RU2366702C2 - Nutrient listeria recovery medium - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: nutrient medium contains pancreatic digest of fresh-water fish, pancreatic digest of infusion broth waste, agar, sodium chloride, sodium carbonate, lithium chloride, vitamin B₁, esculin, ammoniacal ferrum citrate, polymyxin B sulphate, ceftazidime, acriflavine hydrochloride and distilled water.

EFFECT: improved inhibiting and differentiating effect of the medium, reduced time for Listeria recovery.

3 tbl, 2 ex

Description

The invention relates to medical microbiology and biotechnology and can be used to isolate listeria from infected material.

Improving the laboratory diagnosis of listeriosis is associated with the use of various differential diagnostic culture media for the isolation of listeria. But the growth time of the pathogen on the media used is quite long, and therefore there is a need to develop nutrient media that ensure the growth of Listeria colonies that have characteristic signs for determination after 24-42 hours when 10 microbial cells are seeded while suppressing the growth of extraneous microflora.

There is a known differential diagnostic medium for the isolation of listeria, containing pancreatic casein hydrolyzate, pancreatic fish meal hydrolyzate, baker's yeast extract, polymyxin B sulfate, acriflavine, esculin, iron salt, lithium chloride, sodium chloride, agar-agar, nalidixic acid and distilled water ( patent RU 2223313, С12N 1/20, С12Q 1/04, published on 04/10/2004).

A disadvantage of the known medium is the low growth rate of listeria and the late emergence of differentiating features - the growth of colonies with a diameter of 0.3-0.5 mm, having differentiating features (olive color and black halo around the colonies), is observed only after 42-48 hours. , the inhibitory properties of the medium do not allow to suppress the growth of Staphylococcus aureus when plating 500 microbial cells.

The aim of the invention is to increase the inhibitory and differentiating properties of the medium, as well as increasing the growth rate of listeria cultured on it.

This goal is achieved in that the nutrient medium for the isolation of listeria contains a nutrient base (pancreatic river fish hydrolyzate, pancreatic meat production waste hydrolyzate), mineral salts (sodium chloride, sodium carbonate, lithium chloride), vitamin B ₁ , esculin, ammonium citrate , agar-agar, polymyxin B sulfate, ceftazidime, acriflavine hydrochloride and distilled water in the following quantitative ratio of components, g / l:

Pancreatic river fish hydrolyzate 11.0-11.5 meat production waste hydrolyzate water pancreatic  9.0-9.5 sodium chloride 5.0-5.5 sodium carbonate 0.65-0.68 lithium chloride 10.0-10.5 vitamin B ₁ 0.05-0.06 ammonium citrate 0.5-0.55 esculin 0.8-0.85 agar agar 9.0-9.5 polymyxin B sulfate 0.03-0.035 ceftazidime 0.015-0.02 acriflavine hydrochloride 0.0015-0.002 distilled water up to 1 l

Comparative analysis with the prototype showed that the proposed nutrient medium differs from the known one in that it contains hydrolysates of river fish and meat water production waste, sodium carbonate, vitamin B ₁ , ammonium iron citrate, ceftazidime in the proposed amounts. Thus, this nutrient medium meets the criteria of the invention of "novelty."

The analysis of patent and scientific and technical literature showed that the proposed technical solution differs not only from the prototype, but also from other technical solutions in this and related fields. So, the authors did not find a nutrient medium for the isolation of Listeria, which would contain the proposed components in the proposed quantitative ratio. This composition of the medium allows one to enhance its inhibitory and differentiating properties, as well as to increase the growth rate of listeria and reduce the time of appearance of differentiating listeria signs. The qualitative and quantitative composition of the components of the proposed environment allows you to completely inhibit the growth of concomitant microflora, while not directly inhibiting the growth of listeria, but even accelerating it. The medium provides, when 10 microbial cells of the test strain L. monocytogenes 766 are seeded, after 18 hours of incubation at 37 ° C, round, convex, light gray dewy listeria colonies are formed, surrounded by a black halo. After 24 hours, colonies with a diameter of up to 0.3 mm acquire an olive color, and after 42 hours with a diameter of 1.2-1.3 mm, a deepening of the center becomes.

