Differential diagnostic media: list the main types, fundamental composition, purpose and use in practice. Medical encyclopedia - differential diagnostic environments What are differential diagnostic environments used for?
Endo environment. To 100 ml of neutral molten 3% meat peptone agar, add 1 ml of a 10% aqueous solution of crystalline sodium carbonate, keep in a flowing steam apparatus for 10 minutes at a temperature of 100 ° C, cool to 60 ° C and sterilely add 1 g of chemically pure lactose, dissolved in 5 ml of sterile water, and a mixture of fuchsin and anhydrous sodium sulfite. The mixture is prepared as follows. Dissolve 0.5 g of sodium sulfite in 5 ml of sterile water and add to 1 ml of a saturated alcohol solution of basic crystalline fuchsin until the liquid becomes colorless or slightly pinkish. A decolorized mixture of fuchsin and sulfite is added to the molten agar, and the latter takes on a pinkish color. After thorough mixing (make sure that no foam forms), the medium is poured into cups. When cooled, the medium becomes colored; in thick layers it has a pinkish tint. On this medium you can easily distinguish Escherichia coli and paratyphoid bacteria.
Prepare the medium before sowing. To avoid redness, protect it from light. Available in dry powder form.
The preparation method is indicated on the label. Wednesday Gissa . To make an indicator Andrede is taken
The Andrede indicator medium is prepared as follows. Prepare peptone water. To do this, take 1% peptone and 0.5% table salt for a certain volume of distilled water. Boil until the peptone dissolves, filter through a folded paper filter, and set the pH to 7.0-7.2. After alkalization with a 10% NaOH solution, boil again for 10 minutes, filter and add 1% Andrede indicator to the finished 1% peptone water (pH 7.0-7.2). The required carbohydrates or polyhydric alcohols are added to the medium with the indicator in an amount of 0.5% - 1% (lactose, glucose, sucrose, mannitol, dulcite, etc.). The media are poured into test tubes equipped with fermentation tubes to account for gas formation. Instead of tubes, pieces of cotton wool are sometimes placed - 0.02 g per test tube. Fractional sterilization is carried out at a temperature of 100 ° C for 20 minutes for three days in a row.
Properly prepared medium is straw-yellow in color.
Kessler environment. Add 50 ml of fresh bovine bile and 10 g of peptone to 1 liter of tap water. Boil in a water bath for 15 minutes, shaking constantly. After complete dissolution, the medium is filtered through cotton wool and 10 g of lactose is added. Adjust pH to 7.7. To 1 liter of medium, add 4 ml of a 1% aqueous solution of gentian violet and pour into test tubes with floats. Sterilize in an autoclave at an excess pressure of 0.1 MPa for 15 minutes.
Determination of pH of nutrient media
For the cultivation of microbes on artificial nutrient media, the concentration of hydrogen ions is of particular importance, since each type of microbe can develop only at a certain acidity or alkalinity. Most bacteria are adapted to growth and reproduction in neutral or slightly alkaline environment (pH 7.0-7.6).
To determine the reaction of the nutrient medium, two methods are used: electrometric (using a pH meter) and colorimetric. In laboratory practice, the simplest Michaelis colorimetric method is most often used, based on a change in the color of the indicator due to its dissociation depending on the concentration of hydrogen ions in the medium. When determining pH according to Michaelis, indicators of the nitrophenol series are used. Due to the fact that slightly alkaline reaction media are prepared for growing microbes, when determining the pH of the medium, methannitrophenol is used as an indicator, which makes it possible to determine the pH in the range from 6.8 to 8.4. Indicator solutions are prepared in distilled water and stored in dark glass bottles with a ground-in stopper.
Control questions:
Composition of nutrient media.
How are microorganisms cultivated in the laboratory?
What are the different consistency of nutrient media?
How are nutrient media differentiated by origin?
Solid nutrient media and their characteristics.
Dry nutrient media and their characteristics.
Carbohydrate nutrient media, their characteristics.
Autoclaving.
Sterilization with flowing steam.
Pasteurization.
Sterilization by filtration.
How are MPB, MPZh, MPA prepared?
Differential diagnostic nutrient media for the cultivation of bacteria. Media containing proteins. Media containing carbohydrates. Media for determining the reducing ability of bacteria.
