Independent work of students.

1. Bacterioscopy of smears of diphtheria bacillus stained according to Neisser.

2. Seeding material from the pharynx on Clauberg's medium.
3. Pisu test (determination of the enzyme cystinase).
4. Determination of toxigenicity of diphtheria culture.
5. Microbiological diagnosis of whooping cough and parapertussis
(bacteriological, bacterioscopic and serological methods)

Microbiological diagnosis of diphtheria

1. Bacterioscopy of smears of diphtheria bacillus stained according to Neisser:

The material is taken from the slant agar, placed in a drop physiological
solution on a glass slide, a thin smear is made, and it is dried in the air, fixed on the
flames and stain according to Neisser.

Diphtheria bacilli are located in the smear lean at an angle to each other in the form
of outstretched fingers. The body of the stick turns yellow, and the ends turn
darker; they are called Babesh-Ernst bodies, representing the accumulation of
reserve nutrient - volutin.

Volutin granules
2. Sowing material from the pharynx on Clauberg's medium:

Material for research (thicke only from the pharynx and nose) are taken with
sterile cotton swab. Right after taking material should produce sowing, time
between taking material and sowing should not exceed 5-6 hours.
Diphtheria bacilli grow well on Clauberg's medium will support Na, К tellurium,
Sowing put in a thermostate 370C. After 18-20 hours growth on the surface of the
medium, small colonies appear with a slight compaction in the center or in the
form of large flat colonies with a jagged edge (type gravis) or in the form of round,
convex, shiny colonies with a smooth edge (type gentle). Colonies turn black or
grey-black

Colonies of C. diphtheriae in Clauberg's medium.

 3. Pisa test (determination of the enzyme cystinase):

Produce in a column of agar with cysteine injection method then test-tube will
be placed in the thermostate at370From at 20-24 h. On the place of injection sein
blackening environment. In presence corynobacterium diphtheria around grow that
a depth of 1 cm from the surface y appears brown "cloud".

Diphtheria and pseudodiphtheria corynobacteria may cause weak blackening

of the medium in angle, no never form "cloud".

4. Determination of toxigenicity Corynbacterium diphtheria

On a cup of nutrient medium put of filter paper (0.5-6cm) impregnated with

antitoxic serum. On either side of the paper strip, perpendicular to it sow slant 2-3
round plaques at a distance of 3.6-0.8 cm from 1-1.5 cm from each other.
Crops are placed in a thermostate at 370C. The result is taken into account after 24
hours and 48 hours.
The presence of white lines (“whiskers”) indicates the image culture of the
toxin. These lines are due to the interaction of soluble antigens not associated with
exotoxin, but present in it, with antibodies to them.
5. Microbiological diagnosis of whooping cough and parapertussis
(bacteriological, bacterioscopic and serological methods)

1. bacteriological method.

Research material for whooping cough and parapertusis cough is mucus out
deep airway sand excreted by coughing.
For the taking slime swab, without touching the mucous membrane of the
tongue, enter swab over the soft palate and remove the mucus from the back of the
throat. Then seeding is done on Borde-Gengu medium (potato– glycerine before
sowing with a tampon at the surface of the nutrient media with penicillin. Cups
with sowing contain in the thermostate at 370C- 3 days. Colonies of pertussis and
parapertussis rods are smooth, convex, moist, shiny, gray in color, easily removed.

Casein Charcoal Agar Medium Bordet-Gengou

2. Bacterioscopic method.

Smear - Gram stain.

3. Serological method.

At late stage of disease at clinical unexpressed erased forms of diagnosis

whooping cough can be determined using serological methods. RA on glass - 1
drop of pertussis serum is instilled on a glass slide, pertussis serum, then a pure
culture is taken with a bacterial loop and emulsified in these drops at room
temperature.

Accounting: In which drop agglutination occurs, this species is the causative

agent.

Pure pertussis serum parapertussis

Culture