Differential diagnostic nutrient medium for identification of yersinia bacterium. RU patent 2511436.

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology, microbiology, and concerns the recovery and identification of pseudotuberculosis and intestinal yersiniosis agents (Y. Pseudotuberculosis and Y. Enterocolytica). A nutrient medium contains microbiological agar (dry), lactose, glucose, urea, calcium chloride, 1% alcoholic phenol red, 1% alcoholic methylene blue and distilled water in specific proportions.

EFFECT: invention enables reducing the length of studies.

3 dwg, 2 tbl

The invention relates to medicine, in particular to Microbiology, and for the allocation and identification of pathogens of pseudotuberculosis and intestinal yersiniose (K. pseudotuberculosis and Y. enterocolytica).

Pseudotuberculosis and intestinal yersiniosis is infectious diseases caused by Yersinia spp., transmitted through nutritional and characterized by the polymorphism of clinical manifestations and a variety of clinical forms. On the territory of the Russian Federation and registered each year in the world group and sporadic cases of yersiniosis arising in preschool institutions, schools, orphanages, country groups, military units, enterprises and educational institutions, United by a common power source.

The most reliable results, confirming the diagnosis, receive microbiological allocation of Yersinia from pathological material ("gold standard" for diagnosis). However, the isolation and identification of Yersinia associated with certain difficulties and first of all with the length of the proceedings. This implies the need to improve methods of isolation and identification of Y. pseudotuberculosis and Y. enterocolytica by creating effective differential diagnostic growth media.

The scheme microbiological release of Yersinia, adopted at present, consists of consecutive stages: enrichment, the initial seeding on Petri dishes with nutrient media (such environments Serov, №67, Endo, Levin, Maurice and others), secondary screening with solid nutrient media for tubes with one differential media containing urea, mitred column (Kligler, Altnickol and others). In these environments, iersinii grow as Matt dry RAID on sloping surfaces and during injection over the entire height of the column with degradation of urea and education alkali (post after 24 hours of incubation gets a crimson color). Cultures are further used for further identification of biochemical tests and serological temirovna (Fig.1).

Known nutrient medium for the diagnosis of bacteria of the genus YERSINIA.

1)Differential diagnostic combined nutrient medium for selection of Yersinia (SBTS)manufactured by the state research Institute of applied Microbiology (Obolensk, Moscow region).

The composition of the medium: nutrient agar 7,8; for cultivation of microorganisms - 35,0 g

bile purified - 6,0 g

glucose - 10, 0 g

urea-5,0 g

bronchiology blue water-soluble - 0,128 g

phosphate disubstituted potassium (Kanrad) - 1,0 g

Method of preparation: components contribute to 1.0 l of distilled water, heated to boiling and boil for 5 minutes on low heat. Then filtered through cotton-gauze filter, again boil for 1-2 minutes, cool up to 45-50 C and bottled in Petri dishes. Keep the environment within 7-10 days at 4-7°N

2) Environment with urea on Prius (in modification of ZNIIS them. I.I. Mechnikova) indicator bromtimol blue and Christensen indicator bromophenol red.

The use of these environments has made more reliable detection of urease activity of bacteria, which simultaneously with sowing in combination environment do sowing in a test tube with the environment Christensen (prepa).

3) Environment Christensen with urea, composition:

The Solution I. Peptone - 1.0 g

Sodium chloride - 5,0 g

Glucose - 1.0 g

Potassium phosphate one-deputizing - 2.0 g

Phenol red (solution 1:500) - 6,0 ml

Urea - 20,0 g

Distilled water - 100,0 ml

The solution II.

Agar-agar - 15,0 g

Distilled water - 900,0 ml

Sterile 0.5 ATM 15 minutes.

The preparation. It cooled down to 50-55°With the solution II add 100 ml of solution I, mix, pour aseptically into a sterile tube, environment cooled in an inclined position to obtain the column and the jamb. Ready environment yellow.

The research.

