RU2265056C2 - Selective nutrient medium for isolation of klebsiella - Google Patents

Abstract

FIELD: medicinal microbiology.

SUBSTANCE: invention relates to bacteriological diagnosis of diseases caused by Klebsiella and to sanitary microbiology also. Invention relates to a solid agar medium on which Klebsilla cells show good growth. The composition of nutrient medium is depleted and organic nitrogen substrate is replaced for mineral one. Medium comprises antibiotic carbenicillin but doesn't comprise sodium chloride for enhancing conditions that are unfavorable for growth of foreign microflora. Sucrose is a source for carbohydrate nutrition and in common with bromothymol blue forms the indicator system owing to the medium shows blue color. Colonies of Klebsiella show orange color on this medium. The sensitivity of medium is 10 cells. The growth of accompanying microflora is inhibited or absent. Using the proposed medium simplifies identification of Klebsiella cells that acquire specific color in growth on the proposed medium.

EFFECT: improved and valuable properties of medium.

2 tbl, 2 ex

Description

The invention relates to medicine, namely to medical microbiology, and can be used in laboratories for bacteriological diagnosis of diseases caused by Klebsiella, as well as for the isolation of these bacteria from various objects during sanitary-microbiological studies.

Klebsiella often are causative agents of human diseases (pneumonia, ozena, otitis media, cystitis, gastroenteritis, etc.) and animals (mastitis, pneumonia, septicemia, etc.). Klebsiella also belong to the group of pathogens of nosocomial infections. The clinical manifestations of these diseases are diverse, which determines the significance of the results of bacteriological studies. Due to the high variability of antibiotic resistance and other biological properties of Klebsiella, the isolation of pure cultures is necessary for the diagnosis, assessment of their pathogenicity, development of rational treatment tactics, etc. (Semina N.A., Kovaleva E.P., Tikhomirov E.D. Nosocomial infections / / Guide to the epidemiology of infectious diseases. - M: Medicine, 1983, - Volume 1, - p. 463-465.)

From the practice of medicine it is known that for the isolation of Klebsiella, Endo differential diagnostic medium containing nutrient agar, lactose and a buffer system is used. The medium is intended for the isolation of enterobacteria, has an inhibitory effect only on gram-positive microflora.

On this medium, Klebsiella form lactose-positive colonies resembling colonies of other bacteria. Klebsiella colonies can be of various sizes, shapes and colors (colorless, white, beige, yellow).

The disadvantage of this medium is that in addition to Klebsiella, Escherichia, Proteus, and other bacteria grow well on this medium, which makes it difficult to select colonies for further identification (B. S. Kiseleva in the book Enterobacteria. M .: Medicine, 1985, p. 171) .

A known method for the isolation of Klebsiella in Ploskirev's medium, used to isolate mainly shigella and salmonella. The composition of the medium includes nutrient agar, lactose, neutral red, a buffer system and inhibitory substances (bile salts, iodine, brilliant green) that inhibit the growth of gram-positive microflora, delay the swarm of Proteus, inhibit the reproduction of Escherichia and some other bacteria (Medical Microbiology. Under Edited by V.I. Pokrovsky and OK GEOTAR, Moscow: Medicine, 1998, pp. 392-393; N.A. Khomenko in the book of Enterobacteria. Moscow: Medicine, 1985, pp. 265-266).

The disadvantage of this medium is that in addition to Klebsiella, enterobacteria and other bacteria grow well, forming colonies of similar size and color, which makes their identification difficult (B. S. Kiseleva in the book. Enterobacteria. M .: Medicine 1985, p. 178).

Closest to the claimed is the agar medium K-2. K-2 medium contains agar-agar, sodium chloride, magnesium sulfate, potassium sulfate, disubstituted potassium phosphate, monosubstituted potassium phosphate, raffinose, bromothymol blue, crystalline violet, urea. On this medium, after 16-18 hours of incubation, Klebsiella form shiny mucous colonies. Colony coloration varies from yellow or green with a yellow center to blue (G.P. Kalina. Narrowly targeted media for the detection of Klebsiella // Journal of Microbiology, Epidemiology and Immunobiology. - 1980, - No. 6, - p. 28-32) .

The similarity of this medium with the proposed one lies in the fact that both of them are special media for the isolation of Klebsiella and are based on the ability of these bacteria to multiply in the medium with the only sources of nitrogen and carbohydrate nutrition.

