Blood agar (BA) is used for the selective cultivation and isolation of organisms including but not limited to Streptococcus pyogenes, Staphylococcus aureus, and Neisseria. It 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 (Figure 1). This is because, bacteria that are haemolytic in nature such as Streptococcus and Staphylococcus can grow on BA – while producing colonies that shows haemolysis.
Components of Blood agar
The components of Blood agar base required for Blood agar preparation include:
- Blood agar base- which is the solidifying agent
- Beef/meat extract – which provide sources of nitrogen, carbon, and vitamins
- pH – which is usually adjusted to 7.0 at 25 °C (or 77 °F)
- Peptone – which serves as source of vitamins
- Sodium chloride – for maintaining a balanced physiological environment as obtainable in living systems
- pH – which is usually adjusted to 7.3 at 25 °C (or 77 °F)
You require these materials to prepare your Blood agar: blood agar base (usually comes in 500 g), 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
- Weigh out 40.00 g of Blood agar powder using the weighing balance.
- Suspend the 40.00 g of Blood agar powder in 1 litre (1000 ml) of distilled water.
- Mix the solution by stirring to dissolve the agar.
- Heat the mixture by boiling to dissolve the SSA powder completely. Monitor the boiling process closely in order to avoid charring the agar.
- 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.
- Transfer the conical flask containing the boiled/mixed nutrient agar suspension to the autoclave.
- Sterilize the medium at 121 degrees Celsius at 15 psi (or 15 lbs of pressure) for 15 min in the autoclave.
- 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 prepared molten medium which might pour out. More so, you can also be affected by the high pressure built up in the autoclave.
- Allow molten SSA medium to cool to about 45-50 degrees Celsius.
- Add about 5-10 % of sterile defibrinated sheep blood.
- Stir /mix the molten blood agar vigorously.
- Pour prepared molten medium into sterile Petri dish plates.
- Allow the poured plates on the bench to solidify.
- Do sterility check by incubating the poured plates in the incubator at 37 degrees Celsius for 18-24 h.
- At the end of incubation, check the plates for any sign of microbial growth (which is usually indicated by the presence of colony).
- Absence of colony on the plate means that your sterilization is good.
- You can now use your prepared Blood agar plates for your experiment OR store in the refrigerator at 4 degrees Celsius until use.
Brooks G.F., Butel J.S and Morse S.A (2004). Medical Microbiology, 23rd edition. McGraw Hill Publishers. USA.
Cheesbrough M (2006). District Laboratory Practice in Tropical Countries. Part 2 . Cambridge University Press, UK.
Goldman E and Green L.H (2008). Practical Handbook of Microbiology, Second Edition. CRC Press, Taylor and Francis Group, USA.
Madigan M.T., Martinko J.M., Dunlap P.V and Clark D.P (2009). Brock Biology of Microorganisms, 12th edition. Pearson Benjamin Cummings Inc, USA.
Mahon C. R, Lehman D.C and Manuselis G (2011). Textbook of Diagnostic Microbiology. Fourth edition. Saunders Publishers, USA.
Patrick R. Murray, Ellen Jo Baron, James H. Jorgensen, Marie Louise Landry, Michael A. Pfaller (2007). Manual of Clinical Microbiology, 9th ed.: American Society for Microbiology.
Wilson B. A, Salyers A.A, Whitt D.D and Winkler M.E (2011). Bacterial Pathogenesis: A molecular Approach. Third edition. American Society of Microbiology Press, USA.
Woods GL and Washington JA (1995). The Clinician and the Microbiology Laboratory. Mandell GL, Bennett JE, Dolin R (eds): Principles and Practice of Infectious Diseases. 4th ed. Churchill Livingstone, New York.