LIQUID NITROGEN FREEZING IN MICROBIOLOGICAL ASSAY SYSTEMS. I. PRESERVATION OF LACTOBACILLUS LEICHMANNII FOR DIRECT USE IN THE VITAMIN B12 ASSAY.

Suspensions of Lactobacillus leichmannii were stored in liquid nitrogen and were used as direct inocula in vitamin B(12) assays. Complete recovery of viable cells was obtained when the suspensions in basal B(12) medium were rapidly frozen by direct immersion into liquid nitrogen and rapidly thawed by agitating the suspensions in a water bath at 40 C. Greater than 90% destruction occurred when the suspensions were in saline. However, both suspensions were usable in the B(12) assay system. Assay results on a number of test materials indicated good correlation between freshly prepared suspensions and frozen suspensions in basal medium stored 3 months. Suspensions in saline stored for 1 year in liquid nitrogen showed no detectable difference from the first day after freezing. Suspensions frozen slowly at the rate of 1 degree per min from 4 to -40 C and subsequently immersed in liquid nitrogen had a longer lag period of growth and were not usable in the 18-hr assay incubation system. A major advantage of a stored inoculum for direct use in a microbiological assay is the reduced day-to-day variation in the inoculum.