Topic P02: Diagnostics of streptococci Table for major results of Task 1 to Task 5 (to be filled step by step): Strain K L M N P Q R S Gram stain – Task 1 Catalase test Task 2a Slanetz-Bartley medium – Task 2b Culture (blod agar) Task 3 Size Colour Shape Profile Agar changes Other PARTIAL CONCLUSION Task 4a: Optochin (viridans strep only) 4b: STREPTOtest 16 (oral strep only) Task 5a: PYR test (haem. strep only) Task 5b: CAMP (haem. strep only) FINAL CONCLUSION Task 1: Microscopy of suspicious strains There are letter-labelled strains on the table. Gram-stain them and assess, which one is NOT a gram-positive coccus. To avoid confusion, label the slides using a marker. Write your results to the table. Task 2: Basic culture and biochemical tests – genus determination a) Catalase test for differentiation of staphylococci Perform catalase test with all strains from Task 1 with exception of the strain proven not to be a G + coccus. Staphylococci should be catalase positive, streptococci and enterococci should be catalase negative. b) Growth on Slanetz-Bartley agar for differentiation of enterococci On the agar plate you have inoculated all strains, each in one sector. Nevetheless, only one of them is growing. This one should be an Enterococcus – so it is not a streptococcus. Write the results of 2a and 2b to the table. Note: The same thing can be done with Bile-aesculin medium, too, but the colour of colonies is different. Task 3: Blood agar culture On plates with blood agar, once more, you have all strains. Observe all of them, but describe only the strains that were not excluded by tasks 1 and 2. Describe colony morphology, and especially the haemolysis, partial haemolysis or viridation. Write your findings to the table. Now write „partial conclusion“ to your table. To each strain K to S, write „NO STREP“ (no streptococcus) „HAEM STREP“ (partial or total haemolysis) or „VIR STREP“ (viridation) Task 4: More detailed diagnostics of streptococi with viridation a): Optochin test Your task is to evaluate the result of optochin test in two strains shown to be streptococci with viridation. Optochin test is a test, that is not different from a normal diffusion disc test; the only difference is that the effective drug (optochin) is no more used for treatment. Strain with presence of inhibition zone around disc is S. pneumoniae, strain without zone is an „oral streptococcus“. Draw your result, and write „+“ or „–“ to the table. b): Biochemical determination of the „oral“ streptococcus In strain, found in Task 4a to be an „oral streptococcus“ evaluate the results of a biochemical microtest (STREPTOtest 16), using approaches known from summer semester. Tube First row with 8 wells Second row with 8 wells VPT 1H 1G 1F 1E 1D 1C 1B 1A 2H 2G 2F 2E 2D 2C 2B 2A 1 2 4 1 2 4 1 2 4 1 2 4 1 2 4 1 2 Code: Identification Streptococcus ___________ % of probability T index Task 5: Diagnostics of streptococci with partial or total haemolysis This task will be done with three strains proven to be streptococci with beta-haemolysis (parts a, b); the last part will be only done with one of them proven to be „non-A-non-B“. a): PYR test PYR test is a strip-test, simillar to the oxidase test. For reading the colour result it is necessary to wait about five minutes, then add a drop of „Reagent for PYR test“ and wait another 30 s. Positive reaction is the red colour of the reaction zone. This strain is again positive in S. pyogenes (and in Enterococcus, too). Negative result can be seen in S. agalactiae and in non-A-non-B streptococci. Note: Formerly Bacitracin test was used inetead of PYR-test. Its principle was identical with that of optochin test, only with another antibiotic. Unfortunatelly, it is not specific enough, so we do not use it any more. Fill the following table, including drawing a result of PYR test in all three tested strains. Strain (write the letter) Strain (write the letter) Strain (write the letter) Interpretation: negative – positive Interpretation: negative – positive Interpretation: negative – positive b): CAMP test. Note: This test has nothing to do with cyclic adenosinmonofosfate, therefore it is CAMP test and not cAMP test. Its name is derived from names of its inventors. CAMP test is based on haemolytical synergism between S. aureus beta-haemolyzin producing strain, and S. agalactiae strain. Positive result has the form of two triangular zones („butterfly shape“) of complete hemolysis at crossing of both two strains. A small zone of another shape is considered negative. Draw your result (picture is on the following page): c)Agglutination test for detailed diagnostics of mainly non-A-non-B streptococci Task 6: Antibiotic susceptibility tests in streptococci Evaluate the susceptibility tests (diffusion disc tests) for antibiotics in the strains of S. pyogenes and S. agalactiae. To the table, write the abbreviations of antibiotics and for all tested strains measure the susceptibility zone in mm. On the card, you can see the borderline values of diameters – according to them, interprete the zones as susceptlible (S), resistant (R) or dubious (D). Strain à Antibiotic (full name) Zone Æ (mm) Interpr. Zone Æ (mm) Interpr. Task 7: Diagnostics of late sequellae of streptococcal infections – ASO determintion On a side table in a wet chamber you will find a microtitration plate. It includes a positive control, sera 1 to 5. Determine the titers and interprete the risk for pacients. (For precise instructions, see the spring term – J08)