Blood Agar: Introduction, Composition, Principle, Preparation Requirements, Test Procedure, Result -Interpretation, Uses, Keynotes, and Blood Agar Footages

Introduction of Blood Agar

Blood agar is an enriched medium for the cultivation of bacteria. Fastidious organisms, such as streptococci, do not grow well on ordinary growth media. It is a type of growth medium i.e. trypticase soy agar enriched with 5% sheep blood or blood agar base with 5-10 % sterile sheep blood that encourages the growth of bacteria and their hemolysis, such as streptococci, that otherwise wouldn’t grow. 5% sheep blood agar plate is also called BAP.

Composition of Sheep Blood Agar Base

         

Ingredients	Gms / Litre
Casein enzymic hydrolysate	14.0
Peptic digest of animal tissue	4.5
Yeast extract	4.5
Sodium chloride	5.0
Agar	12.5
Final pH (at 25°C)	7.3±0.2

Procedure for the Preparation of BAP

1. Suspend 40.5 grams in 1000 ml of distilled water or deionized 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 45-50°C and aseptically add 7% sterile sheep blood.
5. Mix well and pour into sterile Petri plates. Avoid the formation of air bubbles. You must have warmed the blood to room temperature at the time of dispensing it to a molten agar base.
6. Dispense 15 ml amounts to sterile Petri plates aseptically
7. Label the medium with the date of preparation and give it a batch number (if necessary).

Storage and Shelf Life

Store below 30°C in a tightly closed container and the prepared medium at 2-8°C, preferably in sealed plastic bags to prevent loss of moisture. The shelf life of this prepared BAP is up to four weeks. Use before the expiry date on the label.

Principle and Interpretation

Hemolysins are exotoxins produced by bacteria that lyse red blood cells. The hemolytic reaction can be visualized on BAP observed through the bright transmitted light. On BAP colonies of hemolytic bacteria may be surrounded by a clear, colorless zone where the red blood cells have been lysed and the hemoglobin destroyed to a colorless compound and which is beta hemolysis. Other types of bacteria can reduce hemoglobin to methemoglobin which produces a greenish zone around the colonies and is called alpha hemolysis. Gamma hemolysis is lacking hemolysis where no change in the medium is observed.

Sheep blood agar base with added sheep blood was developed to allow maximum recovery of organisms without interfering with their hemolytic reactions. The sheep blood agar base was formulated to be compatible with sheep blood and give improved hemolytic reactions of organisms. Casein enzymic hydrolysate and yeast extract provide nitrogen, carbon, amino acids, and vitamins. Peptic digest of animal tissue (PDAT) is the nitrogen source. Sodium chloride (NaCl) maintains the osmotic balance. Sheep blood agar base showed considerable improvement and the expected beta-hemolytic reactions with S. pyogenes in comparison to other blood agar bases supplemented with blood

Quality control

• Inoculate Streptococcus pneumoniae and Streptococcus pyogenes into a prepared blood agar.
• Observation of Cultural characteristics after incubation at 35-37°C for 18-24 hours with added 7% v/v sterile sheep blood.

Organisms	Growth	Hemolysis
Streptococcus pneumoniae ATCC 6303	luxuriant	alpha
Streptococcus pyogenes ATCC 19615	luxuriant	beta

• This is an indicator of good quality control of prepared blood agar.

Uses of BAP

BAP has the following uses:

1. Determine the type of hemolysis, whether alpha, beta, or gamma.
2. For the culture of streptococci as well as antimicrobial susceptibility testing (AST).
3. Use of optochin disc for presumptive identification of Streptococcus pneumoniae.
4. Similarly use of bacitracin disc (0.04U) for presumptive identification of Streptococcus pyogenes.
5. To perform the CAMP test for Streptococcus agalactiae.
6. To perform satellitism test for Haemophilus.
7. It is also used for the isolation and cultivation of other than streptococci like Neisseria and other fastidious microorganisms.
8. It is also useful for the preparation of Salmonella Typhi antigens.

