Nutrient culture medium for swine erysipelas strain erysipelothrix rhuisipathie

FIELD: biotechnology.

SUBSTANCE: nutrient culture medium for swine erysipelas strain Erysipelothrix rhuisipathie refers to general biotechnology and veterinary microbiology. As a nitrogenous nutrition source, the nutrient medium contains a mixture of fish autolysate and alkaline mussel hydrolysate in the following proportions: alkaline mussel hydrolysate 20-50%; fermentation peptone 1%; potassium phosphate 0.3%; sodium phosphate 1.8%; fish autolysate - the rest.

EFFECT: method can be used for preparing the microbiological nutrient media for erysipelas strain upstream.

 

The invention relates to a total of biotechnology and veterinary Microbiology and can be used for preparing microbiological culture media for growing the biomass of the strain of the causative agent of swine erysipelas.

The causative agent of swine erysipelas Erysipelothrix rhuisipathie is culture, demanding in terms of power. When the activation strain after long-term storage is of great importance, the rate of increase of biomass. As a rule, for scientific and commercial purposes using traditional nutrient medium on the basis of raw meat. Mastopathy broth BCH and ΜΠΑ from parivara of Hottinger provide good growth of microorganisms without changing their cultural and biological properties. Hydrolysates of sardines and fish meal are also widely used in the manufacture of dry nutrient media for cultivation of microorganisms, because fish proteins according to their structure and amino acid composition is most similar to proteins of meat and can meet the needs of microorganisms in organic nitrogen compounds. However, finding high quality and inexpensive culture media is an important task.

Known Nutrient medium for cultivation of microorganisms (see patent No. 2089609, C1, C12N1/20, EN), contains a source of nitrogen nutrition, sodium chloride, agar. As the nitrogen nutrition use pancr eticheski hydrolyzed fishmeal content of amino nitrogen 4,4±0,2%, total nitrogen 12,±0,5%. A significant drawback is that the hydrolysate has a high cost, because of the need to import from abroad. The closest set of features of the claimed invention is "Poivre seredova "Aquamed" for kultivovana microorganisms (divas. Pat. No. 34711 U, C12N1/20, UA), where nutrient medium is a mixture of the alkaline hydrolysate of shellfish with acid hydrolysate from fish raw materials in a ratio of 3:1 to 1:3. The final concentration of amino nitrogen was 60-120 mg %. In the composition of the medium also includes glucose or maltose, peptone, and agar. A disadvantage of the known environment is insufficient for the cultivation of the strain of the causative agent of swine erysipelas end concentrada content of amino nitrogen.

Characteristics in common with the essential features of the invention, the Nutrient medium for culturing the strain of the agent of swine erysipelas Erysipelothrix rhuisipathie is the fact that the environment consists of peptone is an enzymatic and alkaline hydrolysate of marine raw materials.

The basis of the invention the culture medium for culturing the strain of the agent of swine erysipelas Erysipelothrix rhuisipathie the task through the use of products of marine origin to construct a nutrient medium, which will ensure optimal growth of strain of the causative agent of swine erysipelas Erysipelothrix rhuisipathie for the cooking the vaccine.

This object is achieved in that for the cultivation of vaccine strain of the causative agent of swine erysipelas Erysipelothrix rhuisipathie prepared nutrient medium, consisting of fish autolysate, which impose additional 20-50% alkaline mussels of the hydrolyzate.

Mussel hydrolysate and autolysate sprat get on with well-known technology (see Kruczenski, Century, Kulyasova Century, I.e. To the question of obtaining protein hydrolysis tov from mollusk Leda//Izvestiya TINRO.- 1976.- 99.- C. 102-104); Lichvar N. A. Dry nutrient medium of fresh and frozen fish waste industry. Scientific notes of Dinevi production of culture media. Makhachkala, 1956, 2.).

The authors investigated the level of biomass formation of an industrial strain of the causative agent of swine erysipelas Erysipelothrix rhuisipathie No. 286 on different environments (table. 1).

As can be seen from the table. 1 culture was expressed in all environments except the reference, low growth rate, which indicates the demand for the nutrition conditions. In some environments also noted polymorphism sticks causative agent of swine erysipelas: elongated chain E. rhuisipathie, which is a sign of unfavorable culture conditions, in particular due to unbalanced nutrition.

