.0 g

MgSO4.7H2O 0.5 g

FeSO4.7H2O 0.03 g

NaCl 0.3 g

MgCO3 10.0 g

K2HP04 1.0 g


pH 7.3

Preparation: Add all ingredients in water. Adjust the pH. Sterilization is not necessary if

the inoculations are made as soon as the medium is made LIP. Sterilization at 121°C for

30 min. is desirable for storage.

Use: It is used for the isolation of Nitrosomonas, i.e , bacteria causing conversion of

ammonia to nitrite.

Pathogen Preservation Medium

(meat extract broth)

Peptone 10.0 g

Meat extract 10.0 g

NaCl 5.0 g


pH 7.4

Preparation: Dissolve the ingredients by heating. Broth may be filtered if precipitate

appears. Sterilize by autoclaving at 121°C for 20 min.

Use: This broth is good for preservation of stock culture of pathogens.

Composition:

(NH4)2SO4 1.0 g

K2HPO4 0.5 g

MgSO4.7H2O 0.2 g

NaCl 2.0 g

NaHCO3 5.0 g

Yeast extract 0.1 g

Organic substrate 1.5 g


pH 7.0

Organic substrate may be malate, succinate or fumarate.


Use: Used for cultivation of photosynthetic bacteria from Athiorhodaceae family.

Photosynthetic Bacteria Medium

Composition:

(NH4)Cl 1.0 g

K2HPO4 1.0 g

MgCl2 1.0 g

NaHCO3 1.0 g

Na2S.9H2O 1.0 g


pH 8.0


Use: Used for cultivation of photosynthetic bacteria from Thiorhodaceae family.

Photosynthetic Bacteria Medium

Composition:

(NH4)Cl 1.0 g

KH2PO4 1.0 g

MgCl2 0.5 g

NaCl 0.3 g

NaHCO3 2.0 g

Na2S.9H2O 1.0 g

Fe 500 µg

Tap water to 7.3

pH 7.3


Use: Used for cultivation of photosynthic bacteria from Chlorobeacae family.

Pneumococcal Medium

(Meat infusion broth)

Composition:

Lean meat, ox heart or beef 500.0 g

water 1000 ml

Peptone 10 to 20g


pH 7.4

Preparation: Remove all fat from the fresh meat and mince it. Add the minced meat to

the water and extract by keeping it in the refrigerator for 24 hrs. Strain through muslin.

Pseudomonas Agar F

Composition:

Peptone 20.0 g

Maltose 10.0 g

K2HPO4 1.5 g


Agar 20.0 g


pH 7.2


Use: Used for cultivation of Pseudomonas.

Pseullomonas Medium

(Cetrimide agar)

Composition:

Peptone 20.0 g

Potassium sulphate 10.0 g


Cetyl trimethy ammonium bromide 0.3 g

Agar 20.0 g


pH 7.2 - 7.4

Use: It is a selective medium used for isolation for Pseudomonas. It inhibits the growth

of coliforms and Staphylococcus. Colonies of Pseudomonas are green due to the water

soluble pigment that diffuses into the medium.

Pseudomonas Medium

(Cetrimide agar)

Composition:

Basal medium

Peptone 20.0 g

Glycerol 10.0 ml

Agar 20.0 g


pH 7.2

Preparation: Dissolve the ingredients. Adjust pH. Sterilize by autoclaving at 1210C for

30 min.

Complete medium

Melted basal medium 1000 ml

K2HPO4 (15%) 10.0 ml

MgSO4.7H2O 10.0 ml

Cetrimide solution (2%) 15.0 ml

K2HPO4, MgSO4.7H2O, and cetrimide solution are sterilized by filtration.

Use: It is a selective medium for isolation of Pseudomonas aeruginosa. Cetrimide 0.03%

make the medium selective. The yellow fluorescence of the colonies of Pseudornonas

may be enhanced by examining the culture plates in a dark box fitted with a source of

UV

irradiation.

Psedomonas Medium

(King, Ward and Raney's medium)

Composition:

Medium A - Pyocayanin

Peptone 20.0 g

Glycergl 10.0 ml

MgC12 (anhydrous) 1.4 g

K2S04 (anhydrous) 10.0 g

Agar 20.0 g


pH 7.2

Medium B - Fluorescein

Peptone 20.0 g

Glycerol 10.0 ml

K2HPO4 (anhydrous) 1.5 g

MgSO4.7H2O 1.5 g

Agar 20.0 g


pH 7.2

Preparation: Dissolve the ingredients by heating. Adjust the pH. Sterilize in autoclave at

121°C for 15 min.

Use: For the cultivation of Psezidonionas aeruginosa. Plates of medium A are incubated

at 370C for 48 hrs or longer. Plates of medium B are incubated at 37

0C for 24 hrs and

then at room temperature for 2-3 days.

Note: Medium A enhances pyocyania (blue) pigment whereas medium B enhances

fluorescein (yellow) pigment.

Rhizobium Medium

(Bergersen's synthetic medium)

Composition:

Mannitol 10.00 g

Na2HPO4.12H2O 0.45 g

Sodium glutamate 1.10 g

MgSO4.7H2O 0.10 g

FeCl3.6H2O 0.02 g

CaC12.6H2O 0.04 g

Thiamine 100 µg

Biotin 200 µg


pH 7.0

Preparation: Vitamin solutions are sterilized by filtration and added to the medium after

sterilization 115°C for 20 minutes.

Use: Used for isolation of Rhizobium.

Rhizobium Medium

(Congored yeast extract mannitol agar)

Composition:

Mannitol 10.0 g

K2HPO4 0.5 g

MgSO4.7H2O 0.2 g

NaCl 0.1 g

Yeast extract 1.0 g

Agar 20.0 g


Congored 1% solution 2.5 ml

Preparation: Dissolve weighed amounts of all constituents in distilled water and

autoclave at 121°C for 20 minutes. Congored solution is to be sterilized separately and

added in the medium at the time of pouring in petriplates.

Use: Used for isolation of Rhizobium.

Rhizobium Medium

(Glucose peptone agar)

Composition:

Glucose 5.0 g

Peptone 10.0 g

Agar 20.0 g

Bromo cresol purple (1 % alcoholic solution) 10.0 ml


pH 7.0


Use: Rhizobium rows poorly in this medium and causes little change of pH.

Rumen Bacterial Medium

(Bryant and Robinson medium)

Composition:

Glucose 0.25 g

Cellobiose 0.25 g


Agar 2.0 g

Resazurin 0.0001 g

Minerals 1 3.75 ml

Minerals 2 3.75 ml

Na2CO3 0.2 g

Cysteine HCl –Na2S (of a solution containing 2.5% w/v of each) 1 ml

Centrifuged rumen fluid 20 ml


Mineral 1

K2HPO4 0.6 g


Mineral 2

KH2PO4 0.6 g

(NH4)2SO4 1.2 g

NaCl 1.2 g

MgSO4.7H2O 0.25 g

CaCl2 0.6 g


Preparation: The medium is sterilized in autoclave at 115°C for 20 min. The medium is

equilibrated with and incubated under a gas phase of 50% CO2 and 50% H2.

Use: Used for isolation of rumen bacteria.

Rumen Bacteria Medium

Composition:

Casitone 1.0 g


Mineral I 15 ml

Mineral I1 15 ml

Centrifuged (clarified) rumen fluid 20 ml

Agar 2.0 g

Resazurin 0.0001 g

Sodium lactate (70% w/v) 1.0 g

Glucose 0.2 g

Maltose 0.2 g

Cellobiose 0.2 g

Crysteine HCl 0.05 g



Mineral I

K2HPO4 3.0 g


Mineral II

KH2PO4 3.0 g

(NH4)2SO4 6.0 g

NaCl 6.0 g

MgSO4.7H2O 0.6 g

CaCl2 0.6 g


Preparation: Sterilize the medium at 100°C for 1 hr in autoclave. The medium is

equilibrated with and incubated under 100% CO2.

Use: Used for isolation of rumen bacteria.

Salmonella - Shigella Agar

Composition:

Peptone 5.0 g

Beef extract 5.0 g

Lactose 10.0 g

Bile salt mixture 8.5 g



Brilliant green 0.033 g

Neutral red 0.025 g

Agar 20.0 g


pH 7.0

Preparation: Add all components except agar and indicator. Adjust the pH. Add

indicators then agar. Sterilize at 115OC for 30 min.

Use: Used for selective isolation of Salnronella and Shigella.

Salmonella Medium

(Bismuth sulphite agar)

Composition:

Peptone 10.0 g

Beef extract 5.0 g

Disodium phosphate 4.0 g


Bismuth sulphite 8.0 g


Agar 20.0 g


pH 7.5

Preparation: Dissolve by boiling the ingredients in water. Do not 1 sterilize. Pour in

sterile petriplates.

Use: It is a selective medium used for cultivation of Sabnonella typhi. The colonies of

Saln1017ella appear black.

Salmonella Medium

(Brilliant green agar)

Peptone 10.0 g

Yeast extract 3.0 g

Lactose 10.0 g

Sucrose 10.0 g

NaCl 5.0 g

Agar 20.0 g


Phenol red 0.08 g


pH 7.2

Preparation: Steam sterile at 115°C for 30 min.

Use: Used for isolation' of Salntonella, as Salmonella is non lactose fermenting, colonies

will be red.

Scrlmonella and Shigella Medium

(Dexycholate citrate agar - Hyne's modification of Leifson)

Composition:

Medium A

Meat extract 5.0 g

Peptone 5.0 g

Agar 22.0 g

Neutral red solution [2% in 50% ethenol] 1.25 ml

Lactose 10.0 g


pH 7.4

Preparation: Dissolve the meat extract in 50 ml water. Make just alkaline to

phenolphthalein with 50% NaOH. Boil at 100°C and filter. Adjust the pH. Make the

volume 50 ml and add peptone. Dissolve the agar in 950 ml water by steaming at 100°C

for 1 hour. Mix two solutions. Add neutral red and lactose. Sterilize at 115°C for 15

minutes.

Solution B



Ferric ammonium citrate 4.0 g

Sterile water 100 ml

Dissolve by heating at 60°C.

Solution C

Sodium deoxycholate 10.0 g


Dissolve by heating at 60°C.

Complete medium

Medium A 100 ml

Solution B 5 ml

Solution C 5 ml

Melt the medium A and add solution B and C in this order under sterile condition.

Precaution: The medium should be poured immediately otherwise it

tends to become very soft in presence of deoxycholate.

Use: It is a selective medium for isolation of Salmonella and Shigello.

Salmonella Medium

(Glycerol saline transport medium)

Composition:

Glycerol 300 ml

NaCl 4.2 g

Na2HP04, anhydrous 10 g

Phenol red, (0.02% aqueous) About 15 ml


Preparation: Dissolve the sodium chloride in the water and add the glycerol. Add the

phosphate and heat to dissolve. Then add phenol red to give a purple-pink colour.

Distribute in tubes.

Use: It is used as transport medium for carrying specimens of faeces to laboratory for

isolation of enteric fever bacilli. The medium supresses the growth of other intestinal

bacteria.

Salmorzella Medium

(Kauffmann - Muller tetrathionate broth)

Composition:

(a) Thiosulphate solution

Sodium thiosulphate Na2S2O3.5H2O 50.0 g


Preparation: Same as in tetrathionate medium.

(b) Iodine solution

Potassium iodide 25.0 g

Iodine 20.0 g

Sterile water to 100 ml


(c) Ox bile solution

Desiccated ox bile 0.5 g

Sterile water 5 ml

Dissolve with sterile precautions.

Complete medium

Nutrient broth (pH 7.4) 90 ml

CaCO3 5.0 g

Brilliant green (1 in 1000 aq. solution) 1 ml

Thiosulphate solution 10 ml

Iodine solution 2 ml

Ox bile solution 5 ml


Use: Used for selective isolation of Salmonella.

Salmonell Medium

(Selenite F broth)

Composition:

Sodium acid selenite [NaHseO3] 4.0 g

Peptone 5.0 g

Lactose 4.0 g


Sodium dihydrogen phosphate 0.5 g

Sterile distilled water to 1000 ml

pH 7.1

Preparation: Dissolve the ingredients with sterile precautions in water. Adjust the pH

and distribute in sterile tubes. Autoclave at 100°C for 30 min.

Precaution: Excessive heat is detrimental to the medium. Salts of selenium are very toxic

for animals and man.

Use: For the enrichment of typhoid and paratyphoid group from faeces.

Significance: This medium intibits coliform bacilli. But it may permits the growth of

Proteus.

Salmonella Medium

(Selenite cystine broth)

Composition:

Tryptone 5.0 g

Lactose 4.0 g

Na2HPO4 10.0 g

Sodium acid selenite 4.0 g

L-cystine 0.01 g


pH 7.0

Preparation: Dissolve ingredients in sterile water aseptically. Steam at 100°C for 30 min.

Use: This is an enrichment medium for salmonellae, add 1 ml o sample to 9 ml of

selenite cystine broth.

Precaution: Salts of selenium are very toxic for man and animal.

Salmonella Medium

(Terathionate broth)

Composition:

(a) Thiosulphate solution

Sodium thiosulphate Na2S2O3.5H2O 24.8 g


Add the salt in water with sterile precautions. Steam at 100°C for 30 minutes.

(b) Iodine solution


Iodine 15.7 g


With sterile precautions dissolve the potassium iodide and iodine.

Complete medium

CACO3 2.5 g

Nutrient broth (pH 7.4) 78 ml

Thiosulphate solution 15 ml

Iodine solution 4 ml

Phenol red (0.02 % in 20% ethanol) 3 ml

Add the CaCO3 to the nutrient broth. Sterilize it by autoclaving at 121°C for 20 min.

Cool and add the thiosulphate, iodine and phenol red solutions with sterile precautions.

Distribute in 10 ml amounts in sterile tubes.

Use: The tetrathionate broth inhibits the coliform bacilli and allow the growth of the

typhoid - paratyphoid group. However, it can’t inhibit the growth of Proteus groups.

Salmonella Medium

(Wilson and Blair's medium)

Composition:

Solution A-Bismuth sulphite glucose phosphate mixture

Bismuth ammonio citrate 3.0 g

Sodium sulphite 10.0 g

Disodium hydrogen phosphate (12 H2O) 10.0 g

Glucose 5.0 g


With sterile precautions dissolve the bismuth ammonio-citrate in 25 ml boiling water and

sodium sulphite in 50 ml boiling water. Mix two solutions and boil. Add the sodium

phosphate crystals while boiling the mixture. Cool. Add the glucose dissolve in 25 ml

boiling water and cooled.

Solution B - Iron citrate brilliant green mixture

Ferric citrate solution (1% in sterile distilled water) 200 ml

Brilliant green solution (1% in sterile distilled water) 25 ml

Mix together

Complete medium

Sterile 3% nutrient agar 100 ml

Solution A 20 ml

Solution B 4.5 ml

Melt the agar and cool to 60°C. Add both solutions with sterile precautions and pour

plates.

Use: It is selective medium for the isolation Salmonella. Briliant green makes the

medium selective. It is also a differential as 5. paratyphi A produce green coloured

colonies while S. typhi and S paratyphi B produce black coloured colonies by reducing

sulphite to sulphate.

Salmonella Medium

(Wilson and Blair's medium-Reed's modification)

Composition:

Peptone (2% solution) 8.8 ml

Sea salt mixture 1.2 ml

Mannose (10%) 1.0 ml

Liquid bismuth 0.12 ml

Sodium sulphite (20%) 1.2 ml

Absolute alcohol 0.2 ml

Mercuric perchloride (1 : 10,000) 0.8 ml

Preparation: Sterilize the medium without alcohol at 121°C for 30 min. Add alcohol.

Sea salt water

CaC12 27.0 g

KC1 01.0 g

MgC12.6H2O 03.0 g

MgSO4.7H2O 1.75 g


Use: It is liquid enrichment medium for V. cholerae.

Note: Instead for seat salt mixture bazaar common salt may be used.

Soil Microorganisms Medium

(Asparagine mannitol agar)

Composition:



MgSO4.7H2O 0.2 g

CaCl2.6H2O 0.1 g


FeCl2.6H2O Trace

Asparagine 0.5 g

Agar 15.0 g


Preparation: Add the agar and salt. Dissolve by heating. Add the mannitol. Adjust the

pH. Sterilize in autoclave at 121°C for 20 min.

Use: For the isolation of soil microorganisms.

Soil Microorganisms Medium

(Soil extract agar)

Composition:

Glucose 1.0 g

K2HPO4 0.5 g

Agar 20.0 g

Soil extract (stock) 100 ml


pH 7.2

Preparation: 1000 g of sieved garden soil is mixed thoroughly with 1000 ml of tap

water. A small amount of CaCO3 is added. Mixed throughly and filtered through paper.

Dissolve the agar in 900 ml by heating. Add other components. Adjust the pH and steam

sterilize at 1210C for 20 minutes.

Use: Used for isolation of microorganisms from soil.

Spirochete Medium

(Noguchi's medium)

Composition:

Sterile normal saline 80 ml

Fresh rabbits serum 10 ml

Nutrient agar (sterile melted 3%) 10 ml

Rabbits haemoglobin 12 ml

Procedure: Add rabbits serum to saline at 50°C. To the mixture add sterile nutrient agar

cooled to 50°C. Add rabbits haemoglobin. (3 ml of rabbits blood to 9 ml of sterile

distilled water.) Mix. Distribute in tubes and place at once in the refrigerator until the

agar gels. Incubate to test the sterility.

Use: Used for cultivation of spirochetes.

Staphylococcus Medium 110

Composition:

Protein digest 1.0 g


Gelatin 3.0 g

D-mannitol 1.0 g

Lactose 0.2 g

NaCl 7.5 g

K2HPO4 0.5 g

Agar 2.5 g


pH 7.0

Use: It is a selective medium for the isolation of Staphylococcus aureus. Phenol red may

be added as an indicator. Staphylococcus ferment mannitol and produce acid.

Staphylococcus Medium

(Baird Parker medium)

Composition:

Tryptone 10.0 g

Beef extract 5.0 g

Yeast extract 1.0 g

Sodium pyruvate 10.0 g

Glycine 12.0 g

Lithium chloride 5.0 g

Agar 20.0 g


pH 7.0

Egg yolk emulsion Potassium tellurite 50 ml

solution (3.5%) 3.0 ml

Preparation: Dissolve all the ingredients in 950 ml distilled water. Srerilize by autoclave

at 121°C for 15 minutes. Cool to 50°C and add septically egg yolk emulsion and sterile

potassium tellurite solution. Mix and pour into plates.

Use: It is a selective medium for isolation of coagulase positive staphylococci from pus

and wou?d. The colonies of staphylococci appear black owing to reduction of potassium

tellurite to metallic telllurium (black).


(Brain heart infusion broth)

Composition:

Infusion from calf brains 200.0 g

Infusion from beef heart 250.0 g

Peptone 10.0 g

Dextrose 2.0 g

NaCl 5.0 g

Na2HPO4 2.5 g


pH 7.4

Preparation: Autoclave at 1 1 5OC for 30 minutes.

Use: Used for cultivation of staphylococci specifically when the culture is to be used for

performing coagulase test.


(a) (Glycerol monoacetate agar)

Composition:

Heart infusion broth 100 ml

Glycerol monoacetate 1.0 g

Agar 2.0 g

Preparation: Dissolve the agar in broth by heating. Add the glycerol monoacetate and

autoclave at 1210C for 15 min.

Use: For the cultivation of Staphylococci.

Significance: Colonies of coagulase position staphylococci on this medium are orange,

yellow or buff whereas coagulase negative strains are porcelain white. There is good

differentiation after 48 hr incubation.

(b) (Salt glycerol monoacetate agar)

5% NaCl is .added hl Glycerol monoacelate agar.

Significance: Staphylococci grow in sodium chloride concentration that are high enough

to be inhibitory to many other bacteria. Thus it makes the medium selective.

Staphylococcus Meduim

(Mannitol salt agar)

Composition:

Mannitol 10.0 g

Peptone 10.0 g


Beef extract 1.0 g

Phenol red 0.025 g

Agar 20.0 g


pH 7.4

Preparation: Autoclave at 115°C for 3 minutes.

Use: It is useful for the selective isolation of pathogenic staphylococci. Since most other

bacteria are inhibited by the high salt concentration. Colonies of pathogenic

staphylococci are surrounded by a yellou halo indicating mannitol fermentation.


(Milk agar)

Composition;

Fresh milk (Sterile) Sterile nutrient agar 100 ml

(3.5% agar) 200 ml

pH 7.2

Preparation: Sterilize the milk by autoclaving at 121°C for 20 min. Melt the sterile agar.

Cool to 50°C. Mix with the milk and pour plates.

Use: For the isolation of Staphylococczrs.

Significance: Pigmentation of staphylococcal colonies is marked and easily recognised

against opaque white background.


(Salt milk agar)

Milk agar (given above) with 7-10% sodium chloride.

Use: It is a selective medium for staphylococci. High salt inhibit the other organisms.


(Sodium chloride selective medium)

Composition:

Peptone 1.0 g

Meat extract 0.3 g

NaCl 10.0 g


pH 7.2


Use: The medium is selective for the isolation of Staphylococcus aureus.


(Vogel-Johnson agar)

Composition:

LiCl 5.0 g

Tryptone 10.0 g

Yeast extract 5.0 g

Mannitol 10.0 g

K2HPO4 5.0 g

Glycine 10.0 g

Agar 15.0 g

Phenol red 0.025 g



Use: Used for isolation of Staphylococcus.

Streptococcus Medium

(Crystal violet blood agar)

Composition:

Sterile nutrient agar 90 ml

Sterile horse blood 10 ml

Crystal violet (1 in 1000 aq. solution) 0.2 ml

Preparation: Melt the agar, cool to approx. 50°C. Add the blood and crystal violet with

sterile precaution.

Use: For the cultivation of Streptococcus pyogenes.

Significance: Crystal violet inhibits the growth of some bacteria like Staphylococci.


(Glucose sodium azide glycerol agar)

Composition:

Tryptone 20.0 g

D-Glucose 5.0 g



Sodium azide 0.5 g




pH 7.0


Use: It is used for selective isolation for Streptococcus faecalis.


(Hartley's broth)

Composition:

Pancreatic extract

Fresh pig pancreas 50.0 g


Absolut&lcoho l50 ml

Concentrated hydrochloric acid about 2 ml

Preparation: Remove fat, mince the pancreas and mix it with the water and alcohol.

Shake the mixture thoroughly in a large stoppered bottle. Allow it to stand for 3 days at

room temperature, shaking occasionally. Strain through muslin and filter through paper.

Measure the volume ofthe filtrate and add 0.1% hydrochloric acid. This extract

can be stored for about two months in stopped bottles in the refrigerator.

Complete medium

Lean meat, ox heart or beef 150 g


Sodium carbonate 0.8% solution 250 ml

Pancreatic extract 5 ml

Chloroform 5 ml

Concentrated HCl 4 ml

Preparation: Mix the minced meat and water and heat them at a temperature of 80°C.

The add the sodium carbonate. Cool to 450C and add pancreatic extract and chloroform.

Incubate the mixture at 45°C for 3 hr. stirring frequently. Add acid, heat at 100°C for 30

min. Filter. Store the broth in an acid condition in bottle with 0.25% chloroform. Shake

vigorously and frequently in the next two or three days. Store in cool, dark place. Before

use, adjust the pH to 8.0 and heat at 100°C for 1 hr. to precipitate phosphate. Filter while

hot and allow to cool. Adjust the pH to 7.6, distri~utean d autoclave at 11 5OC

for 20 minutes.

Note: Used for obtaining luxuriant growth of exacting organisms. Streptococci grow well

in this medium. The medium is used specially for the production of diphtheria toxin.


Composition: (Horse flesh digest medium)

Horse flesh 90.0 g

Water 350 ml

Sodium carbonate 1.2 g

Pancreatin 1.75 g

Concentrated hydrochloric acid 2.0 ml

Peptone 3.5 g


Sodium biocarbonate 0.7 g

pH 8.0

Preparation: Mince the meat and mix it with 150 ml of cold water, raising the

temperature to 80°C. Add the remainder of the cold water and the sodium carbonate.

Adjust the pH. Add the pancreatic. Heat the mixture at 56°C for 6 hr. Then add the acid

and again boil at 100°C for 30 min. Filter, add the peptone and filter. Add the sodium

bicarbonate Sterilize by filtration.

Use: It is used for cultivation of haemolytic streptococci.


(Sodium azide medium)

Composition:

Peptone 10.0 g




Glucose 5.0 g

Yeast extract 3.0 g

Sodium azide, (NaN3) 0.25 g

Bromocresol purple (1.6% solution in ethanol) 2.0 ml


pH (self adjusted) 6.8

Preparation: Dissolve the ingredients in water. Sterilize in the autoclave at 121°C for 15

min.

Use: This medium is used for the isolation of Streptococcus fecalis.


(Todd-Hewitt broth)

Composition:

Fat free minced beef 450 g


pH 7.6

Keep in the refrigerator overnight. Heat at 85°C. Filter. Then add 2% peptone. Adjust the

pH. Add

Glucose 0.2%

NaCl 0.2%

Preparation: Boil the mixture for 20 min. Filter. Autoclave at 1150C for 20 min. The

final pH should be 7.8.

Use: This broth is used for grouping and typing Streptococcus pyogenes.

Sulphate Reducing Bacteria Medium

Composition:

KH2PO4 0.5 g

NH4Cl 1.0 g

Na2SO4 4.5 g

CaCl2.6H2O 0.06 g

MgSO4.7H2O 0.06 g


FeSO4.7H2O 0.004 g

Yeast extract 1.0 g

Sodium citrate 2H2O 5.0 g


pH 7.5 ± 0.2

Preparation: Sterilize by autoclaving 20 minutes at 1150C..

Use: Particularly useful for growing cells in continuous culture, for biochemical work

and for manometric studies. It contains no sediment and ferrous sulphate is not

precipitated during growth. Normally used in liquid form.


(Medium E-Postage 1966)

Composition:

KH2PO4 0.5 g

NH4Cl 1.0 g

Na2SO4 1.0 g

CaC12.6H2O 1.0 g

MgSO4.7H2O 2.0 g


FeSO4.7H2O 0.5 g

Yeast extract 1.0 g

Thioglycollic acid 1.0 g

Ascorbic acid 1.0 g

Agar 20.0 g


pH 7.6

Preparation: Ingredient dissolve by boiling, adjust pH. Sterilize by autoclaving for 20

min. at 1150C. The thioglycollic acid should be sterilized by membrane filtration.

Use: Used for enrichment of.sulphate reducing bacteria. For enumeration, used in agar

form.