Keynotes

1. Blood is a good constituent of enriched medium for fastidious organisms even though contains inhibitors for certain bacteria such as Neisseria and Haemophilus genera and the blood agar must be heated to inactivate these inhibitors and to release essential growth factors (e.g., V factor). The heating of blood agar converts it into chocolate agar (75°C for 15 minutes turns a chocolate color) and supports the growth of these bacteria.
2. Hemolysis on blood agar: Mainly three types of hemolysis are produced in Sheep blood agar by Streptococci namely; Alpha hemolysis, Beta hemolysis, and gamma hemolysis but sometimes alpha prime or wide zone alpha hemolysis may be encountered. Hemolysis is best observed by examining colonies grown under anaerobic conditions or inspecting sub-surface colonies. How does one know if the colonies they are observing on a plate have caused alpha hemolysis or beta hemolysis or gamma ?-Follow up principle and interpretation.
3. To check the type of blood agar hemolysis, the blood agar plate must be held up to a bright transmitted light source and observed with the light coming from behind.
4. Alpha hemolysis: Streptococcus pneumoniae
5. Beta Hemolysis: Group A beta-hemolytic streptococci-Streptococcus pyogenes and Group B, beta-hemolytic streptococci-Streptococcus agalactiace. For the group, streptococci maximal activity of both the hemolysins; oxygen labile SLO, and oxygen-stable SLS hemolysins is observed only in anaerobic conditions.
6. Gamma or non-hemolysis: Enterococcus species
7. Alpha prime or wide zone alpha hemolysis: A small zone of intact erythrocytes immediately adjacent to bacterial colony, with a zone of complete red-cell hemolysis surrounding the zone of intact erythrocytes. This type of hemolysis may be confused with Beta hemolysis.
8. The double zone of hemolysis on it is also seen by some organisms like Clostridium perfringens and Aeromonas hydrophilia called target hemolysis.
9. If you are planning to prepare a batch of blood agar plates, prepare a few blood agar plates first to ensure that blood is sterile.

BAP Footages

Staphylococcus aureus beta-hemolytic colony on BAP

Streptococcus pneumoniae Draughtsman colony on BAP

Streptococcus agalactiae colony morphology on BAP

Streptococcus pyogenes beta-hemolytic colony morphology on BAP

Enterococcus colony morphology on BAP showing gamma hemolysis

Further Readings

1. Bailey & Scott’s Diagnostic Microbiology. Editors: Bettey A. Forbes, Daniel F. Sahm & Alice S. Weissfeld, 12th ed 2007, Publisher Elsevier.
2. Pelczar M. J. Jr., Reid R. D., Chan E. C. S., 1977, Microbiology, 4th Ed., Tata McGraw-Hill Publishing Company Ltd, New Delhi.
3. Clinical Microbiology Procedure Handbook, Chief in editor H.D. Isenberg, Albert Einstein College of Medicine, New York, Publisher ASM (American Society for Microbiology), Washington DC.
4. Koneman E. W., Allen S. D., Janda W. M., Schreckenberger P. C., Winn W. C. Jr., 1992, Colour Atlas and Textbook of Diagnostic Microbiology, 4th Ed., J. B. Lippincott Company.
5. Spector W. S., (Ed.), 1961, Handbook of Biological Data, W. B. Saunders Company, Philadelphia and London.
6. https://www.asm.org/getattachment/7ec0de2b-bb16-4f6e-ba07-2aea25a43e76/protocol-2885.pdf
7. Colour Atlas and Textbook of Diagnostic Microbiology. Editors: Koneman E.W., Allen D.D., Dowell V.R. Jr, and Sommers H.M.
8. https://www.bd.com/europe/regulatory/Assets/IFU/Difco_BBL/211086.pdf
9. Mackie and Mc Cartney Practical Medical Microbiology. Editors: J.G. Colle, A.G. Fraser, B.P. Marmion, A. Simmous, 4th ed, Publisher Churchill Living Stone, New York, Melborne, Sans Franscisco 1996.
10. Textbook of Diagnostic Microbiology. Editors: Connie R. Mahon, Donald G. Lehman & George Manuselis, 3rd edition2007, Publisher Elsevier.
11. Manual of Clinical Microbiology. Editors: P.R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover and R. H. Yolken, 7th ed 2005, Publisher ASM, USA