Method of culturing tuberculosis mycobacteria

FIELD: medical microbiology.

SUBSTANCE: tuberculosis mycobacteria M. tuberculosis, which can be used for bacteriological diagnostics of tuberculosis, are cultured on enriched Shkolnikova nutrient medium, to which perfluorodecaline substrate is added at 1:1 ratio, which forms lower phase to improve gas exchange and enhancing growth of mycobacteria. Concentration of free ions in perfluorodecaline substrate is below 10-6 and amount of incompletely fluorinated admixtures below 0.003 wt %. Biphasic medium for culturing mycobacteria is prepared by placing 2 ml sterile perfluorodecaline into test tube followed by placing 2 ml Shkolnikova medium over it to form system, wherein lower phase occupies heavier perfluorodecaline and higher phase nutritive broth. Culturing of virulent laboratory strain H37Rv on above medium revealed considerable increase in growth of mycobacteria compared to conventional Shkolnikova medium. On the 7th day after inoculation, amount of M. tuberculosis in biphasic medium was superior to that in conventional Shkolnikova medium by a factor of 1.7 and on the 10th day by a factor of 3.27.

EFFECT: enhanced growth of mycobacteria.

1 tbl

 

The invention relates to the field of Microbiology and can be used for culturing M. tuberculosis, with microbiological diagnosis of tuberculosis.

There is a method of culturing M. tuberculosis by inoculation of microbial cells in a liquid enriched environment Shkolnikova (Drabkina P.O. Microbiology TB): Medgiz, 1963, - 225 C.)

In the known method the visible growth of mycobacteria appears on day 14 after inoculation inoculum in a nutrient medium. The effectiveness of growth seeding small and depends on the characteristics of the crop.

The aim of the proposed solution is to develop a method of cultivation of mycobacteria, which can increase the output cells of Mycobacterium tuberculosis and to improve the efficiency of growth of M.tuberculosis.

This goal is achieved by creating a 2-phase medium with the lower phase - getparentname substrate in which use perpendicular with a concentration of free ions of less than 10-6M and the number adaptirovannyh impurities less of 0.003 wt.% when the ratio of layers enriched environment Shkolnikova to performanceline equal to 1:1, and the planting of mycobacteria carried out on a dividing line.

Perpendicular (hereinafter referred to PFD) refers to the perfluorocarbons, which are fluorinated hydrocarbons, in which the hydrogen atoms is full of the TEW replaced by fluorine.

PFD is a chemically inert, hydrophobic liquid is 1.5 times heavier than water, can dissolve large amounts of oxygen and carbon dioxide and easily release these gases when changing their content in the environment. Under normal barometric pressure and temperature 37°With 100 ml PFD is dissolved in 42 ml of O2and 134 ml of CO2. The concentration of free fluorine is less than 10-6M, and the number adaptirovannyh impurities less of 0.003 wt.%.

The proposed method by improving gas exchange stimulates the growth of mycobacteria, resulting in the release of microbial cells increased more than 3 times.

PFD effectively support gas exchange in living systems and biological objects. When connecting a liquid medium with PFD in vitro formed two-phase system, because of its greater weight PFD is located under the environment without mixing with it, and at the phase boundary are created optimal conditions for growth of microorganisms due to improved gas exchange and adsorption of nutrients on the surface of the PFD. The presence in the system of cultivation substrate of the PFD with high gas capacity, above all, gives the opportunity to improve the aeration of the growing culture and to slow the development of acidosis due to gas exchange between the environment and getparentname substrate.

Two-phase medium, the th is may for the cultivation of mycobacteria, was prepared as follows. Sterile PFD obtained from the Institute of theoretical and experimental Biophysics RAS, was placed in a test tube 2 ml, it was layered with 2 ml of enriched environment Shkolnikova. Thus was formed a two-phase system, the lower phase which was more severe PFD, and the top - nutrient broth - enriched environment Shkolnikova.

The object of the study served as a laboratory strain M. tuberculosis H37Rv with standard cultural properties, which were prepared microbial suspension in saline with 0.05% tween-80 in accordance with the optical the turbidity standard No. 5 gisk named after. Laurasia. Inoculation of microbial suspensions were made in 0.1 ml in a test tube.

Growth assessment was carried out by counting the absolute number of bacilli in smears according to the method of Shepard. Samples of the material to count the number of mycobacteria took 4; 7 and 10 days after sowing.

Comparison of the results of cultivation of the virulent laboratory strain H37Rv conducted on the developed two-phase environment and an enriched environment Shkolnikova, showed an increase in the yield of cells of mycobacteria at first. The absolute number of bacteria in 1 ml of medium at different duration of cultivation are presented in table 1.

Table 1

The results of counting the number of cells of Mycobacterium tuberculosis
Wednesday cultivationn Number of M.tuberculosis in 1 ml medium (M±m)
 4 days7 days*10 days*
Cf. Shkolnikova20 (0,09±0,005)×107(2025±0,1)×107(63,18±0,06)×10
Cf. Shkolnikova+PFD20 (0,14±0,2)×107(3,89±0,03)×107(to 206.6±0,05)×10
*Differences between groups significant (P<0,01)

A feature of the growth of mycobacteria on the developed two-phase liquid medium is the predominant location of microcolonies in the zone of separation of the phases. The growth of bacterial cells in test tubes with PFD was more significant than in test tubes without PFD. The table shows that on day 7 after inoculation the number of M.tuberculosis in two phase medium was exceeded one in the normal environment SHK 1.7 times, and on 10 day - in is 3.27 times.

Morphology of microcolonies and individual cells of mycobacteria grown using a two-phase medium, with microscopy was identical cells with the environment SHK, acid resistance at colouring method Zn-staining persisted.

p> The method of cultivation of M. tuberculosis in liquid medium School with the substrate of the PFD allowed, compared to control, to increase the growth rate and the growth of cells, that is, to increase the efficiency of cultivation.

The method of cultivation of Mycobacterium tubercuLosis by inoculation of microbial cells in a liquid enriched environment Shkolnikova, characterized in that the planting is carried out in a double-Wednesday, bottom layer which is perpendicular with a concentration of free ions of less than 10-6M and the number adaptirovannyh impurities less of 0.003 wt.%, and the top layer is enriched environment Shkolnikova when the ratio of the layers is 1:1.



 

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