RU1781299C - Method for identification of typhoid fever pathogen - Google Patents

Abstract

Usage: microbiologists, identification of enterobacteria, bacteriological diagnosis. SUMMARY OF THE INVENTION: lactose-positive ones are additionally selected on combined 2 and 3 sugar media. crops that do not form gas and hydrogen sulfide. Re-subculture them on a lactose medium, beveled with a column, containing (g / l): agar 8.9; peptone 2-3; carbohydrate 10-15; sodium chloride 4-5; potassium dihydrogen phosphate 0.3-0.4; sodium thiosulfate 0.2-0.3, bromothymol blue (0.2% aqueous solution) 11-12 ml, distilled water to 1 L, pH 7.2-7.4. The crops are incubated for 24-72 hours at 37 ° C, cultures that give a lactose-negative reaction are selected, they are transferred to a differentiating biochemical series, serological and phagolysable properties are determined, and cultures are identified by the combination of characteristics characteristic of abdominal bacteria typhoid. The method makes it possible to detect a previously not diagnosed by a known method, a significant group of typical bacteria of typhoid fever with the activity of proteolytic enzymes and the thiosulfate reductase enzyme slightly reduced within the limits of natural variability. 5 tab. (L C

Description

The invention relates to microbiology and can be used to identify enterobacteria.

A classic method for identifying the causative agent of typhoid fever is known, which we have chosen for the prototype (Guidelines for the microbiological diagnosis of diseases caused by enterobacteria.

The method is carried out by seeding the test material on enrichment media, followed by reseeding on selective platelet media, sowing the grown lactose-negative colonies on 2- or 3-sugar combined media and identifying the cultures by the combination of biochemical, serological and phage-isolating properties.

The disadvantage of this method is the inability to diagnose a significant group of typical bacteria of typhoid fever with the proteolytic activity of the thiosulfate reductase enzyme slightly reduced within the range of natural variability, giving a false positive assessment of the lactose trait and false negative trait of hydrogen sulfide production on combined media.

The aim of the invention is to improve the accuracy of the method.

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Yu Yu

Yu

The goal is achieved by additional selection from the combined media lactose-positive, not forming gas and hydrogen sulfide crops, replanting on a mowed with a column lactose medium containing, g / l:

Agar 8-9

Pepton2-3

Carbohydrate 10-15

Sodium Chloride 4-5

Potassium dihydrogen phosphate 0.3-0.4

Sodium thiosulfate 0.2-0.3

Bromothymol blue (0.2% aqueous solution) 11-12

The water is distilled to 1 liter, pH 7.2-7.4. incubation of inoculation at 37 ° C (24-72 h), reseeding of cultures, which were considered lactose-negative on the proposed medium, on biochemical media in which the saccharolytic properties are determined on mono-carbohydrate mowed with a column (mannitol, sucrose, dulcite, arabinose, etc.), and a sign of the formation of hydrogen sulfide on a Kligler type medium when cultured at 37 ° C for at least 4-5 days with daily monitoring, determination of serological and phagolysable traits, and identification of the culture by the combination of traits characteristic of Salm onella typhi.

Example 1. The test material is seeded in storage media, incubated for 18-24 hours at 37 ° C. Subcultured on plate selective media. Cultivate at 37 ° C for 20-24 hours. The grown lactose-negative colonies are subcultured into 2 or 3 sugar combined media of the Kligler type. Cultivate at 37 ° C for a day. Lectose-positive cultures that do not form gas and hydrogen sulfide are selected and transplanted onto a lactose-rich nutrient medium slanting with a column, containing, g / l: Agar8-9

Pepton2-3

Carbohydrate 10-15

- Sodium chloride 4-5

Potassium dihydrogen phosphate 0.3-0.4

Sodium thiosulfate 0.2-0.3 Bromothymol blue (0.2% aqueous solution) 11-12

Distilled water to 1 liter pH 7.2-7.4

Crops are incubated for 24-72 hours at 37 ° C, lactose-negative cultures are selected, and they are seeded on a biochemical series in which the breakdown of mannitol, sucrose,

5 xyloses, arabinoses, dulcite, sorbitol are determined by inoculation on monocarbohydrate media slanted with a column, and the production of hydrogen sulfide by incubating the culture on Kligler medium with daily monitoring is not

0 less than 4-5 days. Serological and phagolizable traits are determined and culture is identified by the totality of all traits.

