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The development of a clear understanding of the physiology of marine prokaryotes is complicated by the difficulties inherent in resolving the activity of various components of natural microbial communities. Application of appropriate molecular biological techniques offers a means of overcoming some of these problems. In this regard, we have used direct probing of bulk RNA purified from selective size fractions to examine variations in the rRNA content of heterotrophic communities and Synechococcus populations on the southeastern U.S. continental shelf. Heterotrophic communities in natural seawater cultures amended with selected substrates were examined. Synechococcus populations were isolated from the water column by differential filtration. The total cellular rRNA content of the target populations was assayed by probing RNA purified from these samples with an oligonucleotide complementing a universally conserved region in the eubacterial 16S rRNA (heterotrophs) or with a 1.5-kbp fragment encoding the Synechococcus sp. strain WH 7803 16S rRNA (cyanobacteria). The analyses revealed that heterotrophic bacteria responded to the addition of glucose and trace nutrients after a 6-h lag period. However, no response was detected after amino acids were added. The cellular rRNA content increased 48-fold before dropping to a value 20 times that detected before nutrients were added. Variations in the rRNA content from Synechococcus spp. followed a distinct diel pattern imposed by the phasing of cell division within the irradiance cycle. The results indicate that careful application of these appropriate molecular biological techniques can be of great use in discerning basic physiological characteristics of selected natural populations and the mechanisms which regulate growth at the subcellular level.

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Bathophenanthrolinedisulfonic acid (4,7-diphenyl-1,10-phenanthrolinedisulfonic acid [BPS]) and 3-(2-pyridyl)-5,6-bis(4-phenylsulfonic acid)-1,2,4-triazine (ferrozine), chelators of ferrous iron, are often used to determine iron(II) concentrations in various samples and for identifying or measuring iron reduction in biological systems. In this study, the efficacy of ferrozine and BPS to chelate iron(II) reduced from Fe(3+)-ligands by selected reducing agents was determined. Our results indicate that (i) BPS and ferrozine are not equivalent as kinetic indicators of iron reducing activity; (ii) apparent initial rates of reduction of Fe(3+)-ligands by dithiothreitol, as indicated by formation of complexes of iron(II) with either BPS or ferrozine, differed by a factor of 50; and (iii) nonspecific reduction of some Fe(3+)-ligands by both BPS and ferrozine occurred. Under identical conditions, rates of formation of Fe(2+)-ferrozine generally were slower than rates of formation of Fe(2+)-BPS. These data suggest careful consideration should be given in the design of any experiments where kinetics of iron reduction are monitored with BPS or ferrozine.

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Thirty-five American Type Culture Collection type strains of marine bacteria were used to evaluate the Rapid NFT system (API Analab Products, Plainview, N.Y.) for use in identifying heterotrophic marine bacteria. The 21 biochemical and assimilation tests on the Rapid NFT test strips were treated according to the manufacturer's protocol, which included use of AUX medium (provided with the Rapid NFT system) for preparing assimilation tests, and by substituting phenol red broth base (BBL Microbiology Systems, Cockeysville, Md.) with and without an oil overlay for the AUX medium. A seven-digit numerical profile was obtained for each NFT test strip from each of the three procedures and matched to its corresponding number in the Rapid NFT identification codebook. Also, all biochemical and assimilation test results were analyzed with SASTAXAN and SAS/GRAPH programs (SAS Institute, Inc., Cary, N.C.); similarity matrices were computed for all 35 strains. For comparison purposes, bacterial strains were grouped at a similarity level of 70%. The results indicated a low efficacy of identification for all three procedures. In addition, similarity matrix analysis showed more cohesive grouping based on results of phenol red broth base-treated strains than for the AUX medium provided by the manufacturer. However, none of the three treatments provided exclusive grouping of type strains at the genus level. Thus, the reliability of the data obtained from the NFT system and modifications thereof should be evaluated carefully when environmental isolates are characterized.

