Microscopic enumeration of attached marine bacteria of seawater, marine sediment, fecal matter, and kelp blade samples following pyrophosphate and ultrasound treatments

Abstract

Seawater, marine surface sediment, fecal material of the sea cucumber Parastichopus californicus, and kelp blades of Macrocystis integrifolia were treated with formaldehyde to strengthen the bacterial cells, followed by tetrasodium pyrophosphate addition and sonication to disperse these same cells. These cells were then stained with 4′,6-diamidino-2-phenylindole and counted by epifluorescent microscopy. The technique of strengthening the cells with an aldehyde and then dispersing them in a suspending medium using a deflocculent (pyrophosphate) and ultrasound caused the bacteria to be randomly distributed when filtered onto Nuclepore membranes with a lower variance within and similar variance between subsamples as compared with untreated samples. In most cases the coefficient of variation stabilized after the bacterial cells in 10 randomly chosen microscopic fields had been counted. Sediment bacterial numbers (±SE) determined by the standard technique were 5.20 (±0.36) × 1010 per gram wet weight; by this dispersion technique they were 11.30 (±0.24) × 1011 per gram, a greater than twofold increase. On young and intermediate aged kelp blades, the increased counts which resulted from this dispersion technique ranged from two- to five-fold. Bacterial cells which were epiphytic on a senescent blade and could not be counted at all by the standard technique were determined to be 3.77 (±0.14) × 106 cm−2 using this dispersion method.