(A.P.I. medium-American Petroleum Institute, 1965)

Composition:

K2HPO4 (anhydrous) 0.01 g

NaCl 10.0 g

MgSO4.7H2O 0.2 g

Sodium lactate 4.0 ml

FeSO4(NH4)2SO4.6H2O 0.2 g

Ascorbic acid 0.1 g

Yeast agar 1.0 g

Agar 20.0 g


pH 7.3

Use: Dissolve ingredients with heating. Adjust the pH. Sterilize by autoclaving at 121°C

for 10 min.


(Asparagine sodium lactate gelatine medium)

Composition:

Asparagine 1.0 g


K2HPO4 5.0 g

MgSO4.7H2O 1.0 g

FeSO4(NH4)2.6H2O Trace

Gelatine 120 g


Preparation: Sterilize in an autoclave at 1090C for 20 minutes.

Use: Used for isolation of sulphate reducing bacteria.

Sulphur Oxidizing Bacteria Medium

(Thiosulphate agar)

Composition:

Na2S2O3.SH2O 5.0 g

K2HPO4 0.1 g

NaHCO3 0.2 g

NH4Cl 0.1 g

Agar 20.0 g


Preparation: Steam and sterilize at 121°C for 20 minutes. Add an excess of CaCO3

sterilized separately.

Use: Used for isolation of sulphur oxidizing bacteria.

Sulphur Oxidizing Bacteria Medium

(Sulphur phosphate medium)

Composition:

(NH4)2SO4 0.20 g

KH2PO4 3.00 g

MgSO4 .7H2O 0.50 g

CaC12.6H2O 0.25 g

FeSO4.7H2O trace

Sulphur powdered 10.00 g


Preparation: The medium is sterilized in flowing steam for 3 consecutive days. The pH

of medium is about 4.0.

Use: Used for isolation of sulphur oxidizing bacteria.

Treponema Medium

(a) (Ascitic fluid agar)

Composition:

Glucose agar 100 ml

Ascitic fluid 20 ml

Preparation: Sterilize the glucose agar at 121°C for 20 min. Cool at 45°C. Add sterile

ascitic fluid.

(b) Hydrocele fluid agar

Composition and preparation: Same as above except use hydrocele fluid instead of

asciticfluid.

Use: Used for isolation of a virulent strain of Treponema pallidum.

Vibrio Medium

(Alkaline peptone water)

Composition:

Peptone 10.0 g

NaCl 5.0 g


pH 9.0

Preparation: Add the components in distilled water. Steam sterilize the medium at 1210C

for 20 min.

Use: Used for cultivation of alkaliphilic bacteria (including Vibrio species.)

Vibrio Medium

(Aronson's medium)

Composition:

(a) Agar base

Peptone 10.0 g

Beef extract 30.0 g



pH 7.2


Complete Medium

Anhydrous sodium carbonate (10% solution) 6 ml

Sucrose (20% solution) 5 ml

Glucose (20% solution) 5 ml

Sodium sulphite (10% solution) 2 ml

Basic fuchsine 0.4 ml

(Saturated alcoholic solution) Melted sterile agar base 100 ml

Preparation: Except agar base all the solutions are sterilized by steaming for 30 min for

three successive days.

Add carbonate solution to melted agar base. Heat for 10 min. at 100°C. While hot

a add other solutions. Heat for 20 min. more. The mixture is allowed to stand to settle the

precipitate. Pour plates.

Use: V. cholerae grow with formation of colony, pouched egg in appearance with reddish

centre and paler periphery. The medium is alkaline, it is selective for the cultivation of V.

cholerae.

Vbrio Medium

(Bile peptone transport medium)

Composition:

Peptone 1.0 g




pH 8.5

Preparation: Dissolve the ingredients, adjust the pH. Autoclave at 121°C for 15 minutes.

Precaution:

1) Usually subcultures should be made within 6 hours.

2) In order to make medium more selective for the vibriob, sterile potassium tellurite

solution may be added after autoclaving to give a final concentration of 1 in

2,00,000 as for Monsur's medium.

Use: Useful for maintaining the viability of Vibrio cholerae and preventing overgrowth

by other organisms during transportation of clinical sample.

Vibrio Transport Medium

(Carry-Blair medium)

Composition:

Sodium thoglycollate 0.75 g



Calcium chloride (1%) 4.5 ml


pH 8.4

Preparation: Heat the medium in boiling water bath. Cool to 50°C and add 4.5 ml of

freshly prepared calcium chloride solution. Adjust the pH. Sterilize by autoclaving at

1210C for 15 minutes.

Use: It is used as transport medium for Vibrio, Salnronella and Shigella.

Vibrio Medium

(Gelatin agar)

Composition:

Trypticase 1.0 g


Gelatin 3.0 g

Agar 1.5 g


pH 7.2

Preparation: Dissolve the ingredients by heating. Adjust the pH. Autoclave at 121°C for

20 minutes.

Use: This medium is complementary to Monsur's medium. But it is not inhibitory to

coliform bacilli. Used for primary isolation of Vibrio cholerae.

Vibrio Medium

(Monsur’s medium)

Composition:

(a) Bile Salt gelatin agar

Tryptose 1.0 g




Gelatin 3.0 g

Agar 2.0 g


pH 8.5

Preparation: Dissolve the ingredient by heating. Adjust the pH. Sterilize by autoclaving

at 121°C for 20 minutes.

(b) Stock potassium tellurite solution

Potassium tellurite 0.5 g


Autoclave at 1150C for 20 min.

Complete medium

Bile salt gelatin agar 100 ml

Potassium tellurite solution 1 ml

Preparation: Make 1 in 10 dilution of the stock potassium tellurite solution by using

sterile distilled water. Add it to the melted and cooled agar medium. The final pH of the

medium must be 8.5 to 9.3.

Precaution: Use the medium when fresh because its pH drops after storage.

Use: This medium is used for the selective isolation of cholera and other vibrios. A

medium without gelatin and agar is used for enrichment. 'The potassiun~te llurite inhibits

most coliforrn bacilli.

Vibrio Medium

Composition:

Boric acid, H3BO3 3.101 g

KCl 3.728 g

NaOH (0.2 M solution) 133.5 ml

Dried sea salt 200 g


pH 8.5

Preparation: Dissolve the boric acid and potassium chloride in 20 ml hot water. Cool

and make up the volunie to 250 ml. Add the sodium hydroxide, make up the volume to 1

litre and add the salt. Filter the solution through paper and autoclave at 121°C for 20 min.

Use: Used for maintaining viability of V. cholerae.

Vibrio Medium

(Thiosulphate citrate bile sucrose agar/TCBS medium)

(1) Basal agar medium

Composition:

Yeast extract 5.0 g

Peptone 10.0 g


Agar 20.0 g

Thymol blue solution (2% in 50% ethnol) 2.0 ml

Sucrose 10.0 g

Water 800 ml

pH 8.5

Preparation: Dissolve the yeast extract, peptone and salt in 200 ml water and adjust the

pH to 8.5. Ilissolve the agar in 600 ml water by heating. Add the indicators and sucrose

mixing again and steam sterilize at 1150C for 15 minutes.

(2) Solution A

Sodium citrate Na3C6H5O7.H2O 10.0 g

Sodium thousulphate Na2S2O3.5H2O 10.0 g


Distilled water (sterile) 100 ml

(3) Solution B

Ox bile, desiccated 8.0 g

Distilled water (sterile) 100 ml

Prepare these solutions with sterile precautions, heating if required.


Basal agar medium 80 ml

Solution A 10 ml

Solution B 10 ml

Melt the agar and add solution A and B in this order, with sterile precaution. Four plates

immediately.

Use: It is highly selective for V. parahaemolyticus and may be used for the isolation of

V. chloerae.

Significance: It is inhibitory to gram-positive organisms, most coliforllls and many

strains of Proteus. V. paraheamolyticus from colonies with green or blue centre.

Xanthomonas Medium

(Beef peptone agar)

Composition:

Beef extract 3.0 g

Peptone 5.0 g

Dextrose 10.0 g

Yeast extract 5.0 g

Agar 20.0 g


pH 7.2

Use: Used for isolated of Xanthomonas.

Xanthomonas Medium

Composition:

Chalk 4.0 g

Glucose 0.5 g

Yeast extract 0.5 g

Agar 2.0 g



Use: For cultivation of Xanthomonas.

Plant Tissue Culture Medium

[Murashige Skoog (MS) tissue culture medium]

Composition:

Macronutrients:

NH4NO3 1650.0 mg

KNO3 1900.0 mg

CaCl2.2H2O 440.0 mg

MgSO4.7H2O 370.0 mg

KH2PO3 170.0 mg

Micronutrients:

KI 0.83 mg

H3BO3 6.2 mg

MnSO4.4H2O 22.3 mg

ZnSO4.7H2O 8.6 mg

Na2MoO4.2H2O 0.025 mg

CuSO4.5H2O 0.025 mg

CoCl2.6H2O 2.025 mg

Fe-Versenate (EDTA) 43.0 mg

Vitamins and hormones

Inositol 100.0 mg

Nicotinic acid (Vitamin 9,) 0.5 mg

Pyridoxine-HC1 0.5 mg

Thiamine-KC1 0.1 mg

Indole acetic acid (IAA) 1.30 mg

Kinetin 0.01-10.0 mg

Carbon source

Sucrose 30.0 g

Distilled water to 0.2 g

pH 0.2 g

Use: Commonly used medium for plant tissue cultures. For solidifying, agar is added at

the rate of 0.6%.

Seedlings Medium

(Jensen's medium-modified)

Composition:

CaHPO4 1.0 g

K2HPO4 0.2 g

MgSO4.7H2O 0.2 g

NaCl 0.2 g

FeCl3 0.1 g

Trace elements solution 1.0 ml


pH 7.0

A stock solution of trace elements

Bo 500 mg

Mn 500 mg

Zn 50 mg

Mo 50 mg

Cu 20 mg


Add agar at the rate of 20 g/litre when solid medium is needed.

Use: Used to growing seedlings to test root nodulation in legumes.

2. Fungi

Alternaria Medium

(Lukens and Sisler synthetic medium)

Composition:

Glucose 20.0 g


Magnesium sulphate (MgSO4.7H2O) 0.25 g

Monobasic potassium phosphate 3.0 g

Glycine 1.0 g

Thiacinc HCI 2.0 µg

Niacin 30 µg

Biotin 1 µg

I-inositol 200 µg

Pyridoxine 10 µg

Folic acid 10 µg

pH 6.0

Preparation: Add Bo, Mn, Zn, Cu, Mo and Fe in trace. Steam sterilize at 100°C for 1 hr.

Use: Used for cultivation of Allernaria.

Ascomycetes Medium

(Claussen's medium)

Composition:


Ammonium nitrate 0.05 g


Ferrous phosphate 0.001 g

Inulin 2.0 g

Agar 3.0 g


Preparation: Steam sterilize at 121°C for 20 min.

Use: Used for cultivation of Asconrycetes.

Ascomycetes Medium

(Yeast extract agar)

Composition:

Yeast extract 4.0 g

Malt extract 10.0 g

Dextrose 4.0 g

Agar 15.0 g


Use: Used for cultivation of Acomycete.

Aspergillus Medium

(Pailey, Stafamak, Olson and Johnson's medium)

Composition:

Glycerol 7.5 g

Cane sugar 7.5 g

Peptone 5.0 g




Agar 20.0 g


Preparation: Add glycerol, cane sugar, magnesium sulphate, potassium dihydrogen

phosphate to 200 ml water. Mix peptone and salt to a paste with 200 ml water, at 60°C

and add to the first mixtde. Dissolve agar in 600 ml, then mix all together, pH does not

need adjustment. Autoclave 121°C at for 20 min.

Use: Used for cultivation of AspergilIus auvezrs.

Aspergillzis Medium

Composition:

Glucose 20.0 g

(NH4)2NO3 1.0 g

KH2PO4 0.68 g

MgSO4 0.5 g

FeCl3 0.016 g

ZnSO4 0.005 g


pH 5.4

Add CaCO3 toll effervescence stops.


Use: Used for cultivation of Aspergillus.

Basdiomycetes Medium

Composition :

Basal medium


Magnisum sulphate (MgSO4.7H2O) 0.5 g

Dextrose 20.0 g

Casein sulphate 2.0 g

Adenine sulphate 9.09 mg

Cytosine 2.22 mg

Guanine HCl 4.11 mg

Hypoxanthine 2.72 mg

Thymine 0.25 mg

Uracil 2.24 mg

Xanthine 0.06 mg

Choline Cl 5.58 mg

Orotic acid 0.62 mg

Thiamine HCl 0.337 mg

Riboflavin 0.376 mg

Pyridoxine 0.205 mg

Nicotinic acid 0.123 mg

Calcium pantothenate 0.476 mg

Para-aminobenzoic acid 0.137 mg

m-inisitol 216.19 mg

Folic acid 1 mg

Biotin 0.01 mg

Vitamin B12 0.01 mg

B 0.005 mg

Cu 0.02 mg

Fe 0.1 mg

Ga 0.01 mg

Mn 0.01 mg

Mo 0.01 mg

Zn 0.09 mg


Neutralize with calcium carbonate (CaCO3)

Steam sterilize at 100°C for 1 hr.

Wood extract

Autoclave 500 g dry beech wood (ground in a mill) in 5 litre of distilled water. Reduce

the filtrate on a hot plate to 1 litre.

Tomato extract

Filter canned tomato juice through muslin.

Complete medium

Basal medium 100 ml

Wood/Tomato extract 8 ml

Use: Used for cultivation of basidiomycetes.

Botrytis Separation Agar

Composition


Potassium diflydrogen phosphate 1.5 g




Yeast extract 3.0 g

Glycerol 5.0 g

L-Sorbose 2.5 g

Agar 20.0 g

Tap water to 1000 ml.


Use: A selective medium to distinguish between Botrytis allii and B. cinera on onion.

The former species is severely restricted by sorbose.

Candida Medium

(Corn meal agar)

Composition:

Corn meal 40.0 g

Agar 20.0 g

Water to 1000 ml

pH 6.8

Preparation: Heat the corn meal in the water at about 60°C for 1000 ml. Filter through

filter paper. Make the volume again 1000 ml by adding water. Add the agar. Autoclave at

121°C for 30 minutes. The pH is self-adjusted.

Use: This medium is used for the production of chlamydospores. In the case of Candida

albicans the appearance of the chlamydospores is diagnostic.

Candida Medium

(Molybdenum medium)

Composition:

Peptone 10.0 g

Sucrose 40.0 g

Agar 20.0 g


pH 7.6

Preparation: Autoclave at 109°C for 20 minutes. Cool to 500-55°C and 15 ml of a 12.5%

aclueous soln. of Merck phosphomolybdic acid. (Final concentration 1.9 mglml)

Use: Used for cultivation of Candida albican.

Candida Medium

(Rice starch agar)

Composition:

Rice flour 10.0 g

Agar 20.0 g

Tween 80 10 ml


Preparation: Boil the water and mix with the rice flour. Boil for more 30 sec. Stand for a

few seconds and filter through cotton guaze. Add water to re-adjust the volume. Add the

agar and tween 80 and autoclave at 121°C for 30-minutes.

Use: This medium is used to stimulate chlamydospore formation by Candida albicans.

Celluloytic Fungal Medium

(Cellulose yeast extract agar)

Composition:

Filter paper 12.0 g

Yeast extract 4.0 g

Agar 10.0 g


Preparation: Tear paper into small pieces and macerate in some of the water until the

fibers are separated. Add this to the remainder of the water and dissolve the yeast extract

and agar. Autoclave at 1210C for 20 minutes.

Use: Used for cultivation of cellulolytic fungi.

Cellulolytic Fungal Medium

(Czapek medium-modified)

Composition:




Dipotassiuln hydrogen phosphate 1.0 g


Cellulose 10.0 g


pH 6.4 to 7.0

Preparation: Autoclave at 115°C for 15 minutes to prevent break down of cellulose.

Use: For assaying fungal cellulase.

Note: Substitute cellulose with pectin for assaying pectinase.

Chaetomium Medium

(Knop's Solution)

Composition:

Calcium nitrate 0.5 g



Potassium phosphate 0.125 g

Ferrous chloride 0.005 g



Use: Filter paper saturated with Knop's solution (with agar) is used for maintaining

chaeromium spp.

Chaetomium Medium

(Potato malt agar with cellulose)

Composition:

Potatoes 30.0 g

Malt syrup 5.0 g

Agar 20.0 g


Preparation: Prepare potatoes extract by boiling sliced in water. Autoclave at 121°C for

20 minutes. Strips of sterilized paper are placed on the surface of the medium in petri

dishes or tubes.

Use: Used for cultivation of cellulolytic fungi (Chaetomium)

Dermatophyte Test Medium

Composition:

Phenol red solution

Phenol red 0.5 g

Sodium hydroxide (0.1 N) 15 ml


Complete medium

Peptone 10.0 g

Glucose 10.0 g

Agar 20.0 g

Phenol red solution 40 ml

Hydrochloric acid (0.8 N) 6 ml

Cycloheximide (25% w/v) in acetone 2 ml

Gentamycin sulphate (5% W/V aqu.) 2 ml

Chlorttracycline (0.4% wlv in sterile water) 25 ml


Preparation: Dissolve the peptone glucose and agar in the water by heating. Add the

phenol red solution, acid, cyclohexmide and gentamycin to the hot medium. Steam

sterilize at 1150C for 15 min and cool. Add the chlortetracycline.

Use: The medium is highly selective for dematophytes. Significance: Antibiotics inhibit

most bacteria and cycloheximide restrains bacteria, other molds and yeasts.

Dermatophytes produce enough alkali to tun1 the medium red within 2 weeks at 280C.

Fungus Preservation Medium

Composition:

Peptone 30.0 g

Agar 20.0 g

Water to 1000 ml

pH 5.4


Use: Used as preservation medium for fungi.

Fungal Medium

(Bread crumb agar)

Composition:

Commercial bread crumbs (without preservations) 100.0 g

Agar 20.0 g


Use: Used to permit the luminescence of various fleshy fungi.

Fungal Medium

(Carrot agar)

Composition:

Whole carrot 300.0 g

Agar 30.0 g


Preparation: Cook carrot in 500 ml distilled water, macerate. Add 1000 ml distilled

water and 30 g. agar an4 dissolve. Autoclave at 121°C for 15 minutes.

Use: Used for cultivation of fungi.

Fungal Medium

(Cherry agar)

Composition:

Cherry extract 300 ml

Agar 20.0 g


pH 3.8 – 4.6

Preparation: Dissolve agar in water, add cherry extract. Distribute into pre-sterilized

bottles or tubes and sterilize at 102OC for 5 minutes. Overheating should be avoided, as

the acid reaction will prevent the setting of the medium.

Cherry extract

Stone chemes (red variety). To each 200 g pulp, add 1 litre water for 2 hr. Strain through

cloth and sterilize at 110°C for 1 hr.


Fungal Medium

[Cornmeal (maize) peptone yeast-extract agar]

Composition:

Cornmeal 20.0 g

Dextrose 10.0 g

Peptone 10.0 g

Yeast extract 4.0 g

Agar 20.0 g


Preparation: Boil cornmeal in 700 ml of water for 10 minutes. Strain through cloth.

Dissolve peptone, dextrose, yeast extract and agar to filtrate and make the volume to 1 lit.

Autoclave at 121°C for 20 minutes. .

Use: Used for cultivation of b g i .

Fungal Medium

(Martin rose Bengal agar)

Composition:

Dextrose 10.0 g

peptone 5.0 g


Magnesium sulphate (MgSO4.H2O) 0.5 g

Rose Bengal 0.035 g

Agar 20.0 g


Aueomycin (chlortetracycline) 35 µg/ml

Preparation: Autoclave at 1210C for 20 minutes. Add antibiotic after sterilization of the

medium.

Use: Used for selective isolation of fungi.

Note: Auremycin may be replaced by streptomycin (30.0 mg).

Fungal Medium

(Elliott's agar)

Composition:



Magaesium sulphate 0.50-g

Dextrose 5.0 g

Asparagine 1.0 g

Agar 20.0 g



Fungal Medium

(Emmon's culture medium for fungi)

Composition:

Glucose 20.0 g

Peptone 10.0 g

Agar 20.0 g

Chloramphenicol 40mg


pH 7.2

Preparation: Dissolve the glucose, peptone, and agar by heating. Add the

chloramphenicol which has been suspended in 10 ml of 95% alcohol. Remove alcohol by

heating medium. Autoclave at 121°C for only 10 minutes.

Use: Selective medium for isolation of fungi, I

Significance: It’s principal advantage is that a neutral pH does not inhibit certain molds

that have difficulty in growing at pH 5.6.

Fungal Medium

(Fish broth medium)

Composition:

Fresh cod fish 1.0 kg

Peptone 5.0 g

Sodium chloride 200 g

Wheat flour 400-500 g


Preparation: Macerate the fish in the water and allow to stand for 4 hr. Strain through

muslin. Add peptone and sodium chloride and autoclave at 10g°C for 20 minutes. Add

the flour and strain through muslin. Autoclave at 1210C for 20 minutes.


Fungal Medium

(Hay extract agar)

Composition:

Hay 200.0 g

Glucose 5.0 g

Agar 20.0 g

Distilled water to l000 ml

Preparation: Boil the hay for 30 minutes in 500 ml of the water. Filter and add the

glucose. Make up to 1 litre, then add the agar and heat until dissolved. Sterilize at' 1210C

for 20 minutes.


Fungal Medium

(Honey peptone medium)

Composition:

Honey 60.0 g

Peptone 10.0 g

Agar 20.0 g

Water 1000 ml

Preparation: Dissolve the agar before addition of honey. Steam sterilize at 100°C for 1

hr.

Use: The pH is favorable to fungal growth and inhibitory to most bacteria.

Fungal Medium

(Kauffman's agar)

Composition:

Maltose 5.0 g


Calcium nitrate (4H2O) 0.50 g


Agar 20.0 g


Preparation: Dissolve the ingredients in water. Sterilize at 109°C for 20 minutes.


Fungal Medium

(Malt salt agar)

Composition:

Malt extract 100.0 g


Agar 20.0 g


Preparation: Dissolve be ingredients in the water and autoclave at 1210C for 20 minutes:

Use: For halophilic yeast and molds.

Fungal Medium

(Malt agar)

Composition:

Malt extract powder 30.0 g

Glucose 16.0 g

Agar 15.0 g


Melt and acidify to pH 3.5 with sterile lactic acid solution prior to use.

Use: Medium is selective for mold growth.

Fungal Medium

(Oatmeal agar)

Composition:

Oatmeat 20.0 g

Agar 18.0 g

Trace salt solution 1.0 g


Trace salt solution

FeSO4.7H2O 0.1 g

MnCl2.4H2O 0.1 g

ZnSO4.7H2O 0.l.g

Distilled water 1000.0 ml



Fungal Medium

(Onion-asparagine agar)

Composition:

Onion 100.0 g

Asparagines 0.25 g

Peptone 0.5 g

Agar 20.0 g


Preparation: Peel and cut up onions and boil in water bath in 500 ml of the water. Strain

and add agar and remainder of ingredients dissolved in the other 500 ml water. Autoclave

at 115°C for 15 min. Use: Used for cultivation of fungi.

Fungal Medium

(Potato dextrose agar)

Composition:

Potato (peeled) 200.0 g

Dextrose 20.0 g

Agar 15.0 g


Preparation: Peel off the skin of potatoes, cut into small pieces and boil in 500 ml. of

water till they are easily penetrated by a glass rod. Filter through cheesecloth. Add

dextrose to the filtrate. Dissolve agar in water and bring upto the required volume by the

addition of water. pH is self adjusted. Sterilize at 121°C for 20 minutes.

Use: Medium is selective for isolation of fungi.

Fungal Medium

(Prune agar)

Composition:

Prunes 30.0 g

Sucrose 40.0 g

Agar 30.0 g


Preparation: Boil prunes in water, then pass through sieve to get extract. Add agar, boil

to dissolve, add sucrose and stir until dissolved. Autoclave at 121eC for 20 minutes.


Fungal Medium

(Sabouraud's glucose agar)

Composition:

Glucose 4.0 g

Peptone 1.0 g

Agar 20.0 g

Water to 100 ml

pH 5.4

Preparation: Dissolve the glucose and peptone in water. Adjust the pH. Add agar.

Autoclave at 115°C for 15 minutes.

Use: For the isolation of fungi.

Significance: The low pH arid high sugar content prevent the bacterial growth. It is

useful to add cyclohexiplide for the isolation of dermatophytes.

Fungal Medium

(Tellurite malt agar)

Composition:

(a) Basal medium

Malt extract 4.0 g

Agar 2.0. g

Water to 100 ml

pH 5.4

Preparation: Dissolve the malt extract and agar by heating. Adjust the pH. autoclave at

115°C for 20 minutes.

(b) Potassium tellurite solution

Potassium tellurite 0.5 g


Preparation: Dissolve the potassium tellurite. Autoclave at 115°C for 20 minutes. Store

in the dark.


Basal medium 100 ml

Potassium tellurite solution 1-3 ml

Cool the melted basal medium to 5S°C. Mix the tellurite solution and distribute.

Use: It is a selective medium for the isolation of fungi.

Fungal Medium

(Waksman's medinm)

Composition:

Glucose 10.0 g

Peptone 5.0 g


Magnisum sulphate (MgSO4.7H2O) 0.5 g

Agar 25.0 g


pH 4.0

Adjust pH 4.0 by addition of 1 N H2SO4

Use: Used for isolation of soil fungi. (plate count agar for fungi.)

FusmonumM edium

(Bihi medium, modification by Joffe)

Composition:



Magnisum sulphate 0.5 g


Starch powder 0.2 g

Glucose 0.2 g

Sucrose 0.2 g


Use: Strips of cellulose lens paper are added. This medium always induces conidial

formation in Fusarium.

Fusarium Medium

(Coon's medium)

Composition:

Saccharose 7.2 g

Dextrose 3.6 g


Potassium dihydrogen sulphate 2.72 g


Agar 20.0 g


Preparation: Add to this malachite green to make 1: 40,000 solution or gentian violet to

make 1 :26,000 solution.

Use: Used for cultivation of Fusarium.

Fusmiwn Medium

(Malehite greencaptan medium, a modification of Czapek-Dox)

Composition:






Sucrose 30 g


Malachite green 50 mg

Captain 100 mg

Dicrysticin (mixture of Streptomycin sulphate

Procain pencillin G and Sodium penicillin G) 0.75 mg

Preparation: Add fiesh solutions of malachite green, captan and dicrysticin after

autoclaving and before pouring the plates.

