RU2196827C1 - Method of differentiation of listeria monocytogenes culture - Google Patents

Abstract

FIELD: microbiology. SUBSTANCE: as species Listeria monocytogenes is a single species of genus Listeria with pathogenic properties for humans and animals it is necessary to differentiate this species from relates ones exactly. Method of differentiation of L. monocytogenes involves hydrolysis of lecithin in nutrient medium with addition of activated carbon and without addition. Method involves addition of 5 ml of 10% yolk egg suspension in physiological solution to 100 ml of medium GRM-1 (Obolenks) and lecithin hydrolysis is assayed in the presence of 0.5% of activated carbon but without additional supplements. L. monocytogenes shows hydrolytic activity with respect to lecithin in the presence of activated carbon only in contrast to other species of listeria. Method is specific, rapid and low-cost. Invention can be used for sanitary-epidemiological control of foodstuffs infectivity in centers of State sanitary and epidemiology inspection and food industry. EFFECT: improved method of culture differentiation. 2 tbl

Description

 The invention relates to microbiology and can be used for sanitary-epidemiological control of food contamination in the centers of the Sanitary Inspection and the food industry.

 The species Listeria monocytogenes is the only pathogen for humans and animals of the genus Listeria, therefore, it is necessary to clearly differentiate this species from closely related ones. Differentiation of listeria is possible based on their biochemical characteristics, for example, on the basis of differences in the ability to hydrolyze carbohydrates and phospholipids (lecithin).

A known method of differentiating an isolated culture of Listeria monocytogenes from other representatives of the genus Listeria, comprising determining the hydrolytic activity of 3 carbohydrates. One colony of the isolated culture is seeded in 2 ml of purple carbohydrate nutrient medium containing phenol red dye and each of the following carbohydrates at a concentration of 0.5%: mannitol, rhamnose, xylose. The culture is incubated at 37 ^o C for 7 days, the presence of fermentation is determined by the color change of the indicator from red to yellow. L.monocytogenes ferments rhamnose and does not ferment mannitol and xylose (1).

 However, this method is expensive and also not specific enough, it does not allow, in particular, to reliably differentiate L. monocytogenes from the non-pathogenic species L.innocua.

 The aim of the invention is to simplify and increase the specificity of the method of differentiation of the causative agent of listeriosis L.monocytogenes from other Listeria spp.

 The essence is that the differentiation of L.monocytogenes is carried out by determining the hydrolysis of lecithin in a nutrient medium containing 0.5% emulsion of the yolk of a chicken egg with the addition of 0.5% activated carbon.

Example. The agarized timing medium 1 (Obolensk) is prepared in two different containers by adding 100 ml of distilled water per 39.5 g of medium to each of the containers. Activated carbon in powder in the amount of 0.5 g per 100 ml of water is added to one of the containers. After autoclaving, which is carried out at 121 ^{° C.} for 15 minutes, 5 ml of a sterile 10% emulsion of chicken egg yolk in physiological saline (0.9% sodium chloride solution in distilled water) is added to each container as a lecithin source; and the medium is sterilely poured into Petri dishes.

The culture under study is streaked onto the plates. Inoculated cultures are incubated for 48 hours at 37 ^° C. After this time, hydrolysis of lecithin is observed in a medium containing activated carbon around the culture of L. monocytogenes and L. ivanovii, but not other species of Listeria spp., Which appears as a dense halo 0.2-1 cm wide. No lecithinase activity was detected in L. monocytogenes in L. monocytogenes, activity reaches the same level in L. ivanovii as in other species of carbon in other species of Listeria spp. activity is not detected (table. 1). In the table. 2 shows a method of intra-genus differentiation of Listeria monocytogenes based on the reaction of hydrolysis of carbohydrates (analogue). This method requires a longer incubation time, is expensive, because requires the use of expensive carbohydrates, it is not specific enough for the differentiation of L.monocytogenes from the non-pathogenic species L.innocua.

 Thus, the method of differentiating L.monocytogenes from other listeria by comparing the lecithin activity of a culture grown on a nutrient medium containing and not containing activated carbon is specific, cheap, and fast. Differentiation is performed by comparing lecithinase activity on an environment containing activated carbon and not containing: in L.monocytogenes, lecithinase activity is observed only in the presence of activated carbon, in L. ivanovii, lecithinase activity is observed regardless of the presence of coal, and in other types of listeria, lecithinase activity is not detected neither in the presence of coal, nor in its absence.

Literature
1. FDA Bacteriological Analysis Guide, Part 10, Anthony D. Hitchins, 8th Edition, 1995

Claims (1)

 A method for differentiating Listeria monocytogenes culture from other Listeria spp. including culture inoculation, determination of hydrolytic activity, characterized in that the hydrolytic activity is determined for lecithin in a culture medium containing 0.5% emulsion of the yolk of the chicken embryo, with the addition of 0.5% activated carbon.

Images