UA29973U - Microbiological medium for accumulation of bacteria of the genus listeria - Google Patents

Abstract

A microbiological medium for bacteria accumulation of the genus Listeria. A medium contains pancreatic hydrolyzate of kasein (PHK), yeast extract (YE), lithium chloride, sodium chloride, ammonium iron citrate, distilled water, and as the source of nitrogen - pancreatic hydrolyzate of sprat with retention of ammine nitrogen 0.15-0.20 g/% and pH of the medium is 7.2-7.4.

Description

Description of the invention

The useful model refers to human and veterinary microbiology, biotechnology and can be used for bacteriological diagnosis of listeriosis, for the maintenance and storage of museum strains of cultures and the accumulation of bacterial mass in production conditions.

Listeriosis is a common problem for medical and veterinary specialists, because infection occurs through the alimentary route when using food products and feed of animal and plant origin.

The low concentration of Listeria in pathological material compared to the amount of accompanying microflora 70 prevents identification of the causative agent by the method of direct sowing on a differential diagnostic medium. To increase the number of the listeriosis microbe in the studied samples, a means of accumulating the culture of the pathogen on enrichment media is used.

There is a means of accumulation of listeria at a temperature of (4-6)2C for 14 days in an enrichment medium - a phosphate-salt buffer (I.A. Veleneva, Dissertation of Candidate of Biological Sciences - Vladivostok, 1996. - 19 p.).

The medium is made on the basis of distilled water with the addition of Man RO), dehydrated - 2.Zg/l and

Mag" HRO, dehydrated - 12.5 g/l, pH 7 4.

For the isolation of listeria from seafood and seawater, an enrichment medium containing a solvent and a nutrient base is used (Patent KO 2268307 cl. C1201/04 Method of Accumulation of Listeriosis Microbes (Ivyegia Toposziodepev) dated 04.07.2004). Sterile seawater is used as a solvent, and mineral salts in 5 mother solutions are used as a nutrient base.

The above-mentioned environments cannot be used for the accumulation of bacterial biomass in biotechnological production.

Also, dense nutrient media are often used for the cultivation and diagnosis of bacteria of the I iviegia genus, for example, a nutrient medium based on enzymatic hydrolyzate of blood (patent KO 2118363 29 kl, С12М1/20, С12012/04 dated 19.06.97 Microbiological medium for the cultivation and diagnosis of bacteria genus I isiegia), which also cannot be used in biotechnological production, because the use of dense environments complicates the technological process of bacterial mass accumulation.

The closest microbiological medium is a differential medium for bacteriological diagnosis of listeriosis (patent 48) 2223313 Cl. С12М1/20,01251/04 dated 04/07/2004

Differential-diagnostic medium for the elimination of listeria), which consists of the following components:

Pancreatic fish meal hydrolyzate 12.0-18.0; b"

Pancreatic casein hydrolyzate 8.0-12.0; Those!

Baker's yeast extract 1.5-2.0;

From Lithium chloride 14.0-16.0; with

Sodium chloride 3.0-4.0;

Esculin 0.7-0.9;

Ammonium iron citrate 0.45-0.55; "

Polymyxin B sulfate 0.005-0.015; 70 Nalidixic acid 0.015-0.025; o, c Acriflavin 0.005-0.015; ; » Agar-agar 12.0-14.0;

Distilled water is the rest.

The disadvantage of this medium is that this multi-component medium is differentially dense, has a high cost and is not suitable for the accumulation of Listeria bacterial mass. "se The purpose of the useful model is to develop a microbiological environment for the accumulation of bacteria of the I isiegia genus, which includes pancreatic casein hydrolyzate (PCH), baker's yeast extract (EPD), lithium chloride, sodium chloride, ammonium iron citrate, distilled water , by adding -I 20 as a source of nitrogen-pancreatic hydrolyzate of several with an amino nitrogen content of 0.15-0.20g/90 at a medium pH of 7.2-1 A with the following ratio of components, g/l:

That's it

EPD 1.8-2.3; pc 9.5-10.5;

Lithium chloride 14.0-15.5; c Sodium chloride 2.5-3.5;

Ammonium iron citrate 0.4-0.6;

Pancreatic hydrolyzate number 14.0-16.0;

Distilled water is the rest. 60 to ensure the effectiveness of the microbiological environment.

Pancreatic kelp hydrolyzate, which is offered as a nutritional base, is a product of deep cleavage of proteins with a wide range of amino acids. In addition, the content of amino nitrogen in this nutrient medium is supplemented by pancreatic casein hydrolyzate and meat-peptone broth. The introduction of hydrolyzate of yeast contributes to the intensive growth of listeria, because it is a source of vitamins of group B. A comparative analysis of the technical solution with the prototype allows us to conclude that the microbiological environment differs from -D-

simplification of the existing composition, does not contain expensive components, has more pronounced growth properties, which meets the criterion of "novelty"

Example 1.

The medium is prepared as follows: take the following amounts of components, g/l.

