Levina-GRM Obolensk Nutrient medium with eosin-methylene blue for the isolation and differentiation of pathogenic and conditionally pathogenic enterobacteria, as well as for the isolation of staphylococci, dry.
Price per package 0.25 kg
INSTRUCTIONSon the use of a nutrient medium with eosin-methylene blue dry -Wednesday Levin-GRMObolensk
- PURPOSE
Levin-GRM Obolensk environment is intended for bacteriological research in sanitary and clinical microbiology in order to isolate and differentiate pathogenic and conditionally pathogenic enterobacteria, as well as to isolate staphylococci.
- characteristic
Wednesday Levina-GRM Obolensk is a fine, hygroscopic, photosensitive powder of light lilac color.
Produced in polyethylene cans of 250 g.
2.1. OPERATING PRINCIPLE
The presence of eosin and methylene blue in the medium give it selective properties.
The differentiating ability of the medium is based on the change in the pH of the medium under the action of the acid formed during the fermentation of lactose by bacteria. The complex of indicators in the acidic zone stains the colonies of acid-forming bacteria in a dark purple color, some colonies have a greenish metallic luster.
2.2. COMPOUND
Levin-GRM medium Obolensk is a mixture of dry components at the rate, g/l:
- ANALYTICAL AND DIAGNOSTIC CHARACTERISTICS
The Levin-GRM medium should ensure the growth of test strains of Shigella flexneri 1a 8516 and Escherichia coli 168/59 (O111:K58) on all inoculated Petri dishes after 18-20 hours of incubation at a temperature of (37±1) °C when inoculated at 0, 1 ml of microbial suspension of the culture of each test strain from a dilution of 10 -7, and the growth of the test strain Staphylococcus aureus 209-P (ATCC 6538-P) after 48 hours of incubation at a temperature of (37 ± 1) ° C with inoculation of 0.1 ml of microbial suspension culture of the test strain from a dilution of 10 -5 .
Differentiating properties of the environment. The nutrient medium should provide a clear differentiation of Shigella from Escherichia on all inoculated cups when inoculated with 0.1 ml of a microbial mixture of S. flexneri 1a 8516 and E. coli 168/59 (O111:K58) from a dilution of 10 -6 (in a ratio of 1: 1 ) after 18-20 hours of incubation at a temperature of (37 ± 1) °C.
- PRECAUTIONARY MEASURES
Potential risk of using a nutrient medium in accordance with the Order
Ministry of Health of the Russian Federation No. 4n dated 06.06.2012 - class 2 a.
- EQUIPMENT AND MATERIALS
- Thermostat providing temperature 37±1 °С
- Scales laboratory 2 classes of accuracy
- Autoclave
- Glass test tubes
- Glass pipettes allowing to take liquid volumes of 1 and 2 ml
- Glass measuring cylinder with a capacity of 1000 ml
- Petri dishes sterile
- Distilled water
- flasks
- Glass funnels
- SAMPLES ANALYZED
Objects of research in clinical laboratory diagnostics (blood, bile, urine, pus, etc.).
- Carrying out the ANALYSIS
The study is carried out in a bacteriological laboratory by medical specialists.
7.1. Preparation of Levin-GRM medium Obolensk.
The powder in the amount indicated on the label for the preparation of a specific series of nutrient medium is mixed in 1 liter of distilled water, boiled for 3 minutes, filtered through a cotton-gauze filter and sterilized by autoclaving at a temperature of 110 °C for 20 minutes. The sterile medium is cooled to a temperature
45-50 °C, poured into sterile Petri dishes with a layer of 5-6 mm. After solidification of the medium, the cups are dried at a temperature of (37±1)°C for 40-60 minutes. The prepared medium in Petri dishes is transparent, from light lilac to reddish brown.
The sterile medium can be used within 3 days if stored at 2-8°C.