In addition, the growth of Escherichia coli ATCC 25922, Proteus vulgaris 14, Staphylococcus aureus ATCC 6538, Salmonella cholerae suis 424, Streptococcus viridans 41 when seeding 10,000 microbial cells is completely suppressed.

This medium can be used in medical microbiology to isolate listeria during food control for contamination by pathogenic microorganisms in the order of sanitary and epidemiological surveillance according to SanPiN 2.3.2.1078-01.

Thus, the proposed nutrient medium meets the criteria of "inventive step" and "industrial applicability".

The nutrient medium is prepared by mixing the components in the following quantitative ratio, g / l:

Pancreatic river fish hydrolyzate 11.0-11.5 hydrolyzed meat production waste pancreatic 9.0-9.5 sodium chloride 5.0-5.5 sodium carbonate 0.65-0.68 lithium chloride 10.0-10.5 Vitamin B ₁ 0.05-0.06 ammonium citrate 0.5-0.55 esculin 0.8-0.85 agar agar 9.0-9.5 polymyxin B sulfate 0.03-0.035 ceftazidime 0.015-0.02 acriflavine hydrochloride 0.0015-0.002 distilled water up to 1 l

The medium is prepared as follows: a sample containing pancreatic river fish hydrolyzate, pancreatic meat water production waste hydrolyzate, sodium chloride, sodium carbonate, lithium chloride, vitamin B ₁ , esculin, ammonium iron citrate, agar-agar, are mixed in 950 ml of distilled water, bring to a boil with stirring, boil for 5-7 minutes, cool to a temperature of 35-40 ° C, then make a selective additive containing the above amounts of polymyxin B sulfate, ceft idim, acriflavine hydrochloride, previously dissolved in 5 ml of sterile distilled water, thoroughly stirred, adjusted to 1 liter with sterile distilled water, and poured into sterile Petri dishes. To remove condensate, Petri dishes with the medium are slightly opened and incubated for (40 ± 5) min at a temperature of (21 ± 2) ° С.

In this case, the pancreatic river fish hydrolyzate was prepared as follows: the river fish (roach) was used as a substrate, the cattle pancreas was used as a source of proteolytic enzymes at an enzyme-substrate ratio of 1:10. Hydrolysis of fish was carried out by a reactor method with periodic or constant stirring at a temperature of 45 ° C, pH 8.0, for 6-7 days. For preservation, one percent chloroform was added (v / v). The content of amino nitrogen in the resulting hydrolyzate is 500-560 mg%.

The pancreatic meat water production hydrolyzate was obtained in accordance with industrial regulation No. 01898090-03-06 (registration number of GISK named after L. A. Tarasevich No. 1733-06): 128 l of drinking water was poured into the reactor, 1.2 l was added to it 20% sodium hydroxide solution (to bring the pH to 7.8-8.0), 32 kg of crushed waste products for the production of meat water, 11.7 kg of crushed pancreas and 1.28 kg of chloroform. Hydrolysis was carried out at a temperature of 43 ± 2 ° C for 7 days. After filtration (for conservation), 2.28 L of chloroform was added to the finished hydrolyzate per 122 L of hydrolyzate. Amine nitrogen content 6.0 ± 0.25 mg / ml.

The growth rate of listeria on the proposed medium and its differentiating properties are estimated by plating 10 and 100 microbial cells of the daily agar culture of the strain L. monocytogenes 766. The grown colonies are counted after 18-24-42 hours of incubation at a temperature of (37 ± 1) ° С. At the same time, the appearance of differentiating characters in the Listeria colonies (olive color, black halo, deepening of the center) is noted.

