Normal Flora Staphylococci

FERMENTATION ON MANNITOL SALT AGAR (MSA)
MSA is a selective medium for the recovery of staphylococci from mixed cultures. This medium takes advantage of the ability of staphylococci to grow in the presence of 7.5% NaCl and differential fermentation of mannitol. A pH indicator (phenol red) is incorporated into the agar. The agar color turns from deep pink to yellow in the presence of acidic fermentation products. The test is diagnostic for S. aureus: growth positive + fermentation positive = Staphylococcus aureus.
MATERIALS:
• MSA plates
• Pure cultures from the previous lab period, one confirmed Staphylococcus aureus by latex agglutination; one Staphylococcus culture confirmed NOT to be S. aureus by latex agglutination. Both should be confirmed Gram-positive by staining.
Streak the two Staphylococcus cultures on opposite sides of an MSA plate. Incubate at 37oC and observe agar appearance after 24 hours. DO NOT INCUBATE FERMENTATION TESTS IN THE PRESENCE OF CO2, BECAUSE THE COLOR CHANGE WON'T WORK.
CATALASE TEST
Discriminates between Staph and Streptococcus species. The basis for the test is:
H202 + catalase = H20 + 02 (bubbled off)
MATERIALS:
• Pure cultures from the previous lab period, one confirmed Staphylococcus aureus by latex agglutination; one Staphylococcus culture confirmed NOT to be S. aureus by latex agglutination. Both should be confirmed Gram-positive by staining.
• Glass microscope slides
• 1 tube containing 3% H202
PROCEDURE:
1. With an inoculating needle, pick the center of an 18 to 24 hr pure colony and place on a clean glass slide.
2. Test is not reliable if blood agar is introduced into the H202.
3. Add a drop of 3% H202 to cover the organism or slide.
4. Observe for immediate bubbling (gas liberation); record the results.
COAGULASE TEST
The laboratory test based on detecting the production of the enzyme coagulase by Staphylococcus species is used to differentiate between staphylococci and streptococci, as well as between coagulase-negative staphylococci and Staphylococcus aureus. Coagulase is secreted extracellularly by the bacteria and reacts with the coagulase-reacting factor present in plasma. Clot formation in the plasma is mediated by the complex formed in this reaction.
MATERIALS:
• 2 tubes of citrate plasma
• Microscope Slides
• Pure cultures from the previous lab period, one confirmed Staphylococcus aureus by latex agglutination; one Staphylococcus culture confirmed NOT to be S. aureus by latex agglutination. Both should be confirmed Gram-positive by staining.
1. Pick a colony of putative staphylococci using the inoculating needle. Emulsify the colony in citrate plasma. Repeat for a second organism (one not suspected to be a staphylococcus).
2. Cover the tube and incubate for 4 hours at 37oC. Observe for clot formation by gently tipping the tube.
3. If no clot has formed, incubate the tube again for 18 hours and observe. Weak coagulase producers may require this prolonged incubation period to develop clots.
HEMOLYSIS ON BLOOD AGAR (BAP)
MATERIALS:
• BAP plates
• Pure cultures as above, one confirmed Staphylococcus aureus by latex agglutination; one Staphylococcus culture confirmed NOT to be S. aureus by latex agglutination. Both should be confirmed Gram-positive by staining.
Streak the two organisms on opposite sides of the BAP plate. Incubate the plates for 24-48 hours at 37oC in the presence of a 5% CO2 atmosphere. Observe the zone of color change around the colonies.
Lectures on applied clinical microbiology