1. Harvest parasite spores according to the protocol for maintenance in vivo.
2. Centrifuge at 1400 x g for 10 min.
3. While spores are centrifuging prepare a 75% (v/v) solution of sterile glycerol in fresh Hank's Balanced Salt Solution (HBSS) (ATCC cat. 30-2101) or sterile distilled water.
4. Pool the pellets and adjust the concentration to 2.0 - 4.0 x 109 spores/ml with fresh HBSS or sterile distilled water.
NOTE: If the concentration is too low centrifuge at 1400 x g for 10 min and resuspend in the volume of HBSS required to yield the desired concentration.
5. Mix the cell preparation and glycerol solution in a 1:3 ratio. The final concentration will be 1.0 - 2.0 x 109 spores/ml and 50% glycerol. The time from the mixing of the cell preparation and cryoprotective solution to the start of the freezing process should be no less than 15 min. and no more than 30 min.
6. Dispense in 0.5 ml aliquots to 1.0-2.0 ml sterile plastic screw-capped cryules (special plastic vials for cryopreservation).
7. Place the vials in a controlled rate freezing unit. From room temperature cool at -1°C/min to -40°C. If the freezing unit can compensate for the heat of fusion, maintain rate at -1°C/min through the heat of fusion. At -40°C plunge into liquid nitrogen. Alternatively, place the vials in a Nalgene 1°C freezing apparatus. Place the apparatus at -80°C for 1.5 to 2 hours and then plunge ampules into liquid nitrogen. (The cooling rate in this apparatus is approximately
8. Store in either the vapor or liquid phase of a nitrogen refrigerator.
9. To thaw a frozen ampule, place it in a 35°C water bath such that the lip of the ampule remains above the water line. Thawing time is approximately 2 to 3 minutes. Do not agitate the ampule. Do not leave ampule in water bath after thawed.
10. When completely thawed, infect lepidopteran larvae either by placing spores on the surface of their food plant or artificial diet, or by force-feeding with a syringe mounted on a micro-applicator. Follow the protocol for maintenance in vivo.