Alkaline agar for the isolation of Vibrio cholerae. Culture media

Is is a fine, hygroscopic and photosensitive yellow powder.

The set of reagents should ensure the growth of test stamps Vibrio cholerae cholerae 01 group P-1 (145), Vibrio cholerae eltor 01 group M-878 (890), Vibrio cholerae non 01 P-9741 when inoculating 0.1 ml of microbial suspension of each test -strain after 12-14 hours of incubation at a temperature of 37oC in the form of smooth, translucent colonies with a bluish tint in transmitted light. with a diameter of at least 1.0 mm.

Designed for isolating the pertussis microbe from infected material and culturing strains.

Compound: pancreatic casein hydrolyzate, sodium chloride, sodium carbonate, anhydrous disodium phosphate, feed yeast extract, microbiological agar.

Preparation: the nutrient medium in the amount indicated on the label is thoroughly mixed in 1 liter of distilled water, boiled for 2-3 minutes, stirring occasionally, until the agar is completely melted. Filter through a cotton-gauze filter, pour into vials and sterilize by autoclaving for 20 minutes at a temperature of 120oC.

Description 10733-00005

Nutrient medium Agar alkaline description

Nutrient medium alkaline agar- a dense nutrient medium for the isolation of Vibrio cholerae.
High-quality composition, quite nutritious and promotes the rapid formation of Vibrio colonies, while sodium pyrosulfite partially suppresses the accompanying microflora.
The selective factor is an increased pH level, which ultimately suppresses the development of foreign microorganisms and does not affect the growth of the cholera pathogen.

Nutrient medium Alkaline agar technical characteristics

The selective factor is an increased pH level, which ultimately suppresses the development of foreign microorganisms and does not affect the growth of the cholera pathogen.
Cultivation is carried out at 37 ° C for 12-14 hours.

Alkaline Agar composition :
Pancreatic hydrolyzate of fish meal, dry enzymatic peptone, yeast extract, glucose, disodium phosphate, sodium chloride, sodium metabisulfite, sodium carbonate, agar.

Alkaline Agar preparation:
The drug in the amount necessary for the preparation of a specific series of nutrient medium is mixed in 1 liter of distilled water, boiled until the agar is completely melted, filtered through a cotton gauze filter, poured into bottles and sterilized by autoclaving for 20 minutes at temperature
(121±1) °C.
The prepared nutrient medium is transparent and yellow in color. Slight opalescence is allowed.
pH 8.0±0.2
The prepared medium can be stored for 10-14 days at a temperature of 2-8 °C in a place protected from light.

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Vitamins in precisely prescribed dosages. They use amino acids as nitrogen sources. The advantage of these media is that they have a constant composition; they can be used to determine the needs of microbes for certain nutrients.

Solid nutrient media are prepared from liquid media with the addition of a sealant. Agar-agar is usually used as a sealant. Agar-agar- a product obtained from seaweed, which is a yellowish powder or plates, contains high molecular weight polysaccharides, is not broken down by most microorganisms, is not destroyed during autoclaving, does not change the nutritional value of the media, and does not suppress the growth of microbes. For immunological and bacteriological fields, frozen, clarified agar is used, which, when boiled or autoclaving a mixture of powder and water, melts at a temperature of 85-100°C, and when cooled to 45-48°C forms a gel.

To prepare dense nutrient media, agar-agar is added in a concentration of 1.5 to 3%.

Simple environments.

Meat-peptone broth (MPB) is the protein basis of all media.

There are several ways to prepare MPB:

a) in meat water with the addition of ready-made peptone - this is the so-called meat-peptone broth;

b) on the digestion of hydrolysis products of raw materials using enzymes ( trypsin- Hottinger broth, pepsin- Martin's broth).

They are convenient for transportation, can be stored for a long time, relieve laboratories from the enormous process of preparing media, and bring them closer to resolving the issue of media standardization. The medical industry produces dry media Endo, Levin, Ploskirev, bismuth sulfite agar, nutrient agar, carbohydrates with a BP indicator and others.

Thermostats

Thermostats are used for cultivating microorganisms.

Thermostat- This is a device in which a constant temperature is maintained. The device consists of a heater, chamber, double walls, between which air or water circulates. The temperature is regulated by a thermostat. The optimal temperature for the reproduction of most microorganisms is 37°C.

Method for preparing plate agar

MPA is melted in a water bath, then cooled to 50-55°C. The neck of the bottle is burned in the flame of an alcohol lamp, the Petri dishes are opened so that the neck of the bottle fits in without touching the edges of the dish, 10-15 ml of MPA is poured in, the lid is closed, the dish is shaken so that the medium is evenly distributed, and left on a horizontal surface until it hardens. After drying, plate agar plates are stored in the cold.

