3. Lecithinase activity

On the egg-yolk-salt agar most virulent staphylococci induce lecithin (yolk) reaction manifested in the formation of a turbulent zone with opalescent halo on the periphery around the colony.

• Examine the plate with egg-yolk-salt agar and different strains of staphylococci growth. Note the presence(absence) of the turbulent zone characteristic for lecithinase activity.

4. Capsules

Microscopic examination of the simple stained encapsulated bacteria reveals unstained capsules, which contain red bacterial cells against pink background (if stained with fuchsin).

• Examine microscopically the demonstration preparations of

• S.pneumoniae in tissues (Fuchsin Staining). They are arranged in pairs, their cells are elongated cocci and each pair of cells is surrounded with a capsule (colorless halo).

• K.pneumoniae in tissues (Fuchsin Staining). They are also arranged in pairs, but they are medium-sized encapsulated rods.

3. Phagocytosis (complete phagocytosis and incomplete phagocytosis).

• Examine microscopically the demonstration preparations of:

• S. aureus in pus (Gram Stain). Staphylococci can be observed inside and among the pus cells.

• Neisseria gonorrhoeae in pus (Methylene Blue Staining). The material (pus) for microscopic examination was taken from the urethra of a patient with gonorrhea. Neisseria gonorrhoeae appears as bean-shaped diplococci situated within the leukocyte cytoplasm. We can see dark blue leukocytes with numerous small dark-blue cocci arranged in clusters and pairs within the phagocyte.

Practical tasks

1. Determine the LD 50 for K.pneumoniae tested. Copy Table 11-2 and fill in with the results of your calculations. Make a conclusion.

2. Draw in your protocol notebook the resultant growth of S.aureus on blood agar and show the zones of hemolysis (notify if it is α- or β-hemolysis).

3. Put down in the protocol notebook the resultant growth of S.aureus on egg yolk-salt agar and show the zones of lecitinase activity.

4. Draw in the protocol notebook the results of observation of microbial coagulase activity (note the tube with liquid citrate plasma and the tube with the plasma clot).

5. Draw in the protocol notebook the results of your observations of simple stained encapsulated bacteria: K.pneumoniae and S.pneumoniae (in tissues).

6. Draw in the protocol notebook the results of microscopic examination of phagocytosis. (Complete phagocytosis of S.aureus and incomplete phagocytosis of N.gonorrhoeae).

LESSON 12

ANTIGEN - ANTIBODY REACTIONS. AGGLUTINATION TEST and PRECIPITATION TEST

Prelab conference. Topics for discussion:

1. Antigens, their nature, properties, application.

2. Bacterial antigens.

3. Antibodies (immunoglobulins). Structure and properties of immunoglobulins.

4. Classes of immunoglobulins, their characteristics.

5. Serological diagnostic tests based on the antigen-antibody interactions.

6. Agglutination test. Mechanism, components, application.

7. Indirect (passive) hemagglutination test (IHA-test).

8. Incomplete antibodies. Coomb’s test.

9. Precipitation test, mechanism, components, procedure, and application.

10. The components of serological reactions (diagnosticums, diagnostic antibody-containing antisera).

11. Pre-absorbed immune antisera. Preparation and application (Castellani technique).