Use: Used for selective isolation of Fusarium fiom soil.

Fusarium Medium

(Potato sucrose agar)

Composition:

Potato extract (see page 162) 500 ml

Sucrose 20.0 g

Agar 20.0 g


pH 6.5


Use: Used far cultivation of fungi. (Fusarium)

Fusarium Medium

(Park's medium/Liquid)

Composition:

Glucose 0.7.g


Potassium dihydrogen phosphate 0.2 5



Preparation: Steam sterilize at 1 l5°C for 30 ininutes.

Use: Used for cultivation of Fusarium.

Fusarium Medium

(PCNB medium-modified)

Composition:

Peptone 15.0 g

PCNB (Terraclor, a commercial product is 75% active) 0:5 g

Oxgall 0.5 g

Agar 20.0 g

Chlorotetracycline HCl 50.0 mg

Streptomycin sulphate 100.0 mg

Distilled water to 1000 ml .

Preparation: Steam, sterilizer at 121°C for 20 minutes.

Note: Chlorotetracyline and streptomycin sulphates are heat labile. Sterilize separately.

PCNB: Penta chloronitrobenzene.

Use: To isolate fusaria from soil. It need not to be sterilized if made up fiesh.

Lignolytic Fungi Medium

Composition:

K2HPO4 1.0 g

KCl 0.5 g

MgSO4.7H2O 0.5 g

FeSO4 0.1 g

Lignin 25.0 g

Agar 20.0 g


pH 5.4


Note: By substituting cellulose 'for lignin, the same medium can be used for cellulolytic

fungi.

Use: Used for isolation of lignolytic fungi.

Mucor-synthetic Medium

(Hesseltine, 1954)

Composition:

Dextrose 40.0 g

Asparaghe 2.0 g



Thiamine chloride 0.005 g

Agar 20.0 g


Use: Used for cultivation of Mucor.

Neurospora Minimal Medium

(Beadle and Tatum, 1945)

Composition:

Ammonium tartrate 5.0 g






Sucrose 15.0 g

Biotin 5 x 10-6

g

Bo 0.01 mg

Cu 0.1 mg

Fe 0.2 mg

Mn 0.02 mg

Mo 0.02 mg

Zn 2.0 mg


Use: Used for cultivation of Neutospora.

Neurospora 'complete' Medium

(Beadle and Tatum, 1945)

Composition:

Glucose 5.0 g

Sucrose 5.0 g

Hydrolized casein 5.0 ml

Yeast extract 2.5 g

Spray-dried malt syrup 5.0 g

Vitamin solution 10 ml

Agar 20 g


Vitamin solution

Thiamin 100 mg

Riboflavin 50 mg

Pyridoxin 50 mg

Pantothenic acid 200 mg

p-Aminobenzoic acid 50 mg

Nicotinarnide 200 mg

Choline 200 mg

Inositol 400 mg

Alkali hydrolysed yeast nucleic acid 500 mg

Folic acid 4 µg


The casein hydrolysate in prepared by HC1 hydrolysis and made up to the

equivalent of 50 mg casein per litre.

Use: Used for cultivation of Neutospora.

Penkillium Medium

(Glucose asparagine agar-Krainsky's medium)

Composition:

Glucose 10.0 g

Asparagine 0.5 g


Agar 15.0 g


Preparation: Dissolve glucose, asparagines and potassium phosphate in the water and

add agar. Boil until dissolve. Autoclave at 1150C for 30 minutes.

Use: Used for stimulating production of perithecia in Penicilfum. Also used for

cultivation of actinomycetes.

Penicillium Medium

(Fermentation medium)

Composition:

Lactose 30.0 g

Glucose 10.0 g

Starch 15.0 g


Ethyl mine 3.0 g

Citric acid 10.0 g

Acetic acid 2.5 g

Phenyl acetate 0.5 g


pH 6.8

Preparation: Steam sterilize at 121°C for minutes.

Use: Used for penicillin production by Penicillium chrysogenum.

Penicillum Medium

(Orange fluid medium)

Composition:

Glucose 50.0 g

Ammonium citrate 1.9 g

Potasium dihydrogen phosphate 1.0 g




Extract of 1 orange to 1000 ml

pH 4.5

Orange extract: For 1 litre medium, macerate one good sized orange in 250 ml water.

Make up to 500 ml and boil for ten minutes with constant stirring. Strain through muslin.

Stir up pulp with another 250 mi water and strain again. Make the volume 1 litre.


Pythium Medium

(Glucose nitrate medium)

Composition:

Glucose 5.4 g



Magnesium sulphate (MgS0,.7H,O) 0.5 g

Agar 20.0 g

Thiamine hydrochloride (2ml of 1000 ppm stock sol)


pH 6.0

Preparation: Adjust pH. Autoclave at 1210C for 10 minutes.

Use: Used for reproduction of Pythium aad Phytophthora species.

Phyfophora Medium

(Petri solution)

Composition:

Calcium nitraSe 0.4 g





Preparation: The solution be kept in a refrigerator and used unsterile.

Use: Used for,the induction of sporangia of phytophthore.

Rhizoctonia Agar

Composition:

Saccharose 10.0 g


Agar 20 g

Distilledwaterto 1000 ml


Use: Used for isolation of Rhizoctonia.

Sclerotium Medium

(Garlic agar)

Composition:

Garlic 300.0 g

Agar 20.0 g


Preparation: Peel and cut up garlic and boil in the water for 1 hr. Filter and restore to

original volume with more distilled water. Add agar and sterilize at 121°C for 20

minutes.

Use: Used for cultivation of Sclerotium.

Slime Mold Medium

(Peptone-yeast-extract dextrose agar)

Composition:

Peptone 2.0 g

Yeast extract 2.0 g

Dextrose 5.0 g

Agar 15.0 g

Distilled water to 1000 ml 1000 ml

pH 6.4 – 7.0


Use: Slime mold grow forming translucent patches and producing wheel like

psuedoplasmodia. Slime molds are phagotrophic. Bacterial colonies grow in the

petriplates and slime molds grow on this.

Semisynthetic Fungal Medium

(Czapek-Dox agar)

Composition:





Ferrous sulphatb. 0.01 g

Sucrose 30.0 g

Zinc sulphate Trace

Copper sulphate Trace

Agar 20.0 g


pH 6.4 – 7.0

Preparation: Dissolve all the ingredients except phosphate in half of the water and the

sucrose added. Dissolve the phosphate separately and add to the rest. Make volume to

1000 ml. Sterilize by autoclaving at 121°C for 15 minutes.

Use: Used for isolation of fungi. Czapek's broth is a synthetic medium.

Yeast Medium

(Penicillin streptomycin blood agar)

Composition:

Nutrient agar 90.0 ml

Blood 10.0 ml

Pencillin 300 units

Streptomycin 300 µg

Preparation: Prepare sterile solutions of penicillin and of streptomycin. Melt the sterile

nutrient agar, cool it to 550C. Add the blood collected aseptically and the antibiotic

solutions. Pour plates.

Use: The medium is used for selective cultivation of pathogenic yeast and fungi.

Yeast Medium

(Malt extract agar)

Composition:

Malt 15.0 g


Ammonium chloride 1.0 g

Citric acid (0.1 N) 15.0 ml

Agar 20.0 g



Use: Used for cultivation of yeast and Acetobacter.

Yeast Medium

(Cysteine glucose blood agar)

Composition:

Nutrient agar (2.5% agar sterile) 83 ml

Glucose (20% sterile) 5 ml

Cystine solution (5% sterile) 2 ml

Blood (sterile) 10 ml

Preparation: Melt the agar, cool to 50°C add the remaining ingredients and pour plates.

Cystine solution is sterilized by filtration.

Use: This medium is useful for cultivating the yeast forms of dimorphic fungi.

Yeast Medium

(Hansen's medium)

Composition:

Peptone 1.0 g

Maltose 5.9 g





Use: Used for cultivation of yeasts.

Yeart Medium

(Malt carrot agar)

Composition:

Carrot 250.0 g

Malt extract 5.0 g

Agar 25.0 g


Preparation: Boil carrot until soft. Pass through a fine sieve,

squeezing through as much pulp as possible. Dissolve agar by heating.

Add malt extract. Autoclave at 121°C for 20 minutes.

Use: Used for cultivation of yeast.

3. Algae

Algal Medium

Composition:






Ferric chloride 6H2O 0.01 g


Trace metal solution 10 ml


pH 7.0

Trace metal solution

Ethylenediamintetra acetic acid 0.5 g

Managanese sulphate 6.0 g

Zinc sulphate 11.0 g

Ferrous ammonium sulphate 2.0 g

Cobalt chloride 0.5 g

Calcium sulphate 0.05 g



pH 7.0


Use: For isolation of algae fiom soil and water.

Note: Add agar 20gAitre to produce solid medium.

Algal Medium

(ASM-I medium)

Composition:

Sodium nitrate Dipotassium hydrogen 170.0 mg

Phosphate 17.4 mg

Disodium hydrogen phosphate 14.2 mg

Magnesium chloride 40.7 mg

Magnesium sulphate 49.3 mg

Calcium chloride 22.2 mg

Femc chloride 1.1 mg

Sodium ethylene diarnine tetracetate (N% EDTA) 6.7 mg

Boric acid 2.5 mg

Manganese chloride 1.4 mg

Zinc chloride 0.4 mg

Cobalt chloride 0.02 mg

Cupric chloride 0.00014 mg


pH 7.0 - 7.5

Preparation: Sterilize at 121°C in autoclave for 15 minutes.

Use: For cultivation of freshwater planktonic algae.

Algal Medium

(Beneck's broth)

Composition:

KNO3 0.2 g

MgSO4.7H2O 0.2 g

K2HPO4 or KH2PO4 0.2 g

CaCO3 0.1 g

FeCl3 (1%) 2.0 drops


Agar 25 g

pH 7.0 – 7.5


Precaution: Phosphate should be autoclaved separately from the other components and

mixed aseptically upon cooling.

Use: Used for isolation of algae from soil.

Algal Medium

(Chu's medium No. 10 modified)

Composition:

Calcium nitrate 0.04 g



Magnesium sulphate 7H20 0.025 g

Sodium silicate 0.25 g


Citric acid 0.003 g

A5 trace elements stock solution (Optional) 1.0 ml


A5 Trace elements stock solution

Boric acid 2.86 g

Manganese chloride 1.81 g

Zinc sulphate 0.222 g

Molybdenum trioxide (85%) 0.0177 g

Cupric sulphate 0.079 g


pH 8.5 - 9.0


Use: For cultivation of common freshwater and soil algae.

Precaution: Sterilize phosphate separately.

Algal Medium

(Modified Bristol's medium)

KH2PO4 0.50 g

NaNO3 0.50 g

MgSO4.7H2O 0.15 g

CaCl2.6H2O 0.05 g

NaCl 0.05 g

FeCl3.6H2O 0 0.01 g


Preparation: Steam sterilize at 12 1°C for 15 minutes.

Use: Fro isolation and cultivation of soil algae.

Blue Green Algal Medium

Composition:

K2HPO4 0.2 g

KNO3 1.0 g

Ca(NO3)2 0.1 g

MgSO4.7H2O 0.1 g

FeCl3 0.001 g



Composition:

K2HPO4 0.4 g

(NH4)2HPO4 0.8 g

MgSO4.7H2O 0.4 g

CaSO4 0.4 g

FeSO4.7H2O 0.001 g



Composition:

K2HPO4 0.2 g

MgSO4.7H2O 0.2 g

CaC12.6H2O 0.116 g

NaHCO3 0.2 g


pH 7.8


Composition:

KH2PO4 0.25 g

MgSO4.7H2O 0.125 g

NaCl 0.125 g

CaCO3 5.5 g

FeSO4.7H2O 0.0025 g

Microelements 1 ml


pH 7.3

Microelements:

MnSO4.4H2O 2.5 g

Na2MoO4.2H2O 0.25 g



(Allen and arnon's medium-modified)

Composition:





A5 trace elements stock solution (see page 178) 1.0 ml


PH 8.5 - 9.0

Use: For isolation of nitrogen fixing blue algae.


(Gerloff medium)

Composition:

Ca(NO3)2 0.04 g

K2HPO4 0.01 g

MgSO4.7H2O 0.025 g

Na2CO3 0.02 g

Na2SiO3 .9H2O 0.025 g


Citric acid 0.003 g


Chromatium Agar Shake Deeps

Composition:

Solution A

NH4Cl 0.1 g

KH2PO4 0.1 g

MgCl2 0.05 g

Agar 20.0 g


Solution B

NaHCO3 2.0 g


Solution C

Na2S.9H2O 1.0 g


Preparation: After steam sterilizing each of the three solutions at 121°C for 15 minutes,

cool them to 50°C and pour solutions B and C in to flask A. Dipense aseptically to sterile

tubes.

Use: For purification of Chrimatium.

The use of enrichment media never produces a pure culture. To get pure isolates it

is necessary to serial dilute the mixed cultures 1 with sterile agar shake tubes.

Chlorobium Enrichment Media

Composition:

Solution A

NH4C1 1.0 g

KH2PO4 1.0 g

MgC12 0.5 g


Solution B

NaHCO3 2.0 g


Solution C

Na2S.9H2O 1.0 g


Solution D

FeCl3.6H2O 5.0 g


Solution E

1 N H3PO4

Reparation: Sterilize all five solution at 1210C for 15 minutes. Cool them to 50°C. Add

solution B, C and D into solution A. Adjust the pH at 7.3 by using solution E. Dispense

aseptically in to tubes.

Use: For enrichment of Chlorobiurn.

Mastigocladus Agar

Composition:

MgSO4.7H2O

K2HPO4

NaHCO3 0.02 g

CaC13 0.01 g

Soil extract 10 g

Ferrous ammonium citrate (0.1 %) 1.0 ml

Glass distilled water to 100 ml

Phormidium Agar

Compositwn:


Soil extract 10 ml

KNO3 0.02 g

K2HPO4 0.002 g

Sodium glutamate 0.05 g

Glycine 0.05 g

Liver infusion (Dehydrated) 0.002

Trace elemet stock solution 0.1 ml

Artificial sea water (double strength) 36 ml

Trace element stock solutions

Na2B2O7.10H2O 20 mg

CaSO4.5H2O 100 mg

Fe3PO4 150 mg

MnSO4.4H2O 20 mg

Na2MoO4.2H2O 10 mg

ZnSO4.7H2O 100 mg



Use: Used for cultivation of Phormidium.

Porphyridium Agar

Composition: Yeast extract 0.1 g

Tryptone 0.1 g

K2HPO4 0.002 g

MgSO4.7H2O 0.002 g

KNO3 0.02 g

Soil extract 10 ml

Artificial sea water (double strength) 36 ml


Preparation: Autoclaved at 121°C for 3 minutes.

Use: Used for cultivation of Porphyridium.

Note: Longer autoclaving has been found to result in detractive changes in the media and

resulting solutions do not permit the optimal growth.

4. Protozoa

Entamoeba medium

(Balamuth's medium)

Composition:

Liver, infusion from 27.2 g

Rice powder 1.42 g

Agar 1.1 g

Peptone 0.55 g

Sodium glycerol phosphate 0.3 g

NaCl 0.27 g

Horse serum (sterile) 5.0 ml


Preparation: Add all components, except horse serum and rice powder in water.

Dissolve by heating. Steam sterilize at 121°C for 20 minutes. Sterilize the rice powder at

160°C for 60 minutes. Add (0-lg) rice powder and (0.35ml) horse serum in tubes

containing

medium (7.0ml).

Use: Used for cultivation of Entamoeba species.

Trichomonas Medium

(Diamond's medium - modified)

Composition:


Yeast extract 1.0 g

L-Cysteine HCI.H20 0.5 g

Maltose 0.5 g

L-Ascorbic acid 0.02 g

Horse serum, inactivated 100 ml

Antibiotic solution 10 ml


pH 6.5

Antibiotic solution

Streptomycin sulphate 1.5 g

Amphotericin B 2.0 mg

Penicillin G 10,00,000 units

Distilled water to Sterilize by filtration. 100 ml

Preparation: Add all components except horse serum and antibiotic solution in distilled

water. Steam sterilize at 121°C for 20 minutes. Mix all solutions aseptically. Distribute in

sterile tubes.

Use: Used for the selective cultivation of Trichmonas.

Trichimonas Medium

(Modified CPLM medium - Cysteinej Peptone, Liver Infusion, Maltose medium)

Composition:

(a) Basal medium

Peptone 32.0 g

Maltose 1.6 g

Liver digest 20.0 g


Ringer’s solution to (1/4 strength) 1000 ml

NaOH about 9 ml to adjust pH 6.0

Dissolve the ingredients. Adjust the pH. Steam sterilize at 100°C for 30 minutes

and filter with Whatman no. 1 paper. Autoclave at 115°C for 15 minutes.

(b) Pencillin streptomycin solution

Pencillin 1,00,000 units

Streptomycin 0.1 g

Sterile water 10 ml

(c) Nystatin solution

Nystatin 50,000 units

Sterile water 10 ml


Basal medium 90 ml

Sterile inactivated 10 ml

Serum (Horse/human/rabbit) Penicillin

Streptomycin solution 1 ml

Nystation solution 1 ml

Before use add the serum and antibiotics.

Use: Used for cultivation of Trichmonas vaginalis.

This medium support the growth of I: vaginalis under strictly anaerobic condition.

Leishmania Medium

(Tobie, Von Brand and Mehlman's medium)

Composition:

Solid phase

(a) Basal medium

Meat extract 1.5 g

Peptone 2.5 g

NaCl 4.0 g

Agar 7.5 g


pH 7.2 – 7.4

Autoclave at 121°C for 25 minutes.

(b) Citrated blood

Rabbits blood collected in sterile sodium citrate and inactivated at 56°C for 30 minutes.


Basal medium 75 ml

Blood 25 ml

Melt the basal medium cool to 450C. Add the blood and distribute approx. 5 ml in

sterile tubes. Prepare slants.

Liquid phase

NaCl 8.0 g

KCl 0.2 g

CaCl2 0.2 g

KH2PO4 0.3 g

Glucose 2.5 g


Dissolve the ingredients and autoclave at 121°C for 15 min. with sterile

precautions add 2 ml to test tubes containing the solid medium.

Use: Used for cultivation of Lekhmania and Trypanosomas.

5. Baceriophage

A great many culture media have been devised for the growth of bacteriophage. These

media have to satisfy two main criteria.

1. They must be adequate for the growth of the host bacteria.

2. They must provide suitable conditions for the attachment, penetration,

multiplication and release of the phase.

Soft Agar

Composition:

Agar 0.7 g


Preparation: Sterilize at 121°C for 15 minutes.

Use: Used as a superimposed layer over firm agar.

Phage Broth

Bactotryptone 0.5 g

Bactopepone 0.5 g

Glucose 0.1 g


pH 7.2


Use: Used for dilution of sample for isolation of bacteriophages.

Phage Agar

Composition:

Peptone 0.5 g

Beef extract 0.3 g

Agar 1.5 g

CaCl2 0.002 M

Note: NaCl is committed to prevent spreading of Proteus. Preparation: Previously

sterilized CaCl2 is to be added after autoclaving the medium 121°C for 20 minutes.

Use: For preparation of phage typing plates for Proteus.

Tryptone Broth

Composition:

Tryptone 10.0 g


Calcium chloride (1M) solution 1.0 ml


pH 7.2

Preparation: Previously sterilized CaC12 is too added after autoclaving the medium

121°C for 20 minutes.

Use: Tryptone broth is used for dilution of samples for isolation of bacteriophages.

Tryptone Soft Agar

Composition:

Tryptone broth 1000 ml

Agar 11.0 g

Preparation: As above.

Use: Tryptone soft agar is used as a superimposed layer over firm agar for isolation of

bacteriophages.

Tryptone Yeast Extract Agar

Composition:

Tryptone 10.0 g

Yeast extract 5.0 g


Sucrose 50 g

Agar 15 g


pH 7.4

Preparation: Steam sterilize the medium at 100°C for 1 hr.

Use: In phase typing, bacterial suspension to be tested is swabbed over an agar surface.

The bottom of the plate is marked off in squares and labelled to indicate which phase

types are going to be used. A small drop of each phase type is added to their respective

squares.

Deca Strength Phase Broth (DSPB)

Composition:

Peptone 100 g



K2HP04 80.0 g


pH 7.6

Preparation: It is sterilized in autoclave at 121°C for 20 minutes.

Use: 5 ml broth + 5ml Escherichia coli is added to 45 ml of sewage hr the enrichment of

bacteriophase during its isolation.

Significance: DSPB medium is ten times as strong as ordinary broth to accommodate

dilution with 45 ml of sewage.

"F" Medium (Anderson's)

(Defined lactate ammonium medium)

Composition:

NH4Cl 1.0 g

MgSO4.7H2O 0.1 g

KH2PO4 1.5 g

Na2HPO4 3.5 g

Lactic acid 9.0 g


pH 6.5

Preparation: Autoclave at 121°C for 20 minutes. Magnesium sulphate and lactic acid

solution is sterilized separately and then added aseptically.

Use: Used for growing E. coli and the T-even phages.

"N" Medium (Anderson's)

Composition:

Difco nutrient broth 8.0 g

NaCl 5.0 g



Use: Used for growing E. coli and T-even phages.

"M9" Medium (Adam's)

Composition:

NH4Cl 1.0 g

MgSO4.7H2O 0.13 g

KH2PO4 3.0 g

Na2HPO4 6.0 g

Glucose 4.0 g


Preparation: Autoclave at 1210C for 20 minutes. Magnesium sulphate solution is

sterilized separately and then added aseptically.

Use: Used for growing E. coli and the T-even phases.

Peptone Broth (Hershey, Dixon and Chase)

Composition:

Peptone 10.0 g

Nacl 3.0

Glucose 1.0 g

MgSO4 1 mm

CaC12 0.1 mm

P (as phosphate buffer, pH 7.0) 5 mg


pH 6.8

Preparation: Autoclave at 121°C for 20 minutes. Sterilize MgSO4 and glucose

separately.

Use: Used for growing E. coli and the T-even phages.

MS Broth (Davis and Sinsheimer)

Composition:

Tryptone 10.0 g

Nacl 8.0 g

Yeast extract 1.0 g


Glucose solution, (1 0%) sterile 10 ml

CaCl2 (M) solution, sterile 2 ml

Thiamine hydrochloride 10 mg/ml, sterile 1 ml


Use: Used for preparing coliphases MS2.

H Medium (Maaloe and Hannawalt)

Composition:

Tris 12.0 g

KCl 2.0 g

MgCl2 2.0 g

MgCl2.6H2O 0.5 g

Na2HPO4 0.05 g

Na2SO4 0.02 g

HC1 (conc.) 7.5 ml

Glucose 0.5 g



Use: Used for growing filamentous coliphages M13.

KC Broth (Sinsheimer et al)

Composition:

KCl 100 mg

Tryptone 200 mg

Cacl2 (0.01 M) 1 ml



Use: Used for growing coliphage X174.

MGM Medium (Lannni)

Composition:

Maleic acid 5 x 10-2

M

Glucose 5.0 g

NH4Cl 1.0 g

P (as orthophsphate) 40 mg

S (as Na2SO4) 27 mg

KCl 2.0 g

Gelatin 0.1 g

FeSO4.7H2O 0.5 mg

MgSO4 10-4

M


pH 7.15

Use: Used for growing phages T5.

L Broth (Davision and Freifelder)

Composition:

Tryptone 1.0 g

Nacl 0.5 g

MgCl2 0.01 M


Use: Used for preparation of h phase.

"L" Broth (Levine)

Composition:

Trypone 1.0 g

Yeast extract 0.5 g

NaCl 0.05 g

Glucose 0.01 g


pH 7.0

Use: Used for growing Escherichia coli and for isolation of bacteriophage.

3

Staining Methods

Bacterial Staining

Simple Staining (Monochrome staining)

Solution required

(a) Loeffler' methylene blue

Methylene blue chloride 0.3 g

95% Ethanol 30 ml

0.1 % KOH 100 ml

Dissolve methlylene blue chloride in ethanol. Add 0.1% KOH. Filter the solution before

use. Store at room temperature.

OR

a. 1% Crystal violet

OR

a. Carbol fuchsin (see acid fast staining). Dilute 10 times before use.

Procedure

1. Prepare the smear and heat fix

2. Treat the smear with 5-7 drops of staining solution.

3. Allow the smear to react as follows

Loeffler's methylene blue for 120 to 150 seconds.

OR

per cent crystal violet for 60 to 120 seconds.

OR

Diluted carbol fuchsin for 15 to 30 seconds.

4. Pour off the staining solution and wash the slide by gentle

5. flow of tap water.

6. Dry the slide in air.

7. Examine under oil immersion lens.

Result: Bacteria appear blue or violet or red by respective stains.

Simple Staining Methods for Bacteria in Milk

(Breed's smear count method)

Solution Required

a. Xylene or chloroform

b. 95% ethyl alcohol

c. Breed's methylene blue.

Methylene blue chloride 0.3 g

95% Ethyl alcohol 30.0 ml

2% phenol in water 100.0 ml

Dissolve methylene blue chloride in ethanol. Then add above solution to phenol in

water. Mix

d. 90% alcohol.

Procedure

1. Mark a clean slide with glass marking pencil to make one centimetre square.

2. Place 0.01 ml of milk sample in the centre of the square.

3. Spread the sampfe with inoculating needle to form uniform smear covering the

square.

4. Heat fix the smear and treat the slide with solution a for about 1 minute.

5. Treat the smear with solution b for 3 minutes.

6. Treat the smear with solution c for 2 minutes.

7. Wash the smear with solution d till smear appears faintly blue. (approx 30

seconds)

8. Dry in air and examine under oil immersion lens.

Result: Bacteria appear blue in colour.

Staining of Azotobacter Cysts

Solution required

a. Staining solution

Glacial acetic acid 8.5 ml

Sodium sulphate (anhydrous) 3.25 g

Neutral red 200.0 g

Light green S.F. yellowish 200.0 mg

Ethanol 50.0 ml

After 15 minutes of incubation remove amorphous precipitate by filtering through

0.5 pm membrane filter.

Procedure

1. Suspend the growth of the bacteria in the solution a for wet mount preparation.

2. Observe under oil immersion lens.

Result

1. Vegetative cens appear light yellowish green.

2. The early stage of encystment shows dark green cytoplasm.

3. Cyst: Intine appears colourless

Exine appears brownish red.

Cytoplasm appears green.

Staining of Actinomycetes

Solutions required

a. Absolute methanol

b. Hucker's crystal violet (see grams staining)

Procedure

1. Treat the growth on the coverslip with few drops of solution a for 15 minutes.

2. Wash with tap water and blot dry.

3. Stain with solution b for 1 minute.

4. Wash with tap water, dry in air.

5. Observe under oil immersion lens.

Results: Mycelium and spores appears violet in colour.

