Nutrient medium for determination of toxigenicity of diphtheria microbes dry (Korinethoxagar) 

1. PURPOSE

"Corinethoxagar" is intended to determine the toxigenicity of diphtheria microbes in the diagnosis of infectious diseases "in vitro".

2. KIT CHARACTERISTIC

"Corinethoxagar" is a mixture of dry components in the form of a finely dispersed hygroscopic powder of light yellow color.

Produced in polyethylene cans for 250 g.

2.1. OPERATING PRINCIPLE

The totality of the components included in the kit provides nutritional requirements for the growth of all types of corynebacteria and the production of toxin by toxigenic strains.

The method of determining the toxigenicity of the corynebacteria of diphtheria is the process of counterimmunodiffusion of toxin and antitoxic antibodies in a dense nutrient medium. In places of the optimal quantitative ratio between the toxin produced by measles-non-bacteria and the antitoxin deposited on the filter paper, they interact with the formation of precipitate in the form of a white line or whiskers.

2.2. COMPOSITION OF THE KIT

"Korinethoxagar" is a mixture of dry components at the rate of g / l:

Pancreatic pollock hydrolyzate .................. 20.0

Sodium chloride …….............................................. . ... ... 5.0

Sodium carbonate ................................................ ......... .. 0.6-0.1

Agar microbiological .............................. .. 10.0 ± 3.0

3. ANALYTICAL AND DIAGNOSTIC CHARACTERISTICS

 "Corinethoxagar" provides on all seeded Petri dishes the growth of all types of measles-non-bacteria and toxin formation by toxin test strains of Corynebacterium diphtheriae gravis 665, Corynebacterium diphtheriae mitis 6765 and Corynebacterium diphtheriae intermedius 619, manifested in the formation of precipitation lines after 22-24 h of incubation at temperature (371) C when viewing crops with a magnifying glass in transmitted light, against a dark background. After 44-48 hours of incubation at a temperature of (371) C, the precipitation lines become more compact and should be visible to the naked eye.

The growth of non-toxic strains of Corynebacterium diphtheriae gravis nontoxigenic 4895, Corynebacterium diphtheriae mitis nontoxigenic 3689 and Corynebacterium diphtheriae intermedius nontoxigenic 7227 on all seeded dishes is not accompanied by the formation of precipitation lines.

4. PRECAUTIONS

Compliance with the "Rules of the device, safety, industrial sanitation, anti-epidemic regimen and personal hygiene when working in the laboratories (offices, departments) of sanitary-epidemiological institutions of the Ministry of Health" (Moscow, 1981).

5. EQUIPMENT AND MATERIALS

• Thermostat providing a temperature of 37 ± 1 ° C

• Laboratory scales of 2 classes of accuracy

• Autoclave

• Glass tubes with a capacity of 10 ml

• Glass pipettes allowing to take volumes of liquid 1 and 2 ml

• Cylinder glass measuring capacity of 1000 ml

• Petri dishes are sterile

• Distilled water

• Flasks

• Funnels made of glass

6. ANALYZED SAMPLES

Objects of research in sanitary and clinical microbiology.

7. PREPARATION FOR ANALYSIS

Preparation of "Korinethoxagar".

The preparation in the amount indicated on the label for the preparation of a specific series of media, stir in 1 liter of distilled water, boil for 3 minutes until the agar is completely dissolved. The medium is dispensed into sterile bottles in a regular manner, sterilized by autoclaving at a temperature of 110 ° C for 30 minutes. After autoclaving and cooling to 45-50 ° C, 20% of the blood serum of cattle or horse normal serum is added, mixed and poured into sterile Petri dishes.

The prepared medium in Petri dishes is transparent, yellow in color.

Before applying serum, the medium can be used for 14 days at a storage temperature of 2-8 ° C. The medium with serum can be used for 5 days at a storage temperature of 2-8 ° C.

Preparation of paper strips.

Strips of filter paper with a size of 80x15 mm are wrapped in paper bags of 3 pieces and sterilized by autoclaving at a temperature of 120 ° C for 30 minutes. Paper strips with sterile tweezers are placed in a sterile Petri dish and moistened with diphtheria antitoxin, previously dissolved in 1 ml of sterile distilled water or sterile 0.9% sodium chloride solution (0.25 ml of diphtheria antitoxin solution is wetted on one paper strip). The moistened paper is transferred by sterile forceps to the surface of the nutrient medium in a petri dish. The cups are dried in an open form upside down in a thermostat at a temperature of (371) С for 15-20 minutes.