EPD 1.8 pc 9.5

Lithium chloride 14.0; 70 Sodium chloride 2.5;

Ammonium iron citrate O,4;

Pancreatic hydrolysis number 14.5

Pancreatic hydrolyzate is prepared several times according to Hottinger, according to the methodology (Handbook of microbiological and virological research methods, Birger O.M. / M.: 1967. - P.53-54). t5 To one liter of kelp hydrolyzate, which contains 14.5 g of dry pancreatic hydrolyzate, add measuring spoons, boil until the components dissolve. After cooling, bring the pH to 7.2-7.4, filter and sterilize

Oh, 5 atm. within 30 minutes.

Bacterial control of the environment was carried out in accordance with example C (table).

Example 2. 720 Prepare a medium according to the prescription of example 1 with the following ratio of components, g/l:

EPD 21 pc 10.5

Lithium chloride 15.5;

Sodium chloride 3.5; shsh

Ammonium iron citrate 0.6;

Pancreatic hydrolysis number 16.0

To one liter of kelp hydrolyzate, which contains 16.0 g of dry pancreatic hydrolyzate, add a measuring cup, Me, and boil until the components dissolve. After cooling, bring the pH to 7.2-7.4, filter and sterilize according to m.

Oh, 5 atm. within 30 minutes.

Bacterial control of the environment was carried out in accordance with example C (table). Me,

Example 3. Fo

The effectiveness of the medium was checked using test strains: Iv(egia toposuiodepez Mo924002, Iv(egia 32 mapmii Me924004, Iv(egia hyposia Mo924005.

To compare the growth properties of different media, MPB, Hottinger's broth, Marten's medium, MPB with the addition of 1595 casein hydrolyzate and 0.290 PGK were used.

To determine the growth properties, each test strain was first inoculated on a cut MPA. "

A microbial suspension was prepared in a sterile 0.995 sodium chloride solution, which according to the turbidity standard of 70 corresponded to 50Omln. m.c./cm3. c) c Next, each culture was seeded at 0.1 cm3 per bcm3 of each liquid medium: MPB, MPB with the addition of 195% casein hydrolyzate and 0.295% yeast hydrolyzate, Marten's medium, and Hottinger's broth (3 test tubes).

Incubated for 24 hours at a temperature of (35--0.5)2С. Next, tenfold dilutions were prepared from each test tube of each liquid medium in a sterile physiological solution to a dilution of 10 8. From dilutions 1072-1078, 0.1 cm of microbial suspension was inoculated onto a Petri dish with MPA, (item 3).

The crops were incubated for 48 hours at a temperature of (35--0.5)20. Colonies of Ivyegia toposuiodepev, Ivyegia imapmii, and Ivyega iposcpa on MPA were round transparent with

He has a gray-white shade, size 0.2-1.0 mm.

The results were calculated using a colony counting device (table). - The intensity of bacterial biomass growth was evaluated in time dynamics, the first two hours every 30 minutes, then after 3, 4, 5, 6, 8, 12, and 24 hours by the optical density of the suspension of microbial cells, which was recorded in the corresponding period, photometrically at a wavelength of 59 ohms.

It can be seen from the table that the intended environment has high growth properties. Thus, its adaptation phase (lag phase) took 4.8 times less time in comparison with Marten’s broth and 4.1 times with broth

Hottinger, and the phase of active growth is 2.7 and 1.3 times larger, respectively. c Thus, the proposed liquid nutrient medium for the accumulation of Listeria biomass has the following advantages: - it does not contain expensive components; 60 - environment components are produced at an industrial level, so they are standardized; - the composition of the environment is simplified, while the growth properties exceed those of known bacterial environments; - on the basis of the proposed medium, it is possible to produce differential liquid and dense nutrient bacterial medium. b5

Table

Microbiological environment for the accumulation of bacteria of the genus Ivyegia

Type of nutrient medium Indicators of the listeria cultivation process

Concentration of microorganisms after 24 hours. KUSISm Z, 19

MPBE 1.096PGKya 0.2 95 EPD 1.3-40.08 5.340.3 5.76-40.23

Martin's broth 1.4-0.15 6.1-0.45 7.67-0.18

Hottinger broth 1.201 12.1--0.7 8.55-0.34

Technical solution, 0.29-0.03 16.3-0.6 10.24-0.57 example 1

Technical solution, 0.31-0.04 16.0-0.5 10.32-0.43 example 2

Claims (1)

The formula of the invention Microbiological medium for the accumulation of bacteria of the type I isiegia, which includes pancreatic casein hydrolyzate (PCH), baker's yeast extract (EPD), lithium chloride, sodium chloride, ammonium iron citrate, distilled water, which is distinguished by the fact that it additionally contains as source of nitrogen - pancreatic hydrolyzate of kelp with an amino nitrogen content of 0.15-0.20g/95 at a medium pH of 7.2-7.4 with the following ratio of components, g/l: EPD 1.8-2.3 in pc 9, 5-10.5 lithium chloride 14.0-15.5 shsh sodium chloride 2.5-3.5 ammonium iron citrate 0.4-0.6 pancreatic hydrolyzate few 14.0-16.0 s distilled water the rest. che Official Bulletin "Industrial Property". Book 1 "Inventions, useful models, topographies of integrated microcircuits", 2008, M 3, 11.02.2008. State Department of Intellectual Property of the Ministry of Education and Science of Ukraine. From the village - and? ko (Se) (Se) - Se 60 b5