Blood agar preparation

Blood agar (BA) is used for the selective cultivation and isolation of organisms including but not limited to Streptococcus pyogenes, Staphylococcus aureus, and Neisseria. Blood agaris an enrichment growth media. BA is also used for the isolation of both pathogenic and non pathogenic bacteria from both clinical and non-clinical samples. Blood agar contains about 5 % sheep blood; and the presence of sheep blood in it, makes this medium partly differential in nature. This is because, bacteria that are haemolytic in nature such as Streptococcus and Satphylococcus can grow on BA – while producing colonies that shows different types of haemolysis. Blood agar also supports the growth of fastidious bacteria such as Streptococcus. Fastidious bacteria are organisms that require additional growth nutrients such as growth factors present in blood for their growth. Blood agar is an enrichment growth media as aforesaid, and this is because it contains blood (a source of important growth factors for fastidious bacteria) – which allows for the isolation of fastidious bacteria from clinical and non-clinical samples.

This image shows how Blood agar looks like after preparation

Components of Blood agar base

The components of Blood agar base required for Blood agar preparation include:

  1. Blood agar base- which is the solidifying agent. NOTE: In cases where you do not have a blood agar base; nutrient agar (which is a general purpose media) can also serve as your blood agar base. The most important thing is to have an agar base, for which the use or incorporation of blood will be supported. And nutrient agar base is most efficient in this case, because it is a general purpose media.
  2. Beef/meat extract – which provide sources of nitrogen, carbon, and vitamins
  3. pH – which is usually adjusted to 7.3 at 25 °C (or 77 °F)
  4. Peptone – which serves as source of vitamins
  5. Sodium chloride – for maintaining a balanced physiological environment as obtainable in living systems

You require these materials to prepare your Blood agar: blood agar base or nutrient agar base (usually comes in 500 g), 5-10 % of sterile defibrinated sheep blood, autoclave, conical flask, measuring cylinder, beaker, stirring rod, Bunsen burner, incubator, refrigerator, wire gauze, spatula, weighing balance, timer, cotton wool, aluminium foil, distilled water, Petri dish

STEP BY STEP PROTOCOL TO PREPARE BLOOD AGAR

  1. Weigh out 40.00 g of blood agar powder using the weighing balance.
  2. Suspend the 40.00 g of blood agar powder in 1 litre (1000 ml) of distilled water in a conical flask.
  3. Mix the solution by stirring to dissolve the agar.
  4. Bring the mixture to boil, by mild boiling of the mixture over a Bunsen burner flame. This helps to dissolve the agar completely. Monitor the boiling process closely in order to avoid charring the agar.
  5. Transfer the conical flask containing the boiled/mixed agar suspension to the autoclave.
  6. Sterilize the medium at 121 degrees Celsius at 15 psi (or 15 lbs of pressure) for 15 min in the autoclave.
  7. At the end of sterilization, allow the autoclave to return to normal (zero point) before opening. Otherwise the pressure built up in the autoclave will affect the quality and quantity of the prepared molten medium. More so, you may be affected by the steam from the autoclave due to the high pressure built up in the autoclave.
  8. Allow molten blood agar medium to cool to about 45-50 degrees Celsius.
  9. Add about 5-10 % of sterile defibrinated sheep blood.
  10. Stir / mix the molten blood agar vigorously.
  11. Pour prepared molten medium into sterile Petri dish plates.
  12. Allow the poured plates on the bench to solidify.
  13. Do sterility check by incubating the poured plates in the incubator at 37 degrees Celsius for 18-24 h.
  14. At the end of incubation, check the plates for any sign of microbial growth (which is usually indicated by the presence of colony).
  15. Absence of colony on the plate means that your sterilization is good.
  16. You can now use your prepared blood agar plates for your experiment OR store in the refrigerator at 4 degrees Celsius until use.

REFERENCES

  1. Cheesbrough M (2006). District Laboratory Practice in Tropical Countries. Part 2 . Cambridge University Press, UK.

Leave a Reply

Your email address will not be published. Required fields are marked *