RU2255974C1 - Method of culturing tuberculosis mycobacteria - Google Patents

Abstract

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 7^th 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 10^th day by a factor of 3.27.

EFFECT: enhanced growth of mycobacteria.

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Description

The invention relates to the field of microbiology and can be used for the cultivation of M tuberculosis, in the microbiological diagnosis of tuberculosis.

A known method of cultivating M. tuberculosis by seeding microbial cells in a liquid enriched medium Shkolnikova (Drabkina P.O. Microbiology of tuberculosis, - M .: Medgiz, 1963, - 225 S.)

In the known method, the visible growth of mycobacteria appears on the 14th day after inoculation of seed in a nutrient medium. At the same time, the efficiency of sowing growth is small and depends on the characteristics of the cultivated crop.

The aim of the proposed solution is to develop a method for the cultivation of mycobacteria, which allows to increase the output of cells of the causative agent of tuberculosis and increase the growth efficiency of M. tuberculosis.

This goal is achieved by creating a 2-phase medium with a lower phase - a gas-transporting substrate - which is used perfluorodecalin with a concentration of free ions of less than 10 ^-6 M and an amount of underfluorinated impurities of less than 0.003 wt.% With a ratio of layers of the enriched medium of Shkolnikova to perfluorodecalin equal to 1 : 1, and the sowing of mycobacteria is carried out on the dividing line.

Perfluorodecaline (hereinafter - PFD) refers to perfluorocarbons, which are fluorinated hydrocarbons in which the hydrogen atoms are completely replaced by fluorine.

PFD is a chemically inert, hydrophobic liquid 1.5 times heavier than water, capable of dissolving large quantities of oxygen and carbon dioxide in itself and easily releasing these gases when their content in the environment changes. At normal barometric pressure and a temperature of 37 ° C, 100 ml of PFD dissolves 42 ml of O ₂ and 134 ml of CO ₂ . The concentration of free fluorine ions is less than 10 ^-6 M, and the amount of under-fluorinated impurities is less than 0.003 wt.%.

The proposed method, by improving gas exchange, stimulates the growth of mycobacteria, as a result of which the output of microbial bodies increases by more than 3 times.

PFD effectively supports gas exchange in living systems and biological objects. When a liquid medium is connected with PFD, a two-phase system is formed in a test tube, due to its greater weight, PFD is located under the medium without mixing with it, and optimal conditions are created at the phase boundary for the multiplication of microorganisms due to improved gas exchange and adsorption of nutrients on the PFD surface. The presence in the cultivation system of a substrate made of PFD with a high gas capacity, first of all, makes it possible to improve the aeration of a growing culture and slow down the development of acidosis due to gas exchange between the medium and the gas transfer substrate.

The two-phase medium proposed for the cultivation of mycobacteria was prepared as follows. Sterile PFD obtained from the Institute of Theoretical and Experimental Biophysics of the Russian Academy of Sciences was placed in a 2 ml tube, 2 ml of Shkolnikova's enriched medium was layered on it. Thus, a two-phase system was formed, the lower phase of which was the heavier PFD, and the upper phase was the nutrient broth, the enriched medium of Shkolnikova.

The object of the study was a laboratory strain of M. tuberculosis H37Rv, with standard cultural properties, from which microbial suspension was prepared in physiological saline with 0.05% tween-80 in accordance with the optical turbidity standard No. 5 of GISK named after L.A. Tarasevich. Microbial suspension was inoculated with 0.1 ml per tube.

Growth was assessed by counting the absolute number of mycobacteria in smears according to the Shepard method. Samples of material for counting the number of mycobacteria were taken at 4; 7 and 10 days after sowing.

A comparison of the cultivation results of the virulent laboratory strain H37Rv, carried out on the two-phase medium we developed and on the Shkolnikova enriched medium, showed an increase in the yield of mycobacterial cells in the first. The absolute number of mycobacteria in 1 ml of medium for various cultivation durations is presented in table 1.

Table 1
Mycobacterium tuberculosis cell count results Cultivation medium n Amount of M. tuberculosis in 1 ml medium (M ± m) 4th day 7th day * 10th day * Wed Schoolboy 20 (0.09 ± 0.005) × 10 ⁷ (2025 ± 0.1) × 10 ⁷ (63.18 ± 0.06) × 10 Wed Schoolboy + PFD 20 (0.14 ± 0.2) × 10 ⁷ (3.89 ± 0.03) × 10 ⁷ (206.6 ± 0.05) × 10 * Differences between groups are significant (P <0.01)

A feature of the growth of mycobacteria on the two-phase liquid medium that we have developed is the advantageous arrangement of microcolonies in the phase separation zone. The growth of bacterial cells in tubes with PFD was more significant than in tubes without PFD. From the table it follows that on the 7th day after inoculation, the amount of M. tuberculosis in the two-phase medium exceeded that in the usual Shk medium 1.7 times, and on the 10th day - 3.27 times.

The morphology of microcolonies and individual cells of mycobacteria grown using a two-phase medium during bacterioscopy was identical to cells from Shk medium; acid resistance during staining by the Ziehl-Nielsen method was maintained.

The method of cultivating M. tuberculosis on liquid Sk with a substrate from PFD made it possible, in comparison with the control, to increase the growth rate and cell growth, that is, to increase the efficiency of cultivation.

Claims (1)

A method of cultivating Mycobacterium tubercuLosis by plating microbial cells in a Shkolnikova enriched liquid medium, characterized in that the plating is carried out in a two-layer medium, the lower layer of which is perfluorodecalin with a free ion concentration of less than 10 ^-6 M and an amount of under-fluorinated impurities of less than 0.003 wt.%, And the top layer is Shkolnikova’s enriched medium with a layer ratio of 1: 1.