Differential diagnostic environments(For example, environment Hiss, Clark) are used to study and identify individual types, species and groups of bacteria. Various organic and inorganic compounds, casein hydrolysates, peptone water, Hottinger-Martin broth, supplemented with carbohydrates, alcohols, urea and other substances; when they are broken down, the pH shifts to the acidic (carbohydrates, alcohols, lipids) or alkaline (proteins) side. Accordingly, media with carbohydrates and alcohols, media with urea, media for determining indole formation, media for determining proteolytic activity and combined (polytropic) media are distinguished. Such media are also often supplemented with various indicators (eg, bromothymol blue, Andrade's indicator, bromocresol purple, and cresol red) to help visually identify pH changes characteristic of various microorganisms. In particular, a shift to the acidic side causes the medium with Andrade's reagent to turn red or yellow when using a medium with bromothymol blue, whereas when alkalized, the Andrade's reagent and the bromothymol blue indicator do not change the color of the medium. All differential diagnostic environments are divided into four main groups.
Media containing carbohydrates or polyhydric alcohols. Enzymatic breakdown of substrates leads to a pH shift and a change in the color of the medium, and sometimes the formation of gas. The most common colored media with various carbohydrates (for example, with bromothymol blue, a BP indicator), litmus milk ( Minkevich environment) And Hiss environment. The most commonly used carbohydrates are monosaccharides (xylose, arabinose, glucose, fructose, mannose, galactose), disaccharides (lactose, maltose, sucrose), polysaccharides (starch, glycogen, inulin, dextrin), alcohols (dulcitol, mannitol, sorbitol, glycerin) and glycosides (adonitol, inositol, salicin, amygdalin).
Media for determining reducing ability. This group includes media with dyes that become discolored upon reduction (for example, methylene blue, neutral red, indigo carmine), as well as media with nitrates to determine the denitrifying activity of bacteria (if the result is positive, the media turn blue).
Media containing substances that are assimilated only by a specific group of bacteria. The most famous are nitrate Simmons agar And Coser's citrate medium.
Endo environment. Consists of MPA with the addition of 1% lactose and basic fuchsin (indicator) decolorized with sodium sulfite. Endo medium has a slightly pink color. Used in the diagnosis of intestinal infections to differentiate bacteria that decompose lactose to form acidic products from bacteria that do not have this ability. Colonies of lactose-positive microbes (Escherichia coli) are red due to the reduction of fuchsin. Colonies of lactose-negative microorganisms - salmonella, shigella, etc. - are colorless.
Differential diagnostic environments include a short and an extended motley series. It consists of media with carbohydrates (Hiss media), MPB, milk, and meat-peptone gelatin.
Hiss media is prepared on the basis of peptone water, to which chemically pure mono-, di- or polysaccharides (glucose, lactose, starch, etc.) are added.
To detect pH shifts as a result of the formation of acids and the decomposition of carbohydrates, an indicator is added to the media. With a deeper breakdown of carbohydrates, gaseous products (CO 2, CH 4, etc.) are formed, which are captured using floats - small test tubes lowered upside down into the medium. Media with carbohydrates can also be prepared as dense ones - with the addition of 0.5-1% agar-agar. Then gas formation is detected by the formation of bubbles (breaks) in the column of the medium.
On the MPB, which is part of the motley series, products formed during the breakdown of amino acids and peptones (indole, hydrogen sulfide) are found. Hydrogen sulfide is detected by placing a strip of filter paper soaked in a solution of lead acetate into the MPB after sowing the culture. When amino acids containing sulfur are broken down, hydrogen sulfide is released, and the paper turns black due to the formation of lead sulfide. A complex indicator can be used to determine indole. Indole is formed by the breakdown of tryptophan and can be detected when this indicator is added to a culture grown on MPB. In the presence of indole, MPB turns green or blue.
In practical bacteriological laboratories, micro- and express methods are widely used for an indicative study of the biochemical properties of microorganisms. There are many test systems for this purpose. The most commonly used system of indicator papers (SIB). SIBs are disks of filter paper impregnated with solutions of sugars or other substrates in combination with indicators. Such disks are lowered into a test tube with a culture grown in a liquid nutrient medium. The change in color of the disk with the substrate is used to judge the functioning of the enzyme. Micro-test systems for studying the identification of enterobacteria are represented by disposable plastic containers with media containing various substrates with the addition of indicators. Sowing a pure culture of microorganisms into such test systems allows you to quickly identify the ability of bacteria to utilize citrates, glucose, sucrose, release ammonia, indole, decompose urea, lysine, phenylalanine, etc.
Dry environments. Nutrient agar, as well as the main differential diagnostic media, are currently produced in the form of dry preparations containing all the necessary components. To such powders you only need to add water and boil them, and then, after pouring, sterilize them.
