Practical work

1. Determination of Klebsiella pneumoniae virulence.

Bacterial virulence (the degree of pathogenicity) may be determined by biological examination. We usually use mice, rats, rabbits, guinea pigs, and other animals for this purpose. The experimental animals can be infected via different routes: intramuscularly, intravenously, pernasaly, intraperitonealy, etc.

To determine K.pneumoniae virulence the following experiment was performed.

Groups of experimental mice have been infected intraperitoneally with different doses of K.pneumoniae suspension. (The series of ten- fold bacterial dilutions have been used). Mice’ survival has been noted after an appropriate period of time. Then the internal organs of dead mice (spleen, liver, etc.) were examined microscopically and bacteriologically to determine the presence of K.pneumoniae. (The presence of microbe in the internal organs is a result of generalized infection, so the causative agent responsible for the mice’ death is detected).

The results are shown in Table 11-2.

TASK. Determine the LD₅₀ for K.pneumoniae tested. To make calculations use the formula:

lg LD50 = lg DN – δ ( ∑ Li – 0,5 )

D_N - the largest dosage used for infecting animals

δ – Logarithm of ratio of the second dosage to the first one

Li – the ratio of the dead mice number to the total number of infected mice (in each group)

∑Li – the sum of Li for all groups

0,5 – coefficient

Table 11-2

Determination of k.Pneumoniae virulence

Group #	Dose (microbial cells/ml)	Number of infected animals (in a group)	Number of dead animals (in a group)	Li	∑Li	Micro scopy	Bacteriological examination
1	10 9	17	17			Incapsulated gram-negative rods	The microbe isolated from the internal organs of dead mice was identified as K.pneumoniae
2	10 8	17	16
3	10 7	17	14
4	10 6	17	12
5	10 5	17	10
6	10 4	17	9

2. Determination of bacterial virulence factors:

1. Hemolysins (hemolytic activity)

Virulent strains of staphylococci, streptococci, etc. form zones of α - or β-hemolysis around the colonies on blood agar. (α –hemolysis is characterized by appearing of greenish zones around the colonies on blood agar, as a result of methemoglobin production. β -hemolysis is characterized by production of the clear zones around the colonies on blood agar).

• Notify if the microbe tested can produce hemolysins. Make a conclusion about the type of hemolysis.

2. Coagulase activity

Virulent strains of staphylococci possess the ability to coagulate plasma. The isolated pure culture is introduced into a test tube with rabbit citrate plasma and then incubated at 37 °C.

It usually takes about 2 hours to coagulate the plasma in the tube and you can see the plasma clot inside the tube.

• Note the presence (absence) of the plasma clot inside the tube.