RU2268307C1 - Method for accumulation of listeriosis microorganism (listeria monocytogenes) - Google Patents

Abstract

FIELD: microbiology.

SUBSTANCE: invention relates to methods of sanitary-hygienic control over infectivity of marine water. Method for accumulation of listeriosis microorganisms (Listeria monocytogenes) involves culturing listeria in enrichment medium at temperature 18-20°C wherein the enrichment medium comprises a solvent and nutrient base. Sterile marine water is used as a solvent, and mineral salts in mother solutions are used as the nutrient base as mg/l of distilled water wherein 1 l of the enrichment medium consists of 5 mother solutions and sterile marine water. Invention provides the more complete detection and recording extrabody populations of listeria inhabiting in marine water.

EFFECT: improved accumulating method.

1 dwg, 1 ex

Description

The invention relates to microbiology, in particular to sanitary-hygienic control of the infection of Listeria monocytogenes with sea water and seafood.

The aquatic environment is one of the ecological niches for L.monocytogenes. From the end of the 80s to the present, numerous outbreaks of listeriosis in people associated with the consumption of infected seafood have been observed in several countries in Europe and America. In this regard, the problem of isolation of strains of listeriosis microbe from the marine environment is quite relevant.

The very low concentration of listeria in comparison with the amount of concomitant saprophytic microflora in the studied samples makes it difficult to isolate the pathogen by direct seeding on a differential diagnostic medium. To increase the number of listeriosis microbe in the studied samples, a method of accumulating the pathogen culture on enrichment media is used.

A known method of accumulation of listeriosis microbe at a temperature of 4-6 ° C for 14 days on an enrichment medium - phosphate-buffered saline (FSB) [Beleneva I.A. Abstract. dis. Cand. biol. sciences. - Vladivostok, 1996. - 197 p.]. The medium is prepared on the basis of distilled water with the addition of NaH ₂ PO ₄ anhydrous. - 2.3 g / l and Na ₂ HPO ₄ anhydrous. - 12.5 g / l, NaCl - 5.8 g / l, pH 7.4. Because the accumulation of culture is carried out at a temperature of 4-6 ° C, this method is called "cold" enrichment.

The principle of the method lies in the fact that when the test samples taken from a warm-blooded organism are kept in the “cold”, the concomitant mesophilic microflora is suppressed. In the absence of competitive microflora, Listeria, which have psychrophilic properties, actively multiply, which leads to an increase in the concentration of the pathogen in the studied material. Given the fact that in samples taken from the marine environment, the accompanying microflora are psychrophilic microorganisms, the method of "cold" enrichment is ineffective.

The disadvantages of this method are:

1. The absence of a factor restraining the reproduction of concomitant psychrophilic flora.

2. The inability to isolate L.monocytogenes strains from the marine environment in an unchanged (natural) form when using an unadapted enrichment medium, due to the known variability of the listeriotic microbe.

3. Incomplete identification and recording of extraorganismal L.monocytogenes populations from the marine environment.

The objective of the invention is to more fully identify and account for the extraorganismal populations of Listeria living in the marine environment, by inhibiting the propagation of the concomitant psychrophilic flora and preventing variability of the Listeriosis microbe.

This is achieved by the fact that the accumulation of the listeriotic microbe in the studied samples taken from the marine environment is carried out at a temperature of 18-20 ° C for 14 days on the enrichment medium for L.monocytogenes, which includes a solvent and a nutrient base. Sterile sea water is used as a solvent, and mineral salts are used as a nutrient base in the following ratio of components in mother liquors, mg / l of distilled water:

Solution No. 1:

NaH ₂ PO ₄ - 10

NaNO ₃ - 150

Solution No. 2:

Na ₂ SiO ₃ × 9H ₂ O - 30-60

Solution No. 3:

CuSO ₄ × 5H ₂ O - 0.0196

ZnSO ₄ × 7H ₂ O - 0.044

CoCl ₂ × 6H ₂ O - 0.020

MnCl ₂ × 4H ₂ O - 0.360

Na ₂ MoO ₄ × 2H ₂ O - 0.0126

Solution No. 4:

Na ₂ EDTA - 6.8

Solution No. 5:

FeCl ₃ × 6H ₂ O - 5.3

Moreover, 1 liter of enrichment medium consists of mother liquors in the following quantities:

Solution No. 1 - 0.5-2 ml

Solution No. 2 - 0.5-2 ml

Solution No. 3 - 0.5-2 ml

Solution No. 4 - 0.5-2 ml

Solution No. 5 - 0.5-2 ml,

The rest is sterile sea water

The essential features of the proposal should be considered the accumulation of listeriotic microbe at a temperature of 18-20 ° C, which ensures the suppression of the concomitant psychrophilic microflora in the studied samples; the use of an enrichment medium that is different from that known for its qualitative and quantitative composition.

Going beyond the declared values of the components of the medium leads either to an overuse of the reagents used, or to a decrease in growth parameters of the medium and the appearance of undesirable variability of the listeriotic microbe.

The limits of the content of minerals and vitamins in the nutritional basis of the proposed enrichment medium are optimal and determine the relative balance of the cost of the medium with its growth characteristics.

The use of sea water as a solvent allows avoiding changes in the biological properties of the isolated strains that originally live in the marine environment, and increases the likelihood of detecting a listeriosis microbe in the studied samples.

