RU2272834C2 - Nutrient biphase medium for isolation of trichomonads - Google Patents

Abstract

FIELD: biotechnology, microbiology.

SUBSTANCE: invention proposes a nutrient medium comprising solid and liquid phases. A solid phase represents coagulated serum, and a liquid phase comprises the nutrient medium 199, benzylpenicillin sodium salt and streptomycin solution. Invention provides enhancing the sensitivity of nutrient medium for isolation of trichomonads. Invention can be used in cultural diagnosis of trichomoniasis.

EFFECT: improved and valuable properties of medium.

2 tbl, 3 ex

Description

The invention relates to medicine, namely to microbiology, and can be used, in particular, for the cultural diagnosis of trichomoniasis.

Due to the insufficient sensitivity, subjectivity during microscopic diagnosis of “trichomoniasis” and in accordance with numerous Orders and Instructions, identification of Trichomonas vaginalis is carried out by the cultural method, which, according to most researchers, is the “gold standard”: highly sensitive and specific. To a large extent, its effectiveness depends on the quality of the nutrient media used. The composition of the used media includes meat and peptone broth (BCH), liver preparations, mineral salts, sugars, human or animal blood serum, hydrochloric acid cysteine and other ingredients. 77 g. No. 10-8 / 23).

So, for example, starting from the 40s of the last century, Johnson-Trussell medium (CPLM) was widely used, which, as modified by TsNIIKVI, is a greenish liquid medium with high growth qualities, but it is non-standard (prepared in laboratory conditions), it has limited shelf life.

In the 70 - 80s in the Department of Microbiology of TsNIIKVI developed original culture media for the cultural diagnosis of trichomoniasis, which have been successfully used for several years. At the same time, they all suffer from the same shortcomings as the Johnson-Trussel environment, and in recent years they have practically not been applied in practice. The composition of these media includes: a mixture of casein hydrolyzate and hydrolysin or aminopeptide, pharmaceutical industry waste in the manufacture of lysozyme, enzymatic biomass hydrolysates, microorganisms, as well as fodder doge extract. These and other modifications are described in Order No. 936 and Manual “Laboratory Diagnosis of Genitourinary Trichomoniasis” (G. Dmitriev Laboratory Diagnosis of Bacterial Urogenital Infections. - Moscow: Medical Book, N. Novgorod: Publishing House NGMA, 2003. P.44-67, 161-182, 242-254).

By the mid-90s, a paradoxical situation had developed: in an epidemic of sexually transmitted infections (STIs), including trichomoniasis, in Russia there was virtually no production output of standard culture media for the cultural diagnosis of this disease. In practice, we used media prepared in laboratories, which, of course, affected their quality.

At the same time, there is an urgent need to make wider use of the cultural diagnosis of trichomoniasis, which is necessary when making a diagnosis, especially with dubious microscopic results, as well as with a chronic recurrent infectious process, examination of children, pregnant women, and a number of other cases.

The use of a nutrient medium with high growth indicators, standard composition and ease of preparation will undoubtedly lead to an adequate diagnosis of such a widespread disease burdened with various complications, such as urogenital trichomoniasis.

The closest composition to the proposed medium is the medium of the following composition: nutrient medium 199 - 10 ml, saline solution - 100 ml, yeast autolysate - 10 ml, serum of cattle or horse - 30 ml, 20% maltose solution - 10 ml penicillin - 160,000 units and streptomycin - 160,000 units (G. Dmitriev Laboratory diagnosis of bacterial urogenital infections. - Moscow: Medical Book, N. Novgorod: Publishing House NGMA, 2003. C.180-181).

However, the use of this medium does not exclude the need for subsequent microscopy in order to establish the presence of trichomonads in the test material. The aim of the invention is to increase the sensitivity of the medium for the isolation of Trichomonas.

The goal is achieved in that the proposed two-phase nutrient medium contains a solid phase represented by coagulated serum in an amount of 5 ml and a liquid phase containing a nutrient medium 199 in an amount of 5 ml with a solution of sodium benzyl penicillin 10000-30000 UNITS / ml and a solution streptomycin 10000-30000 mcg / ml - 1 ml, respectively.

The proposed medium was tested at the Department of General and Bioorganic Chemistry of the Astrakhan State Medical Academy on 50 samples of material from patients during 2003.

