Use of the 5-cyano-2,3-ditolyl tetrazolium chloride reduction test to assess respiring marine bacteria and grazing effects by flow cytometry during linear alkylbenzene sulfonate degradation

Abstract

Viable, total and metabolically active bacteria were determined during linear alkylbenzene sulfonate degradation in coastal seawater. Viable bacteria were estimated by plate counts on marine agar media while the total and metabolically active bacteria were determined with the nucleic acid stain SYTO‐13 and the tetrazolium salt 5‐cyano‐2,3‐ditolyl tetrazolium chloride, respectively, in double stain procedures analyzed by flow cytometry. The double stain SYTO‐13/5‐cyano‐2,3‐ditolyl tetrazolium chloride is a rapid and simple method that discriminates bacterioplankton populations according to nucleic acid content and formazan formation. Linear alkylbenzene sulfonate degradation was monitored by high‐performance liquid chromatography analysis. Bacterioplankton degraded linear alkylbenzene sulfonate by growing to communities with a high percentage of viable and metabolically active bacteria. The bacteria produced were rapidly grazed by protozoa; however, the grazing took place mostly on metabolically active cells, which were larger than the rest of the population.

Morphology and DNA content of bacterioplankton in the northern Gulf of Mexico: analysis by epifluorescence microscopy and flow cytometry

“…The latter result is consistent with the reported size-and activity-selective grazing behavior of flagellates (Jürgens & Güde 1994, del Giorgio et al 1996, López-Amorós et al 1998. The share of B-III ('high DNA') bacteria shall, therefore, correspond to that of 'active' bacteria as derived from the Live/ Dead fluorescence kit (Molecular Probes), autoradiography and rRNA probes (Gasol & del Giorgio 2000).…”

Using flow cytometry for counting natural planktonic bacteria and understanding the structure of planktonic bacterial communities

“…Even though the bulk of the population is clearly above threshold, there are some cells with weak red fluorescence due to CTC that have been excluded by the red threshold. This same phenomenon has been encountered in different types of samples (López-Amorós et al, 1998;Sieracki et al, 1999) and suggests that the number of active cells obtained by this method underestimates the actual number of cells with low degree of metabolism. In this respect, the main problem of CTC reduction is that, like with most other physiological probes, the threshold of bacterial activity that it detects is not known, so it is difficult to a priori assign ecological meaning to the results.…”

“…To these, some others should be added: the procedure is fast (~ 1 minute per sample, 100 samples can be processed -data analysis included-in a morning's work), allows counting in very small volumes (down to 1 µl, Troussellier et al, 1993), allows physiological probing simultaneously to enumeration (e.g. López-Amorós et al 1998), and sample processing can be automated (e.g. Jacquet et al, 1998a).…”

“…There is some evidence that CTC may be toxic to some bacteria (Kaprelyants and Kell, 1993a;Ullrich et al, 1996) at the concentrations used, and might not work in some circumstances (Thom et al, 1993), but has been satisfactorily used to stain active bacteria from cultures (Kaprelyants and Kell, 1993a;López-Amorós et al, 1995aMcFeters et al, 1995), freshwater plankton (del Giorgio et al, 1997b;Yamaguchi and Nasu, 1997) and marine plankton (López-Amorós et al, 1998;Sieracki et al, 1999). A double staining protocol with SYTO 13 and CTC has been devised but requires a flow cytometer with double laser capabilities (López-Amorós et al, 1998).…”

Dissecting the microbial food web: structure and function in the absence of autotrophs

“…This apparent discrepancy might indicate that Gasol's (1994) model is not applicable to these particular environments as data sets from such environments were not included in the original model. Another explanation of the observed pattern might be that a large fraction of the DAPI-stained particles was inactive or dead cells, not grazed or grazed only at a lower efficiency by HNF than highly active cells (Sherr et al 1992, López-Amorós et al 1998). In addition, the protozoans observed in these layers could complement their diet with detritus or colloidal DOC (Sherr 1988).…”