PRI me R 2. Preparation of mono-carbon water mowed with a column of medium.

A portion of sodium chloride, carbohydrate, peptone and disubstituted potassium phosphate, hyposulphite is previously dissolved in a small amount of distilled

0 water. The agar was dissolved in the remaining distilled water with stirring and heating until boiling. Then all the ingredients are combined, mixed and an indicator is added. Install

5, 2-7.4. Pour into 7-8 ml tubes, sterilize at 0.5 excess atmosphere at 112 ° C for 20 minutes. After sterilization, the tubes are left in a slightly inclined position to obtain a neat column. The finished medium has a grassy color.

Application of the medium: The test culture is inoculated with a bevel stroke and an injection in a medium column. After cultivation at

5 37 ° C during the day the test is taken into account. The following options are possible depending on the degree of activity of the determined saccharolytic enzyme at the time of accounting:

0 With a high degree of activity - the environment is in a bevel and in a column; beveled colonies turn yellow.

With an average degree of activity, only colonies are stained yellow

5 on the sloping part of the medium.

With a weak degree of activity in the case of delayed fermentation, the medium retains a green color and the final test should be taken into account after 48-72 hours.

0 From the data table. Figure 2 shows that in some test cultures, carbohydrate (lactose) fermentation occurs only under relatively anaerobic conditions of the medium column. In this case, the test is evaluated as

55 positive for (color transition in the medium column from green to bright yellow with a simultaneous appearance of blue color in the beveled part. Gas production is taken into account in the column for the appearance of bubbles, medium breaks.

Enterobacteria, which do not break down the corresponding carbohydrate, color the skew of the medium blue due to the formation of a small amount of alkaline products of proteolysis, which proceeds more intensively under aerobic conditions of the skew. Preservation of a green color or slight nonspecific yellowing of a column while staining the slant of the medium in blue is evaluated as the absence of the corresponding enzyme in the tested bacteria.

Example 3. Medium with minimal ingredients.

The medium is prepared according to Example 2. The obtained medium has the following composition, g / l: Agar8

Peptone2

Lactose10

Sodium chloride4

Sodium thiosulfate 0.2

Potassium dihydrogen phosphate 0.3

Bromothymol blue (0.2% aqueous

solution) 11

Distilled water Up to 1 L

pH 7.2-7.4

Data on the identification properties of the medium, see table. 1.

Example 4 Medium with Optimum Ingredient Values

The medium is prepared according to Example 2. The resulting medium has the following composition, g / l: Agar 8.5

Peptone 2.5

Lactose 12.5

Sodium chloride 4.5

Sodium Thiosulfate 0.25

Potassium dihydrogen phosphate 0.35

Bromothymol

blue (0.2% aqueous solution) 11.5

Distilled water up to 1 liter

pH 7.2-7.4

The data on the identification properties of the medium, see table. 1.

PRI me R 5. The environment with the maximum

the meaning of the ingredients.

The medium is prepared according to example 2. Received

on Wednesday has the following composition (g / l): Agar9

Peptone3

Lactose15

Sodium chloride5

Sodium thiosulfate 0.3

Potassium dihydrogen phosphate 0.4

Bromothymol blue 12

Distilled water Up to 1 L

pH 7.2-7.4

From the data of Table 1 it can be seen that the following medium composition is optimal, g / l: Agar 8-9

Peptone .2-3

Carbohydrate 10-15

Sodium Chloride 4-5

Potassium dihydrogen phosphate 0.3-0.4

Sodium thiosulfate 0.2-0.3

Bromothymol blue (0.2% aqueous solution) 11-12

Distilled water Up to 1 L

pH 7.2-7.4

A decrease in the concentration of ingredients is not favorable for the viability and for the detection of the enzymatic abilities of microbes. An increase in the concentration of ingredients leads to excessive osmotic pressure in the medium, which also complicates the viability and detection of the enzymatic ability of microorganisms.

