MICROBIOLOGY MEDIA COMPOSITION PROCEDURE.pptx

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 In a beaker, 28 grams of the dehydrated powder or lab-prepared media is added to 1000 milliliters of distilled or deionized water. The suspension is then heated to boiling to dissolve the medium completely. The dissolved medium is then autoclaved at 15 lbs pressure (121°C) for 15 minutes. Once the autoclaving process is complete, the beaker is taken out and cooled to a temperature of about 40-45°C. If enrichment is desired, the addition of blood or biological fluids can be done after the autoclaving process. The media is then poured into sterile Petri plates under sterile conditions. 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  species red minute, round Staphylococcus  species pale pink opaque Pseudomonas aeruginosa green-brown fluorescent growth

5. Procedure Suspend 49.53 grams of dehydrated medium in 1000 ml of distilled water. Heat to boiling to dissolve the medium completely. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes.  Cool to 45°C -50°C. 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 Suspend 28 g of nutrient agar powder in 1 litre of distilled water. Heat this mixture while stirring to fully dissolve all components. Autoclave the dissolved mixture at 121 degrees Celsius for 15 minutes. Once the nutrient agar has been autoclaved, allow it to cool but not solidify. 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. Avoid Air bubbles. 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 Suspend 111 grams of Mannitol Salt Agar in 1000 ml of distilled water. Boil to dissolve the medium completely. Sterilize by autoclaving at 15 lbs. pressure (121°C) for 15 minutes. If desired, sterile Egg Yolk Emulsion (E7899) can be added to a final concentration of 5% v/v after autoclaving. 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 Suspend 35.96 grams in 1000 ml distilled water. Mix until the suspension is uniform. Heat to boiling to dissolve the medium completely. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes.  AVOID OVERHEATING. 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. Pour into sterile Petri plates. Allow plates to warm to room temperature. The agar surface should be dry before inoculating. Inoculate and streak the specimen as soon as possible after collection. 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. Incubate plates aerobically at 35-37°C for 18-24 hours and protect from light. 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 Suspend 38 gm of the medium in one liter of distilled water. Heat with frequent agitation and boil for one minute to completely dissolve the medium. Autoclave at 121°C for 15 minutes. Cool to room temperature. Pour cooled Mueller Hinton Agar into sterile petri dishes on a level, horizontal surface to give uniform depth. Allow to cool to room temperature. Check for the final pH 7.3 ± 0.1 at 25ºC. 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 Suspend 89.08 gm of dehydrated medium in 1000ml of distilled or deionized water. Heat to boiling to dissolve the medium completely. Do not autoclave. Cool to 45-50°C. 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 Add 39 gm of Commercial PDA Powder to 1 Litre of Distilled water. Boil while mixing to dissolve. Autoclave 15 min at 121°C.

20. Thank you