Do a lab summary of 300 words.

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Blood agar is a rich complex differential medium which can be used to culture many fastidious (picky, strict growth requirements) bacterial species. Some genera of pathogenic bacteria (Streptococcus, Staphylococcus, and Bacillus in our lab) can be differentiated on the basis of the ability to cause hemolysis of erythrocytes (red blood cells). There are 3 levels of blood hemolysis based upon the production of enzymes or other means by which the organism damages erythrocytes. Beta () hemolysis is the complete lysis or destruction of erythrocytes resulting in a transparent to translucent zone of clearing around colonies. SEE IMAGE “Beta hemolysis”. This damage is due to the production of enzymes called hemolysins (if made by Streptococcus species they are called Streptolysins) which break or lyse erythrocytes. Beta hemolytic species in our lab include Streptococcus pyogenes, Staphyloccus aureus, Bacillus subtilus, and Bacillus cereus.

Alpha () hemolysis is a partial destruction of erythrocytes resulting in an opaque to translucent zone around the colony which is green-brown in color. Alpha hemolysis is not caused by a hemolysin but rather by discoloration due to the removal of potassium from the red blood cells and reduction of hemoglobin to methemoglobin which has a greenish color. Alpha hemolytic species include Streptococcus pneumoniae. We will not work with any alpha hemolytic organisms this semester. No visible affect on the erythrocytes is called gamma () hemolysis. SEE IMAGES “Gamma hemolysis” . Other than the beta hemolytic species, mentioned above, all of our Gram positive organisms are gamma. This makes blood hemolysis a great method for distinguishing the species in our Gram positive genera this semester.

Procedure: Steak blood plates for isolation as you would a TSA plate. Incubate these plates at 37oC for as short of a time period as is necessary to see your hemolysis results. It is wise to examine blood plates frequently if possible, such as at 16-18hrs, 24hrs, 30hrs, etc. Extended incubation (over 36-48 hours) will result in auto-lysing of red blood cells (even on an uninoculated plate) giving false beta hemolysis results. To reiterate, unlike motility or gelatin deeps, you can definitely over-incubate blood agar media.

NOTES:

*We will only use this test to differentiate Gram positive organisms.

* All of our organisms are beta or gamma hemolytic. We have no alpha hemolytic organisms.

* A few of our beta hemolytic organisms, namely S. pyogenes and B. subtilus will, from time to time,

require longer than 24hours incubation to give complete clearing on blood agar. When in doubt,

incubate a few more hours and check the plate again.

* You CAN over-incubate blood plates. The red blood cells will begin to auto-lyse causing gradual

clearing of the agar. This will begin within 48hrs of incubation.

Streptococcus serotyping - Lancefield groups

Streptococci are medically important as the causative agents of various conditions including respiratory tract infections (“strep throat”, pneumonia), scarlet fever, rheumatic fever, impetigo, septicemia, kidney infections, urinary tract infections, and meningitis. Species of the genus Streptococcus have been divided into serological groups based upon antigenic similarity in the species within, and differences between the species in the groups. The groups are called Lancefield groups, named after the scientist Rebecca Lancefield who discovered the unique antigenic characteristics of Streps, and devised the method of classification. Antigens are molecules on the surfaces of cells (and viruses) that serve to identify those cells. “Rapid Strep tests” conducted in hospitals and physician’s offices are based on this principle of antigen recognition. Generally speaking, when the immune system finds a foreign antigen it will respond in part by producing antibodies that specifically recognize that specific antigen. Antibodies are present in your blood serum, hence the term “serotype.” The term serotype implies that the antibody is of the type that recognizes a particular antigen. When antibodies of the correct serotype come in contact with their specific antigen they stick together in a wad called an immune complex. The process of immune complex formation can be detected using various methods, most of which fall into the categories of precipitin or agglutination tests. In either case, immune complex formation is an important diagnostic tool, an example being the rapid strep test.

The sensitivity of these tests can be increased by marking or labeling the antigen (or monoclonal antibody) with colored or radioactive compounds so that the reaction is easier to detect. Such tests include fluorescent antibody (FAB), radioimmuno assays (RIA or a variation known as RAST) and enzyme linked immunosorbent assays (ELISA).

Lancefield groups of Streptococci are designated A through T. Group A includes  hemolytic Streptococcus species (S. pyogenes) which are pathogenic to humans and cause most of the conditions listed at the top of the last paragraph. Group B Strep (S. agalactiae) are mainly problematic in bovine infections but also inhabit the lower gastrointestinal tract and female genital tracts. Here, the organisms can cause septicemia and meningitis in newborns infected as they pass through the colonized birth canal, a condition called "neonatal meningitis." The remaining  hemolytic human pathogenic Streptococci fall under groups C and G. Although they occasionally cause pyogenes-like illnesses, they are much less prominent. “Enterococci” (Streptococcus faecalis) fall under group D. Enterococci are tough! They are resistant to high concentrations of detergents, bile, and beta-lactam antibiotics (-cillins and cephalosporins). Fortunately, enterococci are weakly virulent. They can however cause terminal endocarditis in elderly patients and immune compromised patients. Most serious enterococcal infections are nosocomial, and are caused by a group of organisms called "vancomycin resistant enterococci" (VRE). If treatable at all, VRE can only be killed using combinations of antibiotics. Some Streptococci have variable cell wall constituency and are not groupable, an example being S. pneumoniae.