2-STEP FREEZING OF THE RUMEN CILIATE PROTOZOAN ENTODINIUM-CAUDATUM

A procedure has been developed for the two-step deep freezing of the rumen ciliate protozoan Entodinium caudatum. The average recovery after 24 h of storage in liquid nitrogen (LN2) was 36% under the following conditions: a cell concentration 10(5)-10(6) ml(-1), 5 min equilibration at 25 degrees C with 4% DMSO (dimethylsulfoxide) as a cryoprotective agent and 45 min holding time at -25 degrees C. The samples were immersed into LN2 immediately after the holding phase of cooling. This two-step procedure significantly increases the survival of the rumen protozoan in comparison with one-step freezing. The protozoa preserve their growth ability after one year of storage in liquid nitrogen.