EXPERIMENT 15:
EXAMINATION OF A CLINICAL SPECIMEN FOR
STAPHYLOCOCCI
Unknown swab from boil – per student
1 Trypticase Soy Broth – per student
1 BAP – per student
Gram stained direct smears of boil swab (from instructor)
1 tube plasma – per student
Materials:
Quantitation of PMN, RBC, Monos and bacteria in clinical smears
+
++
+++
++++
Scant or occasional
Few
Moderate
Many
1-2 per several oil immersion fields (o.i.f.)
< 5 per o.i.f.
5-10 per o.i.f.
> 10 per o.i.f.
Procedure:
I.
Examination of Direct Smears (Have you set up Koehler on your microscope?)
Examine already prepared Gram stained smears of your swab (from instructor).
Describe in detail the appearance of the smears using the key above.
Do not look at one field and assume that the rest of the slide is the same as that field.
You must look at 10-15 fields.
Results of Direct Smear to be recorded on p 4-11. Use abbreviations.
Clean oil from the permanently mounted direct smears and replace in tray provided. Do
not discard any permanently mounted smear! Give back to instructor.
II.
Cultivation
(Label BAP with Unknown number near white spot and date at bottom. Label TSB with
unknown number.) Inoculate the unknown dry swab into a tube of TSB, express excess
liquid and roll onto a BAP near top edge or white spot. Streak to obtain isolated colonies
using quadrant method. Incubate for 18-24 hours at 37C, both the TSB and BAP.
Next day:
1. Examine and record description of the isolated colonies on the BAP on p 4-11.
Quantitate growth from 1+ to 4+, depending on how many quadrants have growth
because was cultured from a clinical specimen, i.e., swab. Your grade from the plate
should be close to Direct Smear (DS) grade.
2. Make smears of an isolated colony and the broth culture. Use eyepiece to select a colony
with a sterile straight wire (see p 1-11) and use a loopful of broth culture.
3. Stain by Gram's method and examine microscopically. Record results on p 4-11. Make
certain that morphotypes in the broth culture correlate with morphotypes on direct smear
of isolated colony.
4. Complete identification of the isolate(s) using appropriate tcatalase and slide coagulase).
Record results on p 4-11.


If the organism is a catalase positive and slide coagulase/latex agglutination positive,
it can be reported as Staphylococcus sp or presumptive Staphylococcus aureus
without performing additional tests. Positive tube coagulase would confirm Staph.
aureus identification.
If catalase + , but slide coagulase – , report as Staphylococcus species.
Record results: Boil Swab
Name:
Direct smear:
TSB:
Bap:
Quantity (1+ to 4+)
Genus
Species
__________
*Write final identification in full, no abbreviations.