Make massive planting the whole surface of the casing. Incubated at 37 deg C. Conduct accounting in 2, 4, 6 hours the next day. In some cases, with negative results to see 4-7 days (possible slow reactions). A positive result - red color.

However, the use of additional environments with urea extends the detection and identification of Yersinia for another 1-2 days.

Thus, microbiological identification of Yersinia takes 5 days, which is unacceptable for a long time for making clinical. In addition, significantly increases the cost of bacteriological tests.

As a prototype, the applicant chose a well-known and widely used 3-sugar environment, Altnickol and its modification. The use of this nutrient is regulated by corresponding methodical documents (Epidemiology, laboratory diagnosis of yersiniosis, organization and implementation of preventive and anti-epidemic measures. The statement, approved. The USSR Ministry of health 30.10.1990 year,№15-6/42; Prevention of yersiniosis. Sanitary-epidemiological rules SP 3 3.1.7.2617-10, appr. the resolution of the Chief state sanitary physician of the Russian Federation from 26.04.2010 year,№37; Epidemiological surveillance and prevention of pseudotuberculosis and intestinal yersiniose. Methodical instructions. MU 3.1.12438-2009).

The composition of the medium, Altnickol in classical and modified versions are described in the Order of Ministry of health USSR №535-84 year(table 1)

Table 1 Composition of the medium, Altnickol in classical and modified versions

Ingredients

Modified

Nutrient agar, pH of 7.2 to 7.4, ml

Lactose, g

Sucrose, g

Glucose, g

Sodium thiosulfate (Hypo), g

Ammonium iron (II) sulfate (Salt Mora)

Phenol red, 0,4% odnr-p, ml

Environment of GIS with lactose and Indyk. (BP), g

Distilled water, ml

to 1000,0

Wednesday sterile 0.5 ATM 20 minutes or fluid the ferry 3 days to 20 minutes Wednesday cooled in a slanted position to retrieve column height of 2.5 cm and jamb length 4 see the Finished Wednesday pale pink color.

The course of studies

Sow the bar on the beveled surface and injection in a column. Incubated at 37 C 18-24 hours. Kislotoobrazoutei causes yellow, decomposition of urea (increase pH), reddening.

The disadvantage is that the main purpose of this environment-identification of bacteria family Enterobacteriacea, which includes 12 genera and more than 300 species of microorganisms. Therefore, environment Altnickol does not solve the task differential diagnostic selection of bacteria of the genus Yersinia, (Fig.2).

The objective of the invention is to create a simple differential diagnostic of the nutrient medium with high resolution, which allows you to define affiliation culture allocated to the genus Yersinia without using additional environments, it results in significant reduction of terms of research.

The problem is solved through preparation of differential diagnostic nutrient medium for identification of bacteria of the genus Yersinia containing microbiological agar (dry), lactose, glucose, urea, phenol red dye and distilled water, according to the invention nutrient medium contains methylene blue and chloride calcium in the following ratio, g/100 ml:

Dry agar-3,0

Lactose - 1,0

Glucose -0,15

Urea - 0,3

Calcium chloride - 0,2

Phenol red (1% alcoholic R-R) - 0.2 ml

Methylene blue (1% alcoholic R-R) - 0.1 ml

Distilled water to 100 ml

a pH of 7.0 to 7.2.

As a solvent for paints phenol red and methylene blue used ethyl alcohol.

The development of the proposed a nutrient medium taken into account and used biochemical peculiarities of the genus Yersinia and individual species of this genus.

The simultaneous use of the claimed composition of the nutrient medium alcohol solution of methylene blue and phenol red allows to obtain specific staining (column crimson, beveled part violet) unique to bacteria of the genus Yersinia. Consequently, the proposed culture medium can be considered as a simple and their biochemical properties.

The composition of two colors - differential diagnostic environment with high resolution for bacteria of the genus Yersinia

Introduction into the medium of chloride calcium that is not a component of the power iersini, provides its izotonichnost, prevents bacterial cells from osmotic shock.