The inhibitory properties of the proposed medium are determined by plating on the medium 10,000 microbial cells of the strains of associates (Escherichia coli ATCC 25922, Proteus vulgaris 14, Staphylococcus aureus ATCC 6538, Salmonella cholerae suis 424, Streptococcus viridans 41). After 48 h of incubation at a temperature of (37 ± 1) ° С, the presence or absence of growth of associate colonies was noted.

Example 1. The nutrient medium is prepared as described above, while using the following ratio of components, g / l:

Pancreatic river fish hydrolyzate 11.0 meat water production hydrolyzate pancreatic 9.0 sodium chloride 5,0 sodium carbonate 0.65 lithium chloride 10.0 Vitamin B ₁ 0.05 ammonium citrate 0.5 esculin 0.8 agar agar 9.0 polymyxin B sulfate 0,03 ceftazidime 0.015 acriflavine hydrochloride 0.0015 distilled water up to 1 l

After 18 hours of incubation in the proposed medium, a dewy growth of listeria of a light gray color with one differentiating sign, a black halo, was noted.

After 24 hours of incubation, 9 listeria colonies surrounded by a black halo formed on the proposed medium. The appearance of the second differentiating feature, the olive color of the colonies, was noted.

After 42 hours of incubation on the proposed medium, a third differentiating feature appeared in the Listeria colonies — the center deepening.

After 48 hours of incubation on the proposed medium, there was no growth of any of the five strains - associates.

Example 2. The nutrient medium is prepared as described above, while using the following ratio of components, g / l:

Pancreatic river fish hydrolyzate 11.5 meat water production hydrolyzate pancreatic 9.5 sodium chloride 5.5 sodium carbonate 0.68 lithium chloride 10.5 Vitamin B ₁ 0.06 ammonium citrate 0.55 esculin 0.85 agar 9.5 polymyxin B sulfate 0,035 ceftazidime 0.02 acriflavine hydrochloride 0.002 distilled water up to 1 l

After 18 hours of incubation in the proposed medium, a dewy growth of listeria of a light gray color with one differentiating sign, a black halo, was noted.

After 24 hours of incubation, 11 listeria colonies surrounded by a black halo formed on the proposed medium. The appearance of the second differentiating feature, the olive color of the colonies, was noted.

After 42 hours of incubation on the proposed medium, a third differentiating feature appeared in the Listeria colonies — the center deepening.

After 48 hours of incubation on the proposed medium, there was no growth of any of the five strains - associates.

A test of the proposed environment in laboratory conditions in comparison with the environment of the prototype

Growth properties of nutrient media are given in table 1.

Table 1
Growth properties of culture media for L. monocytogenes (incubation for 18-42 hours at a temperature of (37 ± 1) ° С) Culture medium Sowing dose (microbial cells) The number of colonies grown 18 h 24 h 42 h 48 h Proposed 10
one hundred dew growth 10
77 10
77 10
77 Prototype 10 - - 2 2 one hundred - - 9 9 Note: (-) lack of visible growth.

Thus, the proposed nutrient medium for the isolation of Listeria has a significant advantage, since it allows detecting dewy growth after 18 hours, and after 24 hours - the formed Listeria colonies, while on the prototype medium, colony growth appears only after 42 hours.

The differentiating properties of nutrient media are given in table.2

table 2
Differentiating properties of nutrient media (incubation for 18-42 hours at a temperature of (37 ± 1) ° С). Culture medium Differentiating characteristic Colony color Black halo Deepening the center of the colonies 18 h 24 h 42 h 18 h 24 h 42 h 18 h 24 h 42 h Proposed light gray olive olive + + + - - + Prototype - - olive - - + - - - Note: (-) lack of evidence.

