Blood agar plates contain a typical nutrient growth medium enriched with 5% sheep blood. It is useful for encouraging growth of fastidious organisms. It also can show results of hemolytic enzymes produced by some organisms. If an organism grows on blood agar and produces beta-hemolytic enzymes (those which hemolyze red cells and completely break down hemoglobin) there will be a clear zone around the colony after incubation. If the organism is known as alpha-hemolytic (capable of partial breakdown of hemoglobin) there will be a green zone around the colony following incubation.
Mannitol salt agar is a selective medium for isolation of pathogenic staphylococci. Growth of most bacteria other than staphylococci (halophilic) is inhibited by the high concentration of salt in the medium. Pathogenic staphylococci fermenting in the mannitol sugar turns the phenol red indicator bright yellow. Non-pathogenic staphylococci produce small colonies surrounded by red or purple zones.
PREPARATION AND MATERIALS
Provide a TSA 5% Sheep Blood agar plate and a mannitol salt agar plate for each student, as well as sterile cotton swabs and sterile water.
STUDENT INSTRUCTION
THROAT FLORA:
Have a partner take a throat culture with a sterile cotton swab. The swab must go to the back of the pharynx quickly, not touching the tongue or the salivary secretions. Rub gently and withdraw. The gag reflex is expected. Streak the sample for isolation on blood agar. The instructor will demonstrate the simple but effective “candle-jar” incubation technique which provides a higher CO2 concentration and less oxygen (compatible with the pharynx flora requirements). Observe after 48-hour incubation and do a Gram stain on the selected colonies.
Typical flora of the pharynx is alpha-hemolytic Streptococcus viridans which grows best in greater than normal CO2. A few class members may represent those individuals which are beta-hemolytic Streptococcus pyogenes carriers. They do not show inflammation, but harbor this true pathogen. S. pyogenes is often implicated in infectious pharyngitis (“strep throat”).
SKIN AND NASAL FLORA:
Use a grease pencil to mark a mannitol salt plate into two sections. With a cotton swab moistened in sterile water, sample the skin of the forearm, and streak on one-half of the plate. (Be sure to label correctly). With another swab, take a sample from the nasal cavity to streak on the other area of the dish. Incubate 48 hours, observe and Gram stain selected colonies.
Most people have Stapylococcus epidermidis as normal flora on skin and
in the nasal cavity. Some people harbor Staphylococcus aureus in
these areas. Strains of S. aureus can be responsible for various
disease patterns, from purulent skin lesions to exotoxic food poisoning.
NOTE: In a heath care career, a worker is often asked to take samples
(specimen) from a patient and prepare them for transport to the laboratory for
diagnostic testing. It is critical that the specimen be taken and treated
appropriately so that the lab work will be valid. Much emphasis will be
placed on such techniques in the clinical courses of the health care curriculum.