Me. Sieracki et al., Flow cytometric analysis of 5-cyano-2,3-ditolyl tetrazolium chloride activity of marine bacterioplankton in dilution cultures, APPL ENVIR, 65(6), 1999, pp. 2409-2417
The respiratory activity of marine bacteria is an important indication of t
he ecological functioning of these organisms in marine ecosystems. The redo
x dye 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) is reduced intracellul
arly in respiring cells to an insoluble, fluorescent precipitate. This prod
uct is detectable and quantifiable by flow cytometry in individual cells. W
e describe here an evaluation of flow cytometry for measuring CTC activity
in natural assemblages of marine bacteria growing in dilution cultures. We
found that more CTC-positive cells are detected by how cytometry than by vi
sual epifluorescence microscopy. Samples can be stored refrigerated or froz
en in liquid nitrogen for at least 4 weeks without a significant loss of to
tal cells, CTC-positive cells, or CTC fluorescence. Cytometry still may not
detect all active cells, however, since the dimmest fluorescing cells are
not clearly separated from background noise. Reduction of CTC is very fast
in most active cells, and the number of active cells reaches 80% of the max
imum number within 2 to 10 min. The proportion of active cells is correlate
d with the growth rate, while the amount of fluorescence per cell varies in
versely with the growth rate. The CTC reduction kinetics in assemblages bub
bled with nitrogen and in assemblages bubbled with air to vary the oxygen a
vailability were the same, suggesting that CTC can effectively compete with
oxygen for reducing power. A nonbubbled control, however, contained more C
TC-positive cells, and the amount of fluorescence per cell,vas greater. Act
ivity may have been reduced by bubble-induced turbulence. Addition of an ar
tificial reducing agent, sodium dithionite, after CTC incubation and fixati
on resulted in a greater number of positive cells but did not "activate" a
majority of the cells, This indicated that some of the negative cells actua
lly transported CTC across their cell membranes but did not reduce it to a
detectable level. Automated analysis by flow cytometry allows workers to st
udy single-cell variability in marine bacterioplankton activity and changes
in activity on a small temporal or spatial scale.