Loop sowing

Using a sterile cooled loop, take a drop of material, open one edge of the cup with your left hand, bring the loop inside and make a few strokes in one place with a loop at the opposite edge, then tear off the loop and inoculate the material in parallel strokes from one edge of the cup to the other with an interval of 5-6 mm. At the beginning of sowing, when there are a lot of microbes on the loop, they will give confluent growth, but with each stroke there are fewer and fewer microbes on the loop, and they will remain solitary and produce isolated colonies.

Sowing according to the Drigalsky method

This method is used when inoculating material heavily contaminated with microflora (pus, feces, sputum). To sow using the Drigalsky method, take a spatula and several cups (3-4). Putty knife- this is an instrument made of metal wire or glass dart, bent in the shape of a triangle or L-shape. The material is introduced into the first cup with a loop or pipette and evenly distributed with a spatula over the surface of the medium; with the same spatula, without burning it, the material is rubbed into the nutrient medium in the second cup, and then in the third. With such sowing, the first cup will have confluent growth, and isolated colonies will grow in subsequent cups.

Isolation of pure culture according to Shchukevich

For sowing, take a freshly prepared nutrient medium with condensate. The test material is taken with a loop and introduced carefully, observing the rules of asepsis, without touching the medium and walls, into the condensation water. Bacteria with high mobility “crawl” onto the wet surface of the slanted agar.

As a result of independent work, the student should know:

1. Classification, morphology of mushrooms, their methods of studying.

2. Classification and morphology of actinomycetes.

3. Rules of anti-epidemic regimes and safety precautions.

Be able to:

1. Differentiate microorganisms using microscopy.

2. Microscope the stained preparations.

3. Disinfect the material, treat your hands with disinfectants.

4. Prepare preparations from pure cultures.

Blood agar for CAMP test. To 2% nutrient agar, pH 7.4-7.6, add 0.2% glucose and sheep or large red blood cells cattle. Red blood cells from citrated blood are washed three times with isotonic sodium chloride solution to remove antihemolysin that may be contained in the serum, then they are resuspended in the same solution to the original blood volume. Add 5% red blood cell suspension to the agar.

Bile-alkaline agar (hereinafter referred to as BAL). Dry nutrient agar - 35.0 g; yeast autolysate - 20.0 ml; glucose - 10.0 g; distilled water - 600 ml. The agar is melted, filtered, fresh bovine bile - 400.0 ml and sodium carbonate - 5.0 g are added.

Sterilize the prepared medium by autoclaving at 112 0 C for 15 minutes. Before pouring into cups, 50.0 ml of fresh blood (sheep, rabbit or human), 12.5 ml of a 0.01% crystal violet solution and 20 ml of potassium hydroxide solution (hereinafter referred to as KOH) are added to the medium.

Milk with methylene blue. Fresh milk is brought to a boil, left for a day, removed from the cream, boiled a second time, left again for a day and the top layer is removed a second time. The skimmed milk is filtered through a thick layer of cotton wool and alkalized with a 10% sodium carbonate solution to pH 7.2. To 100 ml of milk add 2.0 ml of 1% aqueous solution methylene blue. The medium prepared in this way is poured into 5.0 ml into sterile test tubes and sterilized with flowing steam for 3 days for 30 minutes.

Enterococcal differential diagnostic medium (EDDS). Dry nutrient agar - 35 g, yeast autolysate for preparing nutrient media - 20 ml, glucose - 10 g, distilled water - 800 ml. Melt when heated, filter, sterilize at 112 0 C in an autoclave for 15 minutes, pH 7.2-7.4.

Before pouring into cups, TTX - 0.1 g is added to the nutrient medium; aqueous solution of crystal violet 0.01% - 12.5 ml; nalidixic acid - 0.1 g; sterile skim milk, heated to 44-45 0 C - 200 ml; fresh defibrinated blood (animal or human) - 50 ml. The contents are thoroughly stirred and poured into 7 ml cups.

Sugar-yeast nutrient agar with potassium tellurite. Dry nutrient agar - 35 g, yeast autolysate - 20 ml, glucose - 10 g, distilled water - 1000 ml. The prepared mixture is melted when heated, sodium citrate is added - 5.0 g. The medium is sterilized at 112 0. C for 15 minutes, pH 7.2-7.4. Before pouring the medium into sterile cups, add 50 ml of horse serum, 0.1 g of nalidixic acid and 0.7 g (35 ml of a 2% aqueous solution) of potassium tellurite and mix thoroughly.

Nutritious broth with glucose. To 100 ml of nutrient broth, pH 7.2-7.4, add 0.2 g of glucose.

The prepared medium is sterilized in fractions for 3 days in a row for 20 minutes or once for 15 minutes at 0.5 atm. in an autoclave.

5% blood agar. To 100 ml of 2% nutrient agar, melted and cooled to 45 degrees. C, pH 7.4-7.6, observing the rules of asepsis, add 5 ml of defibrinated lamb, horse or rabbit blood. The mixture is thoroughly mixed and poured into cups in a layer of 3-4 mm.

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