According to the differentiating properties, the proposed nutrient medium for the isolation of Listeria also has a significant advantage in comparison with the medium - the prototype in terms of time of occurrence and the number of differentiating features. The first differentiating feature - a black halo - is noted on the proposed nutrient medium after 18 hours of incubation, the second - the olive color of the colonies - after 24 hours of incubation. On the prototype medium, there is no growth in these periods of time. The third differentiating feature - the deepening of the center of the colonies - is recorded on the proposed nutrient medium after 42 hours of incubation. On this prototype medium, convex colonies of olive-colored listeria with a black halo are formed by this time.

The inhibitory properties of culture media are given in table 3

Table 3
Inhibitory properties of nutrient media (48 h incubation at a temperature of (37 ± 1) ° С). Culture medium The number of grown colonies (sowing dose of 10,000 m.k.) E. coli S. cholerae suis St. viridans P.vulgaris St. aureus Proposed - - - - - Prototype Solid growth Note: (-) lack of visible growth.

The inhibitory properties of the proposed nutrient medium for the isolation of Listeria also has a significant advantage compared with the prototype medium, since the growth of Escherichia coli ATCC 25922, Proteus vulgaris 14, Staphylococcus aureus ATCC 6538, Salmonella cholerae suis 424, Streptococcus viridans 41 is completely inhibited in the medium inoculation of 10,000 microbial cells, while on the prototype medium growth of Staphylococcus aureus ATCC 6538 is observed.

The inhibitory properties of the test medium were determined with respect to mixtures of the five above associates and listeria. The seeding dose of associates was 100 times higher than the dose of L. monocytogenes.

Mixtures of microbes were prepared by combining 1 ml of a suspension of L. monocytogenes with a concentration of 2000 mk and 1 ml of suspension of one of the microbes-associates with a concentration of 200,000 m.k. Of these mixtures, 0.1 ml of the mixture was inoculated onto the proposed medium and the prototype medium. Previously, all associates were tested for purity of growth on elective nutrient media on which growth of colonies typical of them was detected.

After 18 hours of incubation on the proposed medium, the appearance of the growth of colonies with a black halo was recorded. After 24 hours of incubation, the colonies acquired an olive color characteristic of Listeria, and after 42 hours a deepening of the center of the colonies was noted.

On the prototype environment, in two cases, the growth of two types of colonies was observed: when plating from a mixture of L. monocytogenes and P. vulgaris, the lawn was flat and on their background were single convex olive-colored; when plating from a mixture of L. monocytogenes and St. aureus - in the thickness of the lawn of a golden yellow culture, separate colonies appeared (an average of 11 per cup), in the growth area of which the agar acquired a black color. When plating from mixtures of L. monocytogenes and E. coli, L. monocytogenes and St. viridans, and L. monocytogenes and S. cholerae suis the formation of convex colonies of olive color was observed only after 42 hours of incubation.

Thus, the proposed nutrient medium for the isolation of listeria allows L. monocytogenes to be isolated in a short time (after 18 hours) and, thereby, accelerate the detection of listeria in the infected material.

Claims (1)

Nutrient medium for the isolation of listeria, containing a nutrient base, sodium chloride, lithium chloride, ammonium citrate, esculin, agar-agar, polymyxin B sulfate, acriflavine hydrochloride, distilled water, characterized in that it additionally contains sodium carbonate, vitamin B ₁ and ceftazidime, and pancreatic river fish hydrolyzate and pancreatic meat water production waste hydrolyzate as a nutrient base in the following ratio of components, g / l:
Pancreatic river fish hydrolyzate 11.0-11.5 hydrolyzed meat production waste pancreatic 9.0-9.5 sodium chloride 5.0-5.5 sodium carbonate 0.65-0.68 lithium chloride 10.0-10.5 Vitamin B ₁ 0.05-0.06 ammonium citrate 0.5-0.55 esculin 0.8-0.85 agar agar 9.0-9.5 polymyxin B sulfate 0.03-0.035 ceftazidime 0.015-0.02 acriflavine hydrochloride 0.0015-0.002 distilled water up to 1 l