Note: Slide cultures must first be dried by placing them over boiling water for about 5

minutes until agar has dried.

Staining of Actinomycetes

Solutions required


Bismark brown stain (0.1% w/v) 40 ml

Toluidine blue stain (0.1% w/v) 40 ml

Saturated ammonium sulphate solution Mix together 20 ml

Procedure

1. Grow the culture of actinomycetes on the surface of sterile cellophane placed on

placed on solidified nutrient agar medium.

2. Remove the cellophane bearing growth fiom the agar surface.

3. Treat the growth for 2 minutes with the solutions a.

4. Wash the slide with tap water.

5. Air dry and observe under oil immersion lens.

Result: Vegetative mycelium appears light yellow and the spores blue.

Staining of Mycoplasmas Colony

I. Dine's method

Solution required

a. Diene's stain


Azure II 1.25 g


Benzoic acid 0.20 g

Maltose 10.0 g


Procedure

1. Flood the plate containing suspected colonies of mycoplasmas with 1:9 dilued

solution a in distilled water.

3. Remove the stain.

4. Examine the plate under low power microscope.

Result: The colony appears granular, royal blue to greenish blue in colour.

II Staining with cresyl-fast violet

Solution required

a. Cresyl-fast violet solution

Stock solation

Cresyl - fast violet 1.0 g

Distilled water to (pH-3.7, adjusted with Glacial

acetic acid 1-5 drops/100 ml) 100 ml

Allow the solution to ripen for 48 hours.

Working solution

Stock solution 20.0 ml


Maltose 7.0 g

Mix sodium chloride in stock solution. Filter. Add maltose.

Procedure

Similar to that of Dienes method.

Result: Colonies of mycoplasmas appear red to purple.

Staining method for Bruce11 Abortus

Solutions required

a. Carbol fuchsin 10 times diluted (see acid fast staining ZNCF method.)

b. 0.5 acetic acide.

c. Loeffler's methylene blue (see monochrome staining).

Procedure

1. Prepare the smear do not heat fix.

2. Treat the smear for 15 min with solution a.

3. Drain the solution a treat with solution b for 1'2-20 seconds.

4. Wash with water and treat with solution c for 1-2 min.

5. Wash with water, dry and observe under oil immersion lens. .

Results: Brucella abortus appears red in colour. Other organisms appear blue in colour.

Acid Fast Staining

1. Ziehl-Neelsen method

Solutions required

a. Carbol fuchsin (Ziehl-Neelsen)

Basic fuchsin 1.0 g

95% ethanol 10 ml

5% phenol 100 ml

Dissolve basic fuchsin in ethanol then mix with phenol. Allow this solution to

ripen for 1-2 weeks.

b. Sulphuric acid (20% solution)

c. Leffler's methylene blue (see monochrome staining)

OR

1% Malachite green in water.

Procedure

1. Prepare a smear and heat fix.

2. Treat the smear with solution a and heat the slide by gentle flame for five minutes.

(The stain must not be allowed to evaporate and dry on the slide.)

3. Allow the slide to cool.

4. Wash with water.

5. Treat with solution b till red cnlnl~rn o longer comes out (usually for 90 secs.)

6. Wash with water.

7. Treat the smear with solution c for 20 to 30 seconds.

8. Wash, air dry and examine under oil immersion lens.

Results: Acid fast cells stain bright red, while non-acid fast are stained green or blue

colour according to solution c used.

Note:

a. Following decolourisers are usually used for different organisms.

1. 5 per cent sulphuric acid for M. leprae.

2. 3 per cent HCl in 95% ethanol for M. tuberculosis.

3. 1 per cent sulphuric acid to demonstrate acid fast clubs of Actinomyces and

Nocardia.

b. When malachite green is used as a counterstain (solution c), use deep green filter

in the light source for microscopic observasion.

2. Method of Gross

Solutions required

a. Basic fuchsin with Tween 80

Basic fuchsin chloride 2.0 g

Phenol 6.0 ml

95% ethanol 12.5 ml

distilled water to 150.0 ml

Tween 80 15 drops

Dissolve basic fuchsin chloride in phenol at 80°C. Add 95% ethyl alcohol by

stirring. Make the final volume to 150 ml with distilled water. Allow it to ripen for

1-2 weeks. Filter before use and add Tween 80.

b. 3% HCl in ethanol.

c. Loeffler's methylene blue (see monochrome staining).

Procedure

1. Prepare the smear and heat fix.

2. Treat the smear with solution a for 5-10 min.

3. Wash the smear with water.

4. Wash the smear with solution b till red colour not longer comes out (usually 120

seconds).

5. Wash the slide with water.

6. Treat the slide with solution c for 3 min.

7. Wash, air dry and examine under oil immersion lens.

Results: Acid fast organisms appear red. Non acid fast organisms and background appear

blue in colour.

3. Method of Trauant et al.

Solutions required

a. Staining solutions

Auramine "0" 0.3 g

Phenol 3.0 g


Dissolve the phenol in water with gentle heat. Add the auramine slowly. Shake

vigorously until dissolved, filter and store in dark stopped bottle.

b. Traunt's decolourizer.

NaCl 0.5 g

HCl 0.5 ml

75% Ethyl alcohol to 100 ml

c. Potassium permanganate solution (1 to 1000) aqueous. Procedure

Procedure


2. Treat it with solution a for 15 minutes.

3. Wash the smear with water and treat the smear with solution b for 5 minutes.

4. Wash with water then treat the smear with solution c for 30 seconds.

5. Wash air dry & examine under 8 mm dry objective &a high power eye piece (20x).

Result: Tubercle bacilli appear luminescent yellow coloured. Background appears dark.

Negative Staining

Solution required

a. Nigrosin solution

Nigrison (G.T Gurr) 10 g


Dissolve nigrosin in warm distilled water (require an hour) & filter. Add formalin

0.5 % (i.e., formaldehyde 0.19 %) as a preservative.

OR

2 % congored solution.

OR

India ink.

Procedure

1. Take a loopful bacterial suspension and a drop of solution a at one end of clean

glass slide. Mix.

2. Spread this mixture as a film using the another slide.

3. Allow it to air dry and examine under high power and oil immersion lens.

Result: Bacteria appear colourless with dark background, blue black with nigrosin, red

with congored and blue with India ink.

Note: Film should not be too thick or too thin.

Capsule staining

A. Positive staining methods

1. Method of Hiss (modification of Anthony)

Solution required

a. 1% Crystal violet

b. 20% CuSO4.5H2O (aqueous solution)

Procedure:

1. Prepare a smear. Do not heat fm.

2. Treat the slide with solution a for 2 min.

3. Remove the solution a by washing with solution b.

4. Dry and examine under oil immersion lens.

Result: cells appear dark purple, capsules appear pale blue.

2. Method of Moller

Solutions required

a. Moller's fixations (see fixatives)

b. Moller's crystal violet solution

Crystal violet chloride 0.5 g

95% ethyl alcohol 10 ml


Dissolve crystal violet chloride in 95% ethyl alcohol. Then add distilled water.

c. Saturated aqueous CuS04.5H20 solution

Procedure

1. Prepare the smear and treat the smear with solution a for 15 seconds. Pour off the

solution a and dry the smear.

2. Treat the smear with solution b for 2 minutes. Pour off the solution b.

3. Treat the smear with solution c for 10 seconds.

4. Dry the smear and examine under oil immersion lens.

Result: Capsules appear light purpule violet. Bacterial cells appear dark violet.

B. Negative Stain methods

1. Method of Howie an Kirkpatrick (Releif staining)

Solution required

a. Staning solution

10 per cent water soluble eosin, 'Yellowish' or

'bluish' or erythrosine in distilled water 40 ml

Serum (human, rabbit, sheep or ox heated at

56OC for thirty minutes) 100 ml

Crystal of thymol

Mix well. Allow the mixture to stand at room temperature for several days.

Centrifuge and store the supernatant fluid at room temperature.

Procedure

1. Take one drop of suspension and one drop of solution a at Y one end of slide. Mix

well. Wait for 1 minute.

2. Spread this mixture with the help of another slide as a thin

3. Dry without heating and examine under oil immersion lens.

Result: Background and bacteria appear red while capsule appear stained or lightly

stained.

2. Method of Maneval's

Solution required

a 1% congored

b. Maneval's stain I

5% aqueous solution of phenol 30.0 ml

20% acetic acid 10.0 ml

30% FeC12.6H2O 4.0 ml

1% acid fuchsin 2.0 ml

Mix thoroughly.

Procedure

1. Take one drop of bacterial suspension and one drop of solution a at one end of

slide. Mix. Spread over a slide as a thin film with the help of another slide.

2. Air dry the smear.


4. Wash with distilled water, air dry and examine under oil immersion lens.

Dissolve the nigrosin in warm distilled water, add the formalin and filter.

b. Loeffler's alkaline methylene blue. (see monochrome staining).

Procedure

1. Take one looful of culture on a clean slide.

2. Add one looful of freshly filtered solution a.

3. Mix, allow to dry in air and fix with gentle heat.

4. Treat the smear with solution b for 30s.

5. Rinse rapidly in water, air dry and observe under oil immersion lens.

Result: Bacterial cell appear blue, capsule appear colourless unstained against a dark

grey background of nigrosin.

Note: Safranin may be used in place of the methylene blue. (solution b)

5. Dry India ink film method

Solutions required:

a. 6% glucose solution in water.

b. 8% aqueous solution of nigrosin

OR

Indian ink.

a. Methanol or Leishman stain

b. Methyl violet stain (1% aqueous solution).

Procedure: 1. Take one drop of bacterial suspension and one drop of solution a at one end of

slide.

2. Add one drop of solution b to it.

3. Mix and spread the mixture over a slide as a thin film with the help of another

slide.

4. Dry the smear in air.

5. Treat the smear by solution c. Pour off immediately, dry in air.

6. Treat the smear with solution d for 1-2 minutes.

7. Wash with water, dry and examine under oil immersion lens.

Result: Capsules appear colourless, cells appear purpule. Background appear grey-

6.India ink preparation (Wet film)

Solution required:

a. Indian ink

Procedure:

1. Place one loopful of solution a on a perfectly clean glass slide.

2. Emulsify a small portion of solid bacterial culture in the drop of solution a or mix

in a loopful of liquid culture.

3. Cover the mixture with a clean cover glass and press the latter down firmly, to

form a very thin film.

4. Seal the edges of the cover glass with paraffin wax or other suitable medium.

5. Examine the smear under oil-immersion objective.

Result: Bacteria highly refractile, surrounded by a clear zone against a dark grey

background of ink particles. Noncapsulated bacteria do not show this clear zone.

Sprirochete Staining

1. Fontana's method

Solutions required:

a. Fontana's fixative (see fixatives)

b. Absolute ethyl alcohol

c. Fontana's mordant

Phenol (melted)

Tannic acid

Distilled water to

Dissolve tannic acid in water then add melted phenol.

d. Fontana's stain.

Add 10 per cent ammonia solution to 0.5 per cent aqueous silver nitrate solution

dropswise, until the precipitate formed just redissolve. Slowly add more silver nitrate

solution until the light precipitate reformed and remain stable.

Procedure:

1. Prepare the smear and air dry.

2. Treat the smear with solution a for three times, each time for 30 seconds.

3. Wash the smear with solution b and treat with the same solution for 3 min.

4. Remove solution b and dry the slide in air.

5. Treat the smear with solution c and heat the slide from below i till steam rises for

30 seconds. Cool.

6. Wash the slide with distilled water and air dry.

7. Treat the smear with solution d and heat the slide until steam rises and smear

becomes brown in colour.

8. Wash, dry and examine under oil immersion lens.

Result: Spirochetes appear brownish black against yellow background.

Note: Some immersion oils, cause the film to fade at once. Use Canada balsam only.

2 Becker's method (modified)

Solution required:

a. Fontana's fixative (see fontana's method)

b. Mordant (as in fontana's method)

c. staining solution.

Basic fuchsin (Saturated alcoholic solution) 45.0 ml

Shunk's mordant B (95 or 100% ethanol and

aniline oil 4 ml) 18.0 ml


Mix the Shunk's mordant and the alcoholic fuchsin and then add the distilled

water. (the glassware should be dry). Filter before use.

Procedure:

1. Prepare the smear and dry in air.

2. Treat the smear with solution a for 1 to 3. minutes.

3. Wash in water.

4. Treat the smear with solution b for 3-5 minutes, wash in water.

5. Treat the smear with solution c for 3 to 5 minutes.

6. Wash in water and dry. Observe under oil immersion lens.

Result: Spirochetes appear red in colour.

3. Rue's method

Solutions required:

a. l%Formalin

b. 5% NaHCO,

c. Basic fuchsin

Basic fuchsin

95% ethyl alcohol 25.0 ml

Dilute the stain 1:9 before use.

Procedure:

1. Prepare a smear and air dry.

2. Treat the smear with solution a for 1 minute and dry.

3. Place a drop of solution b on the smear and then add 10 drops of solution c. Mix

well and treat the smear with this mixture for 3-5 minutes.


Result: Spirochetes appear red in colour.

4. Levaditi's method (Staining of spirochetes in tissues) pyridine modification

Solutions required:

a. a.10% formalin

b. %-98% alcohol

c. Silver nitrate pyridine solution

i. Pure pyridine 10.0 ml

ii. Silver nitrate (1 % solution) 90.0 ml

d. d.10% pyriding solution

e. Reducing fluid

Acetone (pure) 10.0 ml

Pyridine (pure) 15.0 ml

Formalin (4%) 100.0 ml

Use immediately.

Procedure: 1. Keep the small piece of tissue (1 mm thick) in solution a for 24 hrs.

2. Wash the tissue for 1 hr in water and then place it in solution b for 24 hrs.

3. Treat the tissue by solution c at room temperature for 2 hrs and then at about 50°C

for 4 to 6 hrs.

4. Wash the tissue section with solution d.

5. Treat the tissue immediately by solution e for 48 hrs in the dark.

6. 'Wash with distilled water, dehydrate with increasing strength of alcohol.

7. Proceed for section cutting according to standard procedure.

Flagella Staining

Special care should be taken in cleaning slide and preparing bacterial smear in flagella

staining.

a. Cleaning the slide: Wash the slide with dichromic acid cleaning solution. Wash

with distilled water. Pass the slide through Bunsen flame until flame shows yellow

colour. I

b. Preparation 01 smear: Take agar slant having actively growing young culture. (18-

20 hrs old). Add slowly about 2-4 ml of sterile saline through the side of tube

keeping slant in upward position. Rotate the tube slowly between palms or 5

minutes. Incubate the tube at optimum temperature for 40 minutes. Take a small

drop of saline suspension from the tube by capillary pipette and put at the one end

of slide. Tilt the slide so that the drop will slowly run over the slide and form a

smear. Allow the slide to air dry. Do not heat fix the smear. Stain the slide the

suitable staining method.

1. Method of Bailey (modified)

Solutions required:

a. Bailey's staining solution a.

6% aqueous FeC13.6H2O 6.0 ml

10% solution of tannic acid Mix. Filter before use 18.0 ml

b. Bailey's staining solution b

Bailey's solution a 0.5% basic fuchsin 3.5 ml

in ethyl alcohol 0.5 ml

Concentrated HCl 0.5 ml

Formalin 2.0 ml

Mix. Filter before use. Use freshly.

c. Carbol fuchsin (see acid fast staining)

Procedure:

1. Prepare the smear as given above.

2. Treat the smear by solution a for about 3-5 minutes.

3. Remove the solution a and treat the smear by solution b for 7 minutes.

4. Wash with distilled water. Do not dry.

5. Treat the smear with solution c immediately and heat it from below till steam rises

for 1 min.

6. Wash the slide with water, air dry ad examine.

Result: Flagella and cell wall appear red in colour.

2. Method of Patel, Kulkarni and Gaikwad

Solutions required:

a. Tannic acid solution

Tannic acid 0.2 gm


b. Iodine solution

Iodine crystal 2.0 g

1 N NaOH 10.0 ml

c. Basic fuchsin solution

Basic fuchsin

Absolute alcohol

Distilled water

Procedure

1. Prepare a smear as mentioned earlier.

2. Treat the smear with 4-6 drops of solution a immediately followed by equal drops

of solution b.

3. Heat the smear from below gently for 1-2 minutes.

4. Add 2-3 drops of solution c to the smear, and again heat for

2. Minutes. (do not allow to dry the steam).

5. Wash the slide with water, air dry and examine tinder oil immersion lens.

Result: Bacteria appek red, flagella appear pink in colour.

3. Method of Gray

Solutions required:

a. Gray's staining solution

Solution I

Saturated solution of aluminium potassium sulphate 5.0 ml

20 per cent tannic acid saturated solution of 2.0 ml

Mercuric chloride 2.0 ml

Solution II

Basic fuchsin 6.0 g

Ethanol 100.0 ml

Complete Solution

Solution I -

Solution II 9.0 ml

Prepare fresh 0.4 ml

Procedure:

1. Prepare smear as given earlier.

2. Treat the smear by solution a for 10 minutes.

3. Wash with water air dry and examine under oil immersion lens.

Result: Flagella appear light red and cells appear dark red in colour.

4. Method of Gray-modified

Solutions required:

a. Similar as Gray's staining solution except the concentration of basic fuchsin is doubled.

Procedure:

1. Prepare a bacterial suspension as given earlier.

2. Place a loopful of suspension on a clean slide and place a cover slip over it.

3. After ten minutes place two drops of solution a at one edge of coverslip. Capillary

action will such the solution below the coverslip.

4. Observe the slide after a minute.

Result: Similar as Gray's method.

Note: In this method it is possible to observe the m o w and presence of flagella.

5. Method of Leifson

Solutions required:


Solution I

Tannic acid 3.0 g

Phenol 0.2 ml


Solution II



Solution III

Basic fuchsin 1.2 g

Ethanol 100.0 ml

Adjust the pH of distilled water to 5 before use. Mix I, 11 & III solutions in equal

volumes in the given order. Store at 3-50C.

b. Loeffler's methylene blue. (see monochrome staining).

Procedure:

c. 1: Prepare the smear as given earlier.

3. Treat the smear with solution a for 5-10 minutes. (Allow to react till precipitates

are observed.)

4. Wash with water. Treat the smear with 1:10 diluted solution b and allow to react

for 1 minute.

5. Wash with water, air dry and examine under oil immersion lens.

Result: Flagella appear red while cells appear blue.

Metachromatic Granules (volutin) Staining

1. Method of Gohar

Solution required:

a. Loeffler's methylene blue (see monochrome staiing)

b. 0.1 % H2SO4

c. Gram's iodine (see gram staining)

d. 1% eosin Y.

Procedure:

1. Prepare the smear and heat fm.

2. Treat the smear with solution a for 5 minutes.

3. Wash with water. Treat the smear with solution b for 15-30 seconds

4. Wash with water. Treat smear with solution c for 1 minute.

5. Wash with water. Treat the smear with solution d for 1 min.


Result: Metachromatic granules appear black. Cytoplasm appears pink.

2. Method of Pugh I

Solutions required:


Toluidine blue (C1 No. 52040) 1.0 g

Ethyl alcohol, absolute 20.0 ml

Acetic acid, glacial 50.0 ml


Combine the acetic acid with the distilled water and dissolve the dye in the

alcohol. Mix. Filter.

Procedure:

1. Prepare a smear, air dry and heat fix.

2. Treat the smear with solution a for 2-3 minutes.

3. Wash with water, air dry and observe under immersion lens.

Result: Metachromatic granules appear reddish purple in colour. Remaining organism

appears light blue.

3. Method with methylne blue

Solution required:

a. Loffler's methylene blue (see monochrome staining)

Procedure:


2. Treat the smear with solution a for 5 min.

3. Wash with water. Air dry and examine under oil immersion lens.

Result: Metachromatic granules appear red against pale blue cytoplasm.

4. Method of Albert (Laybourn modification)

Solutions required:

a. Albert's staining solution

Toiuidine blue-0 0.15 g

Methyl green or Malachite green 0.2 g

95% ethyl alcohol 2.0 g



Dissolve toluidine blue and methyl green in ethyl alcohol. Add this to distilled

water containing glacial acetic acid. Store the reagent for one day and then filter.

b. Lugol's iodine (Grams iodine) see gram staining method.

Procedure:



3. Remove the solution a, do not wash-with water.

4. Treat the wet smear with solution b for 1 minute.

5. wash with water, air dry and examine under oil immersion lens.

Result: Metachromatic granules appear black. Cytoplasm appears light green.

Lipid Staining (Budon's method)

Solutions required:

a. Sudan black B staining solution

Sudan black B

70% ethyl alcohol

Shake before use.

b. Xylene

c. 0.5% safranin.

Procedure:


2. Treat the smear with solution anfor about 10.15 minutes.

3. Remove solution a and dry it. Do not was with water.

4. Wash the smear with solution b.

5. Treat the smear with solution c for 5-10 seconds.

6. Wash yith water, air dry and examine under oil immersion lens.

Result: Lipid granules appear deep blue against light pink cytoplasm.

Cytoplasmic Membrane Staining

Solutions required:

a. Potassium nitrate

Distilled water to

b. Bouin's fixative (see fixatives)

c. Victoria blue solution

Victoria blue 0.4 g


Procedure:

1. Prepare a smear and heat fix..

2. Immerse the slide in solution a for 15 minutes.

3. Treat the slide with solution b for 15 minutes.

4. Wash the slide with water. Treat the slide with solution c for 30 seconds.

5. Wash the slide with water. Dry and examine under oil immersion lens.

Result: The cytoplasmic membrane appears as a deep blue outer layer covering a

contracted, irregular shaped body, the cytoplasm.

Cell Wall Staining

(1) Method of Webb

Solutions required:

a. Tannic acid solution

Tannic acid 5.0 g


b. Crystal violet solution



c. Congored solution

Congored 0.5 g

Dsitilled water to 100.0 ml

Procedure: 1. Prepare a smear and heat fix.


3. Wash the slide with water. Do not dry.


5. Wash with water and treat the smear with solution c for 3 minutes.


Result: Cell wall appear blue and cytoplasm appears colourless.

2. Method of Bourin

Solutions required:

a. Bouins fixative (see fixative)

b. Tannic acid solution

Tannic acid 7.0 g


c. Crystal violet solution



Procedire:

1. Cut down agar slab (2mm thick) with growth of organisms.

2. Keep the covership in the centre of petriplate and place the agar slab on to it facing

growth side down.

3. Add solution a in to petri plate till the slab growth immerses in it. Treat it for 3 hrs.

4. Remove the solution a and then agar slab.

5. Wash the covership by allowing water flow in to petridish and overflow it for 2

hrs.

6. Treat the smear with solution b for 20-30 minutes.

7. Wash the covership and flood with solution c for 1 minute.

8. Mount in water and seal with nail varnish and examine under oil immersion lens.

Result: Bacterial cytoplasm appear pale purple while cell wall appear dark brownish

black.

3. Method of Chance

Solutions required:

a. 0.5% Basic fuchsin

b. 0.5% Congo red

Procedure:


2. Treat the smear with solution a for 1 minute.

3. Wash with water.


5. Wash with water.


Result: Cell wall appears pink, cytoplasm appears colourless.

Spore Staining

1. Fuchsin - methylene blue spore staining

Solutions required:

a. Ziehl-Neelsen carbol fuchsin. (see acid fast staining method)

b. Ferric chloride, 30 per cent aqueous solution.

c. Sodium sulphite, 5 per cent aqueous solution.

d. Methylene blue 1 per cent aqueous solution.

Procedure:

a. Prepare a smear, air dry and heat fix.

b. Treat the smear with solution a for 3-5 min. heating the preparation until steam

water.

c. Wash the smear with water.

d. Treat the smear with solution b for 1-2 min.

5. Replace solution b with solution c and allow to act for 30s.

6. Wash with water, Treat the smear with solution d for 1 min.

7. Wash with water, air dry and observe under oil immersion lens.

Result: Spores appear bright red in colour. Vegetative cells appear blue.

2. Fuchsin-nigrosin spore staining (Fleming)

Solutions required:

a. Ziehl-Neelsen carbol -fuchsin. (see acid fast staining method)

b. (i) Nigrosin 1 per cent solution.

(ii) Sodium sulphik, 5 per cent solution.

c. Nigrosin 10 per cent solution.

Procedure:

1. Prepare a smear, air dry and heat fix.

2. Treat the smear with solution a for 5 min heating the preparation d l steam rises.

3. Wash with water, apply solution b (i) for 5-10 min or b (ii) for 5-30s.

4. Wash with water, air dry.

5. Place a small drop of solution c at one end of the slide and spread in an even layer

over the stained preparation with the edge of another slide.

6. Allow to dry and examine.

Result: Spore appears bright red in colour. Remainder of organism colourless against a

dark grey background of nigrosin.

3. Schaeffer and Fulton's method (modified by Ashby)

Solution required:

a. 5% aqueous solution of malachite green.

b. 0.5% S a b i n or 0.05% basic fuchsin.

Procedure:


2. Treat the smear with solution a steam heat the smear for 1 minute. (Do not allow

the smear to dry)


4. Treat the smear with solution b for 30 seconds.


Result: Spores appear green, vegetative bacilli appear red.

4. 'Dorner's method (Synder's modification)

Solutions required:

a. Ziehl-Neelsen carbol fuchsion (see acid fast staining)

b. 95% ethanol.

c. Saturated aqueous solution of nigrosin.

Procedure:

1. Prepare a smear and dry it in air.

2. Treat the smear with solution a. Steam heat the smear for

a. 5-10 minutes. (Do not allow the smear to dry).

3. re at the smear by solution b for approx. 30 second.

4. Wash the slide.

5. Place a drop of solution c at one end of slide and spread it over the smear with the

help of another slide.

6. Dry the slide quickly using gentle heat and examine under oil immersion lens.

Result: Spores appear red, background appears black. Cells appear colourless.

5. Bartholomew and Mittwer's method. (Cold method)

Solutions required:

a. Saturated aqueous malachite green (approx. 7.6%)

b. 0.25% aqueous safranin.

Procedure:

1. Prepare the smear and heat fix the smear by passing through the flame 20 times.

2. Cool the slide and treat the smear with solution a for 10 minutes.




Result: Spores appear green. Cells appears red.

6. Acid fast staining for spores

Solution required:

a. Ziehl-Neelsen carbol fuchsin.(as in acid fast staining)

b. 2% solution of nitric acid in absolute ethyl alcohol.

c. 1% aqueous methylene blue.

