Determination of abundance and biovolume of bacteria in sediments by dual staining with 4 ',6-diamidino-2-phenylindole and acridine orange: Relationship to dispersion treatment and sediment characteristics

We measured the abundance and biovolume of bacteria in intertidal sediments from Tokyo Bay, Japan, by using a dual-staining technique (4',6-diamidino- 2-phenylindole and acridine orange) and several dispersion techniques (ultr asonic cleaner, ultrasonic sonicator, and tissue homogenizer). Dual stainin g reduced serious background fluorescence, particularly when used for silt- , clay-, and detritus-rich sediments, and allowed us to distinguish bacteri a from other objects during both counting and sizing. Within the studied sa mples, the number of bacterial cells ranged from 0.20 x 10(9) to 3.54 x 10( 9) g of wet sediment(-1). With the cleaner and sonicator treatments, the ba cterial numbers for all of the sites initially increased with dispersion ti me and then became constant. For the homogenizer treatments, the highest ba cterial numbers were observed with the shortest (0.5- to 2-min) treatments, and the counts then declined steeply as the homogenization time increased, indicating that cell destruction occurred. The cleaner treatment had the p ossibility of insufficient dispersion of bacteria for fine-grain sediments. Within the studied samples, the bacterial biovolume ranged from 0.07 to 0. 22 mu m(3). With the cleaner and sonicator treatments, the biovolume peaked during the shorter dispersion time. This pattern was caused not by cell de struction but by the incremental portion of dispersed small cells. We concl uded that with the cleaner and sonicator treatments, the longer dispersion time reflected the real size spectrum and was preferable for accurate estim ation of mean bacterial biovolumes.