A simple technique for freeze-drying anaerobes.

Abstract

Many aerobic bacteria, fungi, and yeasts have been preserved successfully by freeze-drying according to the general method of Flosdorf and Mudd (1). Obligately anaerobic bacteria have presented a more difficult problem. Haynes et al. (2) , a t the Northern Utilization Research Branch, USDA, Peoria, Illinois, have had success with aerobic bacteria, fungi, yeasts, and acti~lonzycetes, but stated that attempts to lyophilize anaerobes had been unsuccessful. Recently (Haynes, personal communication) we learned that they now are freeze-drying certain clostridia routinely. Their method consists of centrifuging the cells from a liver broth, resuspending them in bovine serum, and freeze-drying in about 0.1 ml amounts. While others may have developed satisfactory methods for handling anaerobes in their laboratories, the lack of published literature prompts this report of a method which has given good results in our hands. The procedure has been used for preserving a collection of about 200 anaerobes isolated from several marine environments. The growth medium is used as the suspending fluid, eliminating the use of a centrifuge or special additives such as serum or ski111 milk. In our laboratory anaerobic cultures are routinely made in 12-1111 vaccine vials containing 10 n11 Difco Brewer Thioglycollate Medium, with methylene blue redox indicator. The vials are closed with rubber stoppers and aluminuln ring seals, and sterilized in the autoclave. When properly prepared the medium in the vials shows no color c l~a l~ge , even after prolonged storage. Since most of our cultures are marine forms, the medium is made in sea water of the required salinity, the pH adjusted, and any precipitate removed by filtration. In preparation for freeze-drying, 0.5-1.