Digestive tracts of abyssal scavenging amphipods and a deep-sea holothurian were examined for the presence of intestinal microflora capable of rapid proliferation under in situ pressures of 430 to 520 atmospheres (atm) and temperatures of 3-5°C. For two amphipod specimens, population doubling times of 5 and 6 hours were observed under in situ conditions, compared to 8 and 6 hours, respectively, at 1 atm. Growth enhancement under pressure was related inversely to initial population size and directly to concentration of available nutrient. In the case of the deposit-feeding holothurian, attached bacteria scraped from the intestinal lining showed a doubling time, under pressure, of 11 hours, compared to 36 hours for transient sediment bacteria that comprised the gut contents. These data suggest that deep-sea animals possess a commensal gut flora capable of responding to increased nutrient levels, via feeding of the host, without inhibition by the elevated hydrostatic pressures encountered in the deep ocean environment.

A method is reported that combines the microscopic determinations of specific, individual, respiring microorganisms by the detection of electron transport system activity and the total number of organisms of an estuarine population by epifluorescence microscopy. An active cellular electron transport system specifically reduces 2-( p -iodophenyl)-3-( p -nitrophenyl)-5-phenyl tetrazolium chloride (INT) to INT-formazan, which is recognized as opaque intracellular deposits in microorganisms stained with acridine orange. In a comparison of previously described sample preparation techniques, a loss of >70% of the counts of INT-reducing microorganisms was shown to be due to the dissolution of INT-formazan deposits by immersion oil (used in microscopy). In addition, significantly fewer fluorescing microorganisms and INT-formazan deposits, both ≤0.2 μm in size, were found for sample preparations that included a Nuclepore filter. Visual clarity was enhanced, and significantly greater direct counts and counts of INT-reducing microorganisms were recognized by transferring microorganisms from a filter to a gelatin film on a cover glass, followed by coating the sample with additional gelatin to produce a transparent matrix. With this method, the number of INT-reducing microorganisms determined for a Chesapeake Bay water sample was 2-to 10-fold greater than the number of respiring organisms reported previously for marine or freshwater samples. INT-reducing microorganisms constituted 61% of the total direct counts determined for a Chesapeake Bay water sample. This is the highest percentage of metabolically active microorganisms of any aquatic population reported using a method which determines both total counts and specific activity.

A significant number of viable colony-forming bacteria were recovered from deep-ocean bottom water samples passed through a 0.45μm filter. However, these bacteria small enough to pass through a 0.45μm membrane filter and termed "filterable bacteria" were less abundant in open-ocean surface water and coastal water samples. The reduced size of bacterial cells present in deep-ocean bottom water samples was documented by scanning electron microscopy. The concentration of ATP in the water samples was found to be correlated with results of direct counts of bacteria.Numerical taxonomy of bacterial strains isolated from water samples collected at two stations in the deep sea yielded taxonomic clusters grouped according to sample and size fraction. The generic composition of bacterial populations of bottom water filtrates was compared with that of bacteria retained by 0.45μ m filters. Strains ofAlcaligenes, Flavobacterium, Pseudomonas, andVibrio spp. were identified among those retained by, as well as passing through, 0.45μm filters.Two marine isolates obtained from the filtrate of a deep-ocean water sample were incubated for 9 weeks in nutrient-free artificial seawater, during which the cells became rounded and reduced in size. After the 9-week incubation period, more than 10% of the viable cells of both cultures were able to pass through a 0.4μm filter. The viable count at 9 weeks wasca. 10% of that of the initial population, although from direct counts the total population number remained relatively constant throughout the incubation period. From the observed reduction in cell size and increased starvation resistance of cells held under low nutrient conditions, it is concluded that a significant relationship exists between decreased cell size and increased survival of marine bacteria in the deep sea.

We report a method which combines epifluorescence microscopy and microautoradiography to determine both the total number of microorganisms in natural water populations and those individual organisms active in the uptake of specific substrates. After incubation with 3H-labeled substrate, the sample is filtered and, while still on the filter, mounted directly in a film of autoradiographic emulsion on a microscope slide. The microautoradiogram is processed and stained with acridine orange, and, subsequently, the filter is removed before microscopic observation. This novel preparation resulted in increased accuracy in direct counts made from the autoradiogram, improved sensitivity in the recognition of uptake-active (3H-labeled) organisms, and enumeration of a significantly greater number of labeled organisms compared with corresponding samples prepared by a previously reported method.

We used three methods in determination of the metabolically active individual microorganisms for Chesapeake Bay surface and near-bottom populations over a period of a year. Synthetically active bacteria were recognized as enlarged cells in samples amended with nalidixic acid and yeast extract and incubated for 6 h. Microorganisms with active electron transport systems were identified by the reduction of a tetrazolium salt electron acceptor. Microorganisms active in uptake of amino acids, thymidine, and acetate were determined by microautoradiography. In conjunction with enumeration of active organisms, a total direct count was made for each sample preparation by epifluorescence microscopy. For the majority of samples, numbers of amino acid uptake-active organisms were greater than numbers of organisms determined to be active by other direct measurements. Within a sample, the numbers of uptake-active organisms (amino acids or thymidine) and electron transport system-active organisms were significantly different for 68% of the samples. Numbers of synthetically active bacteria were generally less than numbers determined by the other direct activity measurements. The distribution of total counts in the 11 samplings showed a seasonal pattern, with significant dependence on in situ water temperature, increasing from March to September and then decreasing through February. Synthetically active bacteria and amino acid uptake-active organisms showed a significant dependence on in situ temperature, independent of the function of temperature on total counts. Numbers of active organisms determined by at least one of the methods used exceeded 25% of the total population of all samplings, and from June through September, >85% of the total population was found to be active by at least one direct activity measurement. Thus, active rather than dormant organisms compose a major portion of the microbial population in this region of Chesapeake Bay.