7.2. Taking, sowing infected material and recording the results are carried out in accordance with “ Guidelines on microbiological diagnosis of diseases caused by enterobacteria” (M., 1984) and the order of the Ministry of Health of the USSR dated April 22, 1985, No. 535 “On the unification of microbiological (bacteriological) research methods used in clinical diagnostic laboratories of medical institutions”.
7.3. The test material is thoroughly rubbed with a sterile spatula over the entire surface of the medium. Incubate at a temperature of (37±1) °C for 46-48 hours.
- REGISTRATION AND RECORDING OF RESULTS
After 18-20 hours of incubation at a temperature of (37 ± 1) ° C, the growth pattern of S. flexneri 1a 8516 and E. coli 168/59 (O111:K58) cultures is visually taken into account, and after 46-48 hours the growth pattern of S. aureus 209 culture -P (ATCC 6538-P).
Colonies of S.flexneri 1a 8516 should be round, transparent, colorless or slightly pink, 1.0-1.5 mm in diameter.
Colonies of E. coli 168/59 (O111:K58) should be round, dark purple with a green metallic sheen (there may be individual colonies without a metallic sheen), 1.5-2.0 mm in diameter.
Colonies of S. aureus 209-P (ATCC 6538-P) should be round, colorless or light purple with a dark center, up to 1.0 mm in diameter.
- DISPOSAL
Utilization of Levin-GRM Obolensk medium series with an expired shelf life is carried out according to
SanPiN 2.1.7.2790-10 as medical waste belonging to class "A" - epidemiologically safe waste.
The destruction of the Levin-GRM medium after biological control is carried out according to SanPiN 2.1.7.2790-10 as medical waste belonging to class "B" with mandatory preliminary neutralization by autoclaving for 2 hours at a temperature of (133 ± 1) ˚С.
Handling medical waste should be carried out according to the scheme adopted in a particular organization carrying out medical and (or) pharmaceutical activities. This scheme is developed in accordance with the requirements of the above sanitary rules and approved by the head of the organization.
- STORAGE AND OPERATION CONDITIONS
LEVINA WEDNESDAY(M. Levine, American bacteriologist, born in 1889; syn. eosin methylene blue agar) is a colored elective nutrient medium for the differentiation of bacteria of the fam. Enterobacteriaceae is used in the laboratory diagnosis of dysentery, typhoid fever, salmonellosis and other intestinal infections. For the first time, agar with eosin and methylene blue was proposed in 1916 by Holt-Harris and Teague (J. E. Holt-Harris, O. Teague), later, in 1918, the composition of the medium was modified by Levin.
L. s. along with other solid color differential diagnostic nutrient media for enterobacteria has found wide application in the lab. practice. With its help, it is possible in a relatively large percentage of cases to detect pathogenic lactose-negative microflora. Ready L. s. quite stable, does not change its properties in the light or when stored for several days; in its manufacture, it is not necessary to accurately determine the concentration of hydrogen ions (pH). Included in the L. s. organic dyes selectively retard the growth of gram-positive bacteria, due to which it is also an enrichment medium and gives relatively better results with direct inoculation of the test material, even if it contains a relatively small amount of pathogenic bacteria.
Depending on the quality of the individual ingredients (peptone and especially dyes) obtained on L. s. results may vary slightly, therefore, in the manufacture of each new series medium, it is desirable to use materials of the same brand, which greatly facilitates the recognition of colonies.
There are several prescriptions and methods for preparing L. s. (Yu. A. Kozlov, G. Ya. Sinai, O. G. Birger and others). The method is most widely used, with Krom separately preparing the main medium, solutions of lactose and dyes, suitable for preservation for the future and mixed before use.
The basic medium consists of 10 g of bacteriological peptone, 15 g of agar-agar, 2 g of dibasic potassium phosphate (K2HPO4) and 1 liter of distilled water. It is sterilized in an autoclave at 1 atm for 15-20 minutes, the pH is not corrected.