From the comparative data table. 2, 3, using the determination of the cleavage of lactose on Giess lactose with lactose, Kligler medium and the proposed beveled medium with lactose, it is evident that the latter has the highest sensitivity.

From the data table. Figure 3 shows that for the fermentation of carbohydrates (lactose) in some enterobacteria, the degree of anaerobicity of the conditions is important. To prove this situation, test cultures, which were considered lactose-negative on Kligler's medium, are re-plated to evaluate the lactose test for agarized Gissa lactose-agarized medium with column (Makhachkala Scientific Research Institute for the Production of Nutrient Media) and lactose-agarized mowed.

PRI me R 6. A comparative assessment of the fermentation of lactose on media Kligler, Giss and beveled lactose medium (table 2). Example. The effect on the metabolism of lactose is aerobic (canting of the medium) and relatively anaerobic conditions (column of the medium) (Table 3).

In order to maintain the shape, the beveled column was added to the Gissa medium during preparation in an amount of 8 g / l agar.

Based on the data presented in Tables 2 and 3, it can be argued that the use of a mono-carbohydrate medium beveled with a column allows one to more reliably determine the breakdown of carbohydrates during the variable activity of the corresponding enzyme, to obtain a semi-quantitative evaluation of the test and to determine the enzymatic (under relatively anaerobic conditions of the column ) and the respiratory (under conditions of the beveled part) nature of carbohydrate utilization.

A more than seven-fold difference in the concentration of carbohydrate and peptone avoids the masking effect of alkaline products of proteolysis and creates favorable conditions for the resulting acidic products of the breakdown of carbohydrates.

Example 3. Effect of the exposure time on the production of hydrogen sulfide in S. typhl strains on Kligler medium.

EXAMPLE 9. Effect of exposure time on alkalization of the beveled part of Kligler during cultivation of S. typhi.

Thus, the expiration time of S. typhi cultures on Kligler type media at 4-5 days is optimal, because it is sufficient to detect a sign of hydrogen sulfide formation. Reducing the term leads to a false negative assessment of the sign of hydrogen sulfide formation; increased exposure may result in culture death.

Try it on. Patient V., in whom perforation of the intestine with suspected typhoid etiology was established during surgery, was taken a blood sample in an amount of 12 ml. Inoculated in 150 ml of gall broth. After 48 hours of incubation at 37 ° C, plating from the bile broth onto Endo medium was performed. After 18 hours of incubation at 37 ° C, small colorless colonies grew on Endo medium, which were transferred to the Olkelnitsky combined differential medium. After 24 hours of incubation at 37 ° C, small isolated colonies grew on the bevel of the medium. The bevel and column of medium changed the color from pink to yellow, gas production and hydrogen sulfide production were absent. The cultures were evaluated as fermenting glucose, lactose, not producing gas and hydrogen sulfide, and not subject to further investigation according to the generally accepted scheme of the classical method.

The cultures were transferred to a lactose-laden medium, see Example 1. After 24 h of incubation at 37 ° C, the culture was evaluated as lactose-negative (see Example 2) and transferred to Olkelnitsky’s medium and a biochemical differentiating medium, in wherein the carbohydrate degradation test was evaluated on carbohydrate beveled media with the corresponding carbohydrates (mannitol, sucrose, dulcite, xylose, arabinose, sorbitol, rhamnose).

After 72 hours of incubation of the culture on Olkelnitsky’s medium, alkalization of the beveled part of the medium and production of hydrogen sulfide were noted.