Produce:


2. Treat the smear with solution a. Heat the smear for 3-5 min.

3. Wash with water.

4. Dip the slide rapidly in solution b and immediately wash with water.

5. Treat the smear with solution e for 3 minutes.


Result: The spores appear bright red, cytoplasm appears blue.

Polysaccharide Staining

(1) Hotchkiss's method 1

Solution required:

a. Periodite solution

4% aqueous periodic acid solution 20.0 ml

0.2 M aqueous sodium acetate 10.0 ml

Ethyl alcohol 70.0 ml

Mix well, store in brown bottle. Stable for several days.

b. 70% ethyl alcohol.

c. Reducing rinse solution


Sodium Thiosulphate pentahydrate 10.0 g



2N HCI 5.0 ml

Dissolve potassium iodide and sodium thiosulphate pentahydrate in distilled water.

Add ethyl alcohol and HCl. Sulphur precipitates formed allowed to settle down.

d. Fuchsin sulphite solution

Basic fuchsin 2.0 g


2N HCI 10.0 ml

Potassium metabisulphite 4.0 g

Activated charcoal 1.0 g

Dissolve basic fuchsin in distilled water. Dissolve by heating. Cool to 50°C and

filter. Add HCI and potassium metabisulphite to filterate. Incubate overnight in

dark and cool place. Add activated charcoal, mix and filter. Add more HCI

(approximately 10 ml) to this, till mixture when dried on glass does not turn pink.

Store in dark.

e. Sulphite wash solution

Potassium metabisulphite 4.0 g



Prepare fresh.

f. 00.002% malachite green (aqueous)

Procedure:


2. Treat the slide with solution a for 5 minutes.


4. Treat the smear with solution c for 5 minutes.


6. Treat the smear with solution d for 15-45 minutes.

7. Wash the smear three times with freshly prepared solution e and then with water.

8. Treat the slide with solution f for few seconds.


Result: Polysaccharide appears red. Cell appears green in colour.

2. Alcian blue staining method

Solution required:

a. Alcian blue stain

Alcian blue 1.0 g

95% ethanol 100.0 ml

Mix. Dilute ten times distilled water.

b. Carbol fuchsin (see acid fast staining). Dilute ten times with distilled water.

Procedure:


2. Treat the smear with solution a for 1 minute wash with water and aid dry the

smear.

3. Treat the smear with solution b and remove immediately with water.

4. Air dry and examine under oil immersion lens.

Result: Capsules and other polysaccharides appear blue, while cellular material appears

red.

3. Poly-P-Hydroxybutyrate Granules Staining

1. Prepare a smear of a 48-hour culture on a microscope slide.

2. Maintain a positive control by making smear of a known

3. Air dry the smear and then heat fix it.

a. Nile blue staining method.

1. Flood the smear with 1 % aqueous solution of Nile blue

2. Incubate for 10 min at 550C.

3. Drain off the staining solution, wash gently in running tap water and blot dry.

4. Cover the smear with 8% aqueous acetic acid for one minute at room

temperature.

5. Wash gently in running tap water and blot dry.

6. Examine the stained smear with an epifluorescence microscope at 450 nm

under oil immersion lens.

Result: PHB granules appear bright orange fluorescent.

b. Sudan black staining method:

1. Flood the fixed smear with 0.3% Sudan black B solution in 70% ethanol.

2. Incubate for 10 min at room temperature.

3. Wash with tap water and blot dry.

4. Dip the smear in xylol for half a minute and blot dry.

5. Flood the smear with 0.5% (w/v) aqueous safranin far 10 seconds at room

temperature.

6. Wash with tap water and blot dry. Apply a cover slip.

7. Examine the stained smear with a light microscope using transmitted light

under oil immersion lens

Result: PHB granules stain blue-black. The cell wall stains pink.

Nuclear Staining

1. Robinow's method

Solutions required:

a. Schaudinn's fixative (see fixatives)

b. 1 NHCI

c. Giemsa's stain.

(Stock Giemsa's stain-see protozoa stainirig)

Diluted 10 times with phosphate buffer. (pH 7.0)

Procedure:

1. Prepare a smead and air dry.

2. Treat the smear with warm solution a for 5 minutes.

3. Wash with water.

4. Place the slide in solution b at 60°C for 5-10 minutes.

5. Wash the slide with tap water three times and rinse with distilled water.

6. Treat the smear for 30 minutes at 37OC with solution c.

7. Wash, mount in water. Examine under oil immersion lens.

Result: DNA rich area appear red. Cytoplasm appear colourless.

2. Rapid method of Robinow

Solution required:

a. 0.2N HCl

b. 0.1 % Crystal violet

Procedure:

1. Treat unfixed and air dried smear with solution a at boiling temperature for 5

seconds.

2. Wash with water several times.

3. Teat with solution b for 1 minute.


Result: Nuclear material appear purple in colour.

3. Feulgen method

Solution required:


b. lN HCl

c. Schiffs Reagent

Basic fuchsin 1.0 g

Thionyl chloride 1.0 ml


Dissolve basic fuchsin in boiling distilled water. Cool and filter. Add 1 ml thionyl

chloride and allow to stand overnight. Clear the solution by shaking with activated

charcool. Filter and store in refrigerator.

Procedure:

1. This method is similar to Robinow method described above except only 6" step as

follows. Treat the smear for 60 minutes at 15 to 20°C. with solution c. (Schiffs

reagent).

Result: Nuclear material appears red in colour.

Viability Staining for Bacteria

Solutions required

a. Loeffler's methylene blue (see monochrome staining)

b. Dilute 1 : 10 carbol fuchsin (see acid fast staining)

Procedure:


2. Treat the smear with solution a for 10 minutes. Wash the smear with distilled water

until the smear appears pale blue. (about 30 sec.)

3. Run solution b down the slide for a very short time.

4. Wash immediately with water. Air dry and examine under oil immersion lens.

Result:

1. Living bacteria appear purple and dead bacteria appear pink.

2. Living spores appear faintly pink and dead spores appear ' blue in colour.

Gram's Staining

1. Method of Hucker and Conn

Solutions required:

a. Hucker's crystal violet

Solution X

Crystal violet (90% dye content) chloride 2.0 g

Ethyl alcohol (95%) 20.0 ml

Solution Y

Ammonium oxalate 0.8 g


Dissolve crystal violet in ethyl alcohol and the ammonium oxylate in

distilled water. Mix solutions X and Y.

b. Gram's iodine (Lugol's iodine)

Iodine 1.0 g

Potassium iodine 2.0 g


Dissolve potassium iodide in distilled water then add iodine crystals. Dissolve.

c. Ethyl alcohol (95%)

Ethyl alcohol (1 00%) 95.0 ml


d. Safranin

Safranin (2.5% solution in 95% ethyl alcohol 10.0 ml


Filter the solution before use.

e. Basic fuchsin stain

Basic fuchsin 0.5 g


Note: Never use dilute carbol fuchsin because it tends to stain gram negative bacteria so

intensely that they may appear gram positive.

Procedure:


2. Treat the smear with solution a for 1 minute.

3. Remove solution a by solution b and allow the smear to react with solution b for 1

minute.


5. Treat the smear with solution c for 10-1 5 seconds.


7. Treat the smear with solution d for 30 seconds. Wash air dry and examine.

Result: Gram positive bacteria appear violet, gram, negative appear pink in colour.

2. Method of Kopeloff and Beerman

Solutions required:

a. Methyl violet stain

Solution X1

Methyl violet 6B 10.0 g


Solution X2



Mix 600 ml of solution XI and 160 ml of solution X, Prepare freshly.

b. Iodine solution

Iodine 20.0 g

4% aqueous solution of NaOH 100.0 ml


Dissolve the iodine in the NaOH solution and when it is dissolved add the distilled water.

b. Acetone (1 00%)

c. Basic fuchsin stain

Basic fuchsin 0.5 g

Distilled water 1000ml

Procedure:


2. Treat the smear with solution a for 5 min.

3. Add solution b to remove solution a and allow the smear to react with solution b

for 2 min.


5. Wash the smear with solution c for only 2-3 seconds.

6. Wash the smear with water immediately.

7. Treat the smear with solution d for 30 seconds.

8. Wash with water, dry, examine under oil immersion lens.

Result: Same as Hucker's method.

3. Method of Jensen

Solutions required:

a. Methyl violet stain

Methyl violet 6B (or crystal violet) 5.0 g


Filter before use.

b. Iodine solution (see Hucker's modification)

c. Absolute ethyl alcohol

d. Neutral red solution

Neutral red 1.0 g

1 % acetic acid 2.0 ml


Procedure:


2. Treat the smear with solution a for 30 seconds.


for 30 seconds.


5. Wash the smear by solution e for only 10-15 seconds.


7. Treat the slide with solution d for 1 minute.


Result: Same as in Hucker's method.

Note: This method is used especially in examination of exudates for gonococci arid

meningococci. When gonococci and meningococci are scantly, Sandiford's solution

instead of neutral red solution is useful.

Sandifords stain

Malachite green 0.05 g

Pyronine 0.15 g


cells and nuclei stain bluish green. Gram positive organisms are purple black and

gonococci red.

4. Method of Preston and Morrell

Solutions required:

a. Hucker's crystal violet (see Hucker and Conn method)

b. Gram's iodine (see Hucker and Conn method)

c. Iodine - acetone mixture.

Liquor - iodine fortis 7.0 ml

Acetone to make the volume 200.0 ml

Liquor iodine fortis (BP) Iodine crystals 10.0 g

Potassium iodine 6.0 g

Methanol 90.0 ml


Dissolve iodine crystals and potassium iodine in methanol. Add 10 ml distilled

water. Mix.

d. Basic fuchsin (see Hucker and Conn method)

Procedure:


2. Treat the smear with solution a for 30 seconds.


for 30 seconds.

4. Wash the smear with solution c.

5. Treat the smear the same solution c for 30 seconds.

6. Wash with water and treat with solution d for 30 seconds.

7. Wash dry and examine under oil immersion lens.


5. Rapid method for Gram stain

Solutions required:

a. Crystal violet solution (see Hucker and Conn method)

b. Iodine solution (see Hucker. and Conn method)

c. Alcohol-acetone mixture (1 : 1)

d. Safranin (see Hucker and Conn method)

Procedure:


2. Wash the smear with solution a.

3. Then wash the smear with solution b.

4. Wash the slide rapidly with solution c.

5. Wash with water.

6. Wash the smear with solution d for 5 seconds.



Staining of Virus Elementary Bodies and Rickettsiae

1. Method of Gutstein

Solutions required

a. Methyl alcohol

b. Gutsteins stain

Solution (a)

Methyl violet 1.0 g


Solutions (b)



Mix (1) and (2). Prepare freshly.

Procedure:

1. Prepare thin film of smear by using scrapping of skin lesions.

2. Wash with saline and then with distilled water.

3. Treat the smear in solution a for 30 minutes.

4. Treat the smear by solution b for 20-30 minutes at 370C.

5. Wash the slide with distilled water, air dry and examine under oil immersion lens.

Result: Virus elementary bodies appear violet in colour.

Note: This method is useful for staining the elementary bodies of variola - vaccinia group

of viruses.

2. Method of Castaneda (Bedson's modification)

Solutions required:

a. 1.0 N HC1

b. Azure II Solution

Stock Azure II:

Azure II (Gurr) 1.0 g


Filter. Dilute 1:10 with formaldehyde buffer before use.

Formaldehyde buffer:

Commercial formalin 40.0 ml

Sorensen's M/1 5 phosphate 960.0 ml

buffer pH 7.2

Neturalize the formalin with 1.0 N NaOH in the presense of phenol red. Then mix.

c. 0.25% Aqueous solution of safranin.

Procedure:

1. Treat the smear in solution a for 2 min wash with distilled water.

2. Treat the smear with solution b for 20 min.

3. Wash with distilled water.

4. Treat with solution c for 6-8 seconds.

5. Wash with water, air dry and observe under oil immersion lens.

Result: Rickettsiae are stained blue, while cell nuclei and cytoplasm appears red.

Note: This procedure is also useful for elementary bodies of psittacosis.

3. Method of Nicholau

Solutions required:

a. Nicholau's staining solution

Solution 1

Isamine blue 1.0 g

95% ethanol 10.0 ml

Solution 2

Phenol 3.0 g

Distilled water to Mix solution 1 and 2 Store in

brown bottle. 100.0 ml

Procedure:


2. Treat the smear with solution a and heat the slide from below for 5 minutes.

Solution should not be boiled.

3. Wash with water, dry water, examine under oil immersion lens.

Result: Rickettsiae appear blue.

4. Method of Macchiavello (for rickettsia)

Solutions required:


Basic fuchsin 0.25 g


Adjust the pH 7.3 with alkali. Filter the stain before use.

b. 0.5% citric acid.

c. ' 1% aqueous methylene blue.

Procedure:

1. Prepare the film from tissue and air dry.

2. Heat the slide gently and treat for 4 minutes with solution a.

3. Wash with solution b then wash with water.

4. Treat the smear with solution c for few seconds, Blot dry and examine under oil

immersion lens.

Result: The rickettsiae appears red, tissue cells blue.

5. Method of Giemsa

Solutions required:

(see Giemsa stain for protozoa)

Procedure: Similar like protozoal staining by Giemsa's method.

Result: Virus elementary bodies appear purple in colour.

Note: This method is satisfactory for the elementary bodies of vaccinia and psittacosis.

6. Brucella differential staining method

Solutions required:

(see Brucella monochrome staining)

Procedure: Similar like Brucella staining and useful for staining the elementary bodies of

chlamydia.

7. Seller's stain for Negri bodies

Solutions required:

a. Seller's stain

Saturated solution of basic fuchsin in absolute

methyl alcohol 24.0 ml

Saturated solution of methylene blue in methyl

alcohol 15.0 ml

Absolute methyl alcohol (acetone free) 25.0 ml

Mix these solutions.

b. W/50 Phosphate buffer solution with pH 7.0

Procedure:

1. The smear while still moist, is immersed in the solution a in a coplin jar for 5

seconds.

2. Wash with solution b or with distilled water (pH 7)

3. Dry and examine under oil immersion lens.

Result: Negri bodies are stained cherry red and the inner granules deep blue. Cytoplasm

of the nerve cells appear blue to purplish blue whereas nerve fibres are coloured deep

pink but neural sheaths are not stained.

Note: A properly stained smear in transmitted. light should appear reddish violet in thin

portions whereas purpulish blue in thick portions.

8. Paschen's Staining method

Solutions required:

a. Absolute alcohol

b. Loeffler's. mordant

20% aqueous solution of tannic acid 100.0 ml

Saturated aqueous solution of ferrous sulphate 50.0 ml

Saturated aqueous solution of basic fuchsin 10.0 ml

c. Ziehl's fuchsin (see acid fast staining)

Procedure:

1. Prepare films from the infected material or tissue and allow it to dry.

2. Keep in distilled water for minutes and air.

3. Treat the film with solution a for 5 minutes and air dry.

4. Treat with solution b. Gently heat for one minute and allow the solution b to act on

the slide for 10 min.

5. Wash off the solution b with distilled water and stain with solution c, heating

gently for one minute.

6. Rapidly wash the film with water, dry and examine under oil emmersion lens.

Staining of Virus Inclusion Bodies

1. Giemsa staining method

The procedure and staining reagents are the same as described for protozoa. This method

is useful for demonstration of basophilic inclusion bodies.

2. Staining with methylene blue - Eosin

Solutions required:

a. Bouin's fixative (see fixatives)

OR

Zenker's fixative (see fixatives)

b. Mam's methylene blue - Eosin stain

1 per cent aqueous solution of methylene blue 35.0 ml

1 per cent aqueous solution of eosin 45.0 ml

distilled water 100.0 m ml

c. Solution C

Orange - G 0.5 g

70% ethanol 100.0 ml

Procedure:

1. Fix tissues in solution a and cut paraffin sections in routine way.

2. Wash with water.

3. Treat with soiution b for 12 hrs in the incubator at 370C.

4. Wash the section in water, differentiate under the micro scope in solution c.

Dehydrate the sections in alcohol clear in xylene. Mount in oil and observe.

Result: Negri bodies appear red. Nuclei and central granules of Negri bodies appear blue.

Note: This method is specifically useful for staining the specimen for diagnosis of rabies.

3. Lendrum's method

Solutions required:

a. Formal - saline (see saline)

OR

Fromal - corrosive (see fixative)

b. Mayer's haemalum

Haematoxylin 0.1 g

Sodium iodide 0.02 g

Aluminium potassium sulphate 5.0 g


Keep the solution overnight then add

Chloral hydrate 5.0 g

Citric acid 0.1 g

Mix well. Boil for few minutes.

c. Staining solution

Phloxine B or Rose Bengal 0.5 g

CaCl2 0.5 g


Dissolve CaC12 in water then add phloxine B or rose Bengal.

d. Saturated solution of tetrazine in 2-ethoxy ethanol.

e. 95% ethanol.

Procedure:

1. Treat tissues by solution a for 30 minutes and take paraffin sections in routine way.

Wash with water.

2. Treat the sections with solution b for five minutes and then differentiate in water

till they appear blue.

3. Treat the section with solution c for 30 minutes.

4. Wash with water and differentiate by adding dropwise n solution d.

5. Wash with solution e, dehydrate, clear, mount in oil and observe.

Result: Inclusion bodies appear red against yellow background.

Note: This method is specifically applied for staining acidophilic inclusion bodies.

Staining of Fungi

1. Periodic acid - Schiff's stain method (modified) for fungi in tissue sections

Solutions required:

a. Periodic acid solution (see polysaccharide staining)

b. Schiff's reagent (see nuclear staining)

c. Sulphite wash solution (see polysaccharide staining). Freshly prepare.

d. 0.002 per cent malachite green or light green.

Procedure:

1. Wash the section with distilled water and immerse in the solution a for 5 minutes.

2. Wash the sections in tap water for 15 minutes and then wash with distilled water.

3. Immerse the sections in solution b for 15 minutes.

4. Wash three times with solution c for 5 minutes each.

5. Wash with water for 10 minutes.


7. Treat the solution with solution d for 30-60 seconds.

8. Wash with water, dehydrate in absolute alcohol, clear in xylene and mount in oil.

9. Observe under high power microscope.

Result: Fungi appear red in colour. Cytoplasm appears green in colour.

2. Lactophenol cotton blue staining

Solutions required:

a. Lactophenol cotton blue solution

Lactic acid 20.0 ml

Phenol crystals 20.0 g

Glycerol 40.0 ml


Cotton blue (or methyl blue) 0.075 g

Dissolve the phenol crystal in the liquids by gentle warming. Add the dye.

Procedure:

1. Take a drop of solution a in the centre of clean slide.

2. Transfer a small growth of the fungus with spore in to the drop, using a flamed but

cooled needle.

3. Tease slowly the fungal growth using two needles.

4. Put a coverslip over the preparation without entering air bubbles in the stain.

5. Observe under microscope. (high power)

Result: The fungal cytoplasm appear light blue in colour inside the unstained cell wall of

hyphae. Spores and spore bearing structure is surrounded by a light blue background.

Note: If the mycelia are dark coloured, the use of cotton blue in mounting medium may

be omitted.

Staining of Vesicular Arbuscular Mycorrhizae

(VAM fungi)

Solutions required:

a. Formlin acetic acid - alcohol mixture (see fixatives)

b. b.10% KOH

c. Staining solution

80% lactic acid 10.0 ml

Glycerine 10.0 ml

Chlorazol black E 0.01 g


Dissolve chlorazol black E in distilled water. Then add lactic acid and Glycerine to it.

d. Mounting fluid

Chloral hydrate 20.0 g

Gum Arabic 20.0 g

Glycerine 20.0 g

Glucose syrup 3.0 ml

Basic fuchsin (3% in 95% ethyl alcohol) 10 drops


Procedure:

1. Wash VAM infected material with water and treat over night with solution a.

2. Wash with water for several times.

3. Place the sample in to solution-b.

4. Steam sterilise the sample at 121°C for 15 minutes.

3. Wash several times with tap water, wash with distilled water.

5. Treat sample by solution c for 1 hr at 90°C.

6. Keep sample in to glycerine overnight. Mount sample on slide using solution d.

Result: VAM fungi appears brown black.

Note:

1. Prepare reagents several hours before use and keep undistributed to settle

undissolved particles.

2. Do not breath dust from stain.

Nuclear Staining of Filamentous Fungi

Solutions required:

a. Fixative


Acetic acid 10.0 ml

OR


Acetic acid 10.0 ml

Lactic acid 10.0 ml

b. 95% ethyl alcohol

c. 70% ethyl alcohol

d. 1M HCl

e. Phosphate buffer (pH 6.9) - see buffers

f. Dilute Giemsa stain

Stock Giema stain (see protozoa staining) Giemsa stain 1.0 ml

Phosphate buffer (pH 6.9) 15.0 ml

Procedure:

1. Treat spores and mycelia present on thin agar film on glass slide for 10-12 minutes

with solution a.

2. Wash the preparation with solution b, transfer the preparation in solution c (can be

stored for several days in solution c).


4. Treat the preparation for 5 minutes with solution d at room temperature. Transfer it

to solution d at 60°C and keep for 7 minutes.

5. Wash first with water and then with solution e for five times in each.

6. Treat the preparation with solution f for 2 hrs. (Do not allow to dry the smear).

7. Again wash with solution e.

8. Allow the preparation to air dry.

9. Mount in euparal. Cover the preparation with a coverslip.

10. Observer under high power lens.

Result: The nuclei are seen as rose to pink surrounded by colourless to light blue

cytoplasm.

Staining of Protozoa

A. For Protozoa in faeces

1. Iron haematomylin staining method

Solutions required:


b. 50% ethyl alcohol.

b. Grams iodine (see Gram staining by Hucker's method)

c. Weigert's iron haematomylin

Solution 1

Haematoxylin 1.0 g

Absolute alcohol 100.0 ml

Solution 2

Liquor - fem perchlorate (30%) 4.0 ml



Complete solution

Mix equal parts of solutions 1 and 2. Prepare freshly.

Procedure:

1. Prepare wet smear and treat with solution a for 5 minutes.

2. Wash the smear with solution b and apply solution c for 2 minutes.

3. Wash with solution b for 30 seconds.

4. Wash with distilled water and treat the smear with solution d for 12 minutes.

5. Wash with water, dry and observe under oil immersion lens.

Result: Protozoa are stained blue-black in colour.

2. Method of Dobell

Solutions required:

a. Schaudinn's fixative (see fixative)

b. 2% Aqueous ammonium molybdate

c. 0.2% Iron haematoxylin solution (aqueous)

Procedure:

1. Treat at the smear with solution a for 5 minutes.




5. Treat the smear for 10 minutes with freshly prepared solution C.

6. Wash with distilled water and transfer the smear into tap water for about 30

minutes, until the smear appear blue in colour.

7. Mount in water.

Result: Protozoa appear blue in colour.

B. For Protozoa in blood sample

Preparation of blood film

Gently touch a fresh drop of blood on to one end of clean grease free slide. Allow the

drop to spread along it. Holding the slide at 45°C push the spreader along the slide,

drawing the blood behind it, until the whole of the drop has been smeared. The thickness

and even distribution on the cells plays an important part in getting correct result.

1. Leishman's method

Solutions required:

a. Leishaman’s stain

Leishaman's power 0.15 g

Methanol pure (acetone free) 100.0 ml

Grind the crystals to in a clean dry mortar with little amount of methanol and add

the alcohol a little at a time until all stain is dissolved. The final volume is then

made up with methanol. (Prepared stain is easily available in market.)

b. Buffer solution (pH 7.0)

Na2HPO4 (anhydrous) 5.447 g

KH2PO4 (anhydrous) 4.752 g

Dissolve the phosphate in 2 litre distilled water. This gives a pH of 7.0.

Procedure:

1. 1 . Prepare the blood film. Do not heat fix and treat with solution a for 1-2 minutes.

2. Then it is diluted with an equal volume of solution b (or distilled water of 7.0).

Mix by gentle rocking the slide.

3. Allow diluted solution to react for 10 minutes. Wash the slide with distilled water

until the smear appears pink.


Result: Cytoplasm appears blue. Nuclear material appears red.

2. Giemsa's method

Solutions required:

a. Methyl alcohol, absolute

b. Giemsa stain

Giemsa powder

Methanol (acetone free)

Glycerol 25.0 in1 I

Dissolve Giemsa stain in methanol and constant stirring for several hours. Dilute 1 : 10 in

buffer solution before use. (Ready Giemsa stain in available in the market.)

c. Buffer solution (pH 7) (see Leishman staining)

Procedure:

1. Prepare a blood film, air dry.

2. Treat with solution a for 3 min.

3. Treat the smear for 10 minutes with solution b.

4. Wash and differentiate with buffer solution (pH 7).


Result: Chromatin of malarial parasite and trophonuclei of trypanosomes seen red purple.

Cytoplasm of protozoa appears pale blue.

Note: This method gives excellent results with thick blood films.

3. Field's method

Solutions required:

a. Field's solution a


Azure I 0.5 g


KH2PO4 6.25 g


b. Field's solution b

Eosin (yellow) 1.0 g


KH2PO4 6.25 g


The phosphate salts are first dissolved in water and then stains are added. Use the

stain after 24 hrs. Filter before use.

Procedure:

1. Prepare a thick blood film and dry in air. Do not heat fix.

2. Dip the film for one second in solution a.

3. Wash the slide gently with distilled water until stain ceases. to flow from the film.

(for few seconds)

4. Dip the slide for one second in solution b.

5. Rinse by washing gently for 2-3 sec in clean water.

6. Place vertically against a rack to drain and dry.

7. Examine under oil immersion lens.

Result: Same as in Leishman staining method.

4. Simeon's modification of Boye's and Stevenel's method

Solutions required:

a. Methyl alcohol absolute

b. Stevenel's solution

(I) Medicinal ethylene blue 1.0 g


(II) Potassium permangnate 1.5 g


Mix I and II in a flask. A heavy precipitate forms at once. Heat for 30 minutes to

dissolve the precipitate. Filter and use.

c. Eosin solution

Eosin pure 1.0 g


Procedure:

1. Prepare thick blood film. Do not heat fix.

2. Treat with solution a for 1 min. Wash with water for 4 seconds.

3. Treat the film with solution c for 10 seconds wash with water for 4 seconds.

4. Treat the film with solution b for 15 seconds. Wash with water for 4 seconds.

5. Treat the film with solution c for 5 seconds.

6. Wash with water for 4 seconds. Air dry and examine under oil immersion lens.


5. Wright's staining method

Solutions required:

a. Methyl alcohol absolute

b. Wright's stain

Wright's powder 0.2 g

Methanol (pure) 100.0 ml

Prepare the solution few before use.

c. Buffer solution (pH 7.0) (see Leishman staining method)

Procedure:

1. Prepare a blood film air dry. Treat with solution a for 3-5 min.

2. Treat the smear with solution b for 1-2 min.

3. Dilute with an equal amount of solution c.

4. Allow it react for 3 -5 min.

5. Wash and differentiate with solution c.



4

Reagents

Fixatives

1. Bouin's fixative

Saturated aqueous solutions of picric acid 75.0 ml

Formalin 25.0 ml


Use:

1. This fixative is useful for the investigation of virus inclusion bodies.

2. Used in cell wall staining (method of Bouin)

2. Chrome-acetic acid fixative

10% aqueous chromic acid 2.5 ml

10 % aqueous acetic acid 5.0 ml


Use: For fixation of chromosomes, nucleoli and centrioles of fungi.