When preparing a differential medium, for every 100 ml of the molten basic medium, with constant stirring, add prepared in distilled water and pre-sterilized with flowing steam (fractionally, 3 days in a row for 15-20 minutes.) following solutions in the given sequence 5 ml of 20% solution of medical lactose, 2 ml of 2% solution of alkaline eosin (bacteriological), 1.5 ml of 0.5% solution of methylene blue. Ready environment pour into Petri dishes, dry slightly and use as usual. The color of the medium is blue-violet.
Colonies of Escherichia coli on L. page. small, rounded, with a smooth shiny surface, dark blue (to black) in color, sometimes with a metallic sheen. Young colonies are cloudy, opaque, and can only be stained in the center. Colonies of dysentery, typhoid and paratyphoid pathogens are relatively smaller, round, shiny and transparent, usually completely colorless (young) or with a slight pinkish or bluish tint. Proteus colonies do not give creeping growth; they are small, isolated, orange-yellow in color. The medium changes its color only around the Proteus colonies.
Some authors recommend increasing the amount of 0.5% methylene blue solution to 2 ml per 100 ml of the main medium or excluding phosphate buffer from its composition. You can prepare the medium not with peptone, but on the basis of Hottinger digest, at pH 7.2-7.3 with the addition of the appropriate amount of agar and potassium phosphate. The use of meat water for the preparation of the medium is unacceptable, the differentiation of colonies in this case is significantly worsened.
There is a known method of preparing dry HP, which is very stable in quality and especially suitable for long-term storage and in conditions of expeditionary work (NV Ploskirev).
Stable dry L. page. is produced by the industry, instructions for its manufacture and use are sent along with the medium. Most diagnostic laboratories use for the isolation and differentiation of pathogenic enterobacteria high-quality standard elective dry nutrient media of domestic production - HP, baktoagar Zh, Ploskirev's medium (see Ploskirev's medium).
Bibliography: Guidelines for microbiological diagnosis of infectious diseases, ed. K. I. Matveeva, p. 110, Moscow, 1973; Handbook of microbiological and virological research methods, ed. M. O. Birger, p. 58, Moscow, 1973; Levine, M. The effect of concentration of dyes on differentiation of enteric bacteria on eosin-methylene- blue agar, J. Bact., v. 45, p. 471, 1943.
G. P. Belikov.
Price: 2 970.00 RUB
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Practice
dense media
Bismuth Sulfite Agar
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Paratyphoid A Salmonellosis
The prepared medium is opaque pea green. Strictly selective medium for the isolation of pure cultures of Salmonella. Salmonella typhi, Salmonella enteritidis and Salmonella typhimurium usually form on this medium black colonies with a metallic sheen, surrounded by a zone of blackening as a result of the production of hydrogen sulfide and the reduction of sulfite to iron sulfide, which is black. Salmonella paratyphi A forms light green colonies.
Sample response: Small colonies were found on a dense medium "Bismuth-sulfite agar". Smooth with a smooth edge, dark and opaque with a metallic sheen.
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Wednesday Levin
Sample response: On this medium, Levin's medium, small smooth colonies with a smooth edge of blue-violet color with a metallic sheen were found, with fermentation of lactose to acid. The indicator is methylene blue. Preliminary diagnosis - colienteritis caused by Escherichia coli - Escherichia coli.
Sample response: This medium is Levin's medium made to obtain isolated colonies. Small, smooth, transparent, unstained colonies were found on the medium, and lactose fermentation was absent. Prediagnosis - dysentery caused by possible pathogens:
Serogroup A: Shigella dysenteriae(10 serovars)
Serogroup B: S. flexneri(6 serovars and subtypes)
Serogroup C: S. boydii(15 serovars)
Serogroup D: S. sonnei(1 serovars)
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Wednesday Endo
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Sample response: This environment is the Endo environment. Small colorless, transparent, smooth colonies with a smooth edge were found on it. There is no decomposition of lactose to acid, as indicated by the color of the medium. Andrade indicator is used. The suspected diagnosis is typhoid fever caused by Salmonella Typhi.
