Factors influencing the loss of bacteria in preserved seawater samples

Several time course storage experiments with preserved seawater samples were con ducted to study the loss of bacterial cells as a function of storage lime. The number of bacteria decreased by 24 to 50% within 7 to 29 d in samples preserved with 2.5% glutaraldehyde (final cone.). A comparison between epifluorescence and electron microscope counts showed that the decrease was not due to filtration artefacts. Only 0.4 to 0.6% of the bacterial cells were found to be attached to the walls of the sample containers after 1 yr of storage. There was no positive correlation between the frequency of virus-infected cells at the start of the storage experiments and the loss of bacteria as a function of storage time. Numbers of bacteria declined by only 5% the first 9 d in samples preserved in glutaraldehyde and stored at -20 degrees C. By adding phenolmethylsulfonylfluoride (PMSF), a protease inhibitor, prior to the addition of glutaraldehyde, the loss of bacterial cells only 17 to 18% over a 30 to 35 d period. Our study shows that protease activity may be a major cause of bacterial loss in glutaraldehyde preserved samples.