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:

  1. The BAP test may be used to either test a throat culture or an unknown bacterial species for hemolytic activity.
  2. If an unknown species of bacteria is to be tested, divide a BAP plate in thirds.
  3. Inoculate one third with the unknown, one third with a negative control (such as E. coli), and one third with a positve control (such as Bacillus cereus).
  4. It is best to streak your bacteria in a long W pattern using your inoculating needle with plenty of space between the streak lines.  This will put some space between the streaks making it easier to observe the results.  If there is too much growth, which may happen with a swab, waste products may accumulate which may lyse the blood giving the appearance of hemolytic toxins when they are not actually present.
  5. If a candle jar is not available, take a needle and stab a region of heavy inoculation to inoculate bacteria under the agar surface.  This allows for growth of the bacteria under reduced oxygen.  If a candle jar is available (or some other means to reduce the oxygen), do not stab the plates and incubate them in a candle jar.  The cultures are put in the jar, a lit candle is added, the lid is put on tightly, the candle uses up most of the oxygen and goes out, and then the jar is incubated under reduced oxygen.).  Some hemolysins are oxygen sensitive, so the bacteria should be grown and tested under reduced oxygen.  Both the stab and the candle jar reduce the oxygen allowing for the the testing of oxygen sensitive hemolysins.  Oxygen stable hemolysins are not affected by either treatment.
  6. Incubate the plates for 24 hours at 35 degrees. 

Procedure for Testing a Throat Culture:

  1. If using a throat culture, obtain a swab of the throat and inoculate it in a small area on a BAP plate.
  2. It is not required but is a good idea to streak the rest of the plate with a loop to try to isolate colonies from the throat culture.  Alternatively, one may swab the entire plate with the throat culture.
  3. If a candle jar is not available, take a needle and stab a region of heavy inoculation to allow anaerobic growth of the bacteria or at least growth with reduced oxygen.  Otherwise, do not bother with the stabbing and incubate appropriately labeled plates in a candle jar.  (See above.)
  4. Incubate the plates for 24 hours at 35 degrees. 

Results:

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.

References:

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.

Instructor Notes:

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.