Thus the essential features of the proposed suggestions should be considered the qualitative and quantitative composition of the nutrient medium with high resolution capacity, since the magnitude of the content of components in the claimed nutrient medium are optimal and determine the relative balance cost environment, with its growth characteristics. The concept of high resolution nutrient medium, the Complainant implies the possibility of allocation of bacteria of the genus Yersinia in the course of one culture on the claimed nutrient medium without the use of additional tests, resulting in study periods are reduced to 18 to 24 hours.

Nutrient medium prepared as follows.

Dry ingredients environment dissolved in distilled water in any sequence. After dissolution of agar the mixture is heated to boiling and add indicators (phenol red and methylene blue). Establish a pH of around 7.0-7.2 and poured into test tubes 5-6 ml sterilized by autoclaving 15 min at a pressure of 0.5 ATM.

Hot environment has dirty-green colour, and immediately after autoclaving acquires yellow-orange coloration in the recovery of methylene blue.

Test tubes with the environment gently shaken and set at an angle 45-degree angle (for mowing, leaving a vertical column height of 1,5-2,0 cm and oblique surface 4-5 cm). At cooling environment, it again becomes dirty-green colour due to oxidation of the indicator atmospheric oxygen.

Frozen nutrient medium shall within 2-3 days "to Mature" - to buy a plain uniform in colour, which indicates the oxidation of indicator across the entire environment.

Sowing material carried out throughout the beveled surface (above) and the subsequent injection in a column environment.

At cultivation on the environment through the day various members of the family Enterobacteriaceae causes characteristic changes in the color of the proposed environment (table 2).

Table 2 Changes proposed a nutrient medium under cultivation of bacteria of the family Enterobacteriaceae

Bacteria

Beveled part

The nature of growth on the post

The existence of gas

Shigella

Tender, with a blue tint

Salmonella

Lush, with whitish tint

There are

E. 0 124

Lush, with whitish tint

There are

Escherichia

Lush, with whitish or green

There are

Raspberry

Drain, with a lilac shade

Iersinii

Raspberry

Drain, with a lilac shade

Note: *) - during the shot.

Iersinii ferment glucose with the formation of acid (obligatory sign to all members of the family Enterobacteriaceae) break down urea and restore methylene blue light. However, the final chemical and redox changes in the bar and on the beveled surface environment (jamb) significantly differ in color. In the bar there is an increase in pH due to the alkalinity of the environment (breakdown of urea), resulting in a restored methylene blue indicator light will fade. When this column is colored phenol red indicator crimson color (figure 3).

Consequently, the proposed culture medium can be considered as a differential diagnosis. On the sloping part of the column also is the splitting (hydrolysis of urea and, therefore, alkalization environment. Phenol red indicator turns crimson color which mixes with methylene blue, gives intensively-violet protection.

Thus, various representatives of bacteria of the family Enterobacterioceae having different biochemical properties in relation to lactose, glucose, urea and methylene blue, change the color of the environment at different pH values. Iersinii give a clear picture, as a rule, after 18-24 hours, and other representatives of the family is called the characteristic changes in the environment and in a shorter time.

Declare the nutritional environment meets the criteria of the invention:

"novelty" - claimed the medium simultaneously use two phenol red indicator and methylene blue that amplify the differential diagnostic environment properties and at imposing on each other give crimson and purple color, which allows to differentiate the bacteria Yersinia from various representatives of bacteria of the family Enterobacterioceae);

"inventive step" of a particular applicant prior art revealed no known simultaneous use of two dyes for the diagnosis of bacteria-iersinii.

"industrial applicability" - this nutrient medium simple to perform and can be used for both scientific andresearch purposes, and for the diagnosis of diseases in medical institutions.

Differential diagnostic nutrient medium for identification of bacteria of the genus Yersinia containing microbiological agar (dry), lactose, glucose, urea, phenol red dye and distilled water, wherein the nutrient medium contains methylene blue and chloride calcium in the following ratio, g/100 ml

Dry agar

Calcium chloride

Phenol red (1% alcohol solution)

Methylene blue (1% alcohol solution)

Distilled water

up to 100 ml

a pH of 7.0 to 7.2.