Below are the results of testing.

Example No. 1.

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

Solid phase:

Coagulated serum "kosyachkoy" in vitro - 5

Liquid phase:

Wednesday 199 - 5

The solution of the sodium salt of benzyl penicillin 10000 IU / ml - 1

Streptomycin solution 10,000 mcg / ml - 1

Antibiotics are each diluted in 1 ml of a 0.9% sterile sodium chloride solution and aseptically added to medium 199.

Sowing the test material is carried out on the surface of the coagulated serum by the stroke of the bacteriological loop. Then medium 199 with antibiotics is layered onto the serum.

The incubation of crops is carried out at 37 ° C for up to 7 days in the absence of growth during the first days. In the presence of trichomonads in the studied material at their interface, their abundant growth is noted. Non-instrumented visualization of Trichomonas growth is also possible because medium 199 contains a color indicator, which, in the case of cell growth and, as a result of this change in pH of the solution, changes color from red to yellow.

Example No. 2.

The medium is prepared in accordance with example 1, but the components are taken in the following quantities, ml:

Solid phase:

Coagulated serum "kosyachkoy" in vitro - 5

Liquid phase:

Wednesday 199 - 5

The solution of the sodium salt of benzyl penicillin 20,000 IU / ml - 1

Streptomycin solution 20,000 mcg / ml - 1

Antibiotics are each sterilely diluted in 1 ml of a 0.9% sodium chloride solution and aseptically added to medium 199.

In the presence of trichomonads in the studied material at their interface, their abundant growth is noted with a change in the color of the liquid phase.

Example No. 3.

The medium is prepared in accordance with example 1, but the components are taken in the following quantities, ml:

Solid phase:

Coagulated serum "jamb" in vitro - 5

Liquid phase:

Wednesday 199 - 5

The solution of the sodium salt of benzyl penicillin 30,000 IU / ml - 1

Streptomycin solution 30,000 mcg / ml - 1

Antibiotics are each diluted in 1 ml of a 0.9% sterile sodium chloride solution and aseptically added to medium 199.

In the presence of trichomonads in the studied material at their interface, their abundant growth and discoloration of the liquid phase is noted.

When inoculating material from patients on the proposed medium, the growth of concomitant bacterial flora is inhibited.

Table 1 shows the test results of three options for the proposed environment.

The advantage of the proposed environment is the acceleration of the growth of Trichomonas compared with growth on the Bio-Rad medium due to the fact that Medium 199 contains the nutrients necessary for the growth of eukaryotic cells, which include Trichomonas. Table 2 gives the results of the sensitivity of the known and proposed media for sowing Trichomonas.

In addition, the presence of Trichomonas growth when they are sown on the proposed medium is read visually as a result of a change in the color of the pH indicator, and therefore there is no need for subsequent microscopy, which speeds up the diagnosis process and makes the analysis less time consuming and expensive.

Thus, the use of the new medium significantly increases the sensitivity of bacteriological studies aimed at identifying Trichomonas in the material from patients and subjects.

The invention can be recommended for use in practical health care in venereological, urological and andrological institutions, as well as in the scientific work of laboratories of the corresponding profile.

Table 1
Inhibitory effect on concomitant microflora and a stimulating effect on the growth of Trichomonas components of the test medium The content of ingredients in the environment The presence of visual growth of Trichomonas after 24 hours of incubation The presence of growth of concomitant microflora after 24 hours of incubation Minimum Weak growth Single bacterial colonies on the surface of the solid phase Optimal Abundant growth Lack of growth Maximum Moderate growth Lack of growth

table 2
The results of sowing Trichomonas on the test environment and the environment of Bio-Rad Sowing dose, the number of cells in 1 ml The results of crops on the compared medium (the number of hours after which the growth of Trichomonas is fixed visually) Proposed environment Wednesday Bio-Rad one hundred 48 96 500 24 48

Claims (1)

Nutrient two-phase medium for the isolation of trichomonads, containing a solid phase represented by coagulated serum in an amount of 5 ml, and a liquid phase containing a nutrient medium 199, in an amount of 5 ml with a solution of benzyl penicillin sodium salt 10000-30000 IU / ml and streptomycin solution 10000- 30000 μg / ml - 1 ml, respectively.