A simple method for the freeze-preservation of zoospores of the green macroalga Enteromorpha intestinalis

Settled zoospores of the green macroalga Enteromorpha intestinalis were sub jected to several different freezing and storing treatments at both cryogen ic and non-cryogenic temperatures after which their viability was assessed using a spore germination bioassay. Three different cooling rates were test ed: slow cooling at -1 degrees C min(-1) and -0.5 degrees C min(-1) to end temperatures in the range -20 degrees C to -40 degrees C, and a two-step pr ocedure whereby the spores were frozen to -30 degrees C at a rate of -1 deg rees C min(-1) prior to immersion in liquid nitrogen at -196 degrees C. Spo re viability was also investigated using the cryoprotectants glycerol and d imethyl suphoxide (DMSO), a reduced saline medium and various storage times . In the majority of experiments, the use of a cryoprotectant during the fr eezing process significantly increased the viability of the spores, with DM SO affording slightly greater protection than glycerol. All treatments prod uced high viabilities (ranging from 75.3-100.0%) after 5-min storage at the different end temperatures. However, progressively longer storage up to 7 days generally resulted in a marked reduction in viability. This was with t he exception of spores frozen in a reduced saline medium; a medium of 75% s eawater and either 5 or 10% DMSO greatly increased spore viability, with va lues of > 40% recorded for spores stored at -20 degrees C for up to 5 weeks .