Next explored the environment, which in addition of hydrolysates sprat sod the whinnying from About to 50% of mussels hydrolysate (table. 2). Cultivated strain at standard temperature conditions within 48 hours. The optical density was measured at the photo-electrochlorinator at a wavelength of 545 nm and a thickness of 5 mm cuvette Control sample was UN-cropped medium of the same composition.

As can be seen from the table. 2, adding in a nutrient medium mussels of the hydrolyzate in the amount of 20% by volume provided the doubling of biomass as in the reference environment (0,163 against of 0.081), and experience (0,130 against 0,063), and making 50% of mussel hydrolysate of Wednesday tripled growth. The difference between the experienced and the reference environment, despite the absence in the test environment of glucose, with increase in the content of mussels pyrolysate gradually decreased.

Due to the high content of amino nitrogen (an indicator of nutrient media for cultivation of microorganisms) hydrolysates seafood are valuable raw materials for biotechnological production. In the autolysate fish contains 4-10 times more amino nitrogen than is required for the productive growth of the commercial strains. Mussel hydrolysate, when its content in the environment no more than 20%, dual bacterial mass. Thus the Association of fish autolysate and alkaline mussel hydrolysate of allows you to obtain a balanced nutrient medium for cultivation of the causative agent R is LM of pigs.

An example implementation of the method. Cooked experienced a series of vaccine against swine erysipelas Erysipelothrix rhuisipathie No. 286 in volume 3 DM3. Has preliminarily determined that the pH of fish hydrolysate rate of 6.0, a bmict asnago nitrogen -740 mg%. In mussel hydrolysate pH of 7.7, and the content of amine nitrogen mg%. Of these components was prepared liquid and solid nutrient medium according to the standard formulations for the BCH. In a medium composition for culturing industrial strain on the proposed nutrient medium consisted of a mixture of fish autolysate and 20% by volume of the alkaline mussels of the hydrolysate, peptone is an enzymatic - 1% of the total, potassium phosphate and 0.3%, sodium phosphate - 1, 8%, pH was 7.3 to 7.4. The strains were planted in test tubes with liquid medium, then perseval vials with broths, and after culturing under appropriate conditions, perseval vials with the same medium and cultured in appropriate conditions. At the end of the cultivation was determined representative and the purity of the culture microscopy smears of environments and established the concentration of microbial cells.

The industrial culture of the strain of the causative agent of swine erysipelas, received on Wednesday from hydrolysates, compared with environments, obtained with raw meat did not differ on cultural and morphological properties and meet the requirements of the passport data on the strain. The concentration of the microbial cells in 1 cm shall be not less than 50 million The authors have obtained the following data: on nutrient mediums seafood concentration of microbial cells was 50 million, and the media, made from a decoction of Hottinger - 70 million

Inactivation of microbial cultures was performed using formaldehyde in a concentration of 0.4%. To check the completeness of inactivation was performed by crops received the vaccine in the nutrient medium, the microscopy of Gram-stained smears. Control emissions vaccine was conducted in 5 white mice weighing 18-20 g, injecting them intraperitoneally at 0,sm. The results of observation of the animals during the 16 days showed that the vaccine is harmless: experimental animals remained alive, eat healthy, changes in behavior are noted.

Control immunogenic activity conducted on the same white mice vaccinated with vaccine. The mice were infected with control strain of the agent faces in a lethal dose. All control mice (10 animals), which were not inoculated, died within 4-6 days, all vaccinated survived for 10 days. Based on the results of these studies, concluded that manufactured the vaccine is immunogene-active.

Thus, the claimed nutrient Sereda using products of marine origin, and ensures the growth of the strain of the causative agent of swine erysipelas Erysipelothrix rhuisipathie suitable for making effective the nd vaccine.

Nutrient media for cultivation of strains of the causative agent of swine erysipelas Erysipelothrix rhuisipathie, composed of peptone is an enzymatic, potassium phosphate, sodium phosphate, and the hydrolyzate of marine raw materials, characterized in that as the source of nitrogen nutrition use a mixture of fish autolysate and alkaline mussel hydrolysate of the following ratio of components:

alkaline mussel hydrolysate20-50%
peptone, fermentative1%
potassium phosphate0,3%
sodium phosphate1,8%
fish autolysatethe rest of it.



 

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