The method of determining the hemolytic activity of bacillus anthracis

 

(57) Abstract:

The invention is intended to characterize the strains on their biological activity. Prepare double-agrarian culture medium broth of Hottinger with the addition of yeast extract 2 - 2.5 g/l and calcium chloride to 0.30 - 0.33 g/L. the Concentration of agar in the lower layer is 1.0 to 1.5 wt. % and in the upper layer is 0.6 - 0.8 wt.%. Sow the crop on the top layer. Incubated seeding in the presence of human erythrocytes in the amount of 6 - 8% of the volume of medium for 22 - 24 hours are counted results by the presence of zones of hemolysis of erythrocytes. The invention increases the sensitivity and accelerates the analysis and selection of colonies within a single strain. 3 Il.

The invention relates to Microbiology, in particular to methods characteristics of the strains for biological activity.

Known qualitative way to determine hemolytic activity, which is that the culture is plated on mastopathy agar with 5 - 10% defibrinating the blood of the lamb. Crops incubated at 35 - 37oC for 18 - 20 h, after which produce results visually by the presence of clear zone of hemolysis around the colonies (A. Korotich S., Pokerbracelet. A significant density of agar at a relatively high level of production hemolytic substances agar cultures of Bac. anthracis hinders the diffusion of these substances in the column of agar containing erythrocytes. In addition, adding 5 to 10% defibrinating blood approximately half of this volume is blood serum, which has strong inhibitory action against hemolysis Bac. anthracis. Therefore, this method is suitable only for the detection of hemolytic activity of highly active products of hemolysins, which include most of saprophytic representatives of the genus Bacillus. When working with Bac. anthracis is a way hemolytic activity does not reveal.

Known quantitative method for determining hemolytic activity (Tkachenko Century, the technique of the account of the reaction of hemolysis using fotoelektrokalorimetry FEC) Reports Irkutsk anti-plague Institute, V. 1, Ulan-Ude, 1961). This method is intended for determining the hemolytic activity of substances of different chemical nature.

Prepare a standard suspension of washed red blood cells of the Guinea pig hemolysate which, dissolved in 20 times is on FEC-M cells with the thickness of the liquid layer 3,045 - 3,060 mm in green with the TV in each sample control or experience should be 0.4 ml of a standard suspension of erythrocytes, contained in 4 ml of fluid (saline broth). Only when this ratio is 50% hemolysis in the experience will be characterized by the extinction of 0.250% opt.sq. or close to this value, which corresponds to the highest optical sensitivity of the colorimeter FEC-M

Solutions or suspensions containing hemolytic agents, are mixed with a standard suspension of erythrocytes in the previously specified value and thermostatic at 37oC. To stop the reaction of hemolysis test and control samples centrifuged. The supernatant colorimetrate against appropriate controls. The degree of hemolysis is expressed in percentage considering the fact that 50% hemolysis is characterized by an optical density of hemolysate corresponding 0,250% opt.PL.

This method provides a clear quantitative indicators that it is necessary to study the dynamics of the process of hemolysis, the influence of various physical and chemical factors on the activity of hemolysin, etc.

The disadvantage of this method is the need for appropriate equipment (centrifuge, FEC), and that the process is quite time-consuming. In addition, when working with virulent strains of anthrax centrifuge holds is trifuge, the researcher must work in full antiplague suit type 1.

To obtain a sterile filtrates of broth cultures requires high-quality filtering devices. Sterile can be considered the filtrate only after confirming the negative results of control of sowing. The results are considered final only after keeping them for a few days, during which the filtrate is largely loses its hemolytic activity.

This method is not suitable to determine the heterogeneity of the population of strains, because at inoculation with spores of the broth is formed a mixed population of different clones and determined the total hemolytic activity.

The closest to the essence of the present invention is a method of determining the hemolytic activity of Bacillus anthracis, comprising preparing a double-layer agar plates with the addition of sheep red blood cells in the top layer, forming holes in the thickness of the agar plates, making them daily broth culture, incubating samples with subsequent consideration of the results by the presence of zones of hemolysis around the holes (ed.St. USSR N 1597404, C 12 Q 1/04, 07.10.90, bull. N 37).