3. Flemming fluid

Osmic acid 0.1 g

Chromic acid 0.2 g

Glacial acetic acid 01 ml


The osmic and chromic acids when mixed will keep for only3-4 weeks, The acetic acid

should be added immediately before use.

Use: Used as a fixative for tissue section.

4. Fontan's fixative (Ruge's solution)


Formalin (37-40% formaldehyde) 2.0 ml


Use: Used in spirochete staining.

5. Fomalin-acetic acid alcohol (FAA) mixture

60% Alcohol 90.0 ml


Formalin 5.0 ml

Use: For fixation of plant material before staining.

6. Mercuric chloride-formalin fixative

Saturated aqueous 9.0 g

Solution of mercuric chloride 280.0 ml

Commercial formalin 20.0 ml

Use: The minimum amount of destoration and fairly good cytological details are

obtained, particularly in staining of various inclusion bodies if above fixative is used.

7. Moller's fixation

Lead acetate 9.0 g


Formalin 20.0 ml

Precaution: Store in tightly stoppered bottle.

Use: Used in capsule (method of Moller) staining.

8. Newcomer's fixative.

Dioxane 10.0 ml

Acetone 10.0 ml

Petroleum ether 10.0 ml

Propionic acid 30.0 ml

Isopropyl alcohol 60.0 ml

Use: Most acetic and alcohol mixtures result in to loss of nuclei staining property and

dissolution of mitochondria. This fixative overcomes this difficulty during fixation of

smear.

9. Schaudinn's fixative

Absolute ethyl alcohol 33.0 ml


Saturated solution of HgC12

(6.9g/100 ml at 20°C) in water 66.0 ml

Use:

1. Used to fur the smear in staining of protozoa.

2. Used in nuclear staining (method of Robinow)

10. Susa's Fixative

Mercuric chloride 45.0 g



Trichloroacetic acid 20.0 g

Acetic acid (Glacial) 40.0 ml

Formalin (40% formaldehyde) 200 ml

Use: Fixative for normal and pathological specimens before staining. This fixative give

better result.

11. Zenker's fluid


Potassium bicarbonate 2.5 g

Sodium sulphate 1.0 g


Immediately before use, add 5 ml of glacial acetic acid per 100 ml of above mixture.

Use: It is a fixative for animal tissue staining.

12. Zenker-formal fixative

This is similar to Zenker's fluid except that the 5 ml of formalin is added instead of acetic

acid.

Salines

1. Azide saline

Sodium azide 0.8 g

Physiological saline OR buffered saline 100 ml

Use: Azide prevents microbial decomposition. It is used as a diluting fluid.

2. Borate-calcium saline

NaCl 8.0 g

CaCl2 1.0 g

H3BO3 1.2 g

Na2B4O7.10H2O 0.052 g


Use: It is used as a cell suspension and diluting fluid tor haemagglutination experiments,

where calcium is required and phosphate should be absent.

3. Buffered saline

NaCl 8.0 g

K2HPO4 1 : 21 g

KH2PO4 0.34 g


Use: This solution gives a pH of about 7.3 and also provides potassium and phosphate

ions. It is general diluent and suspending fluid. NaCl can be diluted in different buffer

solution giving desired pH.

4. Formal saline

Sodium chloride (0.9%) solution 90.0 ml

40% formaldehyde 10.0 ml

The pH is adjusted at 7.0 by addition of CaCO3 granules.

Use: In staining of virus inclusion bodies.

5. Phenolized saline


Phenol 5.0 g


6. Physiological saline



Use: Used for preparation of bacterial suspensions. It prevents osmolysis of bacteria.

7. VDRL, buffered saline solution (pH 6.0)

Formaldehyde (neutral, c.p.) 0.5 ml

Na2HPO4.12H2O 0.093 g

KH2PO4 0.170 g

NaCl 10.0 g


Dissolve all the constituents in distilled water. Check the pH of the solution. Store the

reagent in screw capped bottles.

Use: Used for preparation of VDRL antigen.

8. Vernol-NaCI diluent

NaCl 8.5 g

Barbitone (diethyl-barbituric acid) 0.575 g

Sodium barbitone 0.2 g

MgCl2.6H2O 0.168 g

CaCl2 0.028 g


Preparation: stock solution concentrated x 5 is made by dissolving 5.75 g barbitone in

500 ml hot distilled water. Add 85 gm, NaCl and make up the volume to about 1400 ml.

Dissolve 2.0 gm sodium barbitone in 500 ml distilled water and add it to the NaCl

barbitone solution. Make up to 2000 ml. Add 1.68 g MgC12.6H20 and 0.28 g CaCl2. For

use dilute 1 in 5 with distilled water.

Use: This saline may be used for complement fixation tests and gives more reproducible

results.

IMViC Test Reagents

1. Kovac's reagent

n-amyl alcohol 75.0 ml

Hydrochloride acid (conc.) 25.0 ml

P-dimethylamino-benzaldehyde 5.0 g

Add aldehyde to a flask containing alcohol and dissolve by gently warming to 5S°C in a

water bath. Cool and add HCI. Store the reagent in a dark glass bottle in a refrigerator.

Use: Kovac's reagent develop red colour in presence of indole.

2. Methyle red solution

Methyl red 0.04 g

Ethyl alcohol (absolute) 40.0 ml


Dissolve methyl red in ethyl alcohol and add water.

Use: Used in methyl red test. Add about 5 drops of the methyl red reagent in to

inoculated and incubated liquid medium. Mix. Positive tests are bright red and negative

are yellow.

3. Barritt's reagent (Voges Proskauer test)

Solution A: 5g alpha-naphthol in 100 ml of 95% ethyl alcohol. Dissolve the alpha

naphthol in the ethyl alcohol with constant stirring.

Solution B: 40g potassium hydroxide in 100 ml water.

Use: Used in Voges Proskauer test. Add 1 ml of Potassium hydroxide and 3 ml of alpha

naphthol in the inoculated and incubated liquid medium. A positive reaction is detected

by the development of an eosin-pink colour usually in 2-5 minutes.

Caution: Avoid all contact with human tissues, alpha-naphthol is considered to be

carcinogenic.

Other Reagents

Andrade's indicator

Preparation: Prepare 0.5% solution of acid fuchsin. Add 1 N NaOH to it, slowly, till

colour turns slight yellow.

Use: Used as an indicator in sugar fermentation tube. It becomes dark red in colour at or

below pH 5.5.

Gelatin Hydrolysis

1. Mercuric chloride solution (Frazier's solution)


Hydrochloric acid (conc.) 20.0 ml


Use: Used for detection of gelatin breakdown. After flooding the plate with mercuric

chloride solution gelatin liquifying microorganisms show clear zone around colony

against opaque background.

2. Tannic acid solution

Tannic acid 1.0 g


Use: To test gelatin hydrolysis. Tannic acid causes relative opacity around gelatin

liquifying colonies, quick to develop but fading as the medium also becomes opaque.

3. Trichloroacetic acid

Trichloroacetic acid (C.P.) 5.0 g


Dissolve the acid in the water with constant stirring.

Use: Used to test gelatin hydrolysis by microorganisms on the gelatin agar.

Pectin Hydrolysis

Hexadecyltrimethyl ammonium 1.0 g

bromde


Use: To detect pectin hydrolysis. It precipitates intact pectin.

Starch Hydrolysis

Lugol's Iodine (Gram's Iodine)

Use: After flooding the plate of starch agar with iodine solution amylolytic

microorganisms show clear zone around the colony against purple background.

Oxidase test reagent

Dimethyl-p-phenylene diamine hydrochloride 1.0 g


The reagent should be made fresh daily. It should not be stored longer than one

week in the refrigerator.

If the preparation becomes darkened, discard. Tetramethyl-p-phelylenediamine

dihydrochloride (1%) is even more sensitive but it is more expensive and difficult to

obtain.

Use: To test presence of certain oxidises in bacteria, the dye is reduced to deep purple

colour if the test is positive.

Methylene blue (1:25000) solution

Methylene blue dye 0.04 g


Preparation: Dissolve the methylene blue in the distilled water and dispense into regular

staining bottles.

Use: Used in methylene blue reduction test (for determining the quality of milk.) Dilute

10 times before use.

Motility studies reagent

Carboxy-methyl-cellulose 2.0 g

Sucrose (0.2 M) 98.0 ml


Preparation: Prepare 0.2 M sucrose solution by adding 68.4 g. of sucrose in 1000 ml of

distilled water.

Use: If a bacterial culture growing on a solid medium is to be examined for motility, a

loopful of culture should be mixed with a drop of 2% CMC at the centre of coverslip.

Methyl cellulose reagent

Carboxy methyl cellulose 10.0 g


Reparation: Dissolve the methyl cellulose in warm distilled water.

Use: For microscopic observation of protozoa.

Significance: Methyl cellulose slows down the movement of protozoa.

Resazurin (1:20,000) solution

Resazurin

Distilled water to

Preparation: Dissolve the resazurin in distilled water.

Use: Used in resazurin reduction test (for determining the quality of milk.)

Cleaning solution for glassware

(Sulphuric acid dichromate solution)

Sodium dichromate 25.0 g

Sulphuric acid (conc.) 1000 ml


Preparation: Dissolve the dichromate crystals in 50 ml of warm water. Cool to, room

temperature and add acid slowly to the preparation. Use: This preparation is used for the

removal of residual organic matter from laboratory glassware. Glassware should be

soaked in this solution for a number of days. Then rinsed in running tap water at least 10

times, then rinsed twice in single distilled water and finally rinsed once in double glass

distilled water.

Ferric chloride reagent

FeCl36H2O 12.0 g

2% Aqueous HCl 100.0 ml

Made up the 2% aq. HCl by adding 5.4 ml of concentrated HCl (37%) to 94.6 ml H2O.

Use: Used in phehyyalanine deaminase test. Development of green colour indicate

positive phenylalanine deaminase test.

Diphenylamine reagent (nitrate test)

Diphenylamine 0.7 g

Sulphuric acid (conc.) 60 ml


Preparation: Dissolve diphenylamine in a mixture of sulphuric acid and water. Cool and

add slowly 11.3 ml of concentrated hydrochloric acid. After the solution has stood for 12

hours some of the base separates, showing that the reagent is saturated.

Use: Used in detection of nitrate in the medium. This reagent produces a blue black

colour in the presence of either nitrites or nitrates. It is necessary to make sure that no

nitrites are present by Trommsdorf -regent when it is used as a test for nitrates.

Trommsdorf's reagent

Zinc, chloride solution (20%) 100.0 ml

Starch 4.0 g

potassium iodide 2.0 g


Preparation: Prepare 100 ml of 20% aqueous ZnC12 solution and add slowly with

constant stirring to a mixture of 4.0 g of starch in water. Dissolve completely by heating.

Dilute with water and add the potassium iodide. Made the volume to 1 litre by additions

of more water, filter and store in brown-stoppered bottle.

Use: For detection of nitrites. This reagent produce intense blue-black colour in present

of nitrite.

Nitrite test reagents

Solution A:

Sulfanilic acid 8.0 g

5 N acetic acid (1 part glacial acetic acid to

2.5 parts water) 1000 ml

Solution B:

Dimethyl-alpha-napthylamine 5.0 g

5 N acetic acid 1000 ml

Immediately before use, mix equal volumes of solution A and solution B.

Use: Add 0.1 nil of test reagent to 5 ml of broth. A development of red colour indicate

presence of nitrite.

Caution: Although at this time it is not know for sure, there is a possibility that

dimethyl-alphanapthylamine in solution B may be carcinogenic. For reasons of safety,

avoid all contact with tissues.

Nessler's reagent (Ammonia test)


Ammonia free distilled water (cold) 35.0 ml

Mercuric chloride solution (saturated) 35.0 ml

Potassium hydroxide (50% aqueous solution) 400 ml


Dissolve potassium iodide in ammonia free distilled water. Add a saturated solution of

mercuric chloride to it. (slight precipitate persists.) Add solution of potassium hydroxide.

Dilute to 1 litre, allow to settle for one week and decant the supernatant for use. Store in

brown bottle.

Use: Used for detection of ammonia. Positive reaction is shown by a colour ranging from

a pale but distinct yellow to a dark brown precipitate.

Buffers

1. Acetic buffer

Solution required:

A: 0.2 M solution of acetic acid (11.55 m1 in 1000 ml).

B: 0.2 M solution of sodium acetate (16.4 g of C2H3O2Na or 27.2 g of C2H3O2Na.3H2O

in 1000 ml). X ml of A + Y ml of B, diluted to a total of 400 ml

X Y pH

185.2 14.8 3.6

176.0 24.0 3.8

164.0 36.0 4.0

147.2 52.8 4.2

122.0 78.0 4.4

102.0 98.0 4.6

80.0 120.0 4.8

59.2 140.8 5.0

42.0 158.0 5.2

35.2 164.8 5.4

19.2 180.8 5.6

2. Barbitone (veronal) buffer

Solutions required:

A: 0.2M solution of sodium barbitone (sodium diethyl barbiturate) (7.37 g in 1000 ml).

B: 0.2M HCl 100 ml of A+ X of B, diluted to a total of 400 ml

X pH

3.0 9.2

5.0 9.0

8.0 8.8

12.0 8.6

18.0 8.4

25.4 8.2

35.0 8.0

45.0 7.8

55.0 7.6

65.0 7.4

78.0 7.2

86.0 7.0

90.0 6.8

3. Bicarbobnate-CO2 buffer

Concentration of CO2 in gaseous phase

5% 10% 20%

Concentration 0.02 M 7.4 7.1 6.8

Of NaHCO3 0.05 M 7.8 7.5 7.2

Above solution give desired pH only at 370C.

4. Borax-NaOX buffer

A: 0.05 M solution of borax (19.05 g in 1000 ml, 0.02 M in terms of sodium borate)

B: 0.2 M NaOH 100 ml of A + X ml B, diluted to a total of 400 ml

X pH

0.0 9.28

14.0 9.35

22.0 9.4

35.2 9.5

46.0 9.6

58.0 9.7

68.0 9.8

77.2 9.9

86.0 10.0

92.0 10.1

5. Boric acid-Borax buffer

Solution required:

A: 0.2 M solution of boric acid (12.4 g in 1000 ml).

B: 0.2 M solution of borax (19.05 g in 1000 ml : 0.2 M in terms of sodium borate).

100 mi of A + X ml of B, diluted to a total of 400 ml.

X pH

4.0 7.6

6.2 7.8

9.8 8.0

14.6 8.2

23.0 8.4

35.0 8.6

60.0 8.8

118.0 9.0

230 9.2

6. Cacodylate buffer

Solutions required:

A: 0.2 M solution of sodium cacodylate [42.8 g of Na (CH3)2 AsO2.3H2O in 1000 ml]/.

B: 0.2 M NaOH 100 ml of A + X ml of B diluted to a total of 400 ml

X pH

5.4 7.4

8.4 7.2

12.6 7.0

18.6 6.8

26.6 6.6

36.6 6.4

47.6 6.2

59.2 6.0

69.6 5.8

78.4 5.6

86.0 5.4

90.0 5.2

94.0 5.0

7. Carbonate-Bicarbonate buffer

Solutions required:

A: 0.2 M solution of anhydrous sodium carbonate. (2.12 g in 100 ml).

B: 0.2 M solution of sodium bicarbonate. (1-68 g in 100 ml).

X ml of A + Y ml of B, diluted to a total of 400 ml

X Y pH

8.0 92.0 9.2

19.0 81.0 9.4

32.0 68.0 9.6

44.0 56.0 9.8

55.0 45.0 10.0

66.0 34.0 10.2

77.0 23.0 10.4

85.0 15.0 10.6

8. Citrate buffer

Solutions required:

A: 0.1 M solution of citric acid (19.21 g in 1000 ml).

B: 0.1 M solution of sodium citrate (29.41 g C6H5O7Na3.2H2O in 1000 ml).

X ml of A + Y ml of B, diluted to a total of 400 ml.

X Y pH

186.0 14.0 3.0

174.8 25.2 3.2

160.0 40.0 3.4

148.0 52.0 3.6

140.0 60.0 3.8

132.0 68.0 4.0

126.0 74.0 4.2

112.0 88.0 4.4

102.0 98.0 4.6

92.0 108.0 5.0

82.0 118.0 5.0

72.0 128.0 5.2

64.0 136.0 5.4

54.8 145.2 5.6

47.2 152.8 5.8

38.0 162.0 6.0

28.8 171.2 6.2

9. Citrate-Phosphate buffer

Solutions required:

A: 0.1 M solution of citric acid (19.21 g in 1000 ml).

B: 0.2 M solution of dibasic sodium phosphate (28.39 g of Na2HPO4 or 71.7 g of

Na2HPO4.12H2O in 1000 ml). X ml of A + Y Ml of B, diluted to a total of 100 ml.

X Y pH

44.6 5.4 2.6

42.2 7.8 2.8

39.8 10.2 3.0

37.7 12.3 3.2

35.9 14.1 3.4

33.9 16.1 3.6

32.3 17.7 3.8

30.7 19.3 4.0

29.4 20.6 4.2

27.8 22.2 4.4

26.7 23.3 4.6

25.2 24.8 4.8

24.3 26.7 5.2

23.3 26.7 5.2

22.2 27.8 5.4

21.0 29.0 5.6

19.7 30.3 5.8

17.9 32.1 6.0

16.9 33.1 6.2

15.4 34.6 6.4

13.6 36.4 6.6

9.1 40.9 6.8

6.4 43.6 7.0

10. Glycine-NaOH buffer

Solutions required:

A: 0.2 M solutions of &cine (15.01 g in 100 ml).

B: 0.2M NaOH 100 ml of A + X ml of B, diluted to a total of 400 ml.

X pH

8.0 8.6

12.0 8.8

17.6 9.0

24.0 9.2

33.6 9.4

44.8 9.6

54.4 9.8

64.0 10.0

77.2 10.4

91.0 10.6

11. Maleate buffer

Solutions required:

A: 0.2M solutions of acid sodium maleate (8 g of NaOH+23.2 g of maleic acid of 19.6 g

of maleic anhydride in 1000 ml).

B: 0.2MNaOH 100 ml of A + X pf B, diluted to a total of 400 ml.

X pH

14.4 5.2

21.0 5.4

30.6 5.6

41.6 5.8

53.8 6.0

66.0 6.2

76.0 6.4

83.2 6.6

88.8 6.8

12. Phosphate buffer

Solutions required:

A: 0.2 M solution of monobasic sodium phosphate. (31.2 g NaH2PO4.2H2O in 1000 ml).

B: 0.2 M solution of dibasic sodium phosphate. (28.39 g of Na2HPO4 or 7 1.7 g of

N3HP0,. 12H20 in 1000 ml). X ml of A + Y of B, diluted to a total of 200 ml.

13. Succinate buffer

X Y pH

92.0 8.0 5.8

87.7 12.3 6.0

81.5 18.5 6.2

73.5 26.5 6.4

62.5 37.5 6.6

51.0 49.0 6.8

39.0 61.0 7.0

28.0 72.0 7.2

19.0 81.0 7.4

13.0 87.0 7.6

8.5 91.5 7.8

5.3 94.7 8.0

Solutions required:

A: 0.2 M solution of succinic acid (23.6 g in 1000 ml)

B: 0.2MNaOH 100 ml of A + X ml of B, diluted to a total of 400 ml.

X pH

30.0 3.8

40.0 4.0

53.0 4.2

66.8 4.4

80.0 4.6

94.0 4.8

106.8 5.0

121.2 5.2

136.8 5.4

150.0 5.6

162.8 5.8

174.0 6.0

14. Tris (hydroxymethyl) aminomethane HCL (Tris HCL buffer)

Solution required:

A: 0.2 M solution of tris (hydroxymethyl) aminomethane (24.2 g in 1000 ml).

B: 0.2 M HCL 100 ml of A + X ml of B, diluted to a total of 400 ml.

X pH

10.0 9.0

16.2 8.8

24.4 8.6

33.0 8.4

43.8 8.2

53.6 8.0

65.0 7.8

76.8 7.6

82.8 7.4

88.4 7.2

5

The Standard Solutions

A standard solution is one of known concentration. Following standard solutions are

frequently required in

microbiology laboratory:

(1) Molar (2) Molal (3) Nomal

(1) Molarity (M) = Molarity is defined as the number of moles

of solute contained in one litre.

A 1 molar (1.0 M) solution of sodium hydroxide contain 1.0

mole or 40 g of sodium hydroxide in one litre of solution because,

NaOH (aq.) Na++OH

1 mole of Na+ = 23 g (from table no. 2)

1 mole of oxygen = 16 g (from table no. 2)

1 mole of hydrogen = 1 g (from table no. 2)

Thus,

1 mole of NaOH = Total 40g

Thus,

When 40g (I mole) of NaOH is dissolved in 1 litre of water it gives 1 molar solution.

(2) Molality (M) = Molality expresses the concentration of a solution in which the

quantity of solute is given in moles, and the quantity of solvent in kilograms. Molality is

defined as the number of moles of solute dissolved in 1000 g (1 kg) of solvent. A one

molal (1.0 m) solution of sodium hydroxide contain 1.0 mole of 40 g of NaOH dissolved

in 1 kilogram of water, that is

(3) Normality (N) = Normality expresses the concentration of a solution in which the

quantity of solute is given in gram equivalent weights and the volume of solution in

litres.

Normality therefore represents the number of gram equivalent weights of solute

dissolved in one litre of solution. That is,

Gram equivalent weight of solutes are dependent on the total positive or negative charges

in the formula of acid, base or salt.

PPM (Parts Per Million Solution)

Gram of solution per million grams of solution or one g of solute1 million ml of solution.

OR

therefore 1 ppm = 1 mg NaCl/100 ml or 1 mg NaCl/ml.

Table 1

The gram equivalent weights of few substances

Compound Lons Lonic

change

1.00 mol.

(in g.)

1.00 gram

equivalent

weight (in g)

HCl H++Cl 1 36.47 36.47

H2SO4 2H++SO4-2 2 98.08 49.04

NaOH Na++OH- 1 40.00 40.00

Ca(OH)2 Ca+2

+2OH 2 74.10 37.05

K3PO4 3K++PO4-3 3 212.28 70.76

Table 2

International atomic weights of the element

Element Symbol Atomic weight

Aluminium Al 26.98

Antimony Sb 121.75

Arsenic As 74.92

Barium Ba 137.34

Beryllium Be 9.01

Bismuth Bi 208.98

Boron B 10.82

Bromine Br 79.90

Cadmium Cd 112.40

Calcium Ca 40.08

Carbon C 12.01

Chlorine Cl 35.45

Chromium Cr 52.00

Cobalt Co 58.93

Copper Cu 63.55

Fluorine F 19.00

Gold Au 196.97

Hydrogen H 1.01

Iodine P 126.90

Iron Fe 55.85

Lead Pb 207.20

Magnesium Mg 24.31

Mercury Hg 200.59

Nickel Ni 58.69

Nitrogen N 14.008

Oxygen O 16.0000

Palladium Pd 106.7

Phosphorus P 30.98

Platinum Pt 195.23

Potassium K 39.096

Radium Ra 22605

Selenium Se 78.96

Silicon Si 28.06

Element Symbol Atomic weight

Silver Ag 107.880

Sodium Na 22.997

Strontium Sr 87.63

Sulfur S 32.066

Tin Sn 118.70

Titanium Ti 47.90

Tungsten W 183.92

Uranium U 238.07

Vanadium V 50.95

Zinc Zn 65.38

Zirconium Zr 91.2

Solution required:

A. 1% aqueous barium chloride solution

B. 1% aqueous sulphuric acid solution.

Add the amount given in the following table to clean, dry ampoules. Ampoules

should have same diameter as the test tube to be used in subsequent density determinates.

Seal the ampoules and label them.

Table 3

McFarland nephelometer barium sulphate standards

Tube No. Barium chloride

1% (ml)

Sulphuric Acid

1% (ml)

Corresponding Approx. Density of

bacteria (million/ml)

1 0.1 9.9 300

2 0.2 9.8 600

3 0.3 9.7 900

4 0.4 9.6 1200

5 0.5 9.5 1500

6 0.6 9.4 1800

7 0.7 9.3 2100

8 0.8 9.2 2400

9 0.9 9.1 2700

10 1.0 9.0 3000

Use: used to determine density of bacteria in suspension. (specially for preparation of

vaccines.)

Table 4

Antimicrobic zone of inhibition evaluation (Kirby Bauermethod).

Significance of zone diameters with disk potency.

Antimicrobial agent Disk

potency

Resistant

mm

Intermidat

e mm

Sensitive

mm

Amikacin 10 mcg <12 12-13 >13

Ampicillin

Gram negative organisms and

enterococci.

Staphylococci

10 mcg

10 mcg

<12

<21

12-13

21-13

>13

>28

Bacitracin 10 units <9 9-12 >12

Carbenicillin

Proteus sp. & E. coli.