Sample response: This medium is used for the accumulation of isolated colonies. Wednesday - Endo. Found small smooth with a smooth edge, red with a metallic sheen due to the decomposition of lactose to acid using the Andrade indicator. Preliminary diagnosis - colienteritis caused by Escherichia coli - Escherichia coli.
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Methods for determining sensitivity to antibiotics
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Sample response: The Petri dish presents a method for determining sensitivity to antibiotics - the paper disc method, in which a bacterial suspension is applied to the surface of agar in a Petri dish and then discs containing a certain amount of antibiotic are placed. Diffusion of the antibiotic into the agar leads to the formation of a zone of inhibition of the growth of microorganisms around the disks. After incubation of the cups in a thermostat at a temperature of 35 about -37 about C during the night, take into account the result by measuring the diameter of the zone around the disk in millimeters.
The isolated culture is highly sensitive to monomycin, the diameter to stunting is 30 mm, neomycin is 26 mm, kanamycin is 20, and levomycetin is absent at all. Characteristic of Escherichia coli - Escherichia coli.
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Seeding from the air
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Sample response: This plate agar was inoculated from the air by the sedimentation method. Indication of indoor air pollution is the total number of bacteria per 1 cubic meter. The number of bacteria is determined in 10 minutes. To do this, a Petri dish with plate MPA is left open in the air for 10 minutes, then incubated in a thermostat at a temperature of 37 degrees for a day. The results are counted by the total number of colonies. The norm in the operating room before the operation is 3-5 colonies. After - 15. There is also an aspiration method, where the air is evaluated with a special Krotovoy device.
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Mixture of microorganisms
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color row
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Dysentery
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MPB (for determining proteolytic sv-in): H2S-; Indole-;
Mannitol: Acid+; Gas-;
Maltose: Acid+; Gas-;
Wednesday Peshkov: growth by injection, the color changed to red, which indicates the fermentation of mannitol to acid. Indicator - Andrade;
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coli
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BCH (for determining proteolytic sv-in): H2S + (blackened); Indole + (blushed);
Glucose: Acid+; Gas+;
Lactose: Acid+; Gas+;
Maltose: Acid+; Gas+;
Sucrose: Acid-; Gas-;
Peshkov's medium: turbidity of the entire column (the culture is mobile), the color has changed to red, which indicates the fermentation of mannitol to acid and the presence of gas bubbles. Indicator - Andrade;
Ressel's medium (Indicator - bromthymol blue): Bevel and column has yellow, and in the thickness - gas, which indicates the decomposition of lactose and fructose to gas and acid.
Typhoid fever
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Hiss Wednesday (Indicator - water-pink):
Glucose: Acid+; Gas-;
Maltose: Acid+; Gas-;
Mannitol: Acid+; Gas-;
Lactose: Acid-; Gas-;
Sucrose: Acid-; Gas-;
Peshkov's medium: turbidity of the entire column (the culture is mobile), the color has changed to red, which indicates the fermentation of mannitol to acid. Gas is missing. Indicator - Andrade;
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salmonellosis
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BCH (for determining proteolytic sv-in): H2S + (blackened); Indole-;
Hiss Wednesday (Indicator - water-pink):
Maltose: Acid+; Gas-;
Sucrose: Acid-; Gas-;
Peshkov's medium: turbidity of the entire column (the culture is mobile), the color has changed to red, which indicates the fermentation of mannitol to acid and gas. Indicator - Andrade;
Ressel's medium (Indicator - bromothymol blue): The bevel is unchanged in color, and the column is yellow. In the thickness - gas, which indicates the decomposition of fructose to gas and acid, lactose is unfermented.
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gas gangrene
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Kitt-Tarozzi medium: liquid medium for culturing anaerobes. To prepare it, the liver (meat) cut into small pieces is poured with three times the amount of MPB, boiled for 30 minutes, filtered. Dried liver pieces are laid out (for oxygen adsorption) in test tubes and poured with broth. Sterilize at 120°C for 30 minutes. Before use, warm and fill with sterile neutral paraffin oil.
