3.5. Long term conservation of vegetative cells
Experimental work using P. larvae requires a readily available source of this bacterium in culture. However, P. larvae quickly dies in culture and some means of preserving an isolate must be used. Although short-term preservation works (see section 3.5.1.), long-term conservation is required to maintain the genetic integrity of the original isolate.
Production of frozen endospore stock suspensions is a very good procedure for long-term preservation. However, some isolates may not sporulate well in vitro and the slow rate of spore germination can hamper the start of experiments. Methods employing ultra-low freezing and lyophilization of vegetative cells (see sections 3.5.2. and 3.5.3. below) are suitable for long-term storage of this microbe.
Use of ultra-low freezing of P. larvae in glycerol has been routinely used and cultures exceeding five years of storage at -80oC remain viable (Douglas W Dingman; personal observations). No known research regarding preservation of P. larvae by lyophilization has been published. However, Haynes et al. (1961) developed a method to preserve P. popilliae by lyophilization and Gordon et al. (1973) imply that this method can be used for P. larvae. The protocol long used to preserve P. larvae strains at the National Center for Agricultural Utilization Research (USDA-ARS Culture Collection; NRRL) is similar to that described by Haynes et al. (1961) for P. popilliae.