Pseudomonas aeruginosa

50 mcg

50 mcg

<13

<15

18-22

13-14

>22

>14

Cefadroxil 30 mcg <15 15-17 >17

Cefotaxime 30 mcg <15 15-22 >22

Ceftriaxone 30 mcg <14 14-20 >20

Cephalothin 30 mcg <15 >14

Chroramphernicol 30 mcg <13 13-17 >17

Ciprofloxacin 5 mcg <16 16-20 >20

Clindamycin 2 mcg <15 15-16 >16

Colistin 10 mcg <9 9-10 >10

Cotrimoxazole 30 mcg <14 14-20 >20

Erythromycin 15 mcg <14 14-17 >17

Gentamicin

Ps. aeruginosa 10 mcg <13 >12

Kanamycin 30 mcg <14 14-17 >17

Lincomycin 2 mcg <17 17-20 >20

Methicillin 5 mcg <10 10-13 >13

Nafcillin 1 mcg <11 11-12 >12

Nalidixic aci 30 mcg <14 14-18 >18

Neomycin 30 mcg <13 13-16 >16

Metilmycin 30 mcg <15 15-16 >16

Mitroflurantoin 30 mcg <15 15-16 >16

Norfloxacin 5 mcg <16 16-20 >20

Oleandomycin 15 mcg <21 12-16 >16

Oxolinic acid 2 mcg <11 >10

Penicillin G

Staphylococci 10 units <21 21-28 >28

Other organisms 10 units <12 12-21 >21

Antimicrobial agent Disk

potency

Resistant

mm

Intermidat

e mm

Sensitive

mm

Polymyxin B 300 units <9 9-11 >11

Rifampicin

Neisseria

Meningitides 5 mcg <25 >24

Streptomycin 10 mcg <12 12-14 >14

Tetracycline 30 mcg <15 15-18 >18

Tobramycin 10 mcg <12 12-13 >13

Triple sulfa 250 mcg <12 13-16 >16

Vancomycin 30 mcg <10 10-11 >11

Table 5

Relation between trasmittancy (T %) & optical density (O.S.)

T % O.D. T % O.D.

100 0.000 50 0.301

99 0.04 49 0.310

98 0.009 48 0.319

97 0.013 47 0.328

96 0.018 46 0.339

95 0.022 45 0.347

94 0.027 44 0.357

93 0.032 43 0367

92 0.036 42 0.377

91 0.041 41 0.387

90 0.046 40 0.398

89 0.051 39 0.409

88 0.056 38 0.120

87 0.061 37 0.432

86 0.066 36 0.444

85 0.071 35 0.456

84 0.076 34 0.459

83 0.081 33 0.482

82 0.086 32 0.495

81 0.092 31 0.509

80 0.097 30 0523

79 0.102 29 0.538

78 0.108 28 0552

T % O.D. T % O.D.

77 1.114 27 0.569

76 0.119 26 0.585

75 0.125 25 0.602

74 0.131 24 0.620

73 0.137 23 0638

72 0.143 22 0.658

71 0149 21 0.678

70 0155 20 0.699

69 0.161 19 0721

68 0.168 18 0.745

67 0.174 17 0.770

66 0.181 16 0.796

65 0.187 15 0.824

64 0194 14 0.854

63 0.201 13 0.886

62 0.208 12 0.921

61 0.215 11 0.950

60 0.222 10 1.000

59 0.229 9 1.046

58 0.237 8 1.097

57 0.244 7 1.155

56 0.252 6 1.222

55 0260 5 1.301

54 0.268 4 1.398

53 0.276 3 1.523

52 0.248 2 1.699

51 0.292 1 2.000

Optical density of given transmittance can be determined by using following formula:

O.D. = 2 – Log % Transmittance

Table 6

Temperature and Pressure Relationship

Steam pressure (psi) Temperature (0C)

1 2

0 100.0

1 101.9

2 103.6

Steam pressure (psi) Temperature (0C)

3 105.3

4 106.9

5 108.4

6 109.8

7 111.3

8 112.6

9 113.9

10 115.2

11 116.4

12 117.6

13 118.8

14 119.9

15 121.0

16 122.0

17 123.0

18 124.1

19 125.0

20 126.0

21 126.9

22 127.8

23 128.9

24 129.6

25 130.4

26 131.3

27 132.1

28 132.9

29 133.7

30 134.5

Around the working range of 15 psi, each raises the autoclave temperature by

approximately 10C.

Table 7

MPN/100 ml using one tube of 50 ml & five tubes of 10 ml

50 ml tube positive 10 ml tubes positive MNP / 100 ml

0 0 0

0 1 1

0 2 2

0 3 4

0 4 5

0 5 7

1 0 2

1 1 3

1 2 6

1 3 9

1 4 16

1 5 18+

Table 8

MPN determination from multiple tubes test (McCrady’s table) for set of nine tubes

Number of tubes giving positive reaction

out of

MPN Index

/ 100 ml

95 % confidence limits

3 of 10 ml

each

3 of 1 ml

each

3 of 0.1 ml

each

Lower Upper

0 0 0 <3 - -

0 0 1 3 <0.5 9

0 1 0 3 <0.5 13

1 0 0 4 <0.5 20

1 0 1 7 1 21

1 1 0 7 1 23

1 1 1 11 3 36

1 2 0 11 3 36

2 0 0 9 1 36

2 0 1 14 3 37

2 1 0 15 3 44

2 1 1 20 7 89

2 2 0 21 4 47

2 2 1 28 10 150

3 0 0 23 4 120

3 0 1 39 7 130

3 0 2 64 15 380

3 1 0 43 7 210

3 1 1 75 14 230

3 1 2 120 30 380

3 2 0 93 15 380

3 2 1 150 30 440

3 2 2 210 35 470

3 3 0 240 36 1300

3 3 1 460 71 2400

3 3 2 1100 150 4800

3 3 3 >2400 - -

Table 9

MPN determination from multiple tube test (McCrady’s table) for set fifteen tubes.


out of

MPN Index

/ 100 ml


3 of 10 ml

each

3 of 1 ml

each

3 of 0.1 ml

each

Lower Upper

0 0 0 <2 - -

0 0 1 2 <0.5 7

0 1 0 2 <0.5 7

0 2 0 4 <0.5 11

1 0 0 2 <0.5 7

1 0 1 4 <0.5 11

1 1 0 4 <0.5 11

1 1 1 6 <0.5 15

1 2 0 6 <0.5 15

2 0 0 5 <0.5 13

2 1 0 7 1.0 17

2 1 0 7 1.0 17

2 1 1 9 2.0 21

2 2 0 9 2.0 21

2 3 0 12 3.0 28

3 0 0 8 1.0 19

3 0 1 11 2.0 25

3 1 0 11 2.0 25

3 1 1 14 4.0 34

3 2 0 14 4.0 34

3 2 1 17 5.0 46

4 0 0 13 3.0 31

4 0 1 17 5.0 46

4 1 0 17 5.0 46

4 1 1 17 7.0 63

4 1 1 21 7.0 63

4 1 2 26 9.0 78

4 2 0 22 7.0 67


out of

MPN Index

/ 100 ml


3 of 10 ml

each

3 of 1 ml

each

3 of 0.1 ml

each

Lower Upper

4 2 1 26 9.0 78

4 3 0 27 9.0 80

4 3 1 33 11 93

4 4 0 34 12 93

5 0 0 23 7 70

5 0 1 31 11 89

5 0 2 43 15 110

5 1 0 33 11 93

5 1 1 46 16 120

5 1 2 63 21 150

5 2 0 49 17 130

5 2 1 70 23 170

5 2 2 94 28 220

5 3 0 779 25 190

5 3 1 110 25 250

5 3 2 140 37 340

5 3 3 180 44 500

5 4 0 130 35 300

5 4 1 170 43 490

5 4 2 220 57 700

5 4 3 280 90 850

5 4 4 350 120 1000

5 5 0 240 68 750

5 5 1 350 120 1000

5 5 2 450 180 1400

5 5 3 920 300 3200

5 5 4 1600 640 5800

5 5 5 >2400 - -

MPN / 100 ml can be calculated by using following formula:

=

√

Table 10

Preparation of indicators

Indicator solution Indicator (g) 965 % Ethanol (ml) Distilled water (ml)

Bromocresol green 0.4 500 500

Bromocresol purple 0.4 500 500

Bromocresol blue 0.4 500 500

Cresol red 0.2 500 500

Methyl red 0.2 500 500

Phenolphthalein 1.0 50 50

Phenol red 0.2 500 500

Thymol blue 0.4 500 500

Preparation: Dissolve the indicator in 95 % ethanol. Add water, Filter before use.

Table 11

Power of units – prefixes

Multiple Prefix Symbol

1012

Tera T

109 Giga G

106 Mega M

103 Kilo K

102 Hector H

10 Deca Da

10-1

Deci D

10-2

Centi C

10-3

Milli M

10-6

Micro µ 10

-9 Nano N

10-12

Pico P

10-15

femto F

Table 12

Indicators with pH range

Indicators Full acid Colour Full alkaline Colour pH range.

1 2 3 4

Cresol red (acid range) Red Yellow 0.2-1.8

Meta cresol purple (acid range) Red Yellow 1.2-2.8

Thymol blue (acid range) Red Yellow 1.2-2.8

Bromo phenol blue Yellow Blue 3.0-4.6

Bromo cresol green Yellow Blue 3.8-5.4

Indicators Full acid Colour Full alkaline Colour pH range.

1 2 3 4

Chlor cresol green Yellow Blue 4.0-5.6

Methyl red Red Yellow 4.4-6.4

Chlor phenol red Yellow Red 4.8-6.4

Bromo cresol purple Yellow Purple 5.2-6.8

Bromo Thymol blue Yellow Blue 6.0-7.6

Neutral red Red Amber 6.8-8.0

Phenol red Yellow Yellow 6.8-8.4

Cresol red (alkaline range) Yellow Yellow 7.2-8.8

Meta cresol purple (alkaline

range)

Yellow Purple 7.4-9.0

Thymol blue (alkaline range) Yellow Blue 8.0-96

Cresolphthalein Colourless Red 8.2-9.8

Phenolphthalein Colourless Red 8.3-10.0

Acyl blue Red Blue 12.0-13.6

Parazo orange Yellow Orange 11.0-12.6

Toly red red yellow 10.0-11.6

Table 13

Test organisms for microbiological assay of antibiotics

Antibiotic Test organism ATC-1

No. NTTC2 No. (NCIB

3 No.)

1 2 3 4

Amikacin Staphylococcus aureus 29737 7447

Antifimial Sacchromyces cerevisiae

Candida albicans

9763

-

10716

-

Bacitracin Micrococcus luteus 10240 7743

Bleomycin Mycobacterium smegmatis 607 -

Carbenicillin Pseudomonas aeruginosa 25619 -

Cephalosporin Bacillus subtilis

Staphylococcus aureus

6633

29737

8236

6571

Chloranphencol S. lutea - 8340

Clindamycin Bacillus subtilis

Staphylococcus aureus

6633

29737

8236

6571

Doxycycline Staphylococcus aureus 29737 7447

Erythromycin Micrococcus luteus 9341 (8553)

Framycetin Bacillus pumilus

Bacillus subtilis

14884

6633

8241

8236, 10400

Gentamicin Staphylococus epidermidis 12228 (8853)

Antibiotic Test organism ATC-1

No. NTTC2 No. (NCIB

3 No.)

1 2 3 4

Bacillus subtilis

Klebsiella edwardsii

6633

-

8236

10896

Kanamycin sulphate Baccillus pumilus 14884 8241

Staphylococcus aureus 29737 7447

Kanamycin B Bacillus subtilis 6633 8236

Neomycin Staphylococcus epidermidis 12228 (8853)

Novobiocin Staphylococcus epidermidis 12228 (8853)

Nystatin Saccharomyces cfrevisie 2601 10716

Oxytetracycline Bacillus cereus var. mycodies 11778 10320

Staphylococcus aureus 29737 7447

Penicillin Bacillus subtilis - 8236

Staphylococcus aureus - 6571

Polymyxin B BordeteNa bronchiseptica 4617 8344

Rifampicin Bacillus subtilis 6633 8236

Streptomycin Bacillus subtilis 6633 8236

Klebsiella pneumoniae 10031 (9111)

Sulphonamides Bacillus pumilis 14884 8241

Trimethoprim Bacillus pumilus 14884 8241

Ticarcillin Pseudomonas aeruginosa - 10490

Tetracycline Bacillus cereus 11778 10320

Staphyoccoccus aureus 29737 7447

Tobramycin Bacillus subtilis 6633 82368236

Klebsiella edwardsii - 10896

1. American Type culture Collection, 2 1301 Park Lawn Drive, Rockville, MD

20852m USA.

2 National Collections of Type Culture, Central Public Health Laboratory, Colindale

Avenue, London NW9 5HT, England.

2. National Collection of Industrial Bacteria, toryy Research Station P.O. Box 31,

135 Abbey Road, Aberdeen 98 DC Scotland.

Table 14

Temperature Celsius-Fehrenheit Relation

Fehrenheit Celsius Fehrenheit Celsius Fehrenheit Celsius Fehrenheit Celsius

1 2 3 4 5 6 7 8

0.0 32.0 26.0 78.8 51.0 123.8 76.0 168.8

1.0 33.8 27.0 80.6 52.0 125.6 77.0 170.6

2.0 35.6 28.0 82.4 53.0 127.4 78.0 1724

3.0 37.4 29.0 842 54.0 129.2 79.0 174.2

4.0 39.2 30.0 86. 55.0 131.0 80.0 176.0

5.0 41.0 31.0 87.8 56.0 132.8 81.0 177.8

6.0 42.8 32.0 89.6 57.0 134.6 82.0 179.6

7.0 44.6 33.0 91.4 58.0 136.4 83.0 181.4

8.0 46.4 34.0 93.2 59.0 138.2 84.0 183.2

9.0 48.2 35.0 95.0 60.0 140.0 85.0 185.0

10.0 50.0 36.0 96.8 61.0 141.8 86.0 186.8

11.0 51.8 37.0 98.6 62.0 143.6 87.0 1886

12.0 53.6 38.0 100.4 63.0 145.4 88.0 190.4

13.0 55.4 39.0 102.2 64.0 147.2 89.0 192.2

14.0 57.2 40.0 104.0 65.0 149.0 90.0 194.0

15.0 59.0 41.0 105.8 66.0 150.8 91.0 195.8

16.0 60.8 42.0 107.6 67.0 152.6 92.0 197.6

17.0 62.6 43.0 109.4 68.0 154.4 93.0 199.0

18.0 64.4 44.0 111.2 69.0 156.2 94.0 201.2

19.0 66.2 45.0 113.0 70.0 158.0 95.0 203.0

20.0 68.0 46.0 114.8 71.0 159.8 96.0 204.8

21.0 69.8 47.0 116.6 72.0 161.6 97.0 206.6

22.0 71.6 48.0 118.4 73.0 163.4 98.0 208.4

23.0 73.4 49.0 120.2 74.0 165.2 99.0 210.2

24.0 75.2 50.0 122.0 75.0 167.0 100.0 212.0

25.0 77.07

0C =

Table 15

Percentages by volumes at 15.600 (60

0F) of ethyl alcohol corresponding to apparent

specific gravity at various temperatures*

Apparent

Specific

Gravity

15.56/

15.56

20/

20

22/

22

24/

24

25/

25

26/

26

28/

28

30/

30

32/

32

34/

34

35/

35

36/

36

1 2 3 4 5 6 7 8 9 10 11 12 13 1.000 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