Differential diagnostic media are special mixtures nutrients, used to determine the species of microbes and study their properties. When bacteria grow on differential diagnostic media, chemical processes, caused by the presence of various microbial cells. Some of them are capable of breaking down, others -, others - causing oxidation and reduction reactions, etc. Thanks to the action of enzymes, corresponding changes occur in the differential diagnostic environment.
Differential diagnostic environments can be divided into four main groups.
Rice. 1-6. Various forms of gelatin breakdown. Rice. 7 - 9. Liquid medium with carbohydrate and Andrade indicator: fig. 7 - no fermentation; rice. 8 - fermentation with acid formation; rice. 9 - fermentation with the formation of acid and gas. Rice. 10 - 12. Semi-liquid medium with carbohydrate and BP indicator (from dry nutrient medium): fig. 10 - no fermentation; rice. 11 - fermentation with acid formation; rice. 12 - fermentation with the formation of acid and gas. Rice. 13-15. Seitz artificial litmus serum: Fig. 13 - no fermentation; rice. 14 - fermentation with acid formation; rice. 15 - fermentation with formation. Rice. 16 and 17. Milk with methylene blue: fig. 16 - lack of reduction; rice. 17 - reduction. Rice. 18 and 19. Simons medium: fig. 18 - lack of citrate assimilation; rice. 19 - citrate assimilation. Rice. 20 - 24. Litmus milk: rice. 20 - no fermentation; rice. 21 - fermentation with acid formation; rice. 22 - fermentation with the formation of alkali; rice. 23 - peptonization; rice. 24 - reduction. Rice. 25. Liquefaction of a coiled product (in transmitted light). Rice. 26. Hemolysis on blood agar (in transmitted light). Rice. 27. Blood medium with potassium tellurite.
1. Media containing protein and revealing the ability of microbes to break down proteins (proteolytic properties): meat-peptone “column”, coagulated horse or bovine serum, milk, blood agar. When inoculating bacteria by puncture into meat-peptone gelatin, in a “column”, in the case of protein breakdown, liquefaction of the medium is observed. When inoculated on a medium with coagulated whey, protein breakdown is determined by the liquefaction of the medium and the formation of depressions on its surface. The breakdown of milk by a microbe is revealed by the clearing or dissolution of the initially curdled milk. The presence of hemolytic activity of the test culture is checked by inoculating it on a special blood agar. As a result of destruction around the colonies (for example, hemolytic or), zones of clearing are formed.
2. Media for identifying the ability of microbes to break down carbohydrates and high-atomic ones (Endo medium, Levin medium, Russell medium, Drigalsky - Conradi medium, Rapoport - Weintraub medium, Shustov medium). To identify these properties of microorganisms, a “variegated” series is also used, that is, a series of test tubes containing various carbohydrates, polyhydric alcohols and an indicator. Litmus tincture or bromothymol blue are used as indicators. The decomposition of any of the carbohydrates with the formation of acid is detected by a change in the color of the indicator, the formation of gas is detected by the filling with gas and the floating of a special glass float in a liquid medium. Or use semi-liquid Hiss media (see) with 0.5% agar with appropriate sugars and Andrade indicator. After inoculating the microbe on these media, the formation of acid is detected by the reddening of the medium, and the formation of gas by the appearance of its bubbles in the agar or by the rupture and upward shift of the agar column. The differential diagnostic media of the second group also include starch agar, which is used to determine the ability of microbes to break down starch, Clark's medium, etc.
3. Media on which the ability of microbes to decolorize dyes added to the broth is revealed: methylene blue, thionine, litmus, neutral red or others (Rothberger’s medium, Omelyansky’s medium). The third group also includes media with nitrates, which are used to determine the ability of microbes to reduce nitric acid salts (nitrates) in salt nitrous acid(nitrites) and further into ammonia or free nitrogen.
4. Media that reveal the ability of microbes to assimilate substances that are not digestible by other microbes, for example, a medium with sodium citrate (Simons citrate agar) to distinguish Escherichia coli, which lacks the ability to assimilate this medium, from other bacteria of the intestinal group, or a medium with sodium oleic acid for differentiation diphtheria bacillus from false diphtheria and diphtheroids (Engering's agar).
Differential diagnostic media also include media for differentiating anaerobes, tellurite media for differentiating diphtheria bacteria, media with urea, alkaline media(Dieudonne agar) for the cultivation of Vibrio cholerae, etc. See also Identification of microbes.
(syn. P. s. differential) P. s., which allows one to distinguish one type of bacteria from another by their enzymatic activity or cultural properties.
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