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The degree and temporal context of variations in ribosome content during nutrient starvation of two copiotrophic marine bacteria, Vibrio alginolyticus and Vibrio furnissii, have been examined. The organisms were starved either by nutritional shift-down or by consumption of limiting nutrients resulting from growth into stationary phase. Measurements of the amount of hybridization to 16S rRNA-specific probes revealed that the cells retained between 10 and 26% of their original rRNA content after 15 days of starvation. In V. alginolyticus, losses in stationary-phase cells occurred rapidly (1 to 2 days), whereas cells shifted into starvation remained larger and retained considerably more rRNA. The ability of V. alginolyticus to recover from starvation was assessed after cells were maintained for 2, 8, and 15 days in nutrient-depleted medium. The pattern of recovery at the level of rRNA accumulation depended upon the duration of nutrient deprivation and the manner in which it was imposed. Stationary-phase cells starved for 2 days had only slight relative increases in rRNA levels after excess nutrients were added. As the duration of starvation lengthened to 8 and 15 days, increasingly greater amounts of rRNA (30 and 70 times preenrichment values, respectively) were transcribed after nutrient enrichment. Shift-down cells recovered from 2 and 8 days of starvation without extensive rRNA production. After 15 days, nutrient enrichment caused 16S rRNA levels to increase 30-fold. The results indicate that the mechanisms controlling starvation-survival in these marine bacterial species are linked to the physiological state at the onset of starvation and that the subsequent pattern of recovery will depend upon how starvation was initiated.

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Extracts of the sponge Tedania ignis have been reported to contain several diketopiperazines. As part of an investigation of the commensal and symbiotic microflora of sponges, we have consistently isolated, from specimens of T. ignis, a Micrococcus sp. which produces diketopiperazines in laboratory cultural media. This is the first demonstration that a bacterium associated with a sponge produces secondary metabolites ascribed to the sponge host.

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Sufficient laboratory and field data are now available to hypothesize that enteric pathogens survive for very long periods of time in sea-water. In fact, these Gram-negative bacteria probably enter into dormancy, during which they remain viable and potentially virulent, yet are non-culturable when traditional bacteriological methods are employed. Increasing use of the world's oceans-for discharge of domestic wastes may result in public health problems in the future from the allochthonous human pathogens accumulating in the marine environment at disposal sites.

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Vibrio spp. predominated in the culturable bacterial community of surface waters of the Puerto Rico Trench at the site of disposal for nearly ten years of pharmaceutical wastes. In this area and surrounding waters as far as 1000 km north of the dumpsite and south into the Caribbean Sea, Vibrio spp. comprised up to 100% of the culturable bacteria, with Acinetobacter spp. being the second most prevalent group. Pseudomonas spp., reported to be common in these waters a decade earlier, were virtually absent from all samples examined during a three year study involving 9 cruises. Staphylococcus spp. were also found in water samples collected within the dumpsite. Using cultures isolated from surface water samples collected at the dumpsite, laboratory experiments confirmed that pharmaceutical waste can enrich for Vibrio spp., in preference to Pseudomonas spp., with growth of the strains proportional to the amount of waste added.

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Oyster meats were examined from three commercial processing plants at different stages of processing and storage for four standard indicator bacterial groups, five species of vibrios reported to be associated with shellfish associated gastroenteritis, and Aeromonas hydrophila . Processing reduced the overall microbial load, but the individual bacterial groups examined remained statistically the same throughout processing. Upon storage, the concentration of Vibrio parahaemolyticus significantly declined with a concomitant increase in levels of A. hydrophila , and the levels of all other Vibrio spp. remained statistically the same. The findings suggest that, while processing results in a cleaner appearing product, processing does not eliminate potentially pathogenic vibrios. However, processing and subsequent storage of oyster meats do not appear to increase the levels of vibrios.

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Environmental and clinical strains of Vibrio cholerae were exposed to nutrient-free artificial seawater and filtered natural seawater microcosms for selected time intervals and examined for changes in cell morphology and number. Cells observed by transmission electron and epifluorescence microscopy were found to undergo gross alterations in cell morphology with time of exposure. The vibroid cells decreased in volume by 85% and developed into small coccoid forms surrounded by remnant cell walls. The initial number of cells inoculated into nutrient-free microcosms (culturable count and direct viable count) increased 2.5 log10 within 3 days, and even after 75 days the number of viable cells was still 1 to 2 log10 higher than the initial inoculum size. Nutrient-depleted coccoid-shaped cells were restored to normal size and assumed a bacillary shape within 3 h and began to divide within 5 h after nutrient supplementation. The increase in cell number and decrease in cell volume under nutrient-depleted conditions, as well as the rapid growth response after nutrient supplementation, may describe some of the survival mechanisms of V. cholerae in the aquatic environment.

