Survival of lyophilized vegetative Bacillus popilliae in dry soil and in soil kept in atmospheres with different relative humidities (RH) was determined. Cells survived for at least 1 year when the RH was 22% or lower, but an RH of 33% or higher reduced viability. At 33% RH, no bacteria were recovered after 5 months of storage; at 42% or above, no bacteria were recovered after 1 month of storage. The difficulty of producing large numbers of Bacillus popilliae spores in vitro has stimulated interest in the possibility of using stabilized vegetative cells to control the Japanese beetle. It has been shown that more than 50% of young, actively growing cells of B. popilliae survive lyophilization, and approximately 10% of the original cells will still be viable after 6 months of storage at room temperature (7). Recently, cells lyophilized in our laboratory produced typical milky disease when injected into Japanese beetle larvae (G. St. Julian, personal communication). This study was undertaken to determine survival of lyophilized B. popilliae in dry soil and in in soils stored in atmospheres having different relative humidities (RH). MATERIALS AND METHODS Organism. B. popilliae NRRL B 2-309, originally obtained from the Northern Utilization Research and Development Division, Peoria, Ill., was used in collecting experimental data. Medium. The medium contained 1.5% tryptone

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.

Viable cells detectable in lyophilized preparations of Bacillus popilliae were increased by 72% when dry cells were heated at 60 to 65 C for 2.5 h. ' Contribution no. 1065, Division of Biology, Kansas Agricultural Experiment Station.