Viability of Bacillus popilliae after Lyophilization of Liquid Nitrogen Frozen Cells1

Abstract

The per cent viability of Bacillus popilliae after lyophilization of liquid nitrogen frozen cells was determined. Lyophilization of 9to 12-hr cells which had been suspended in 5% sodium glutamate plus 0.5% gum tragacanth, frozen in liquid nitrogen vapor, and dried 4 to 5 hr with the ampoules exposed to room temperature resulted in survival of 64.6% of the original cells. After storage of these lyophilized preparations for 6 months at room temperature, 10.5% of the original cells were still viable. Spores of Bacillus popilliae Dutky, the causative agent of type A milky disease of the Japanese beetle, Popillia japonica Newman, have been used as a successful biological control agent (2). However, since sporulation of B. popilliae in vitro has not been able to produce spores in quantity, the possibility of infecting larvae with stabilized vegetative cells in soil is of interest. Milky disease bacteria withstand lyophilization without change in their morphological and cultural characteristics, and their ability to initiate disease in susceptible hosts is not destroyed (5). Lyophilized cultures will remain viable for at least 20 months (5), but information on the rate of survival has not been reported. This study was undertaken to determine the viability of B. popilliae that had been frozen in liquid nitrogen and lyophilized.

Heat-Induced Increase in the Number of Viable Cells in Lyophilized Preparations of Bacillus popilliae1

“…They were then placed in 10-ml glass ampoules (American Instrument Co., Inc., Silver Spring, Md.) in 0.5-ml amounts, frozen in liquid nitrogen, and lyophilized according to the method of Lingg et al (8).…”