Bacterial Hemolysis Reactions

Jason Bofinger, M.D.
23 August 2006

The clinical microbiology laboratory utilizes a number of different media for the growth and identification of bacteria. Some of these media contain blood or blood products to enhance the growth of potential pathogens. In addition, the presence of blood in these media allows for discernment of organisms based on their ability to alter or destroy the red blood cells and create a color change in the medium. This process is known as hemolysis and it can be described as alpha- or beta-hemolysis depending on the effect on the red blood cells. In alpha-hemolysis, colonies are surrounded by a zone of intact erythrocytes that have a greenish color. Bacterial peroxide production, which results in the reduction of hemoglobin to methemoglobin, is generally responsible for this appearance. In beta-hemolysis, colonies are surrounded by a clear zone in which few or no intact erythrocytes can be found. Beta-hemolysis is caused by the action of hemolysins, erythrocyte-lysing enzymes, produced by the bacteria. In the photograph below, note how the cross-hatched pattern of the fluorescent panel in the ceiling is visible -- but only in the areas of hemolysis. In the photos below, the plate on the left shows beta-hemolysis and the plate on the right shows alpha-hemolysis.

Beta-hemolysis (transmitted light)Alpha-hemolysis

Perhaps the most useful distinction among species of hemolysis-causing bacteria are in the genus, Streptococcus. Group A streptococci (S. pyogenes) are beta-hemolytic. They produce two hemolysins: streptolysin O and streptolysin S. Streptolysin O is hemolytically active in the reduced stated (when there are available –SH groups), but is rapidly inactivated in the presence of oxygen. It is thus most often responsible for hemolysis observed when growth is found in deep cuts made into the agar (where oxygen is scarce). Streptolysin O is antigenic. Antistreptolysin O is an antibody that appears in humans after infection with Group A streptococci. It blocks hemolysis produced by streptolysin O, and can be quantitatively measured in a person suspected of having recent infection with the organism (especially helpful in the evaluation of possible rheumatic fever). Streptolysin S is responsible for the hemolysis observed around colonies growing on the surface of blood agar plates. While it is not antigenic, it may be inhibited by a non-specific inhibitor this is frequently present in human and other animal sera. The presence of antistreptolysin O and non-specific inhibitors of streptolysin S may explain in part why hemolysis may be present on sheep blood, but not on human blood agar.


Because of these differences sometimes noted on different animal blood agar preparations, the hemolytic reactions of beta-hemolytic streptococci on sheep blood agar are in some parts of the world used as the standard for defining hemolytic reactions of streptococci. Streptococci, however, are not the only hemolytic bacteria. Some of the other bacteria that can produce hemolytic reactions include: Staphylococcus aureus, Enterococcus spp., Bacillus cereus, Clostridium perfringens, Corynebacterium diphtheriae, Listeria monocytogenes, and certain strains of E. coli and Pseudomonas aeruginosa. Differences in patterns of hemolysis can also be observed on different animal blood agars when some of these other bacteria are cultured. For example, in a genomic study of various strains of Enterococcus faecium, beta-hemolytic activity was observed on both sheep and human blood in only one strain, while four strains showed beta-hemolysis on human blood, but not on sheep blood, and the remainder of the strains studied showed no hemolysis on either medium.


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