Experiments on freezing damage with freeze substitution using moth antennae as test objects

SUMMARY Insect antennae can be easily frozen by immersion into propane at 90 K, freeze substituted at 194 K and, after warming up, embedded and sectioned at room temperature. Freezing damage (f.d.) may occur during cooling the specimen down (primary f.d.) or due to re‐crystallization, during warming up, if substitution was not complete (secondary f.d.). Experimental evidence suggests that secondary freezing damage is not likely to occur with freeze substitution, provided the acetone is water free (by adding molecular sieve) and the rate of warming up is low. All freezing damage observed in the specimens therefore most probably is due to primary freezing damage during cooling. Propelling the specimen with high speed into the coolant, instead of merely dropping it in, does not improve the quality of preservation obtainable but increases the yield of well‐frozen specimens.