Long-term preservation and storage of mycobacteria

Under contract with the National Institute of Allergy and Infectious Diseases, the Trudeau Mycobacterial Culture Collection has been greatly expanded to provide for the scientific community a collection of representative strains of mycobacteria of biomedical importance. Problems concerned with the preparation, bottling, and distribution of such organisms have been dealt with and are detailed in this paper. Examination of collected data revealed that the temperature of storage and not the suspending menstruum was more important for prolonged survival of mycobacteria stored at subzero temperatures. For optimum results, mycobacteria may be suspended either in Dubos Tween-albumin broth or in Middlebrook 7H-9 liquid medium supplemented with ADC enrichment (commercial sources used) and stored at -70 C. Either of these suspending fluids supplied a growth-supporting medium for cultures which must be shipped long distances, often without refrigeration. To avoid sublimation of suspending medium during prolonged storage at -70 C (a problem inherent in many screw-capped containers), we have chosen to use vaccine-stoppered serum bottles sealed with aluminum crimp caps. The methods described have provided suspensions with (i) excellent viability over prolonged periods of storage, (ii) stable metabolic activities, and (iii) highly reproducible inocula for animal experiments.