RU2266956C2 - Broth for brucelle isolation - Google Patents

Abstract

FIELD: biotechnology, in particular clinical microbiology.

SUBSTANCE: claimed broth contains nutrient broth, enzymatic peptone, vitamin preparation, D(+)-glucose, sodium chloride, metabisulfite, nalidixic acid, crystalline violet, microbiogical agar, and distilled water. Broth of present invention is useful in bacteriological diagnosis of brucellosis.

EFFECT: broth with increased growth characteristics and decreased cost.

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Description

The invention relates to medicine, namely to clinical microbiology, can most effectively be used for bacteriological diagnosis of brucellosis.

Brucellosis refers to zoonotic diseases in which sick farm animals are the main source of disease in humans.

In the Russian Federation, up to 500 cases of newly diagnosed brucellosis are registered annually.

The variety of clinical manifestations of brucellosis often complicates the correct diagnosis and requires the use of laboratory research methods.

Particular difficulties in the diagnosis of brucellosis are cases with an unclear anamnesis, with an uncharacteristic clinic of early and especially late manifestations of infection, relapse, disease in infected vaccinated and often encountered latent forms of infection. In all these cases, laboratory research methods are often crucial (1).

Nutrient media are known for the isolation of Brucella agar Marten, Albimi agar, serum-dextrose agar, glucose-glycerol agar, Erythritol-agar (2, 3, 4).

These media (serum-dextrose agar, glucose-glycerol agar) are not standard, time-consuming to use, in addition, all of the above media are non-selective and do not allow to isolate the brucella culture from the infected material.

The closest solution to the problem of the same purpose in terms of the set of characteristics and the achieved effect is a nutrient medium for the isolation of Merck brucellas (4), containing a nitrogen source - meat peptone and casein peptone, growth stimulator - yeast extract, as well as D (+) glucose , sodium chloride, agar, as inhibitors of concomitant microflora - bacitracin, polymyxin, cyclohexamide, ethyl violet.

The reasons that impede the achievement of the technical result indicated below when using a known medium adopted as a prototype is that the medium has insufficient growth properties, in addition, this nutrient medium of expensive production is expensive.

The objective of the invention is to increase the growth properties of the medium and reduce the cost of the medium.

The specified technical result in the implementation of the invention is achieved by the fact that the proposed medium additionally contains a nutrient broth for the cultivation of microorganisms as a source of nitrogen nutrition, vitamin EKD and sodium metabisulfite as growth stimulants, and crystalline violet and nalidaxic acid as inhibitors of concomitant microflora in the following ratio of components (5, 6, 7) in g / l of distilled water:

nutritional broth for microorganism cultivation - 14.0-16.0 peptone enzymatic - 9.0-11.0 vitamin preparation "EKD" - 4.0-6.0 D (+) glucose - 0.5-1.5 sodium chloride - 4.0-6.0 sodium metabisulfite - 0.05-0.15 crystal violet - 0.0005-0.0015 nalidixic acid - 0.015-0.025 microbiological agar - 10.0-12.0

The introduction of crystalline violet and nalidixic acid into the composition of the proposed medium completely inhibits the growth of concomitant gram-positive and gram-negative microflora and does not affect the growth of brucella. In addition, the nutrient medium has good growth properties, the growth of brucella colonies by 3 days is noted, while on the prototype medium by 4-5 days. The medium is constructed from domestic, guest components and does not require ex temporae antibiotics after sterilization of the medium.

The environment is prepared as follows:

Example 1. In 1 liter of distilled water, 14.0 g of nutrient broth for the cultivation of microorganisms, 9.0 g of enzymatic peptone, 4.0 g of the vitamin preparation EKD, 0.5 g of D (+) glucose, 4, 0 g of sodium chloride, 0.05 g of sodium metabisulfite, 0.005 g of crystalline violet, 10.0 g of microbiological agar.

The medium is brought to a boil and boiled for 2 minutes. Before sterilization, nalidixic acid is added (0.015 g of nalidixic acid is dissolved in 0.5 ml of a 1% NaOH solution with the addition of 4.5 ml of distilled water) and added to the culture medium. The medium is sterilized by autoclaving at a temperature of 112 ° C for 20 minutes. Poured into sterile Petri dishes, after cooling the medium to 45 ° C. The cups are dried in an oven at 37 ° C for (35 ± 5) minutes.

