Environment for the cultivation of streptococci

 

(57) Abstract:

The invention is intended for accumulation of biomass streptococci. The environment contains components in the following ratio, g on 1 l of distilled water, citric acid 4,9-5,0, potassium phosphate dvuhkamernyi 4,9-5,0, sodium chloride 1,9-2,0 magnesium sulfate 4,9-5,0, iron sulfate 0,04-0,05, asparagine 0,9-1,0, glycocol 0.9 to 1.0 and glycerin 30,0-31,0 ml. Environment provides a good and rapid growth of streptococci from the first day of incubation and allows for 4-5 times passirovannye and sowing density of 90-100 million microbial cells (M. K.) in 1 ml of medium to stabilize and secure the maximum accumulation of biomass to 10-12 billion m K. in 1 ml of culture fluid.

The invention relates to Microbiology, in particular to the development of a nutrient medium for the cultivation of streptococci.

Know the use of broth of Hottinger and nutrient medium for cultivation of enterococci containing pancreatic hydrolysate of cerebration or albumin obtained from waste globulin production (Bosian E. G et al. A. S. N 1412283 and N 1418284, 1986).

The disadvantage is the complexity of the technological process of preparation of the nutrient medium, trudnosti industry for cultivation of Mycobacterium tuberculosis and consisting of the following components in grams in 1 liter distilled water, citric acid 10,0; potassium phosphate dvuhkamernyi - 5,0; ammonium citrate - 5,0; sodium chloride and 0.5; magnesium sulfate is 0.5; the iron sulfate - 0,05; sulfate zinc - 0,1; cobalt chloride - 0,002; glycerol - 50; asparagine - 1.0; glycocol to 1.0.

The medium is a nutrient salt solutions with high nourishing properties for microorganisms.

Attempts to use for the cultivation of streptococci this synthetic environment had a positive result. Persiana with meat-peptone broth (BCH) liquid synthetic media culture streptococci did not provide adequate microbial growth. In a separate test tubes, vials, flasks growth of biomass streptococci accounted for no more than 1 to 2 billion microbial cells in 1 ml, which is inefficient.

The purpose of the present invention is the development environment for the cultivation of streptococci with a high content of microbial cells in 1 ml culture liquid, suitable for the industrial cultivation of streptococci in the production of streptococcal antigens and vaccines.

Designed environment contains components in the following ratio to 1 liter of distilled water: lemon - 4,9 - 5,0; f is let - 0,04 - 0,05; asparagine - 0,9 - 1,0; glycocoll - 0,9 - 1,0 and glycerin 30,0 - 31,0 ml.

The results showed that with the increasing content in the nutrient medium of sodium chloride from 0,5 to 1,5; 2,0; 2,5 g was provided by the sequential accumulation of biomass streptococci respectively 2 - 2,5; 3 - 3,5; 3,5 - 4,0; 3,5 - 4,0 billion microbial cells in 1 ml of culture fluid. Further, starting from 2 - 2.5 g/l, the increase of the content in the nutrient medium of sodium chloride was not accompanied by an increase in biomass streptococci.

The best results in the accumulation of biomass streptococci, up to 8 - 9 billion microbial cells in 1 ml of culture liquid were obtained in variants environments while reducing the amount of citric acid with 10 g to 5, Further reducing the number of citric acid in a nutrient medium to 4.8 - 4.5 g/l was not accompanied by an increase in biomass streptococci.

The increase of the content in the nutrient medium of magnesium sulphate 0.5 g to 1,0; 2,0; 3,0; 4,0; 5,0; 5,5 g/l was provided by the sequential accumulation of biomass streptococci 4 - 4,5; 5 - 6; 6 - 7; 7,5 - 8; 8,5 - 9; 8,5 - 9 billion microbial cells in 1 ml of culture fluid. Further, since 5 - 5.5 g/l, the increase of the content in the nutrient medium of magnesium sulphate is not soprovojdeno or increase, any decrease in the intensity of growth and accumulation of biomass streptococci. At the same time, a further decrease in nutrient medium of glycerin, ranging from 30 to 25 ml, was accompanied by a decrease in the accumulation of biomass streptococci. It was thus established that the optimal amount of glycerol in the medium determined within 30 - 31 ml.

It should be noted that during the search of the experiments we did not observe the effect on the growth of Streptococcus cobalt chloride, citrate of ammonium and sulphate of zinc, and therefore they were excluded from the components of the new environment scenarios.

Thus, the use of this variant of liquid salt synthetic nutrient medium provided good and rapid growth of streptococci from the first day of incubation and allowed by 4-5-fold passirovannye germs on it, when the seeding density to 90 - 100 billion microbial cells in 1 ml of medium, to stabilize and to provide maximum biomass accumulation up to 10 - 12 billion microbial cells in 1 ml of culture fluid.

According to Panin A. N. (Abstract. dis. D. C. N., Streptococcus pigs, 1992, 27 C. ), to prepare vaccine drug biomass accumulation streptococci should not b is suitable for use in the biological industry in the production of streptococcal antigens.

Environment prepared by serial dilution or pre-mixed components in distilled water, followed by neutralization with ammonia to pH 7.0, and 7.1 before autoclaving.

Environment for the cultivation of streptococci containing citric acid, potassium phosphate (disubstituted), sodium chloride, magnesium sulfate, iron sulfate, asparagine, glycocol and glycerin, characterized in that the medium contains components in the following ratio, g on 1 l of distilled water:

Citric acid - 4,9 - 5,0

Phosphate potassium disubstituted - 4,9 - 5,0

Sodium chloride - 1,9 - 2,0

Sulphate magnesium - 4,9 - 5,0

Iron sulfate - 0,04 - 0,05

Asparagine - 0,9 - 1,0

Glycocol - 0,9 - 1,0

Glycerin - 30,0 - 31,0 ml

 

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