Topic P01: Diagnostics of staphylococci

Task 1: Microscopy of infectious material



Description (write names of objects, and connect by arrows with the objects on your picture)






In your microscope, observe Gram stained smear. Describe and draw observed object. Mention presence
of bacteria (their shape, staining and quantity) and also WBCs, epitheliae and their mutual ratio.


Table for major results of Task 2 to Task 7 (to be filled step by step):

Strain

                                                                                      K

                                                                                                   L

                                                                                                    M

                                                                                                     N

Gram stain – Task 2






Task 3

Culture (blod agar)

                   Size






                   Colour






                   Shape






                   Profile






                   Haemo- lysis





                   Other






Task 4: growth on BA + 10% NaCl





Task 5: catalase test

(write + or - )





PARTIAL CONCLUSION





Task 6a: Clumping factor test (+ / -)





Task 6b: Plasma-coagulase test (+/ -)





Task 6c: Test for hyaluronidase





FINAL CONCLUSION






Task 2: Microscopy of microbial cultures


Strain K                            Strain L                                  Strain
M                                   Strain N

Gram stain pure cultures of given organisms, labelled by letters. Draw your findings here and write
the results into the table above.

Task 3: Growth on blood agar (BA)

Fill the table for Task 3. In „Other“ write all other specific characteristics.

Task 4: Bacterial growth on BA with 10 % NaCl

Evaluate growth ability of given strains on BA with 10 % NaCl, that serves as a selective medium
for staphylococci. Write „+“ for presence of growth and „–“ for absent growth.

Task 5: Catalase test

Evaluate presence of catalase enzyme. Using microbiological loop, take several colonies of given
strains and mix them wit a drop of 3% H[2]O[2] on slide. As you already know (Topic J04), positive
reaction is characterized by


____________________________, while ____________________________ is negative. Write „+“ for and
„–“.

Now, fill the row „Partial conclusion“. Write STAPH for strains found to be staphylococci and OTHER
for strains that do not belong to genus Staphylococcus.

Task 6: Tests for S. aureus differentiation

6a) Clumping factor test (test of bound plasmacoagulase)

On the slide, a drop of diluted rabbit plasma was placed. Using microbiological loop suspend the
examinated staphylococcal strain in it. Draw your result here and write the conclusion to the
table. (Boxes for strains proven not to be staphylocci should be left empty).


Strain ______









6b) Plasmacoagulase test (test of free plasmacoagulase)

In 0.5 ml of 10× diluted rabbit plasma several colonies of examined straphylococcal strain was
suspended. The suspension is incubated in a thermostat at 37 °C. The result is evaluated after 1, 2
and 24 hours. The reaction is considered positive, when rabbit plasma in the test tube is
coagulated, i.e. the total volume of the test-tube is „gel-like“, at least partially. Write and
draw the result of this reaction for given strains after 24 h of incubation.



Negative result


Positive result


6c) Hyaluronidase detection

On blood agar, about 2 cm large band of Streptococcus equi, is inoculated. It is a capsule-forming
bacterium. In 90° direction to this band, a line of a given Staphylococcus is to be inoculated.
When the given staphylococcal strain produces hyaluronidase, it diffuses to the vicinity, and the
capsule of Streptococcus equi is lysed overnight. You can see it as half-circular zone without
„mucoidity“ in the strip of Streptococcus equi.

Draw the result of your reaction (including positive and negative reaction) and describe.
Attention! The principle of this tast has NOTHING to do with haemolysis! If you see it, you may
draw it, nevertheless it is not important for this task. Follow teacher‘s instructions and do the
task only after his/her explanation!

Task 7: Determination of staphylococci using biochemical microtest (STAPHYtest 16)

Only for Medical Microbiology students

Task 8: Susceptibility of staphylococci to antibiotics

Assess susceptibility of given strains to chosen antibiotics using diffusion disc test.
Susceptibility to given antibiotics is to be evaluated using the diffusion disc test. Evaluate
susceptibility to given antibiotics using comparison of found diameter of inhibition zone and
reference zone. Write full names of antibiotics, diameter of zones and interpretation.


                             Antibiotic (full name, not abbreviation!)

                                             S. aureus

                                          S. epidermidis

                                        Zone diameter (mm)

                                          Interpretation

                                        Zone diameter (mm)

                                          Interpretation