With whom then melted, cooled and poured into sterile Petri dishes and 20 ml was Also prepared in physiological solution of 0.7% agar, in which, after cooling to 45oC contributed 6% of sediment washed sheep red blood cells, and then was applied to 5 ml on frozen bottom layer of agar. After solidification of the top agar layer in the plate by a punch with a diameter of 8 mm were doing well, the bottom of which was sealed 1 - 2 drops of molten 1% agar. One Cup had 6 - 7 holes. Then preparing a suspension of spores of the studied strains, corresponding to 10% of the protected area.Mut. In a test tube with 3 ml of sterile broth of Hottinger seeded by 0.05 ml (1 drop) prepared suspensions of spores and put them in the unit 36oC for 24 h In the wells were made daily broth culture 0.1 ml Crops were incubated at a temperature of 36oC. records were made after 24 and 48 hours

This method was much more sensitive than qualitative, because the use of washed erythrocytes were eliminated the inhibitory effect of serum, and the process of diffusion hemolytic substances in the hole in the form of a solution (broth culture), were faster, which also contributed to the decrease of the density and thickness of the layer of agar containing erythrocytes.

The method is I.

The disadvantage of this method is the impossibility to assess the hemolytic activity of isolated colonies that not allows the selection of clones with various hemolytic activity within a single strain. The disadvantage is due to the fact that you are using broth culture, resulting in estimated hemolytic activity of the entire population of the studied strain.

The aim of the invention is to increase the sensitivity and acceleration analysis, enabling selection of colonies with different levels of hemolytic activity within a single strain by creating the best conditions for the synthesis and manifestations of the functional activity of hemolysin Bacillus anthracis, as well as through the use of the most sensitive to the action of washed human erythrocytes.

This goal is achieved by the fact that prepare two-layer agar nutrient medium broth of Hottinger with the concentration of agar in the lower layer is 1.0 to 1.5 wt.%, and in the upper layer of 0.6 - 0.8 wt.% with the addition of yeast extract 2 - 2.5 g/l and calcium chloride to 0.30 - 0.33 g/l, sow the crop on the top layer, incubated seeding in the presence of human erythrocytes in the amount of 6 - 8% of the volume creatio the inventive method has the following distinctive features.

Application in the top agar layer more sensitive human erythrocytes, adding stimulator of the synthesis of hemolysin and its functional activity CaCl2and yeast extract in the growth of colonies Bac. anthracis improves the sensitivity of the method.

The principal difference is that in the proposed method, the agar plates are nutrient medium on the surface of which is the growth of the colonies, the secretion of hemolysin, its diffusion through agar with erythrocytes and revealing his actions in the zone of hemolysis around isolated colonies. In the prototype of the double-layer plate with erythrocytes are only a test-system for detection of hemolysin secreted microbial population broth culture in General, with prior cultivation. In this regard, the period of the study is reduced to 24 hours compared to 72 h in the prototype.

The method is as follows. Prepare the double-layer agar plate. The first layer in a Petri dish pour 20 ml of sterile molten agar of the following composition: 100 ml of the broth of Hottinger (pH 7,2-7,4), 1 g of agar, 0.2 g yeast extract (Sigma), 0.3 ml of sterile 10 -% aqueous solution of CaCl2.g of agar, 0.2 g yeast extract (Sigma), 0,30 ml of 10% aqueous solution of CaCl2, in which, after cooling to 45oC add sterile 6% washed three times in physiological solution of NaOH of human erythrocytes. Pre-prepared suspension of spores of the studied strain, while sowing 0.1 ml which on the surface of agar plates in Petri dish formed 25 - 30 colonies. On the surface of agar plates with erythrocytes put 0.1 ml of this suspension of spores, smooth rocking spread over the surface of the agar. After all liquid is absorbed Cup turned over and placed in a thermostat at 37oC. Records produced after 24 hours visually by the presence of clear zones of haemolysis.

The possibility of practical use of the invention is confirmed by examples of specific performance.

Example 1. Preparing double-layer agar plate. The first layer in a Petri dish poured into 20 ml of sterile molten agar of the following composition: 100 ml of the broth of Hottinger (pH 7,4), 1 g of agar, 0.2 g yeast extract (Sigma), 0,31 ml of sterile 10% aqueous solution of CaCl2. After solidification of the lower layer from above poured 5 ml of molten agar: 100 ml broth of Hottinger, 0.7 g of agar, 0.2 g yeast extract (Sigma), 0,31 ml of 10% aqueous solution of CaClthe e NaOH of human erythrocytes.

