University of California, Santa Cruz Student Health Center THROAT CULTURES Method: Determination of presumptive presence or absence of Group A beta-hemolytic streptococci through use of a blood agar plate and bacitracin disc. Materials: Blood agar plate (BAP) (Hardy Diagnostics), sterile Bacitracin Disc, 0.04 unit (Becton, Dickinson and Co.) Inoculating Loop (Fisher Scientific) Disposable Inoculating Loop (Fisher Scientific) Bacti-Cinerator sterilizer Sterile Cotton or Dacron Swabs (McKesson or Fisher) Specimen: Throat exudate collected on a sterile Dacron swab. Specimen must be labeled preferably with a registration sticker, or last, first name and registration number. Procedure: 1. Cultures are collected with a cotton or polyester swab from posterior pharynx, tonsils, tonsillar fossae, or areas of inflammation and exudate, avoiding the lips and tongue. 2. The swab is placed back into the paper packet and transported to the lab. The paper packet is labeled with a patient registration sticker or with patient name and ID number. Specimens without patient identification will be rejected. 3. In the laboratory, the specimen is set up according to the throat culture inoculation procedure. Specimen can be up to 4 hours old without preservatives. 4. Roll swab over top 1/4 of (BAP). Sterilize inoculating loop using Bacti-Cinerator. Hold loop inside core for 5 - 10 seconds and allow to cool completely. Alternatively, a disposable loop may be used. 6. Starting at the top portion of the primary inoculum, streak the inoculum out into 3 more quadrants. Use loop to place two cuts into agar; one in the primary area and one in the first quadrant. Refer to the throat culture inoculation procedure for more information. 7. Using disc applicator, place bacitracin disc (Taxo A) over the primary inoculum. Gently press disc onto agar. This will aid in the detection of presumptive Group A beta-hemolytic streptococci which are sensitive to bacitracin and grow outside the zone of inhibition created by the disc diffusing into the agar. 8. Incubate plate at 36 2 C for 18-24 hours. Exceptions: On Saturdays before normal Mondays(not holidays) cultures will incubate 48 hours and be read on Monday; On Fridays during school breaks throat cultures will be sent to Quest. A Rapid Strep test may be substituted as necessary. Interpretation: 1. A macroscopic reading of the culture plate is usually sufficient. A magnifier is available if necessary. DIRECTOR REVIEW / DATE DIRECTOR REVIEW / DATE DIRECTOR REVIEW / DATE REVISED / DIRECTOR REVIEW / DATE REVISED / DIRECTOR REVIEW / DATE HC LAB (8/08) tc_cult / bacti proc University of California, Santa Cruz Student Health Center THROAT CULTURES -2- 2. Observe plate for beta-hemolysis with a zone of inhibition (no growth, no hemolysis) around bacitracin disc of at least 9 mm, (Bacitracin disc size = 7mm). This is indicative of presumptive Group A beta-hemolytic streptococci. After confirming that catalase test is negative, report out as "(Few, Moderate, Many)Presumptive Group A beta-hemolytic streptococci found by bacitracin." 3. If beta-hemolysis is observed with typical streptococci morphology (see following) but with no zone of inhibition around bacitracin disc, perform a catalase test and if negative, report as "(Few, Moderate, Many) beta-hemolytic streptococci, not Group A." A rapid strep test may be done if the area around the disc is difficult to read or if there are too few bacteria to read disc reliably. 4. Cultures containing no beta-hemolytic streptococci colonies are reported out as "No beta-hemolytic streptococci found." 5. If beta-hemolysis is observed with typical staphylococci morphology but no streptococci, perform a coagulase and catalase test. If catalase and coagulase tests are positive report out as (Few, Moderate, Many) Staphylococcus aureus. Also select from pull down menu, "No beta hemolytic streptococci found." 6. If other bacteria such as gram negative rods are found, work up and report out. 7. After final report is sent out, place all culture plates in biohazard waste container. References: Bailey and Scott, Diagnostic Microbiology, 11th edition, 2002, Mosby, Inc, St. Louis, pp. 303 - 310 and 899 - 906. Product insert, Bacitracin Discs (Taxo A) for Differentiation of Group A Streptococci, BD BBL Microbiology Systems, Becton, Dickinson and Company, 2003. HC LAB (8/08) tc_cult / bacti proc .