The method is as follows. The test material (seawater, sludge, intestinal contents of aquatic organisms, etc.) is introduced into the enrichment medium in a ratio of 1: 1. The inoculated medium is incubated at a temperature of 18-20 ° C for 14 days. After that, the test material is plated on solid differential diagnostic media for L.monocytogenes.

To prepare the enrichment medium, mother liquors, mg / l of distilled water are first prepared:

Solution No. 1:

NaH ₂ PO ₄ - 10

NaNO ₃ - 150

Solution No. 2:

Na ₂ SiO ₃ × 9H ₂ O - 30-60

Solution No. 3:

CuSO ₄ × 5H ₂ O - 0.0196

ZnSO ₄ × 7H ₂ O - 0.044

CoCl ₂ × 6H ₂ O - 0.020

MnCl ₂ × 4H ₂ O - 0.360

Na ₂ MoO ₄ × 2H ₂ O - 0.0126

Solution No. 4:

Na ₂ EDTA - 6.8

Solution No. 5:

FeCl ₃ × 6H ₂ O - 5.3

Before introducing into the enrichment medium, the mother liquors are sterilized with pressure at 1-1.5 atm. Sea water is filtered through a cotton-paper filter and then pasteurized at a temperature of 70-75 ° C in a water bath for 20 minutes three times with an interval of 24 hours.

To mother solutions in the following amounts:

Solution No. 1 - 0.5-2 ml

Solution No. 2 - 0.5-2 ml

Solution No. 3 - 0.5-2 ml

Solution No. 4 - 0.5-2 ml

Solution No. 5 - 0.5-2 ml

add sterile seawater up to 1 liter.

Example

The studied material (sea water, sludge, intestinal contents of aquatic organisms) is introduced into test tubes with an enrichment medium in a ratio of 1: 1 and incubated at a temperature of 18-20 ° C for 14 days. Then the contents of the tubes are vigorously shaken and 0.1 ml of the suspension is inoculated onto a solid differential diagnostic medium for L.monocytogenes. The enrichment medium for L.monocytogenes includes the following components:

Solution No. 1 - 1 ml The composition of the solution No. 1 mg / l of distilled water: NaH ₂ PO ₄ - 10 NaNO ₃ - 150

Solution No. 2 - 1 ml The composition of the solution No. 2, mg / l of distilled water: Na ₂ SiO ₃ × 9H ₂ O - 30-60 Solution No. 3 - 1 ml The composition of the solution No. 3, mg / l of distilled water: CuSO ₄ × 5H ₂ O - 0.0196 ZnSO ₄ × 7H ₂ O - 0.044 CoCl ₂ × 6H ₂ O - 0.020 MnCl ₂ × 4H ₂ O - 0.360 Na ₂ MoO ₄ × 2H ₂ O - 0.0126 Solution No. 4 - 1 ml The composition of the solution No. 4, mg / l of distilled water: Na ₂ EDTA - 6.8 Solution No. 5 - 1 ml The composition of the solution No. 5, mg / l of distilled water: FeCl ₃ × 6H ₂ O - 5.3 Sterile sea water - up to one liter.

The process of propagation of L.monocytogenes (strain 10 CN, 1 / 2a serotype) in the inventive and known enrichment medium (FSB) is shown in the drawing. For 14 days of cultivation in the inventive enrichment medium at a temperature of 18-20 ° C, the concentration of L.monocytogenes (strain 10 CN) increased by 3.6 lg (drawing, curve 1). When cultured in phosphate-buffered saline (prototype) for the same period of time at a temperature of 4-6 ° C, the concentration of the listeriotic microbe increased by 3.3 lg (drawing, curve 2).

The above example shows that the proposed enrichment environment in its growth properties is practically not inferior to the prototype. The advantages of the proposed environment over the known one are expressed in the possibility of isolating strains of the listeriosis microbe in an unchanged (natural) form, as well as the ability to more fully identify and account for the extraorganismal populations of listeria living in the marine environment.

Claims (1)

The method of accumulation of listeriosis microbe (Listeria monocytogenes), including the cultivation of listeria in an enrichment medium, characterized in that the accumulation of bacteria is carried out at a temperature of 18-20 ° C, and the enrichment medium contains a solvent and a nutrient base, and sterile sea water is used as a solvent, and as a nutrient base - mineral salts, and in the following ratio of components in the mother liquor, mg / l of distilled water: Solution No. 1: NaH ₂ PO ₄ - 10 NaNO ₃ - 150 Solution No. 2: Na ₂ SiO ₃ · 9H ₂ O - 30-60 Solution No. 3: CuSO ₄ · 5H ₂ O - 0.0196 ZnSO ₄ · 7H ₂ O - 0.044 CoCl ₂ · 6H ₂ O - 0.020 MnCl ₂ · 4H ₂ O - 0.360 Na ₂ MoO ₄ · 2H ₂ O - 0.0126 Solution No. 4: Na ₂ EDTA - 6.8 Solution No. 5: FeCl ₃ · 6H ₂ O - 5.3, moreover, 1 l of enrichment medium consists of mother liquors in the following quantity: Solution No. 1 - 0.5-2 ml Solution No. 2 - 0.5-2 ml Solution No. 3 - 0.5-2 ml Solution No. 4 - 0.5-2 ml Solution No. 5 - 0.5-2 ml The rest is sterile sea water.