RU1837071C - Method for cultivating bacteria of bacillus anthracis type - Google Patents

Description

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The invention relates to microbiology (and, in particular, to methods for the manufacture of Bacillus anthracis pores, and can be used in research institutions involved in the study of | Iberian ulcer, as well as in the Biological Industry in the manufacture of anti-syssyra vaccines .

The aim of the invention is to increase the level of spore formation and yield of the biomass of the spores of the strains of Bacillus anthracis, shorten the production time, and reduce the cost and labor costs of the production of the target product.

The goal is achieved by the fact that in the manufacture of the spore form of the strains of Bacillus ajnthracls, instead of CPA, a new forum medium containing dry

hydrolyzed fodder yeast 12-15 g / l with a total nitrogen content of 1.2-1.5%, amine nitrogen 0.5-0.7% and additionally agar 3.5-40 g / l. sodium chloride 4-6 g / l, monosubstituted phosphate. potassium 3-7 g / l, disubstituted potassium phosphate 5-12 g / l, oxalic sodium 0.8-1.2 g / l, tween-80 0.02-0, 04% The indicated amount of total and amine nitrogen provides a high level of spore formation (98-99%). In addition, the cultivation of the strains was carried out under other conditions, namely, after 16-18 hours of cultivation at 34-35 ° C, the temperature was lowered to 28-30 ° C and cultivation was continued for 56-64 hours.

Application for the production of spore. material of strains of Bacillus antHracis new nutrient medium, as well as change

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cultivation conditions allow to increase the yield of spores by 15-20 times compared with the prototype, to reduce the time of manufacture, to reduce the cost and labor costs for the production of the target product without changing the biological properties of the strains.

Example. A new sporulation medium is used to prepare the spore material of the strains of Bacillus anthracis, in which a dry hydrolyzate of feed yeast 12-15 g / l is used as a source of nitrogen-containing substances. A decrease in the amount of hydrolyzed fodder yeast in the composition of the nutrient medium below 12 g / l leads to a decrease in the biomass of spores, an increase of more than 15 g / l is impractical because it does not increase the yield of spores, but prolongs the process of spore formation by 20-25 hours.

To create a neutral pH of the nutrient medium (7.2 ± 0.2) per 1 liter of medium, 5-12 g of potassium phosphate disubstituted and 3-7 g of monosubstituted potassium phosphate are added. Changing the amount of these components below or above the tolerances causes a pH shift to the acidic or alkaline side, which adversely affects the growth of anthrax strain of the link. The addition of oxalic sodium (0.8-1.2 g / l) and tween-80 (0.02-0.04%) to the medium promotes rapid spore formation. A change in the content of these substances slows down the formation of spores.

Then, agar (3.5-40 g / l) was added to the nutrient medium, sterilized by autoclaving at 120 ° C for 40 min and poured into 0.5 l in 2.5 l (quarter) bottles. agar solidification of the bottle with the medium is checked for sterility, keeping it for 16-20 hours at.

For the production of spores, the Bacillus anthracis strain is seeded in m-peptone broth, the crops are incubated at Zb-37 ° C for .14-16 hours. Then the broth culture is seeded in bottles with sporulation medium at the rate of 4-6 ml in each bottle. Crops are incubated at for 16-18 hours, then the temperature is lowered to 28-30 ° C and cultivation is continued for 56-64 hours.

After culturing Bacillus anthracis under the described conditions, the spore culture is sucked from the bottles into a glass container through a siphon with a gauze filter. The concentration of viable spores is then determined by sieving on Petri dishes with the proposed growth medium and then counting the grown colonies.

As a result of comparative studies, it was found that the level of spore formation on the proposed sporulation medium by this time was 98-99%, while on the CPA this indicator was 58-60%, only on the 4th-5th day of cultivation on the CPA the level of sporulation was 65 and 70%, respectively. The cultivation of the strain on the developed nutrient medium for more than 80 hours is impractical, since the biomass yield and the level of spore formation do not increase.

Thus, when cultivating a strain on a developed nutrient medium, the level of spore formation is higher than by 1.8-1.9 times on KPA. In addition, when grown on CPA, part of the spores is immature, and about 15-20% of vegetative cells do not form spores, while almost all cells sporulate on the proposed medium (98-99%).

Cultivation conditions are of great importance for increasing spore yield. It was found that lowering the temperature of cultivation to 28-30 ° C on the developed nutrient medium leads to a significant increase in spore yield (8-12 times).

As a result of comparative studies, it was found that the yield of spores from 1 L of a new sporulation medium during cultivation of the vaccine strain 55-VNIIVViM under the described conditions is (60-74) 1010. whereas with the same amount of CPA, (3-4) 1010 was obtained dispute.

The table shows comparative data obtained during the production of 5 series of spore material of the vaccine strain 55-VNIIVViM in parallel experiments on CPA and a new sporulation medium. Given that a single immunizing dose of vaccine for large animals is 2 10 spores, 30,000 to 37,000 doses are received with 1 liter of new sporulation medium. whereas with 1 liter of KPA - 1500-2000 doses of anthrax vaccine.

Thus, the yield of spores of the vaccine strain 55-VNIIVViM from a unit of a new sporulation medium under the developed culturing conditions is 15-20 times higher than in the prototype. °

Claims (1)

The claims A method of cultivating bacteria of the species Bacillus anthracis, comprising sowing and incubating bacteria at 34-35 ° C in a dense sporulation medium containing a nitrogen source, sodium chloride, agar and water, characterized in that, for the purpose of increase the level of spore formation, spore yield and speed up the method, incubation is carried out for 16-18 hours on a medium containing fodder yeast hydrolyzate with a total and amine nitrogen content of 1.2-1.5 and C, 5-0.7%, as a nitrogen source respectively and additionally containing potassium phosphate monosubstituted. disubstituted potassium phosphate, oxalate and tin-80 sodium in the following ratio of com. Spore yield of vaccine strain 55-VNIIVViM with 1 liter of culture medium ponents, g / l: hydrolyzed fodder yeast with a total and amine nitrogen content of 1.2-1.5 and 0.5-0.7%, respectively 12-15; agar 35-40; sodium chloride 4-6; sodium oxalate 0.8-1.2; monosubstituted potassium phosphate 3-7; potassium phosphate disubstituted 5-12; tween-80 0.2-0.4; water to 1 l, then the temperature is lowered to 28-30 ° C and further incubated in the same medium for 56-64 hours