However, the K-2 medium has the following disadvantages:

- insufficient selectivity due to incomplete inhibition of the growth of enterobacteria, cytrobacters and some other bacteria, which, when seeding substrates with an abundance of concomitant microflora, leads to an increase in the number of screenings for identification of colonies;

- the lack of a clear difference in the colony of Klebsiella in color;

- complex multicomponent composition.

The purpose of the invention is to increase the selective and differential diagnostic properties of a dense agar medium designed to isolate Klebsiella.

The present invention solves the main problem - the creation in an agar medium of conditions favorable for the propagation of Klebsiella and unfavorable for the growth of other bacteria. The invention is expressed by a combination of essential features sufficient for the positive result provided by the invention.

The solution to this problem lies in the fact that instead of an organic source of nitrogen (urea), mineral (potassium nitrate) is used to deplete the organic substrate in the composition of the medium. The medium contains the antibiotic carbenicillin and does not contain sodium chloride in order to enhance adverse conditions for the growth of extraneous microflora. Sucrose serves as a source of carbohydrate nutrition and, together with bromothymol blue, forms an indicator system. These features are significant and distinctive from the prototype.

For the preparation of the medium components are used in the following amounts, g:

Agar agar 15.0-20.0 Potassium nitrate 1.8-2.0 Magnesium sulfate 0.15-0.20 Sucrose 15.0-20.0 Bromothymol blue 0.04-0.05 Carbenicillin 0.010-0.015 Distilled water, ml Up to 1000 pH of the medium 7.6-7.8

Samples of all components, with the exception of sucrose, carbenicillin and bromothymol blue, are dissolved in water and brought to a boil. Set the pH and sterilize at 0.5 atm for 20 minutes. In this condition, the medium can be stored at room temperature for 3 months. Before use, the medium is melted, at a temperature of 45-50 ° C, the remaining components are introduced and poured into Petri dishes. The color of the medium is blue. After 18 hours of incubation at 37 ° C, Klebsiella form round-shaped mucous colonies, orange in color, with a diameter of 1.5-2.5 mm, clearly contrasting with the blue background of the medium.

The proposed medium was tested during the study of 147 samples in the microbiological laboratory of the Research Institute of Regional Infectious Pathology of the Astrakhan Medical Academy, in the microbiological laboratory of NPMK "Ecological Medicine" and the laboratory of ecology and indication of causative agents of natural focal infections of the Astrakhan Anti-Plague Station of the Ministry of Health of the Russian Federation during 2000-2002. The following are the results of a comparative assessment of the quality of the proposed environment and the prototype (examples 1 and 2).

Example 1

To determine the growth qualities of the proposed medium, one-day cultures of K. pneumoniae (4 strains) and K.oxytoca (4 strains) were sown on the proposed medium (agar-agar 15.0, potassium nitrate 1.8, magnesium sulfate 0.15, sucrose 15.0, bromothymol blue 0.04, carbenicillin 0.01, distilled water to 1.0 L, pH of the medium 7.6-7.8), K-2 medium, Endo medium and nutrient agar AGV. Sowing dose of 100 cells. Sowing cultures in a volume of 0.2 ml was performed on 5 Petri dishes of each medium. After 18 hours of incubation at 37 ° C, the number of colonies in the proposed medium was 92.0 ± 4.6%; 82.4 ± 3.8% increased on K-2 medium; on Endo medium 88.2 ± 3.6% of the number of colonies formed on nutrient agar.

To assess the selective properties, sowing of the proposed medium and K-2 medium of various types of bacteria in the amount of 100 cells was performed. The results are presented in table 1. The data in table 1 show that the selective qualities of both media are high, but with respect to E. coli, E. cloacae, E. aerogenes, C. freundii, this property of the proposed medium is more pronounced than that of K-2 medium.

When sowing a microbial mixture consisting of E. coli, P.vulgaris, K. pneumoniae in the amount of 10 ⁵ , 10 ⁵ , and 10 ² cells, respectively, on the proposed medium and K-2 medium, after 18 hours only Klebsiella colonies grew on the proposed medium . Colonies of Klebsiella, an insignificant number of very small colonies of Escherichia and single P.vulgaris grew on K-2 medium. Moreover, the isolation of pure cultures of Klebsiella was difficult due to the admixture of ashrichia and proteas.

The results of experiments with pure cultures of bacteria (table 1.) indicate the high growth qualities of the proposed environment for Klebsiella and a significant advantage in comparison with the prototype in its ability to suppress the reproduction of other bacteria.

