1) Crystal violet blood agar:
This is used as a selective medium for Streptococus pyogenes.At a concentration of 1 in 500000, crystal violet inhibits the growth of staphylococcus aureus and reduces the growth of commensals in throat specimens.
preparation procedure of Crystal violet blood agar
- Add 1 ml of 0.02 % w/v aqueous solution of crystal violet to every 100ml of sterile blood agar.
- Mix well and dispense in sterile petri-dishes.
- Label the plates ‘CV’.
- Date the medium and give it a batch number.
- Store the plates for future uses
Quality control
- To test the performance of the medium, inoculate half of the plate with a 5 hour broth culture of Streptococcus pyogenes and the other half with a 5 hour culture containing S. pyogenes and Staphylococcus aureus.
- After overnight incubation in a candle jar at 35-37° C, record the selectivity of the medium and compare with the results of previous performance tests.
2) Neomycin Blood Agar
This is used as a selective medium for obligate anaerobes. At a concentration of 70 mg/ml,neomycin sulphate inhibits facultative anaerobic Gram negative rods.
Note: At concentration over 10 mg/ml, neomycin sulphate mayinhibit the groeth of Bactreroides melaninogenicus. Anaerobic Gram positivegocci may not grow on neomycin (or kanamycin) blood agar.
Preparation procedure of Neomycin blood agar
- Prepare a stock solution containing 70000 mg/ml neomycin by dissolving 0.5 g neomycin suphate containing(containing 0.35 g neomycin base) in 5ml of sterile water.
- From this, prepare a working solution of 17500 mg/ml by mixing 2 ml of stock solution with 6 ml of sterile water.
- Add 1 ml of working neomycin sulphate solution to 250 ml of blood agar to give a concentration of 70 mg/ml.
- Mix well and pour in sterile petridishes
- Label the plates ‘N’.
- Date the medium and give it a batch number.
- Store the plates for future use.
Quality control
- To test the performance of medium,inoculate half of plate with a 5 hour cooked meat broth culture of Bacteroides fragilis and the other half with a 5 hour culture containing B. fragilis and Proteus.
- After 48 hours anaerobic incubation at 35-37° C, record the selectivity of the medium and compare with the results of previous performance test.
3) Kanamycin Blood Agar
This medium may be used as an alternative to neomycin blood agar. At a concentration of 75 mg/ml, kanamycinis selective for obligate anaerobes including Bacteroides melaninogenicus.
Preparation procedure of Kanamycin blood agar
- Prepare a 15 g/l kanamycin solution by dissolving 1.5 g kanamycin sulphate in 100 ml of sterile water.
- Add 1 ml of this solution to every200 ml of blood agar.
- Mix well and pour in sterile petridishes as described for blood agar.
- Label the plates ‘K’.
- Date the medium and give it a batch number.
- Store the plates until uses
Quality control
- Test the performance of the medium in the same way as described for neomycin blood agar.
4) Chocolate (Heated Blood) Agar
Chocolate agar is used to grow Haemophilus influenza and other pathogens that require, or grow better on this highly nutritious medium.
Preparation procedure of chocolate agar
- Prepare as described for blood agarexcept after adding the blood, heat the medium in a 70° C water bath until it becomes brown in color.
- This takes about 10-15 minutes duringwhich time the medium should be mixed gently several times.
- After allowing the medium to cool toabout 45°C,remix and dispense in sterile petri dishes as described forblood agar.
- Date the medium and give it a batch number.
- Store the plates
Note: care must be taken not to over-heat or prolong the heating of the medium because this will cause it to become granular and unfit for use.
Quality control
- Test the performance of the medium by inoculating it with Haemophilus influenzae.
- After overnight incubation in a candle jar at 35-37°C, record the growth and compare the results of previous performance tests.