0.999 0.67 0.66 0.66 0.66 0.66 0.66 0.66 0.66 0.66 0.66 0.66 0.66

0.998 1.34 1.34 1.34 1.34 1.34 1.33 1.33 1.32 1.32 1.32 1.32 1.32

0.997 2.02 2.2 2.01 2.01 201 2.01 2.00 2.00 2.00 1.99 1.99 1.99

0.996 2.71 2.70 2.70 2.70 2.70 2.70 2.70 2.69 2.68 2.67 2.66 2.66

0.995 3.41 3.40 3.40 3.39 3.39 3.39 3.38 3.37 3.36 3.35 3.34 3.34

0.994 4.13 4.11 4.10 4.10 4.09 4.09 4.07 4.06 4.05 4.04 4.03 4.03

0.993 4.86 4.84 4.83 4.82 4.81 4.80 4.79 4.77 4.75 4.74 4.73 4.72

0.992 5.61 5.58 5.56 5.55 5.54 5.53 5.51 5.49 5.47 5.45 5.44 543

0.991 6.38 6.34 6.32 6.30 6.29 6.28 6.25 6.23 6.20 6.17 6.16 6.15

0.990 7.17 7.12 7.09 7.06 7.05 7.03 7.00 6.98 6.94 6.91 6.90 6.88

0.989 7.98 7.90 7.87 7.84 7.83 7.81 7.78 7.74 7.70 7.66 7.64 7.62

0.988 8.82 8.71 8.67 8.63 8.61 859 855 8.50 8.46 8.41 8.39 8.37

0.987 9.66 9.54 9.49 9.43 9.41 9.39 9.33 9.27 9.22 9.17 9.14 9.12

0.986 10.50 10.36 10.30 10.24 10.21 10.18 10.11 10.05 9.99 9.92 9.89 9.86

0.985 11.37 11.19 11.12 11.04 11.00 10.96 10.89 10.82 10.74 10.67 10.63 10.59

0.984 12.25 12.04 11.95 11.86 11.81 11.77 11.68 11.60 11.51 11.42 11.38 11.34

0.983 13.16 12.90 12.79 12.68 11.81 11.77 11.68 11.60 11.51 11.42 11.38 11.34

0.982 14.08 13.77 13.64 13.52 13.46 13.40 13.29 13.18 13.06 1295 12.90 12.85

0.981 15.02 14.66 14.51 14.37 14.60 14.24 14.11 13.98 13.85 13.73 13.67 13.61

0.979 16.92 16.46 16.27 16.09 1600 15.92 15.75 15.59 15.44 15.29 15.22 15.15

0.978 17.91 17.38 17.17 16.97 16.87 16.78 16.59 16.41 16.24 16.08 16.00 15.92

0.977 18.91 18.31 18.08 17.85 17.74 17.63 17.43 17.24 17.05 16.88 16.79 16.71

0.976 19.93 19.26 18.99 18.74 18.62 18.50 19.28 18.07 17.87 17.67 17.58 17.49

0.975 20.93 20.20 19.91 19.64 19.50 19.38 19.13 18.90 18.68 18.47 18.37 18.27

0.974 21.92 21.14 20.82 20.53 20.38 20.25 19.99 19.74 1950 19.28 19.17 19.06

0.973 22.90 22.05 21.72 21.41 21.25 21.11 20.83 20.56 20.31 20.07 19.95 19.84

0.972 23.87 22.96 22.61 22.27 22.11 21.96 21.66 21.38 20.11 20.86 20.73 20.61

0.971 24.81 23.87 23.49 23.13 22.96 22.88 22.49 22.19 21.91 21.64 21.50 21.37

0.970 25.85 24.84 24.44 24.06 23.88 23.72 23.38 23.06 22.77 22.48 22.34 22.21

0.969 26.69 25.62 25.21 24.82 24.63 24.45 24.10 23.77 23.46 23.16 23.01 22.88

0.968 27.60 26.50 2606 25.65 25.45 25.26 45.89 24.54 24.21 23.90 23.76 23.61

0.967 28.50 27.36 26.89 2646 26.26 2606 25.68 25.32 2498 24.66 24.50 24.35

0.966 29.39 28.19 27.72 27.27 27.06 26.85 26.46 26.09 25.74 25.40 25.24 25.08

0.965 30.26 29.03 28.53 28.07 27.85 27.6 27.23 26.85 26.49 26.14 25.97 25.81

0.964 31.11 29.85 29.34 28.86 28.63 28.41 27.99 27.59 27.2 26.86 26.69 36.52

0.963 31.93 30.66 30.12 29.64 29.40 29.18 28.74 28.33 27.95 27.58 27.39 27.92

0.962 32.72 31.44 30.91 30.41 30.17 29.94 29.50 29.07 28.67 28.29 28.10 27.92

0.961 33.50 32.21 - - 30.92 - - 29.80 - - 28.80 -

0.960 34.27 32.96 - - 31.65 - - 30.51 - - 29.50 -

0.959 35.02 33.69 - - 32.37 - - 31.22 - - 30.18 -

0.958 35.75 34.41 - - 33.08 - - 31.91 - - 30.86 -

0.957 36.46 35.12 - - 33.78 - - 32.58 - - 31.53 -

0.956 37.16 35.82 - - 34.46 - - 33.25 - - 32.18 -

0.955 37.84 36.49 - - 35.13 - - 33.92 - - 32.83 -

0.954 38.51 37.16 - - 35.79 - - 34.57 - - 33.46 -

0.953 39.17 37.82 - - 36.44 - - 35.22 - - 34.10 -

0.952 39.82 38.46 - - 37.09 - - 35.85 - - 34.72 -

0.951 40.46 39.10 - - 37.71 - - 36.47 - - 35.34 -

0.950 41.09 39.73 - - 38.33 - - 37.09 - - 35.95 -

0.949 41.70 40.35 - - 38.95 - - 37.7 - - 3655 -

0.948 42.31 40.96 - - 39.56 - - 38.30 - - 3715 -

0.947 42.92 41.56 - - 40.17 - - 38.90 - - 37.74 -

0.946 43.51 4215 - - 40.76 - - 39.49 - - 38.32 -

0.945 43.10 42.74 - - 41.34 - - 40.07 - - 38.90 -

0.944 44.68 43.33 - - 41.92 - - 40.64 - - 39.47 -

0.943 45.25 43.90 - - 42.49 - - 41.21 - - 40.04 -

0.942 45.81 44.46 - - 43.06 - - 41.77 - - 40.59 -

0.941 46.37 45.03 - - 43.62 - - 42.33 - - 41.15 -

0.940 46.92 45.58 - - 44.18 - - 42.89 - - 41.70 -

0.939 47.46 46.12 - - 44.73 - - 43.44 - - 42.24 -

0.938 48.00 46.67 - - 45.27 - - 43.98 - - 42.79 -

0.937 48.53 47.20 - - 45.81 - - 44.52 - - 43.33 -

0.936 49.06 47.73 - - 46.34 - - 45.06 - - 43.86 -

0.935 49.58 48.26 - - 46.87 - - 45.58 - - 44.39 -

0.934 50.09 48.78 - - 47.40 - - 46.11 - - 44.91 -

0.933 50.60 49.80 - - 47.92 - - 4663 - - 45.43 -

0.932 51.11 49.30 - - 48.43 - - 47.15 - - 45.95 -

0.931 51.61 50.31 - - 48.94 - - 47.65 - - 46.46 -

0.930 52.11 50.81 - - 49.44 - - 48.16 - - 46.97 -

0.929 52.61 51.31 - - 49.94 - - 48.66 - - 47.47 -

0.928 53.10 51.80 - - 50.44 - - 49.16 - - 47.97 -

0.927 53.59 52.29 - - 50.93 - - 49.66 - - 48.47 -

0.926 54.08 52.79 - - 51.42 - - 50.15 - - 48.96 -

0.925 54.57 53.27 - - 51.91 - - 50.64 - - 49.44 -

0.924 55.05 53.76 - - 52.40 - - 51.13 - - 49.93 -

0.923 55.52 54.24 - - 52.88 - - 51.61 - - 50.41 -

0.922 5600 54.72 - - 53.36 - - 52.09 - - 50.89 -

0.921 56.47 55.19 - - 53.84 - - 52.57 - - 51.37 -

0.920 56.95 55.67 - - 54.31 - - 53.05 - - 51.84 -

0.919 57.41 56.14 - - 54.78 - - 53.51 - - 52.32 -

0.918 57.88 56.60 - - 55.25 - - 53.98 - - 52.79 -

0.917 58.34 56.07 - - 55.72 - - 54.45 - - 53.26 -

0.916 58.79 57.53 - - 56.18 - - 54.92 - - 53.72 -

0.915 59.25 57.99 - - 56.65 - - 55.38 - - 54.18 -

0.914 59.70 58.44 - - 57.10 - - 55.84 - - 54.65 -

0.913 60.15 59.89 - - 57.56 - - 56.30 - - 55.11 -

0.912 60.59 59.34 - - 58.01 - - 56.76 - - 55.56 -

0.911 6104 59.79 - - 58.46 - - 57.21 - - 56.02 -

0.910 61.48 60.24 - - 58.91 - - 57.66 - - 56.47 -

0.909 61.92 60.68 - - 59.36 - - 58.11 - - 56.93 -

0.908 62.36 61.12 - - 59.80 - - 58.55 - - 59.39 -

0.907 62.79 61.56 - - 60.25 - - 59.00 - - 57.81 -

0.906 63.23 62.00 - - 50.68 - - 59.43 - - 58.26 -

0.905 63.66 62.43 - - 61.12 - - 59.87 - - 58.70 -

0.904 64.09 62.86 - - 61.55 - - 60.31 - - 59.13 -

0.903 64.53 63.30 - - 61.99 - - 60.75 - - 59.57 -

0.902 64.96 63.73 - - 62.42 - - 61.18 - - 60.00 -

0.901 65.38 64.16 - - 62.85 - - 61.61 - - 60.43 -

0.900 65.81 64.58 - - 63.27 - - 62.04 - - 60.86 -

0.899 66.23 65.01 - - 63.70 - - 62.47 - - 61.29 -

0.898 66.65 65.43 - - 64.13 - - 62.89 - - 61.72 -

0.897 67.07 65.85 - - 64.55 - - 63.32 - - 62.14 -

0.896 67.48 66.27 - - 64.97 - - 63.74 - - 62.57 -

0.895 67.90 66.69 - - 65.39 - - 64.16 - - 62.99 -

0.894 68.31 67.10 - - 65.81 - - 64.58 - - 63.41 -

0.893 68.72 67.52 - - 66.23 - - 65.00 - - 63.83 -

0.892 69.13 67.93 - - 66.64 - - 65.42 - - 64.32 -

0.891 69.54 68.34 - - 67.06 - - 65.83 - - 64.67 -

0.890 69.94 68.75 - - 67.47 - - 66.25 - - 65.08 -

0.888 70.75 69.56 - - 68.28 - - 67.07 - - 65.91 -

0.887 71.15 69.96 - - 68.69 - - 67.48 - - 66.32 -

0.886 71.54 70.36 - - 69.10 - - 67.49 - - 66.73 -

0.885 71.94 70.76 - - 69.50 - - 68.29 - - 68.13 -

0.884 72.33 71.16 - - 69.90 - - 68.69 - - 68.54 -

0.883 72.73 71.55 - - 70.29 - - 69.09 - - 68.94 -

0.882 73.12 71.95 - - 70.69 - - 69.49 - - 69.34 -

0.881 73.50 72.34 - - 71.09 - - 69.89 - - 68.74 -

0.880 73.89 72.73 - - 71.48 - - 70.29 - - 69.14 -

0.879 74.28 73.12 - - 72.87 - - 71.68 - - 70.53 -

0.878 74.66 73.50 - - 78.26 - - 71.07 - - 70.93 -

0.877 75.05 73.89 - - 72.65 - - 71.46 - - 72.32 -

0.876 75.43 74.28 - - 73.04 - - 71.85 - - 70.71 -

0.875 75.81 74.66 - - 73.42 - - 72.24 - - 71.10 -

0.874 76.19 75.04 - - 73.81 - - 72.63 - - 71.49 -

0.873 76.56 75.42 - - 73.19 - - 73.01 - - 71.88 -

0.872 76.94 75.80 - - 73.57 - - 73.39 - - 71.27 -

0.871 77.31 76.18 - - 73.95 - - 73.77 - - 71.65 -

0.870 77.68 76.55 - - 75.33 - - 74.16 - - 73.03 -

0.869 78.05 76.92 - - 75.70 - - 74.54 - - 73.41 -

0.868 78.41 77.29 - - 76.08 - - 74.92 - - 73.79 -

0.867 78.78 77.66 - - 76.45 - - 75.29 - - 74.17 -

0.866 79.14 78.03 - - 76.82 - - 75.66 - - 74.55 -

0.865 79.51 78.39 - - 77.19 - - 76.04 - - 74.92 -

0.864 79.87 78.76 - - 77.56 - - 76.41 - - 75.29 -

0.863 80.22 79.12 - - 77.93 - - 76.78 - - 75.67 -

0.862 80.58 79.48 - - 78.29 - - 77.15 - - 76.04 -

0.861 80.94 79.84 - - 78.65 - - 77.51 - - 76.41 -

0.860 81.29 80.20 - - 79.01 - - 77.88 - - 76.78 -

0.859 81.65 80.55 - - 79.37 - - 78.24 - - 77.14 -

0.858 82.00 80.91 - - 79.73 - - 78.60 - - 77.51 -

0.857 82.35 81.26 - - 80.09 - - 78.96 - - 77.87 -

0.856 82.70 81.61 - - 80.44 - - 79.32 - - 78.23 -

0.855 83.04 81.96 - - 80.80 - - 79.67 - - 78.59 -

0.854 83.39 82.31 - - 81.15 - - 80.03 - - 78.95 -

0.853 83.73 82.66 - - 81.50 - - 80.38 - - 79.30 -

0.852 84.07 83.01 - - 81.85 - - 80.73 - - 79.66 -

0.851 84.41 83.35 - - 82.20 - - 81.09 - - 80.01 -

0.850 84.75 83.69 - - 82.54 - - 81.44 - - 80.37 -

0.849 85.09 84.03 - - 82.89 - - 81.78 - - 80.72 -

0.848 85.42 84.37 - - 83.23 - - 82.13 - - 81.07 -

0.847 85.75 84.71 - - 83.57 - - 82.48 - - 81.42 -

0.846 86.08 85.04 - - 83.91 - - 82.82 - - 81.77 -

0.845 86.40 85.38 - - 83.25 - - 82.17 - - 81.11 -

0.844 86.73 85.71 - - 83.59 - - 82.51 - - 81.46 -

0.843 87.05 86.03 - - 84.92 - - 82.85 - - 81.80 -

0.842 87.38 86.36 - - 85.25 - - 84.18 - - 83.14 -

0.841 87.70 86.68 - - 85.59 - - 84.52 - - 83.48 -

0.840 88.02 87.01 - - 85.92 - - 84.85 - - 83.82 -

0.839 88.33 87.33 - - 86.24 - - 85.18 - - 84.16 -

0.838 88.65 87.65 - - 87.57 - - 86.51 - - 85.49 -

0.837 88.96 87.97 - - 87.89 - - 86.84 - - 85.82 -

0.836 89.27 88.29 - - 87.21 - - 86.16 - - 85.15 -

0.835 89.58 88.60 - - 87.53 - - 86.49 - - 85.48 -

0.834 89.58 88.91 - - 87.85 - - 86.81 - - 85.80 -

0.833 90.19 89.22 - - 88.16 - - 88.13 - - 86.13 -

0.832 90.49 89.53 - - 88.48 - - 87.45 - - 86.45 -

0.831 90.78 89.83 - - 88.79 - - 87.77 - - 86.77 -

0.830 91.08 90.14 - - 89.10 - - 88.08 - - 87.10 -

0.829 91.37 90.44 - - 89.41 - - 88.40 - - 87.41 -

0.828 91.66 90.73 - - 89.71 - - 88.71 - - 87.73 -

0.827 91.95 91.03 - - 90.02 - - 89.02 - - 88.05 -

0.826 92.24 91.32 - - 90.32 - - 89.33 - - 88.36 -

0.825 92.53 91.62 - - 90.61 - - 89.64 - - 88.67 -

0.824 92.80 91.90 - - 90.91 - - 89.94 - - 88.98 -

0.823 93.08 92.19 - - 91.21 - - 90.24 - - 89.29 -

*Specific gravity is determined by taking weight of water & sample at the same temp. e.g. 25/250C

0.822 93.36 92.48 - - 91.50 - - 92.54 - - 89.59 -

0.821 93.63 92.76 - - 91.79 - - 92.84 - - 89.90 -

0.820 93.90 93.04 - - 92.07 - - 91.13 - - 90.20 -

0.819 94.16 93.31 - - 92.36 - - 91.42 - - 90.49 -

0.818 94.43 93.58 - - 92.64 - - 91.71 - - 90.79 -

0.817 94.69 93.85 - - 92.92 - - 92.00 - - 91.09 -

0.816 94.95 94.12 - - 93.20 - - 92.28 - - 91.38 -

0.815 95.20 94.38 - - 93.47 - - 92.56 - - 91.66 -

0.814 95.46 94.64 - - 93.74 - - 92.84 - - 91.95 -

0.813 95.71 94.90 - - 94.01 - - 93.12 - - 92.24 -

0.812 95.96 95.16 - - 94.27 - - 93.40 - - 92.53 -

0.811 96.20 95.42 - - 94.53 - - 93.67 - - 92.80 -

0.810 96.45 95.67 - - 94.79 - - 93.94 - - 93.08 -

0.809 96.69 95.92 - - 95.05 - - 94.20 - - 93.36 -

0.808 96.93 96.16 - - 95.31 - - 94.47 - - 93.63 -

0.807 97.16 96.41 - - 95.56 - - 94.73 - - 93.90 -

0.806 97.39 96.65 - - 95.81 - - 94.99 - - 94.16 -

0.805 97.62 96.89 - - 96.06 - - 95.24 - - 94.43 -

0.804 97.85 97.12 - - 96.31 - - 95.50 - - 94.69 -

0.803 98.07 97.36 - - 96.55 - - 95.75 - - 94.95 -

0.802 98.29 97.59 - - 96.79 - - 96.00 - - 95.21 -

0.801 98.50 97.81 - - 97.03 - - 96.25 - - 95.46 -

0.800 98.72 98.03 - - 97.26 - - 96.49 - - 95.72 -

0.799 98.92 98.26 - - 97.50 - - 96.73 - - 95.97 -

0.798 99.13 98.47 - - 97.72 - - 96.97 - - 96.21 -

0.797 99.33 98.68 - - 97.95 - - 97.21 - - 96.46 -

0.796 99.54 98.89 - - 98.17 - - 97.44 - - 96.70 -

0.795 99.73 99.10 - - 98.39 - - 97.67 - - 96.94 -

0.794 99.93 99.30 - - 98.60 - - 97.89 - - 97.18 -

0.793 - 99.50 - - 98.81 - - 98.12 - - 97.41 -

0.792 - 99.70 - - 99.02 - - 98.33 - - 97.64 -

0.791 - 99.90 - - 99.22 - - 98.55 - - 97.86 -

0.790 - - - - 99.43 - - 98.76 - - 98.09 -

0.789 - - - - 99.63 - - 98.97 - - 98.31 -

0.788 - - - - 99.83 99.18 98.53

0.787 - - - - - - - 99.38 - - 98.74 -

0.786 - - - - - - - 99.58 - - 98.95 -

0.785 - - - - - - - 99.78 - - 99.16 -

0.784 - - - - - - - 99.98 - - 99.36 -

0.783 - - - - - - - - - - 99.56 -

0.782 - - - - - - - - - - 99.76 -

0.781 - - - - - - - - - - 99.96 -

0.7809 - - - - - - - - - - 99.98 -

0.7808 - - - - - - - - - - 100.0 -

Table 16

Basic SI units and their abbreviaions

Physical quantity Name Symbol

Lenght Meter M

Mass Kilogram kg

Time Second s

Amount of substance Mole mol

Thermodynamics temp. Kelvin K

Electric current Ampere A

Luminous intensity Candela cd

Table 17

The special names & symbols of some derived SI units

Physical quantity Name Symbol Units

Frequency Hertz Hz S-1

Force Newton N Kg ms-2

Presure Pascal Pa N m-2

Energy or work Joule J Nm

Power Watt W Js-1

Electrical charge Coloumb C As

Electric capacitance Farad F AsV-1

Potential diference Volt V WA-1

Resistance Ohm Ω VA-1

Conductance Siemens S Ω

Radioactivity Becquerel Bq S-1

Absorbed dose of radiation Gray Gy J kg-1

Customary temp. Degree Celcius 0C

0C=K-273.15

Table 18

Notations

10-15

= quardillionth

10-12

= trillionth

10-9

= billionth

10-6

= millionth

10-3

= thousandth

10-2

= hundredth

10-1 = tenth

102 = hundred

103 = thousand

106 = million

109 = billion

Table 19

Refractive indicates of diferent substances used as mounting medium

Substance Refractive index

Glass 1.56

Distilled water 1.33

Cedar wood oil (immersion oil) 1.51

CCL4 1.46

Sandal wod oil 1.51

Olive oil 1.47

Glycerol 1.47

Canada balsam 1.54

Eupapa 1 1.48

Polysterene 1.59

Air 1.00

Naphrax 1.74

Appendies

Appendix-I

List of minimum equipment required for good microbiology laboratory.

1. Microscope with oil immersion lens One for every student in the batch

2. Hot air oven one

3. Autoclave one

4. Bacteriological incubator Two

5. Rotary shaker one

6. Incubator shaker one

7. Calorimeter one

8. Stirror one

9. Laminar air flow unit one

10. Refrigemtor one

11. Monopan balance (digital) one

12. Unit for glass distilled water one

13. W chamber for genetics experiments one

14. Heating mantle one

15. pH meter one

16. Medical centrihge one

17. Laboratory hot plate one

18. Chemical balance one

19. Antibiotic zone diameter reader one

20. Colony counter one

21. Phase contrast microscope one

22. Laboratory fermenter one

Appendix-2

Addresses for getting chemicals, instruments, readyrnade media and cultures required for

microbiology practicals

1. Readymade media

* Himedia Laborat0riePs vt. Ltd.

A-406, Bhaveshwar Plaza,

235 Marg, Mumbai-400 086, India

* LOBA Chemie Pvt. Ltd.

78/80, Babu Genu Road,

P.B. No. 2042

Mumbai-400 002.

2. Chemicals

Poona Chemical Laboratory

207, Magalwar Peth,

Near Gadital

Behind Modi Petrol pump,

Pune - 411011.

S.D. Fine Chen Ltd.

3 15 H.O., T.V. Ind. Estate,

248 Worli Road, Post Box No. 19160

Mumbai-400 030.

Glaxo India Limited

Dr. Annie Besant Road,

Mumbai-400025.

Ranbaxy Fine Chemical Division

12th Floor, Derika Towers 6, Nehru Place

New Delhi-110019.

Borosil Glass works Ltd.

4031404, Kaliandas Udyog Bhavan.

Near Century Bazar, Worli Murnbai-400025

Superfit Continental Private Ltd.

5-6, Old sitaram Building 1st floor.

204 Princess Street, Mumbai-400 002.

Qualigens Fine Chemicals Division.

Glaxo SmithKline Pharmaceuticals Ltd.

Dr.Annie Besant Road, Worli.

Mumbai-400030.

Loba Chemie Pvt. Ltd.

Jehangir Villa, 107 Wode House Road,

Colaba, Mumbai - 400005 (India).

Asgi Enterprises.

10, Dadi Santook Lane, Mumbai-2

Thomas Baker. (Chemicals) Pvt. Ltd,

Head Office.

4/86, Bharat Mahal, Marine Drive, Mumbai 400002 (India).

Universal Laboratories Pvt. Ltd.

Corporate office-507, Raheja Centre,

214, Nariman point, Mumbai-400021. (India).

Biotech Pvt Ltd.

18/1, Madhukunj Society, Panchavati,

Off Pashan Road, Pune-411008.

Bangalore Geni.

No. 6, 6th Main, BDA. Industrial Suburb,

Near SRS Road Peenya,

Bangalore-560058. (India).

3. Instruments

M/S Dynarnicro [Bacteriology Equipment]

M/S Toshniwal Brothers Pvt. Ltd.

198, Jarnshedji Tata Road,

Mumbai-400020.

M/S MICLAB Instruments

Gulmohar 'B', Flat No. 5

428-43013, Gultekadi,

Pune-411009.

M/s TEMPO Industrial Corporation

I, lamington Chambers

394, Dr. Bhadkamkar Marg,

Mumbai-400004.

M/S Ope1 Instruments Pvt. Ltd.

562, Sadashiv Peth, Laxi Road,

Bhanuvilas Chowk,

Pune-411030.

Biotron Healthcare (India) P. Ltd.

30 1, Coral classic, 20h Road.

Chembur, Mumbai400071.

Micro Devices Metrohm Ltd.

Millenium Business park,Mahape,

Navi. Mumiotrobai-400710.

Remi Equipments Ltd.

14, Shah Industrial Estate, Veera Desai Road.

Andheri (W), Mumbai 400053.

SAKSHAM Technologies Pvt.,Ltd.

502, Niti Apartments, Underai Road,

Near New Era.

Cinema, Malad(W), Mumbai-400064.

Thermo Electron Cotpration,

Laboratory Consumables & pipetting

Ratatie 2, P. 0. Box 100.

Fin-o1621 Vantaa.

Finland.

Elico Limited.

B-90, A.P.I.E. Sanathnagar,

Hyderabad-500018, A.P. India.

Millipore (India) F'vt. Ltd.

50-A. I1 Phase, Ring Road, Peenya,

Bangalore-560058, India.

Labindia Instruments Pvt. Ltd.

201, Nand chambers, L.B.S. Marg,

Near Vandana cinema, Thane-400602.

4. Microbial Cultures

Fermentation Technology Laboratory

Indian Institute of Science

Bangalore-3 (Karnataka)

National Collection of Industrial Microorganisms

National Chemical Laboratory,

Council of Scientific and Industrial Research;

Pashan Road,

Pune (Maharashtra)

5. Fungal Cultures

Herbarium Cryptogame India Orientaiks

Division of Mycology and Plant Pathology

Indian Agricultur Raels earch Institute,

New Delhi- 12, India

Microbial Type Culture Collections

I Gene Bank (MTCO) Institute of Microbial Technology

Post box No. 1304, Sector 39-A,

Chandigarh-160014, India.

Agharkar Research Institute

Bhandarkar Road,

Pune.

References

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9. Medical Microbiology, Robert Cruickshank, J.P. Duguid, B.P. Marmion, R.H.A.

Swain Twelfth edition, Vol. 11. Churchill Living Stone, New York (1975).

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Co. New Delhi (1977).

11. Medica Microbiology, N.C. Dey and T.K. Dey. Allied Agency, Kolkata, Ninth

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Ranade and R.V. Gadre Maharashtra Association for Cultivation of Science Pune

(1988).

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Vadhani Industrial Estate, Mumbai, India.

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Washington, D.C. (1978).

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19. Standard Methods for the Examinatiori of Water and Waste Water, 15th ed.

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20. Standard Methoh for the Examination of Water and Waste Water, 11th ed.

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Butterworths, London (1961).

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Delhi (1990).

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Parks. CRC Press Inc., Florida (1993).

24. Practical Pathology and Microbrology, N.C. Dey, T.K. Dey and D. Sinha, New

Central Book Agency Pvt. Ltd. Kolkata, India (1994).

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Company, London (1973).

26. Microbiology Concepts and Application, Pelczar, Chan and Krieg. McGreaw

Hill, Inc., London (1993).

27. Microbiology-Molecular Biology and Pathology, edition Jack Maniloff American

Society for Microbiology, Washington, D.C. (1992).

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Publisher, England (1994).

29. Practical Zoology, K.C. Ghose B. Manna New Central Book Agency,

KoIkata(1991).

30. Pollution Microbiology A Laboratory Manual, Melvin S. Finstein Marcel Dekkar

Inc., New York (1972).

31. Laboratory Manual for Chemical and Bacterial Analysis of Water and Sewage,

Theroux Eldridge and Mallmann Agro Botanical Publishers, India (1992).

32. Laboratory Methods in Food andDairy Microbiology, w.f. Harrigan and Margaret

E. McCance. Academic Press, London (1976).

33. A Text Book of Biotechnology, R.C. Dubey, S. Chand and Company Pvt. Ltd.,

New Delhi (1993).

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London (1989).

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New York (1991).

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Thirteenth ed. by William Horwitz. Published by AOAC Washington DC.

(1980).

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Helnemann Reed Book Service Ltd. Rushden Northants UK(1991).

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Chrchill-Livingstone, London (1981).

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Cummings Publishing Company Inc., London (1948).

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(1991).

41. Industrial Microbiology, Prescott and Dunn 4' ed. CIBS Publisher and

Distributors, Delhi (India) (1987).

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Bufferworth, London (1976).

43. Hugh, R. and Leifgson, F., J. Bact 66 (1953) 24.

44. Will is A.T. and Hobbs, G., J. Path Bacf (1 959) 511.

45. Progress in Microbiological Techniques, Burman, N.P. in Collins C.H. (ed.),

Bufferworths, London (1967).

46. BBL Manual of Products and Laboratory Procedure. 5th edn, Becton Dickismson

and Co. Maryland (1968).

47. Basic and Practical Microbiology, Ronald M. Atlas MacMillan Publishing

Company, New York (1986).

48. Microbiology: A Laboratory Manual, James G. Cappuccino Natalie Sherman

Addition - Wesley Publishing Company London (1982).

Index

Acetate buffer, 254 Aspergillus medium, 146-47

1991 Acetobacter medium, 44-46 Azospirillum semisolid medium,

Ws Acholeplasma medium, 45-46 60-61

Acid fast staining, 196-99 Azotobacter medium, 5740

t and Actinomycetes medium, 39-43

Agrobactarium isolation medium, Bacteriophage, 185-90

46-47 Bagnra, 50

Alcaligenes medium, 46 Balch, 55-56

Algal medium, 173-76 Basal medium, 25

Alternasia medium, 145 Basdiomycets medium, 147-48

Aminoacid decarbexylation test, Bateroides medium, 62

1-2 Bdellovibrio medium, 62-63

Moeller's medium, 1-2 Beadle and Tatum, 165-66

Falkow's medium, 2 Becking's medium, 63

Ammonification medium, 47 Bennet's agar, 39

Ammonium medium, 17 Bile escutin medium, 5

Amylase production test, 2-3 Bile sensitivity test, 3-6

Anaerobic bacteria medium, Bioluminescent bacteria medium,

Antibiotic assay medium, 2 1-22 Blood agar, 82

Ascomycetes medium, 145-46 Blue green algal medium, 176-78

Bordetella medium, 64-65 Dextrose tripone a g e 40

Brycella medium, 65-67 Dine's shining, 194-95

Burk's medium, 59 DNzse agar, 4

DNase test, 4-5

Campylobacter medium, 68 Durham's peptone water, 9- 10

Candida medium, 149-50

Capsule staining, 199-204 Egg yolk agar, 11-12, 13

Carbon utilization medium, 4 1-43 Eijkman text, 5

Carry-Blair medium, 138-39 Eimhjellen medium, 87

Casein hydrolysis, 3 Endo's agar, 73

Catalose test, 35 Entamoeba medium, 18 1

Cellulolytic fungal medium, Enteric bacteria medium, 78-79

150-5 1 Equipments required in

Cellulolytic medium, 68-69 microbial lab, 295

Cell wall stairling, 214-16 Erwinia selective medium,

Chaetomium medium, 15 1-52 79-80

Chitin hydrolyse, 3 Esculin hydrolysis, 5

Christensen's medium, 33-34

Citrate utilization test, 3-4 Falkow's medium, 2

Clostridium medium, 70-72 Fat agar, 12-13

Coaglucose test, 36-37 Filde's extract, 66-67

Coliform medium, 72-73 Flagella staining, 207-1 1

Corder, 54 Flat sour bacteria medium,

Corynebacterium medium, 74-76 80-8 1

Cultivation medium, 39-190 Fontana's staining, 204-05

algae, 173-80 Fungal medium, 153-61

bacteria, 39- 144 Fusarium medium, 16 1-64

bacteriophage, 185-90 Fuibacterium medium, 8 1

fungi, 145-72

protozoa, 1 8 1-84 Garlic abr, 169

Cytopha medium, 76 Gelatin agar, 5-6

Gelatin hydrolysis, 5-6

Deaminase test, 15- 16 General medium, 82-85

Dhedrogenase activity, 38 Gluconate test, 6

Denitrifying bacteria medium, Glucose peptone agar, 114

77-78 Glucose phosphate broth, 34

Dermatophyte test medium, 152 Glycerol yeast extract agar, 40

Derxia medium, 76-77 Gram's staining, 224-29

Gram-negative broth, 85 Lactate agar, 11

Gross's agar, 197-99 Lactate fermentation, 11

Lactic acid bacteria medium,

Haemophilus medium, 86 88-89

Halophiles medium, 86-88 Lactobacilli medium, 89-90

Hankin's medium, 20-21 Lapage, 49

Hartley's broth, 131 Lecithinase production, 11 - 12

Hayflick medium, 106-07 Leishmania medium, 183-84

Hippurate hydrolysis, 7 Leptospira medium, 9 1-93

Homo and heterofermentation Lignolytic fungi medium, 164-65

differentiation, 6-7 Lipid staining, 213- 14

Hoyer's medium, 44 Lipolysis test, 12-13

Hugh and Leifson medium, 20 Litmus milk medium, 13-14

Hungate, 51 Litmus milk test, 13-14

Hydrogen sulphide production: Lysozyme broth, 10

Kligler's iron agar, 8-9 Lysozyme sensitivity test, 10

lysine iron agar, 9

pepton water, 8 MacConkey's broth, 5, 79

SIM agar, 7-8 Malonate utilization and

Hydrolysis of fats, 38 phenylalanine, 15- 16

Malonate utilization test, 14-1 5

India ink staining method, 204 Mandel and Reese medium, 69

Indole test, 9-10 Media for biochemical test, 1-38

International atomic weights of Melanine production, 14

elements, 265-66 Metachromatic granules staining,

Iron haematomylin staining, 239 211-12

Methyl red test, 16

Jensen's medium, 60 Milk agar, 3

Minimal medium, 94-95

Katznelson and base medium, 24 Moeller's medium, 1-2

Kauffmann-Muller broth, 120 Molality, 263-64

Kelton medium, 105 Molarity, 263

King, Ward and Raney's medium, Monsur's medium, 139 i

112-13 Motility agar, 16

Kligler's iron agar, 8-9 Motility and nitrate reduction test,

Knop's solution, 151 17

Korthof's medium, 92 MR-VP broth, 16

Koser's citrate broth, 3-4 Mucor-synthetic medium, 155

Mycobacterium medium, 96-98 Pikovskaya's medium, 24-25

Mycoplasma medium, 10 1-07 Plant tissue culture medium,

Myxobacterium medium, 142-43

98-101 Pneumococcal medium, 110

Positive stainingonethods,

Nagler test, 37 199-200

Negative staining, 199; 200-04 Potassium cyanide test, 25

Neisseria transport medium, Potato-carrot agar, 41

107-08 PPM (Parts Per Minute), 264

Neurospora medium, 165-66 Pseudomonas medium, 111 - 13

Nitrate medium, 18- 19

Nitrate reduction, 18- 19 Reagents, 244-62

Nitrification test, 17- 18 buffers, 254-62

Nitrosomonasm edium, 108 fixatives, 244-47

Noren's medium, 100 IMVIC test, 249

Normality, 264 other, 249-5 1

Nucluear staining, 222-23 salines, 247-49

Oatmeal agar, 40-41 Rhizobium medium, 113-14

ONPG test, 19-20 Rhizoctonia agar, 169

Optochin test, 37 Robertson's broth, 47-48

Oxidase negative, 35-36 Rue's staining, 206

Oxidation fermentation test, 20 Rumen bacteria medium, 1 15- 16

Salmonella medium, 116-24

Pathogen preservation medium, Seedlings medium, 143-44

108-09 Sijpesteijn, 52

Pectin hydrolysis, 20-21 Simmon's citrate medium, 4

Penassay seed agar, 22 Simple staining, 191-96

Penicillin assay, 2 1-22 Sloppy agar medium, 103

Penicillin medium, 167-68 Snyder test, 30-31

Phenolphthalein phosphate agar, Sodium chloride tolerance test,

23 25-26

Phenylalanine deaminase test, 23 Sodium hippurate broth, 7

Phormidium agar, 179-80 Soil microorganisms medium,

Photosynthetic bacteria medium, 124-25

109-10 Solenite broth, 121

Phsphate solubilization, 24-25 Sowers, 53

Phsphotase production, 23 Spirochete medium, 125-26

Spore staining, 216- 19 6-7

Sprirochete staining, 204-07 Transmittaney and optical density:.

Staining methods, 191-243 relation between, 268-70

bacteria, 191-235 Transport medium, 104

fungi, 235-38 Treponema medium, 136

protozoa, 239-43 Trichomonas medium, 181-83

Staining virus elementary bodies. Triple sugar-iron agar, 32-33

229-33 Tryptone nitrate broth, 18

Staining virus inclusion bodies, TSI agar test, 32-33

233-35 Tween pliospliate buffered

Standard solution, 263-94 substrate medium, 11

Staphylococcus medium, 126-30 Tyrosine agar, 14

Starch agar, 2-3

Starcll hydrolysis. 250-54 Urea hydrolysis. 33-34

Streptococcus medium, 130 - 33

Sucrose gelatin agar, 90 Viability staining, 223-24

Sugar fermentation medium, Vibrio medium, 137-42

26-29 Vogel-Johnson agar, 129

Sugar fermentation, 26-29 Voges Proskauer test, 34

Sulfatase test, 29-30

Sulphate reducing bacteria Wayne's sulfatase medium,

medium, 133-35 29-30

Sulphur oxidizing bacteria Wilson and Blair's medium,

medium, 135-36 123-24

Synthetic agar, 42

Xanthine decomposition, 34-35

Temperature and pressure: Xanthomonas medium, 142

relation, 270-71 Xanthine agar, 34

Terathionate broth, 122 Yeast extract-malt extract agar,

Thiocyanate utilization test, 43

31-32 Yeast glucose agar, 42-43

Todol-Hewit broth, 133 Yeast medium, 17 1-72

Tomato juice agar, 90

Tomato juice-gelatin medium, Ziehl-Neelsen staining, 176-97

handbook of media stains and reagents in...

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