MEDIA composition AND PROCEDURE

1. MICROBIOLOGY
different types of media composition
Submitted by
Abhijit padhi

2. NAM (nutrient agar media)
Composition
Yeast extract 3gm/ltr.
Peptone 5gm/ltr.
Nacl 5gm/ltr.
Agar 15gm/ltr.
ph should be 7.2±0.2
• All type of bacteria should be able to grow.

3. Procedure
1.In a beaker, 28 grams of the dehydrated powder or lab-prepared media is
added to 1000 milliliters of distilled or deionized water.
2.The suspension is then heated to boiling to dissolve the medium
completely.
3.The dissolved medium is then autoclaved at 15 lbs pressure (121°C) for
15 minutes.
4.Once the autoclaving process is complete, the beaker is taken out and
cooled to a temperature of about 40-45°C.
5.If enrichment is desired, the addition of blood or biological fluids can be
done after the autoclaving process.
6.The media is then poured into sterile Petri plates under sterile conditions.
7.Once the media solidifies, the plates can be placed in the hot air oven at a
lower heat setting for a few minutes to remove any moisture present on the
plates before use.

4. MacConkey Agar
Composition
Peptone 20gm/1ltr.
Lactose monohydrate 10gm/1ltr.
Bile salts 1.5gm/1ltr.
Sodium chloride 5gm/1ltr.
Neutral red 0.03gm/1ltr.
Crystal Violet 0.001gm/1ltr.
Agar 13.5gm/1ltr.
ph should be 7.1±0.2
• MacConkey agar is used for
the isolation of gram-negative
enteric bacteria.
Escherichia coli red/pink non-mucoid
Aerobacter
aerogenes
pink mucoid
Enterococcus speci
es
red minute, round
Staphylococcus sp
ecies
pale pink opaque
Pseudomonas
aeruginosa
green-brown
fluorescent
growth

5. Procedure
1.Suspend 49.53 grams of dehydrated medium in 1000 ml of
distilled water.
2.Heat to boiling to dissolve the medium completely.
3.Sterilize by autoclaving at 15 lbs pressure (121°C) for 15
minutes.
4.Cool to 45°C -50°C.
5.Mix well before pouring into sterile Petri plates.

6. Blood agar
Composition
Peptone 5gm/1000ml
beef extract/yeast extract 3gm
Agar 15gm
NaCl 5gm
ph should be 7.1±0.2
• 5% Sheep Blood

7. Procedure
1.Suspend 28 g of nutrient agar powder in 1 litre of distilled water.
2.Heat this mixture while stirring to fully dissolve all components.
3.Autoclave the dissolved mixture at 121 degrees Celsius for 15
minutes.
4.Once the nutrient agar has been autoclaved, allow it to cool but not
solidify.
5.When the agar has cooled to 45-50 °C, Add 5% (vol/vol) sterile
defibrinated blood that has been warmed to room temperature
and mix gently but well.
6.Avoid Air bubbles.
7.Dispense into sterile plates while liquid.

8. Chocolate agar
Composition
Casein/Animal Tissue Digest 15gm/1ltr.
Corn-starch 1gm/1ltr.
Sodium chloride 5gm/1ltr.
Dipotassium Phosphate 4gm/1ltr.
Monopotassium Phosphate 1gm/1ltr.
Hemoglobin solution 2%/1ltr.
Koenzyme enrichment 10ml/1ltr.
Agar 10gm/1ltr
ph should be 7.1±0.2
• chocolate media for N. gonorrhea.

9. Procedure
• Preparation of the hemoglobin solution
• Add hemoglobin to distilled water and bring volume to 500 ml.
Mix thoroughly and autoclave for 15 min at 15lbs pressure at
21°C. Cool at 45°C -50°C.
• Preparation of medium
• Add components except for hemoglobin solution to distilled
water and bring volume to 500ml. Mix thoroughly. Gently heat
until boiling. Autoclave for 15 min at 15lbs pressure at 21°C.
Cool at 45°C -50°C. Add 500ml of sterile hemoglobin solution
and mix thoroughly. Pour into sterile Petri dishes or distribute
into sterile tubes.

10. MSA media (Mannitol-Salt agar)
Composition
Peptone 10gm/1ltr.
Nacl 75gm/1ltr.
D-mannitol 10gm/1ltr.
Yeast extract 1gm/1ltr.
Phenol red 0.025gm/1ltr.
Agar 15gm/1ltr.
ph should be 7.2±0.2
Organisms Results
Staphylococcus aureus
Yellow colonies surrounded
by the yellow zone
Staphylococcus epidermidis Pink or Red colonies
Micrococci Red colonies
Escherichia coli No growth

11. Procedure
1.Suspend 111 grams of Mannitol Salt Agar in 1000 ml of distilled
water.
2.Boil to dissolve the medium completely.
3.Sterilize by autoclaving at 15 lbs. pressure (121°C) for 15
minutes.
4.If desired, sterile Egg Yolk Emulsion (E7899) can be added to a
final concentration of 5% v/v after autoclaving.
5.Pour cooled Mannitol Salt Agar into sterile petri dishes and
allow to cool to room temperature.

