Enumerating nucleoid-visible marine bacterioplankton: Bacterial abundance determined after storage of formalin fixed samples agrees with isopropanol rinsing method

Abstract

The effect of storage of formalin-fixed sea water samples on bacterial abundance determined by DAPI staining and epifluorescence microscopy was compared with the abundance of nucleoid-visible bacteria using isopropanol rinsing after DAPI staining to remove non-specific staining of DAPI. While storage of formalin-fixed water samples at 4°C for 7 mo caused total bacterial numbers to decline exponentially until 50 to 70 d, after which they remained stable, storage of sea water had no effect on the number of nucleoid-visible bacteria over a 7 mo period. Extrapolating the bacterial growth curve of nutrient amended sea water cultures by regression analysis to t0 resulted in a number of active bacterial cells similar to that obtained by isopropanol rinsing of DAPI-stained samples. This might indicate that the lag phase commonly detected in sea water cultures is caused by the dominance of inactive bacteria at the time of inoculation. An intensive field study in the Dutch Wadden Sea showed that the number of nucleoid-visible bacteria continuously declined from around 100% of the total bacterial abundance at the end of the phytoplankton bloom to 75% within 2.5 mo. Again a strong correlation was detected between nucleoid-visible bacteria determined by DAPI staining and subsequent isopropanol rinsing and total bacterial abundance detectable in the formalin-fixed sea water samples after 7 mo of storage. Although the exact mechanism of the loss of 'non-nucleoid-visible cells' during sample storage remains unclear, the excellent agreement between the 2 methods indicates that DAPI-stained samples enumerated immediately and after a prolonged storage might be an alternative approach to determine the total bacterial abundance and the number of nucleoid-visible bacteria.