Development of technology for longterm storage of Mycobacterium tuberculosis cultures

Abstract

Objective. To develop a technology for freezing and long-term storage of mycobacteria cultures and implement into a microbiological laboratory routine practice. Materials and Methods. For comparison between different condition, the media for freezing mycobacteria containing 0.9% NaCl or Middlebrook’s 7H9 medium with the addition of glycerol at various concentrations: 0%, 4.0%, 20.0%, 50.0% (by final volume) was evaluated. To freeze suspensions of cultures, two methods were used: one – using a cryo-freezer (cooling rate of 1°C/min), the second – direct placement in a deep freeze at a temperature of -80°C. Suspension of a museum strain of Mycobacterium tuberculosis H37Rv was used for preparing suspension. The efficiency of the chosen storage method was assessed by the proportion of viable cells obtained after thawing the suspensions (total number of cells according to quantitative PCR data/number of CFU in Lowenstein-Jensen medium). Results. After storage under various conditions, from 1.2% to 16.9% of mycobacterial cells remained viable. The maximum percentage of viable cells of 16.9% (95% CI 8.6–42.4%) reached when stored in a freezer at a temperature of -80°C without using a cryo-freezer in a medium consisting of 0.9% NaCl with glycerol (20.0% by final volume). However, no statistically significant differences in viable cells were found in the medium containing 0.9% NaCl solution with the addition of 4.0%, 50.0% glycerol, as well as Middlebrook’s 7H9 medium with and without glycerol. Conclusions. A technology for long-term storage of mycobacterial cultures has been developed, based on freezing a cell suspension at a temperature of -80°C in a medium consisting of a 0.9% NaCl solution with the glycerol (20.0% volume fraction), which provides a high degree of cell viability preservation, is lowcost and can be easily integrated into the routine work of a microbiological laboratory.