1. With your marking pencil, divide the bottom of a BAP and an MSA plate into four segments each.
2. Using the grown broth cultures, inoculate one section of each plate with S. aureus, one with S. epidemidis, one with S. saprophyticus, and one with E. coli. Streak each section carefully, remaining within the assigned space.
3. With heated and cooled forceps, pick up a novobiocin disk, place it in the center of one of the streaked areas of the BAP, and press it gently onto the agar with the forcep tips.
4. Repeat step 3 for the remaining three organisms on the streaked BAP.
5. Place the plates in the 35°C incubator for 24 hours.
6. Perform a coagulase test on each of the three Staphylococcus broth cultures as follows:
a. Using a sterile pipette, measure 0.1 ml of the S. epidemidis broth culture with the aspiration device. Transfer this inoculum to a tube of plasma. Discard the pipette in disinfectant. Label the tube.
b. Inoculate a second and third tube of plasma with 0.1 ml of the S. aureus and S. saprophyticus broth cultures, respectively, as in step 6a.
c. Place all inoculated plasma tubes in the 35°C incubator. After 30 minutes, remove and examine them (close the incubator door while you read them). Hold the tubes in a semihorizontal position to see whether the plasma in the tube is beginning to clot into a solid mass. If so, make a record of the tube showing coagulase activity. Return unclotted tubes to the incubator.
d. Repeat procedure 6c every 30 minutes for 4 hours, if necessary.
7. After 24 hours of incubation of the plate cultures prepared in procedures 1 and 2, examine and record colonial morphology. Make Gram stains of each culture on the BAP and record microscopic morphology. Measure and record the diameter of the zone of inhibition around the novobiocin disks. A zone size greater than 12 mm in diameter is considered susceptible.
8. Following the instructor's directions, place one drop of the latex agglutination reagent onto each of two circles on the card provided. With the special stick contained in the kit or a sterile inoculating loop, pick up several colonies of S. aureus from the blood agar plate you inoculated at the previous laboratory session. Emulsify the colonies in the latex reagent, being careful not to scratch the card. Repeat this procedure with colonies of S. epidemidis. Do not use colonies from the mannitol agar plate as these are difficult to emulsify.
9. Rotate the card gently for 20 seconds, observing the circles for a clearly visible clumping of the latex particles and a clearing of the milky background (see fig. 20.1). This reaction signifies a positive test. Record the results in the chart, then dispose of the reaction card in the disinfectant provided.
Morello-Mizer-Granato: Laboratory Manual and Workbook in Microbiology, 7/e
III. Diagnostic Microbiology in Action
Was this article helpful?