Example 2

Five stool samples of healthy individuals artificially infected K. pneumoniae in the amount of 10, 10 ² and 10 ³ cells in 0.1 ml. Material was sown in a volume of 0.1 ml. on the proposed medium (agar-agar 20.0, potassium nitrate 2.0, magnesium sulfate 0.20, sucrose 20.0, bromothymol blue 0.05, carbenicillin 0.015, distilled water to 1.0 l, pH 7.6-7.8), and K-2 medium. After 18 hours of incubation at 37 ° C, Klebsiella were isolated on both media from samples containing more than 10 cells in 0.1 ml. At a dose of 10 cells, single Klebsiella colonies grew from crops of 5 samples on the proposed medium and 3 on K-2 medium.

Only colonies of Klebsiella grew on the proposed medium, the identification of which was facilitated by their orange color.

To assess the quality of the proposed environment, material was studied from patients with various inflammatory diseases and river water samples. Samples of river water were previously diluted 10 times with boiled tap water and sown in a volume of 0.5 ml per cup.

The results of these studies, presented in table 2, show the advantages of the proposed environment in comparison with the prototype - medium K-2 when sowing material with an abundance of concomitant microflora (feces of patients, river water). When using other, moderately seeded substrates accompanying microflora, no differences were revealed. It should be noted that in all cases, Klebsiella were isolated from the same samples in parallel on two media.

Thus, a selective differential diagnostic dense nutrient medium for the isolation of Klebsiella is proposed, in which the mineral source of nitrogen (potassium nitrate) and carbohydrate (sucrose) create conditions for the propagation of Klebsiella. Carbenicillin and the absence of organic compounds, sodium chloride, inhibit the growth of other bacteria. Differentiation of Klebsiella colonies by color is achieved by the sucrose-bromothymol blue indicator system.

The advantages of the proposed environment in comparison with the prototype are:

- in increasing sensitivity and selectivity;

- the marked difference in the colony of Klebsiella in color, which facilitates identification;

- in fewer components of the environment

The proposed environment can be recommended for the separation of Klebsiella from the material from sick people and other substrates, as well as for the separation of Klebsiella from mixed cultures of various bacteria.

Table 1
The growth rate of bacteria on the proposed environment and the environment K-2 Type of bacteria The number of strains The number of selected crops and the nature of growth Proposed environment Wednesday K-2 Satisfactory Weak Is absent Satisfactory Weak Is absent K.pneumoniae 8 8 0 0 8 0 0 E.coli 8 0 0 8 0 4 4 P.vulgaris 4 0 0 0 0 0 0 P.mirabilis 4 0 0 0 0 0 0 E.cloacae 8 0 1 7 0 6 2 E.aerogenes 6 0 0 6 0 5 1 C.freundii 4 0. 0 4 0 4 0 S.sonnei 4 0 0 4 0 0 4 S.flexneri 4 0 0 4 0 0 4 S.typhimurium 6 0 0 6 0 0 6 St.aureus 10 0 0 10 0 0 10

Note:

Satisfactory growth - the size of the colonies corresponds to those on nutrient agar.

Weak growth - colonies with a diameter of 0.5 mm or less.

table 2
Sowing of Klebsiella from various substrates Test substrate Number of samples The number of positive samples Proposed environment Wednesday K-2 Feces of patients 58 11 (18.9%) 7 (12%) Sputum 22 3 (13.6%) 2 (9%) Urine thirty 4 (13.3%) 3 (10%) Detachable wounds 28 2 (7.1%) 3 (11.7%) Female genital discharge 21 3 (14.3%) 2 (9.5%) Detachable from the ears 14 2 (14.2%) 2 (14.2%) River water twenty 6 (30%) 4 (20%)

Claims (1)

A selective breeding medium for Klebsiella, including agar-agar, magnesium sulfate, bromothymol blue, carbohydrate, a nitrogen source and distilled water, characterized in that it additionally contains the antibiotic carbenicillin, it contains sucrose as a carbohydrate, and potassium nitrate as a nitrogen source in the following proportion of components: Agar agar 15.0-20.0 g Magnesium sulfate 0.15-0.20 g Potassium nitrate 1.8-2.0 g Sucrose 15.0-20.0 g Bromothymol blue, 1% aqueous solution 4.0-5.0 ml Carbenicillin 0.010-0.015 g Distilled water Up to 1000 ml pH 7.6-7.8