12. EMB (Eosin-methylene blue)
Composition
Peptic digest of animal tissue 10gm/1ltr.
Dipotassium phosphate 2gm/1ltr.
Lactose 5gm/1ltr.
Sucrose 5gm/1ltr.
Eosin – Y 0.400gm/1ltr.
Methylene blue 0.065gm/1ltr.
Agar 13.5gm/1ltr.
ph should be 7.2±0.2
Organisms Growth
Escherichia coli
Blue-black bull’s eye; may
have a green metallic sheen
Pseudomonas aeruginosa Colorless
Enterobacter aerogenes
Good growth; pink, without
sheen
Klebsiella pneumoniae Pink, mucoid colonies
Proteus mirabilis
Luxuriant growth; colorless
colonies
Salmonella Typhimurium
Luxuriant growth; colorless
colonies

13. Procedure
1. Suspend 35.96 grams in 1000 ml distilled water.
2. Mix until the suspension is uniform. Heat to boiling to dissolve the medium completely.
3. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes. AVOID OVERHEATING.
4. Cool to 45-50°C and shake the medium in order to oxidize the methylene blue (i.e. to restore its
blue color) and to suspend the flocculent precipitate.
5. Pour into sterile Petri plates.
6. Allow plates to warm to room temperature.
7. The agar surface should be dry before inoculating.
8. Inoculate and streak the specimen as soon as possible after collection.
9. If the specimen to be cultured is on a swab, roll the swab over a small area of the agar surface and
streak for isolation with a sterile loop.
10.Incubate plates aerobically at 35-37°C for 18-24 hours and protect from light.
11.Examine plates for colonial morphology. If negative after 24 hours, reincubate an additional 24
hours.

14. MHA (Mueller Hinton Agar)
Composition
Beef extract 2gm/1ltr.
Acid hydrolysate of casein 17.5gm/1ltr.
Starch 1.5gm/1ltr.
Agar 17gm/1ltr.
ph should be 7.3±0.2
Positive controls: Expected results
Escherichia coli ATCC® 25922
Good growth; pale straw
colored colonies
Pseudomonas
aeruginosa ATCC® 27853
Good growth; straw colored
colonies
Staphylococcus
aureus ATCC® 25923
Good growth; cream colored
colonies
Negative control:
Un-Inoculated medium No change

15. Procedure
1.Suspend 38 gm of the medium in one liter of distilled water.
2.Heat with frequent agitation and boil for one minute to
completely dissolve the medium.
3.Autoclave at 121°C for 15 minutes. Cool to room temperature.
4.Pour cooled Mueller Hinton Agar into sterile petri dishes on a
level, horizontal surface to give uniform depth.
5.Allow to cool to room temperature.
6.Check for the final pH 7.3 ± 0.1 at 25ºC.
7.Store the plates at 2-8 ºC.

16. TCBS (Thiosulfate-citrate-bile salts-
sucrose)
Composition
Proteose peptone 10gm/1ltr.
Yeast extract 5gm/1ltr.
Sodium thiosulphate 10gm/1ltr.
Sodium citrate 10gm/1ltr.
Bile 8gm/1ltr.
Sucrose 20gm/1ltr.
Sodium chloride 10gm/1ltr.
Ferric citrate 1gm/1ltr.
Bromothymol blue 0.04gm/1ltr.
Thymol blue 0.04gm/1ltr.
Agar 15gm/1ltr.
ph should be 8.6±0.2
Microorganisms Characteristics
Vibrio cholera
Flat yellow colonies, 2-3 mm in
diameter
Vibrio alginolyticus Large yellow colonies
Vibrio fluvialis, Vibrio vulnificus Yellow or translucent colonies
Vibrio parahaemolyticus
Colorless colonies with a green
center
Pseudomonas, Aeromonas Blue colonies
Enterobacteria or others Tiny transparent colonies

17. Procedure
1.Suspend 89.08 gm of dehydrated medium in 1000ml of distilled
or deionized water.
2.Heat to boiling to dissolve the medium completely.
3.Do not autoclave.
4.Cool to 45-50°C.
5.Mix well and pour into sterile Petri plates.

18. PDA (Potato Dextrose Agar)
Composition
Potato infusion 200gm/ltr.
Dextrose 20gm/ltr.
Agar 20gm/ltr.
Note: 200 gm of potato infusion is equivalent to 4.0 gm of potato extract.
ph should be 5.6± 0.2

19. Procedure
1.Add 39 gm of Commercial PDA Powder to 1 Litre of Distilled
water.
2.Boil while mixing to dissolve.
3.Autoclave 15 min at 121°C.

20. Thank you