Growth in the form of turbidity was found on this medium. There is also gas formation.
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Botulism
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Wednesday Weinberg: a semi-liquid nutrient medium used for the cultivation of pathogenic anaerobes, which is a meat-peptone broth containing 1% agar and 0.2% glucose;
Small, disc-shaped, fluffy colonies were found in a column of sugar agar. The medium is ruptured due to increased gas production during glucose fermentation.
Make a cut. A part of the colonies is taken with a Pasteur pipette or a needle and placed in the Kitta-Tarozzi medium for accumulation.
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Agglutination reaction (expanded)
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NB!: Friends, do not forget that you need to read the reactions from the end - checking the control of serum and antigen. After you are likely to be asked for a definition titer of agglutinating serum (maximum serum dilution at which agglutination occurs with the corresponding microorganism) and diagnostic serum titer (titer of antibodies to a specific pathogen in the blood serum, detected in patients and not observed in healthy people).
Sample response: This reaction is an extended agglutination reaction. The serum control is presented as a clear liquid and is negative. In the antigen control - turbidity, also negative. We look at a dilution of 1/100 - transparent with an umbrella at the bottom, when shaken - flakes, which means the reaction is positive. And so on up to 1/6400. We look at the dilution of 1/12800, turbidity is noticeable - the reaction is negative.
Therefore, the agglutination reaction is positive up to a dilution of 1/6400, with negative controls, which means the titer of the test serum is 1/6400.
Agglutination reaction - gluing of corpuscles (bacteria, erythrocytes, etc.) with antibodies in the presence of electrolytes - sodium chloride.
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Compliment Binding Response
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CSC is based on complement activation by an antigen-antibody complex.
It takes place in two stages:
Mixture incubation: antigen+antibody+complement
Indicator: detection of one complement by adding a hemolytic system (sheep erythrocytes + hemolytic serum in triple titer)
Sample response: CSCs were registered with the sera of patients 1 and 2 to detect antibodies to the corresponding antigen in them. The reaction with the serum of patient 2 is negative, since in the first stage the antibody and antigen did not match and did not bind, which means they did not bind the complement. Then the free complement combines with the hemolytic system and lysis occurs. In the serum of patient 1, a delay in hemolysis was detected, which means that the “antibody + antigen + complement” complex was formed. Complement is bound and does not join the hemolytic system. Hemolysis is absent. The diagnosis is positive.
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Passive (indirect) hemagglutination reaction
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AT RNGA detection of serum antibodies antigenic erythrocyte diagnosticum, which is erythrocytes with antigens adsorbed on them.
RPGA put in plastic tablets or in test tubes with dilutions of the patient's blood serum, to which an erythrocyte diagnosticum is added.
Erythrocytes (or latex particles) with antigens adsorbed on them interact with the corresponding blood serum antibodies, which causes erythrocytes to stick together and fall out to the bottom of the tube or cell in the form of a scalloped sediment. With a negative reaction, erythrocytes settle in the form of a button.
Levina Wednesday
(lactose eosinmethylene agar) is a differential-selective medium for the isolation of enterobacteria. Allows you to distinguish lactose-fermenting (form dark blue or black colonies) from non-fermenting lactose to-r (colonies of the color of the medium). Proteus forms isolated orange colonies. The medium is prepared by adding to 100 ml of 1.5% MPA, pH 7.2 - 7.4, 2 ml of 0.5% water solution methylene blue, 1.5 ml of 2% eosin solution, 2 g of lactose and 0.2 g of dibasic potassium phosphate, then pour it into Petri dishes. The medium has a light purple color. The industry produces a dry preparation, which is prepared according to the instructions on the label. HP variant - lactose bromthymol medium - does not have inhibitory properties against Klebsiella scleroma.
(Source: Glossary of Microbiology Terms)
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