Recovery of Leptospires After Dry Storage For Ten Years

Abstract

Nine strains of leptospires representing six serotypes were dried in vacuo from the liquid state and stored at 4 C . Viable organisms were recovered from all dried cultures during and after 10 years of storage. Motile forms were not observed immediately after rehydration, but they became apparent after several days of incubation.In their recent paper on the preservation of leptospires by freezing, Alexander et al. (1) reviewed the literature on this topic and also commented that attempts to preserve these organisms by freeze-drying have rarely been successful. Although this may be true, it is difficult to gauge the extent of this failure since there has been little documentation of it. On the other hand, successful recoveries of leptospires after drying and storage have been claimed by Annear (2,3,6) and by Otsuka and Monako (8).In one of these reports (3), it was recorded that dried cultures were prepared by drying liquid leptospiral suspensions in vacuo on the walls of small glass tubes and that viable organisms were recovered from these preparations after storage for 5 years (6). Subsequent tests showed the organisms to be viable after several additional years of storage at 4 C, but these experiments have now terminated since no further tubes remain for testing. However, a second series of experiments was done 10 years ago with a modified drying method and on a wider range of leptospires. The method was derived from previous work on the preservation of other microorganisms (4, 5). Briefly, it involved drying liquid leptospiral suspensions in vacuo on quartz fibers contained in small glass tubes. This paper describes the preparation of these dried cultures and the results from tests on their viability during and at the end of the 1 0-year storage period. MATERIALS AND METHODSOrganisms. Nine strains of leptospires of six different serotypes were dried. The serotypes were icterohaemorrhagiae (two strains), canicola (two strains), pomona (two strains), tarassovi, australis, and zartoni. The organisms were grown in Korthof medium (7) at 30 C and harvested for drying 2 to 3 days after the initial turbidity was observed.Suspensions for drying. Suspensions were prepared from both uncentrifuged cultures and from centrifuged deposits of 20-ml culture volumes. Centrifuging was done for 30 min at 5,000 X g. In the former experiments, 50% (wtlvol) glucose was added to the culture to yield a final carbohydrate concentration of 5% (wt/vol); in the latter experiments, the centrifuged deposits were mixed with equal volumes of 10% (wt/vol) glucose.Drying and storage. Small glass tubes with tufts of quartz wool in the bottoms and cotton-wool plugs in the necks were prepared and sterilized by autoclaving at 15 Ib/in2 for 20 min (4, 5). Twodrop volumes of the suspensions were inoculated on the quartz fibers, and drying was then carried out in vacuo on a manifold over P,O, and at a pressure of approximately lo-* torr. The tubes were exposed externally to ambient atmosphere, and no attempt was made to control the drying temperatur...