Was previously prepared suspension of spores Bac. anthracis STI-1, when sown on the surface of agar plates in Petri dish is formed of 15 to 25 colonies.

On the surface of agar plates with erythrocytes was applied in 0.1 ml of this suspension of spores, smooth rocking distributed over the surface of the agar. After all liquid is absorbed Cup turned down the covers and placed in a thermostat at a temperature of 37oC. analysis was carried out after 24 h visually by the presence of zones of hemolysis around the colonies (Fig.1) and their width.

Example 2. Preparing double-layer agar plate. The first layer is poured into 20 ml of sterile molten agar of the following composition: 100 ml of the broth of Hottinger (pH of 7.2), 1.2 g of agar, 0,23 g yeast extract (Sigma), 0.33 ml of a 10% aqueous solution of CaCl2. After solidification of the lower layer from above poured 5 ml of molten agar: 100 ml broth of Hottinger, 0.8 g of agar, 0,23 g yeast extract (Sigma), 0.33 ml of a 10% aqueous solution of CaCl2, in which, after cooling to 45oC sterile added 7% three times of washed human erythrocytes.

Pre-prepared suspension of spores Bac. anthracis 228/8, when sown on the surface of agar plates in Petri dish was formed 15 - 25 colonies.

oC. Records held after 23 hours the Results are presented in Fig. 2. Clearly visible zone of hemolysis around isolated colonies, as well as differences in their width.

Example 3. Preparing double-layer agar plate. The first layer is poured into 20 ml of sterile molten agar of the following composition: 100 ml of the broth of Hottinger (pH 7,4), 2.3 g of agar, 0.25 g yeast extract /Sigma/, 0,30 ml of 10% aqueous solution of CaCl2. After solidification of the lower layer from above poured 5 ml of molten agar: 100 ml broth of Hottinger, 0.7 g of agar, 0.25 g yeast extract (Sigma), 0,30 ml of 10% aqueous solution of CaCl2, in which, after cooling to 45oC sterile added 8% washed three times with a saline solution of NaOH of human erythrocytes.

Pre-prepared suspension of spores Bac. anthracis Ihtiman, when sown on the surface of agar plates in Petri dish grows 15 to 25 colonies.

On the surface of agar plates with erythrocytes was applied in 0.1 ml of this suspension of spores, smooth rocking cups distributed over the surface of the agar. After all liquid is absorbed Cup turned the covers down and SIP is dry zone of hemolysis around isolated colonies and differences in the width of the individual colonies within a single strain.

As shown by experimental studies, the decrease in the concentration of yeast extract in the medium is less than 2 g/l are not conducive to the rapid formation of a sufficient magnitude colonies of Bacillus anthracis, an increase of more than 2.5 g/l leads to an increased production of a germ of proteolytic enzymes that reduce hemolytic activity.

The decrease in the concentration of CaCl2less than 0.30 g/l leads to reduction of the activating effect of the substance on the synthesis and functional activity of hemolysin, raising more than 0.33 g/l leads to clouding of the environment, complicating analysis.

Less than 22 hours is not enough for the growing needs of the colonies and the formation of clear zones of haemolysis. In the time that is later than 24 h, overlapped with the action of proteolytic enzymes, which may slightly distort the results.

Thus, the invention is practicable, its use will allow for a comparison with the prototype to increase the sensitivity analysis, to speed up the process 3 times, and the possibility of selection of clones with various hemolytic activity within a single strain will create the conditions for guaranteed selection subcultures in the production of vaccines, and e is tion.

The method of determining the hemolytic activity of Bacillus Anthracis, comprising preparing the two-agar nutrient medium with the concentration of agar in the lower layer is 1.0 to 1.5 wt.%, and in the upper layer of 0.6 - 0.8 wt.% with the addition of erythrocytes in the amount of 6 - 8% of the total environment, sowing the studied culture on the top layer, incubation planting with subsequent consideration of the results by the presence of zones of hemolysis of red blood cells, characterized in that the agar nutrient medium is prepared with broth of Hottinger with the addition of yeast extract 2.0 to 2.5 g/l and 0.30 - 0.33 g/l calcium chloride, incubation of sowing is carried out in the presence of human erythrocytes for 22 - 24 hours

 

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