After evaluating the tests on differentiating media, cultures were evaluated

as non-fermenting lactose, sucrose, dulcite, rhamnose, fermenting glucose, xylose, arabinose, mannitol, sorbitol, decarboxylating lysine, not forming indole, not utilizing citrate and malonate

sodium, hydrogen sulfide-producing, motile gram-negative bacilli, agglutinating salmonella serum 09 and VI, Hd, lysing typhoid bacteriophage.

Based on the totality of these signs, they were identified as bacteria of typhoid fever, which was confirmed after repeated identification in the Rostov-on-Don regional baclaboratory,

where the isolated culture was assigned to phagovar E I, the second enzymatic type.

This example illustrates the likelihood of a diagnostic error when using combined media and improving the accuracy of the study using the proposed method.

PRI me R 11. A urine sample in an amount of 50 ml was delivered to the laboratory during the examination of N, who was in contact with patient B.

Direct sowing was performed on Ploskirev's medium, Endo. After 20 h, small colorless colonies grew on these media, which were transferred to Kligler's medium and incubated at 37 ° С. After 24 hours, the removed cultures were evaluated as lactose-positive, not forming gas and hydrogen sulfide. The cultures were transferred to a lactose column-mowed medium and cultured at 37 ° С for 24 h and were evaluated as lactose-negative. The cultures were again redirected to a Kligler type medium (the enterprise of the central NIIVS named after I.I. Mechnikov) and a biochemical differentiating medium. After 48 hours of incubation on Kligler's medium, alkalization of the slant of the medium and production of hydrogen sulfide were noted.

Based on test results on media

of differentiating series, including monocarbohydrate media beveled with a column, cultures according to the totality of biochemical, serological, and phagolizable characters were evaluated as typhoid. After

re-identification in the Rostov-on-Don regional baclaboratory culture was assigned to phagovar E, the second enzyme type.

PRI me R 12. A sample of bowel movements

enterocolitis patient in an amount of 1 g

was added to the selenite broth on plates with Ploskirev and Endo media. After 20 hours of incubation at 37 ° С, small colorless colonies grew on Endo and Ploskirev media, which were transferred to Kligler type media. The cultures were cultured at 37 ° С for 24 h and were taken into account as lactose-positive, not forming gas and hydrogen sulfide, and transferred to a lactose-like medium slanted with a column. After 24 hours of cultivation at 37 ° C, the culture was considered lactose-positive and not further identifiable by typhoid bacteria.

This example illustrates that in this case, the assessment of the lactose test on Kligler's medium was reliable.

Thus, the use of the proposed method makes it possible to identify, by additional selection on the 2nd 3-sugar agar, lactose-positive crops that do not form gas and hydrogen sulfide, and reseeding them on a lactose, mowed with a column culture medium, previously not diagnosed with a significant group of typhoid bacteria and prescribe appropriate etiotropic treatment and take timely necessary anti-epidemic measures.

Claims (1)

The claims A method for identifying the causative agent of typhoid fever by sowing the test material on enrichment media, followed by reseeding on selective plate media, reseeding grown lactose-negative colonies on two or three sugar media and identification of cultures by the combination of biochemical, serological and phagolizable properties, characterized in that, in order to increase the accuracy of the method, they are additionally selected on combined media primary identification lactose-positive, not forming a gas and hydrogen sulfide culture, subcultured on a lactose-containing medium mown with a column, containing, g / l: Agar 8-9 Pepton2-3 Carbohydrate 10-15 Sodium Chloride 4-5 Potassium dihydrogen phosphate 0.3-0.4 Sodium thiosulfate 0.2-0.3 Bromothymol blue 0.2% aqueous solution 11-12 Distilled water up to 1 liter pH 7.2-7.4 incubated for 24-72 hours, lactose-negative cultures were selected, saccharolytic properties were determined on monocarbohydrate media slanted with a column, and hydrogen sulfide production was measured on Kligler-type media for 24-120 hours. Identity Properties Data Table 1 table 2 Continuation of Table 2 Table 3 Table 4 Table 5