Blood Agar Plates (BAP)
Blood agar is actually a couple of related media, all of which are rich formulations containing peptones, yeast extracts, liver or heart extracts (depending on the medium), and blood. The blood is usually sheep's red blood cells (RBC), though horse and other species may be used. Blood agar is used to grow fastidious organisms (species that do not grow easily) requiring a rich media providing many nutrients and growth factors that are largely supplied by blood. It is also a differential media in allowing the detection of hemolysis (destroying the RBC) by cytolytic toxins secreted by some bacteria, such as certain strains of Bacillus, Streptococcus, Enterococcus, Staphylococcus, and Aerococcus.
Blood agar plates are routinely used in the clinic to test for pathenogenic bacteria in throat swabs. These throat pathogens are often Gram positive cocci that may be hemolytic, producing exotoxins called hemolysins that destroy blood cells. One such pathogen that BAP can detect is Streptococcus pyogenes which causes a number of diseases including strep throat, rheumatic fever, and necrotizing fascitis.
Aim of Test: blood agar plates allow for the growth of fastidious organisms and the differentiation of cells according to three hemolytic activities:
The Three Types of Hemolytic Activities:
Procedure for Testing an Unknown:
Procedure for Testing a Throat Culture:
Observe your plates for hemolytic activity and record your results as alpha, beta, or gamma (α, β, γ). Holding the plate up to a light source helps.
Difco Laboratories. 1998. Difco Manual, 11th ed. Difco Laboratories, Sparks, MD, USA.
Harley J.P. and Prescott L.M. 2002. Laboratory Exercises in Microbiology, 5th ed. McGraw-Hill Higher Education, New York, NY, USA.
Pickett M.J. 1989. Methods for identification of flavobacteria. J. Clin. microbiol. 27:2309.
Ruoff, K.L. 1995. Streptococcus, p. 299. In P.R. Murray, E.J. Baron, M.A Pfaller, F.C. Tenover, and R.H. Yoken (eds.). Manual of clinical microbiology, 6th ed. American Society for Microbiology, Washington, D.C.
Different types of blood (sheep versus horse, etc.) can affect the hemolytic activity of strains. Optimal hemolytic activity occurs under reduced or anaerobic conditions, but this is often not required. Hemolytic activity is much easier for students to read and interpret with the use of a candle jar compared to stabbing into the agar, so this method is recommended. Over incubation of the plates may cause overgrowth which may cause waste products to accumulate which may lyse the blood giving the appearance of hemolytic toxins when they are not actually present. Many enterics give false hemolytic activity when overgrown. The plates can be refrigerated if needed to prevent over incubation.