Example 2. In 1 liter of distilled water, 15.0 g of nutrient broth for the cultivation of microorganisms is successively dissolved, 10.0 g of enzymatic peptone, 5.0 g of vitamin EKD preparation, 1.0 g of D (+) glucose, 5, 0 g of sodium chloride, 0.1 g of sodium metabisulfite, 0.001 g of crystalline violet, 11.0 g of microbiological agar.

The medium is brought to a boil and boiled for 2 minutes. Before sterilization, nalidixic acid is added (0.02 g of nalidixic acid is dissolved in 0.5 ml of a 1% NaOH solution with the addition of 4.5 ml of distilled water) and added to the culture medium. Further according to example 1.

Example 3. In 1 liter of distilled water, 16.0 g of nutrient broth for the cultivation of microorganisms are successively dissolved, 11.0 g of enzymatic peptone, 6.0 g of the vitamin preparation EKD, 1.5 g of D (+) glucose, 6, 0 g of sodium chloride, 0.15 g of sodium metabisulfite, 0.0015 g of crystalline violet, 12.0 g of microbiological agar.

The medium is brought to a boil and boiled for 2 minutes. Before sterilization, nalidixic acid is added (0.025 g of nalidixic acid is dissolved in 0.5 ml of a 1% NaOH solution with the addition of 4.5 ml of distilled water) and added to the culture medium. Further, according to examples 1, 2. The quality control of the medium was carried out by seeding test strains of Brucella abortus BA 19 from a dilution of 10 ^-6 and 10 ^-7 , Echerichia coli 168/59, Proteus vulgaris HX 19 N 222, Stafylococcus aureus 209-P from a dilution 10 ^-3 . After 72-120 hours of incubation of the crops in the thermostat at 37 ° C, the presence of growth is determined.

The results of bacteriological control of the proposed and known environments are presented in the table.

The table shows that the proposed environment has higher growth properties. On the proposed medium, brucella form typical colonies on day 3, while on the known medium on day 4-5.

The presented advantages of the proposed environment will improve the diagnosis of brucellosis during bacteriological examination of clinical material for the presence of brucella.

Sources of information

1. G.I. Lyamkin, L.V. Lyapustina, O.V. Maletskaya, I.A. Sokolova, I.F. Taran, L.I. Tkachenko, V.G. Dalvadyants, A.V. Tambovtsev. Status and prospects of laboratory diagnosis of brucellosis. "Clinical Laboratory Diagnostics", 2002, No. 12, p. 46-47.

2. P.A. Vershilova, Brucellosis. Moscow, 1972, p. 55-57.

3. Yu.A. Kozlov, Culture media. Moscow, 1950, p. 62, 169.

4. Catalog of dry microbiological culture media. Makhachkala, 1998, p. 48.

5. Catalog of the company "Merck", 1990, p.66.

6. FS 42-3520-98.

7. FS 42-3G BC-91.

8. Chemical reagents and high-quality chemicals, Catalog, Moscow, Chemistry, 1983, p.145, 365, 284.

Table Comparative characteristics of biological indicators of the proposed and known environments Culture media Test strains Growth rate Sensitivity Inhibition Suggested medium Brucella abortus 19 VA 2-3 days Colonies are colorless, with a pearlescent hue, d = 0.2-0.5 mm from a 10 ^-6 dilution Test strains S.aureus 209-P, E.coli 168/59, Pr.vulgaris HX19 n 222 complete from a dilution of 10 ^-3 Known Brucella culture media + selective supplement Brucella abortus 19 VA 4-5 days Colonies are colorless, with a pearlescent hue, d = 0.2-0.5 mm from a 10 ^-6 dilution Test strains S.aureus 209-P, E.coli 168/59, Pr.vulgaris Hxl9 N 222 from a dilution of 10 ^-3

Claims (1)

Brucella isolation medium containing a nitrogen supply, growth stimulator, microbiological agar, D (+) glucose, sodium chloride, concomitant microflora inhibitors, distilled water, characterized in that it additionally contains a nutrient broth for the cultivation of microorganisms as a nitrogen source and peptone enzymatic, as a growth promoter - vitamin preparation "EKD" and sodium metabisulfite, as an inhibitor of concomitant microflora - nalidixic acid and cris allichesky purple the following component, g / liter of distilled water: Nutrient broth for Microorganism cultivation 14.0-16.0 Peptone enzymatic 9.0-11.0 Vitamin preparation "EKD" 4.0-6.0 D (+) glucose 0.5-1.5 Sodium chloride 4.0-6.0 Sodium metabisulfite 0.05-0.15 Crystal violet 0.0005-0.0015 Nalidixic acid 0.015-0.025 Microbiological agar 10.0-12.0