Task 1. Study the preparation from the pure cultures of Staphylococcus areus and S.epidermidis microscopically; draw them in the protocol.
Task 2. Prepare and stain by Gram method the preparation taken from the blood of the patient suspected of staphylococcal sepsis, draw it in the protocol.
Task 3. Study the growth of S. aureus and S. epidermidis in milk meat infusion agar. Fill in the protocol with their cultural properties.
Task 4. Study the growth of S. aureus and S. Epidermidis in blood meat infusion agar. Fill in the protocol with their cultural properties.
Task 5. Study the growth of S. aureus and S. Saprophiticus in egg yolk salt agar. Fill in the protocol with their cultural properties.
Task 6. Study the growth of other types of Staphylococcus spp. in citrate plasma. Draw the plasma in the test tubes before streaking of Staphylococcus spp. and after 24-hour growth.
Task 7. Study the sensitivity of the isolated staphylococcal culture to antibiotics; draw a conclusion. Write the results in the protocol.
Task 8. Study the sensitivity of the isolated staphylococcal culture to phintonside garlic and make a conclusion. Write the results in the protocol.
Task 9. Study the main antimicrobial drugs used for treatment, prevention and diagnostics of suppurative diseases. Write them in your copybook.
RECOMMENDATIONS FOR PRACTICAL WORK
All types of Staphylococcus spp. have identical staining and morphological properties. Therefore, stained by Gram method of pure culture of S.aureus, S.epidermidis in preparation it is possible to see gram-positive cocci like "clusters of vine".
Direct microscopy of the material taken from the patient with staphylococcal infection is not used. For example, microscopy of the mucus from the nose shows that staphylococci do not have classic morphology. However, in case of sepsis, microscopy of the blood makes it possible to reveal (1) Staphylococcus spp. in a shape of clusters of vine (bacterium propagates itself in the blood) and (2) erythrocytes.
S. aureus in milk meat infusion agar
S. epidermidis in milk meat infusion agar
Milk meat infusion agar is a special medium capable to expose the ability of bacteria to form a pigment. Therefore, S.aureus forms yellow colonies in this medium. That is why they are called golden staphylococci. Other types of staphylococci do not have such a pigment.
Blood agar is a special medium with the ability of bacteria to select hemolysin coming to light on. In blood agar abundant growth of the most staphylococcal species occurs within 18 - 24 hours. Only individual colonies should be picked for preliminary identification testing at this time. Since most species can not be distiguished from each other in the basis of the colony morphology within a 24-hour incubation period, colonies should be allowed to grow for at least additional 2 - 3 days before the primary isolation plate is confirmed for species or strain composition.
S.aureus in a blood agar forms round colonies in which hemolysis is formed. It occurs at the expense of allocation hemolysin by bacteria. The ability of bacteria to allocate hemolysin is one of the pathogenic signs.
S.epidermidis has no ability to synthesize hemolysins. Therefore, in round colonies there is no zone of hemolysis.
Round colonies absent zone hemolysis
Round colonies a zone hemolysis
For revealing lecitinase activity of Staphylococcus spp., cultures of bacteria grow on the special selective medium. This medium is yolk salt agar containing 8-10% of NaCl. Staphylococcus spp. tolerates to sodium chloride in concentrations of 5-10%. Salt-containing media are useful in isolating staphylococci from samples containing large numbers of other bacteria. The bacteria allocating lecitinase destroy yolk lecithin. Therefore round colonies with the turbidity zone (as enzyme diffusion on agar) are formed. Such bacterium in this case is S.aureus.
Other kinds of staphylococci, such as S.epidermidis, S.saprophyticus, do not allocate such enzyme, therefore round colonies do not form lecitovitelase zone.
Negative plasmacoagulase test
Positive plasmacoagulase test
It is known that plasmacoagulase concerns enzymes which promote invasive microbes on a macroorganism and their preservation in it. Plasmocoagulase leads to formation of the inflammatory centre round microbes. It leads to plasma coagulation that interferes them phagositosis and to action complement. For the exposure of coagulase, and coagulase is an enzyme of pathogenicity of bacteria, which rolls up plasma of blood, facilitates penetration of bacteria in tissues, utilize the reaction of plasmacoagulase. Plasmacoagulase is an enzyme that functions like thrombin to convert fibrinogen into fibrin. The ability to clot plasma continues to be the most widely used and generally accepted criterion for the identification of pathogenic staphylococci associated with acute infections.
Measure a zone of absence of growth round a paper disk, study the sensitivity of isolated staphylococcal culture to antibiotic, and make the conclusion. Put down the results in the protocol.
Staphylococcus spp. is known to be sensitive to vegetative antibiotics. In the given experience, because of diffusion phytoncide garlic in an agar the growth of inhibition zone is formed. It specifies the bactericidal action of the given vegetative antibiotics on the given culture.
Staphylococcal toxoid is a vaccine containing inactivated, which help formalin (0,4%) and temperature (40°C) exotoxin S.aureus. It is used specifically to prevent and treat staphylococcal infections.