Freezing Seawater for the Long-term Storage of Bacterial Cells for Microscopic Enumeration

Although enumerating bacterial cells is a fundamental step in understanding microbial ecosystems in marine environments, substantial decrease in bacterial counts with increasing sample storage time hampers the accurate estimation of bacterial biomass. We compared the variations in bacterial cell numbers caused by freezing and thawing of sample bottles or slides. Bacterial counts of seawater samples frozen only once in a sampling bottle yielded approximately 95％ of the original numbers after 90 days, whereas 80％ of the original count was obtained for samples prepared on slides. Only 67％ and 58％ of the original counts were recovered in samples repeatedly frozen and thawed in bottles or on slides, respectively. The results indicated that freezing a seawater sample in a bottle increased the consistency of the epifluorescence microscopic enumeration of bacterial cells.