Alkaline agar. Alkaline agar culture medium for isolation of Vibrio cholerae
CAMP blood agar... To 2% nutrient agar, pH 7.4-7.6, add 0.2% glucose and erythrocytes of sheep or cattle. Erythrocytes from citrated blood are washed three times with isotonic sodium chloride solution in order to remove antihemolysin that may be contained in the serum, then they are resuspended in the same solution to the initial blood volume. 5% suspension of erythrocytes is added to the agar.
Bile - alkaline agar (hereinafter-HA). Dry nutrient agar - 35.0 g; yeast autolysate - 20.0 ml; glucose - 10.0 g; distilled water - 600 ml. The agar is melted, filtered, 400.0 ml of fresh bovine bile and 5.0 g of sodium carbonate are added.
The prepared medium is sterilized by autoclaving at 112 ° C for 15 minutes. Before pouring into cups, 50.0 ml of fresh blood (ram, rabbit or human), 12.5 ml of 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, freed from cream, boiled again, left again for a day and the top layer is removed again. The milk skimmed in this way is filtered through a thick layer of cotton wool, alkalinized with 10% sodium carbonate solution to pH 7.2. To 100 ml of milk add 2.0 ml of a 1% aqueous solution of methylene blue. The medium prepared in this way is poured into sterile test tubes, 5.0 ml, sterilized with flowing steam for 3 days, 30 minutes each.
Enterococcal differential diagnostic environment (EDDS). Dry nutrient agar - 35 g, yeast autolysate for the preparation of nutrient media - 20 ml, glucose - 10 g, distilled water - 800 ml. It is melted when heated, filtered, sterilized at 112 ° 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; an aqueous solution of crystal violet 0.01% - 12.5 ml; nalidixic acid - 0.1 g; sterile skim milk heated to 44-45 ° C - 200 ml; fresh defibrinated blood (animals or humans) - 50 ml. The contents are thoroughly mixed 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 with heating, sodium citrate - 5.0 g is added, the medium is sterilized at 112 0. C for 15 minutes, pH 7.2-7.4. Before pouring the medium into sterile dishes, 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, mix thoroughly.
Nutrient broth with glucose. 0.2 g of glucose is added to 100 ml of nutrient broth, pH 7.2-7.4.
The prepared medium is sterilized fractionally 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. С, 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, poured into cups with a layer of 3-4 mm.
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Description 10733-00005
Agar culture medium alkaline description
Alkaline Agar culture medium- a dense nutrient medium for the isolation of Vibrio cholerae.A quality composition, quite nutritious and promotes the rapid formation of vibrio colonies, while sodium pyrosulfite partially suppresses the accompanying microflora.
An elevated pH level is a selective factor, which finally suppresses the development of foreign microorganisms and does not affect the growth of the cholera pathogen.
Agar culture medium alkaline technical characteristics
An elevated pH level is a selective factor, which finally 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, peptone dry fermentative, yeast extract, glucose, sodium phosphate disubstituted, sodium chloride, sodium metabisulfite, sodium carbonate, agar.
Alkaline Agar preparation:
The drug in the amount required 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 vials and sterilized by an autoclave.
(121 ± 1) ° C.
Ready nutrient medium, transparent, yellow. Light opalescence is allowed.
pH 8.0 ± 0.2
The ready-made medium can be stored for 10-14 days at a temperature of 2-8 ° C, protected from light.
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Vitamins in exactly 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 in certain nutrients.
Solid culture 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, is a yellowish powder or plate, contains high-molecular polysaccharides, is not degraded by most microorganisms, does not decompose during autoclaving, does not change the nutritional value of the media, does not suppress the growth of microbes. For immunological and bacteriological fields, frozen, clarified agar is used, which melts at a temperature of 85-100 ° C when boiling or autoclaving a mixture of powder with water, and forms a gel when cooled to 45-48 ° C.
For the preparation of solid nutrient media, agar-agar is added at a concentration of 1.5 to 3%.
Simple environments.
Meat Peptone Broth (BCH) is the protein basis of all media.
There are several ways to prepare BCH:
a) in meat water with the addition of ready-made peptone - this is the so-called meat-peptone broth;
b) on digestions of the products of hydrolysis of the feedstock using enzymes (trypsin - Hottinger's broth, pepsin - Martin broth).
They are convenient for transportation, can be stored for a long time, relieve laboratories from the huge process of preparing media, and bring them closer to resolving the issue of standardizing media. The medical industry produces dry Endo, Levin, Ploskirev media, bismuth sulfite agar, nutrient agar, carbohydrates with a BP indicator, and others.
Thermostats
For the cultivation of microorganisms, thermostats are used.
A thermostat is a device that maintains a constant temperature. The device consists of a heater, a chamber, double walls, between which air or water circulates. The temperature is regulated by a thermo regulator. The optimum 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 bottles enters, without touching the edges of the cup, 10-15 ml of MPA is poured, closing the lid, shaking the cup so that the medium is evenly distributed, left on a horizontal surface until solidification. After drying, the agar plates are stored in a cold place.
Loop seeding
A drop of material is taken with a sterile cooled loop, one edge of the cup is opened with the left hand, the loop is inserted inside and at the opposite edge, several strokes are looped in one place, then the loop is torn off and the material is inoculated with parallel strokes from one edge of the cup to the other with an interval of 5-6 mm. At the beginning of inoculation, when there are a lot of microbes on the loop, they will give confluent growth, but with each stroke of microbes on the loop, fewer and fewer remain, and they will remain solitary and give isolated colonies.
Sowing according to the Drygalski method
This method is used when sowing material abundantly seeded with microflora (pus, feces, sputum). For sowing according to the Drygalsky method, take a spatula and several cups (3-4). Putty knife- This is an instrument made of metal wire or glass rod, bent in the form of a triangle or L-shaped. The material is introduced with a loop or pipette into the first cup and evenly spread with a spatula over the surface of the medium, with the same spatula, without burning it, rub the material into the nutrient medium in the second cup, and then in the third. With this inoculation, confluent growth will occur in the first dish, and isolated colonies will grow in subsequent dishes.
Isolation of pure culture according to Shchukevich
For sowing, take a freshly prepared nutrient medium with condensate. The material under study 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 agar slant.
As a result of independent work, the student should know:
1. Classification, morphology of fungi, their methods of study.
2. Classification and morphology of actinomycetes.
3. Rules of anti-epidemic regimes and safety precautions.
Be able to:
1. Differentiate microorganisms by microscopy.
2. Microscopic examination of stained preparations.
3. Disinfect material, treat hands with disinfectants.
4. Prepare preparations from pure cultures.
Presents is a fine, hygroscopic and light-sensitive 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 inoculated with 0.1 ml of microbial suspension of each test - the strain after 12-14 hours of incubation at a temperature of 37oС in the form of smooth, translucent colonies with a bluish tinge in transmitted light. with a diameter of at least 1.0 mm.