STORAGE OF MYCOBACTERIAL STRAINS AT FREEZING STATE

Abstract

It is known that most bacteria maintain their viability at freezing state if they are suspended in 5 to 20% glycerol (1.2). Postgate and Hunter (2) reported that storage of Aerobacter aerogenes at -20°C depended on the protective agent used; only glycerol permitted extended storage. They observed that these organisms maintained their viability at 85% after 40 days of freezing at -20°C. Since similar studies on mycobacteria have not been made, the present study has concerned with this, and the storage of mycobacterial strains was extended to 6 months of freezing. The purpose of the study is to find a conventional method for maintainance of stock cultures of mycobacteria.
M. smegmatis Jucho, M. tuberculosis H₃₇Rv, M. bovis BCG, M. kansasii Forbes 84, nonpho tochromogen N 100616, and scotochromogen P-5 were used. E. coli K-12 also was used.
M. smegmatis Jucho was suspended in various solutions at concentration of 5. 5 mg (wet weight) /ml and stored in a refrigerator at -15°C. At different intervals, one tube of each group was taken and diluted with saline to 1: 1 to 1: 1, 000, 000 dilutions. Aliquots (0.02mlsamples) of these dilutions were inoculated to Löwenstein-Jensen medium and the number of colonies was counted after five days of incubation at 37°C. The results are shown in Table 1. When suspended in saline, the organisms decreased their viable numbers, but when suspended in Sauton medium, 3% glycerol broth, and 10% glycerol, all of which contained glycerol, they maintained their viability even after 42 days of storage. Similar experiments were conducted using E. coli. Determination of viable numbers was made similarly but in use of nutrient broth agar medium for counting. The results are shown in Table 2. The resutls showed that stor age in 10% glycerol was most satisfactory to maintain the viability. Suspension of nutrient broth without glycerol decreased the viable number markedly.
In view of the above results, storage of five strains at -20°C was examined in 10% glycerol (aquaeous solution). The observation was made until the end of the sixth month. The results are shown in Table 3. This method of storage maintained the viability of the test strains almost completely until the end of the third month. After six months of storage also, the viability was maintained fairly well in M. tuberculosis and M. bovis and almost satis factorily in other three strains. In view of the results obtained, storage of mycobacterial strains in 10% glycerol at -20°C was considered to be useful to maintain the stock cultures in laboratories.