**3.3 Slow freezing with DMSO and glycerol**

Storage of live nematodes in liquid nitrogen was first demonstrated by Hwang (1970) who reported survival of animals in the genera *Aphelenchoides*, *Panagrellus*, *Turbatrix* and *Caenorhabditis*, after slow-cooling in heat-sealed glass ampoules, using DMSO as cryoprotectants. Later, glycerol was applied to slow cooling of *Caenorhabditis elegans* at the first-larval stage (Brenner, 1974) and since then this cryopreservation procedure has been routinely used for freezing this model organism in laboratories in the past 40 years. Up to 100% of the first-stage larvae survival can be achieved.

On the other hand, a slow-cooling method using DMSO as a cryoprotectant was used for cryopreserving another *Caenorhabditis* species popular in basic scientific research, i.e. *Caenorhabditis briggsae* (Haight et al., 1975). The worms were simply suspended in cooled 5% DMSO for 10 minutes and then slowly cooled at a rate of 0.2C per minute to -100C before storage in liquid nitrogen. About 75% of the animals at the second-larval stage and the third-larval stage, 50% of the forth-stage larvae and 3% of the adult animals survived the freezing/thawing cycle. In contrast to *Caenorhabditis elegans*, no animals at the first-larval stage survived the cryopreservation (Haight et al., 1975). Obviously, traits favoring the cryopreservation procedures are present in different larval stages in both species.

The dog parasitic nematode *Strongyloides stercoralis* can be cryopreserved by slow cooling after incubation for up to 60 minutes in a solution containing 10% DMSO. When thawed, third-stage larvae retain infectivity to dogs and the recovered first-stage larvae develop to the third-larval stage and regain infectivity (Nolan et al., 1988). The cryopreserved sheep nematodes *Haemonchus contortus*, *Trichostrongylus colubriformis* and *Ostertagia circumcincta*, at the first-larval stage, retained their infectivity as well as unfrozen worms, when cooled slowly in 10 % DMSO to -80C before being transferred to liquid nitrogen for storage (Gill and Redwin, 1995).

#### **3.4 Vitrification by an ethylene glycol two-step procedure**

Before being used in mouse and fly embryo vitrification, ethylene glycol had been used for vitrifying the human platyhelminth *Schistosoma mansoni* and the farm animal nematode *Onchocerca microfilariae* in a similar two-stage procedure. *Schistosoma mansoni* were preincubated in 10% ethylene glycol at 37C for 10 minutes, then cooled at 0C for 5 minutes and finally incubated in 35% ethylene glycol for 10 minutes at 0C. Before being rapidly cooled in liquid nitrogen, the worms were spread on a glass sliver (prepared from microscopic coverslips) which acted as an open carrier. About half of the thawed worms survived and remained infective in mice with an efficiency equivalent to half that of

in response to thermal and other environmental stresses (Jagdale & Grew, 2003; Qiu & Bedding, 2002). The trehalose content in *Steinernema carpocapsae* increases from 4% to 8% after being incubated in 22% glycerol for 18 hours, before the animals are further processed for cryopreservation (Popiel & Vasquez, 1991). Production of natural cryoprotectants by the animal itself could, therefore, be the key to good animal survival in this cryopreservation procedure. Exploring an efficient way to induce the production of the natural cryoprotectants can improve cryo-survival. Identifying the molecular pathway responsible for cryoprotectant production may help make cryopreservation of these species simpler.

Storage of live nematodes in liquid nitrogen was first demonstrated by Hwang (1970) who reported survival of animals in the genera *Aphelenchoides*, *Panagrellus*, *Turbatrix* and *Caenorhabditis*, after slow-cooling in heat-sealed glass ampoules, using DMSO as cryoprotectants. Later, glycerol was applied to slow cooling of *Caenorhabditis elegans* at the first-larval stage (Brenner, 1974) and since then this cryopreservation procedure has been routinely used for freezing this model organism in laboratories in the past 40 years. Up to

On the other hand, a slow-cooling method using DMSO as a cryoprotectant was used for cryopreserving another *Caenorhabditis* species popular in basic scientific research, i.e. *Caenorhabditis briggsae* (Haight et al., 1975). The worms were simply suspended in cooled 5% DMSO for 10 minutes and then slowly cooled at a rate of 0.2C per minute to -100C before storage in liquid nitrogen. About 75% of the animals at the second-larval stage and the third-larval stage, 50% of the forth-stage larvae and 3% of the adult animals survived the freezing/thawing cycle. In contrast to *Caenorhabditis elegans*, no animals at the first-larval stage survived the cryopreservation (Haight et al., 1975). Obviously, traits favoring the

The dog parasitic nematode *Strongyloides stercoralis* can be cryopreserved by slow cooling after incubation for up to 60 minutes in a solution containing 10% DMSO. When thawed, third-stage larvae retain infectivity to dogs and the recovered first-stage larvae develop to the third-larval stage and regain infectivity (Nolan et al., 1988). The cryopreserved sheep nematodes *Haemonchus contortus*, *Trichostrongylus colubriformis* and *Ostertagia circumcincta*, at the first-larval stage, retained their infectivity as well as unfrozen worms, when cooled slowly in 10 % DMSO to -80C before being transferred to liquid nitrogen for storage (Gill

Before being used in mouse and fly embryo vitrification, ethylene glycol had been used for vitrifying the human platyhelminth *Schistosoma mansoni* and the farm animal nematode *Onchocerca microfilariae* in a similar two-stage procedure. *Schistosoma mansoni* were preincubated in 10% ethylene glycol at 37C for 10 minutes, then cooled at 0C for 5 minutes and finally incubated in 35% ethylene glycol for 10 minutes at 0C. Before being rapidly cooled in liquid nitrogen, the worms were spread on a glass sliver (prepared from microscopic coverslips) which acted as an open carrier. About half of the thawed worms survived and remained infective in mice with an efficiency equivalent to half that of

cryopreservation procedures are present in different larval stages in both species.

**3.3 Slow freezing with DMSO and glycerol** 

and Redwin, 1995).

100% of the first-stage larvae survival can be achieved.

**3.4 Vitrification by an ethylene glycol two-step procedure** 

unfrozen worms (James, 1981). On the other hand, 70% of the *Onchocerca microfilariae* were viable and remained infective after vitrification and thawing (Ham et al., 1981).

A similar vitrification methodology was also applied to the plant parasitic nematode *Meloidogyne Graminicola*, a rice root-knot nematode (Bridge & Ham 1985). The second-stage larvae were pre-incubated in 10% ethylene glycol at 37C for 15 minutes and then incubated in 40% ethylene glycol for 30-45 minutes. The worms were then rapidly cooled in liquid nitrogen in the same manner as James (1981) on *Schistosoma mansoni*. This two-stage vitrification procedure on plant nematodes was later modified by Triantaphyllou & McCabe (1989) who replaced the glass coverslip slivers with a small strip of chromatography paper as a carrier device. A survival rate of up to 90% was obtained. The author reported that the modified two-step method produced satisfactory results on other plant parasitic nematodes also, such as some *Meloidogyne* and *Heterodera* species (Triantaphyllou & McCabe, 1989).