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Laboratory microecosystems (microcosms) prepared with a chemically defined sea salt solution were used to study effects of selected environmental parameters on growth and activity of Vibrio cholerae. Growth responses under simulated estuarine conditions of 10 strains of V. cholerae, including clinical and environmental isolates as well as serovars O1 and non-O1, were compared, and all strains yielded populations of approximately the same final size. Effects of salinity and temperature on extended survival of V. cholerae demonstrated that, at an estuarine salinity (25%) and a temperature of 10 degrees C, V. cholerae survived (i.e., was culturable) for less than 4 days. Salinity was also found to influence activity, as measured by uptake of 14C-amino acids. Studies on the effect of selected ions on growth and activity of V. cholerae demonstrated that Na+ was required for growth. The results of this study further support the status of V. cholerae as an estuarine bacterium.

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Nine chemically defined inoculation diluents, with compositions ranging from 0.85% NaCl to 35% marine salts, were used to evaluate the influence of diluent composition on the biochemical profiles of 30 marine and estuarine bacterial strains, including species of Vibrio, Aeromonas, Allomonas, and Photobacterium. Results demonstrated that a 20% marine salts diluent enabled the characterization of halophilic strains normally nonreactive by the API 20E system. Furthermore, the use of 20% marine salts showed that certain environmental isolates, identifiable as Vibrio parahaemolyticus by the recommended clinical inoculation procedure, were Vibrio vulnificus. An analysis of the profiles provided by the nine diluents indicates that the API 20E system, modified by the use of a diluent composed of 20% marine salts and incubated at 22 degrees C, can provide a reliable tool for the rapid characterization of marine and estuarine bacterial isolates.

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Laboratory microcosms were employed to evaluate the influence of selected environmental parameters, organic nutrient concentration, and salinity on the growth and survival of a toxigenic strain of Vibrio cholerae LA4808. Over the range conditions tested, this strain of V. cholerae showed maximum response as determined by increased plate counts and direct microscopic counts in microcosms prepared with a chemically defined sea salts solution at a salinity of 25%, but with lower or higher salinity levels, the maximum population size declined. When added organic concentrations of less than 1,000 micrograms/liter were present, a marked salinity effect on the growth of V. cholerae was detected. However, at or above an organic nutrient concentration of 1,000 micrograms/liter, the need for an optimum salinity level was spared. From the results of this study, it is concluded that V. cholerae can grow under conditions of organic nutrient concentration and salinity typical of estuaries. Results obtained support the hypothesis that V. cholerae is an autochthonous member of the estuarine microbial community.

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Plating methods for estimating survival of indicator organisms, such asEscherichia coli, and water-borne pathogens includingVibrio cholerae, have severe limitations when used to estimate viable populations of these organisms in the aquatic environment. By combining the methods of immunofluorescent microscopy, acridine orange direct counting, and direct viable counting, with culture methods such as indirect enumeration by most probable number (MPN) estimation and direct plating, it was shown that bothE. coli andV. cholerae undergo a "nonrecoverable" stage of existence, but remain viable. Following 2-week incubations in saltwater (5-25%o NaCl) microcosms, total counts, measured by direct microscopic examination of fluorescent antibody and acridine orange stained cells, remained unchanged, whereas MPN estimates and plate counts exhibited rapid decline. Results of direct viable counting, a procedure permitting estimate of substrate-responsive viable cells by microscopic examination, revealed that a significant proportion of the nonculturable cells were, indeed, viable. Thus, survival of pathogens in the aquatic environment must be re-assessed. The "die-off" or "decay" concept may not be completely valid. Furthermore, the usefulness of the coliform and fecal coliform indices for evaluating water quality for public health purposes may be seriously compromised, in the light of the finding reported here.

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Three methods employed to distinguish staphylococci from micrococci were compared, using clinical and environmental strains. When these methods are used, misinterpretation of results, as well as erratic results, may occur, and suggestions for eliminating these problems are provided. The most sensitive test that combines ease of use and speed in obtaining results for distinguishing the two genera is the lysostaphin susceptibility test. Two other tests, facultatively anaerobic growth in semisolid thioglycolate agar and fermentation of dextrose, may also be used to distinguish these two genera, but results are often slow in developing, are subject to technical difficulties, and may lead to incorrect assignment of certain species of staphylococci and micrococci to their proper genera.

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A series of cruises during 1979 and 1980 to the pharmaceutical dump site located 64 km north of Arecibo, Puerto Rico, in the Atlantic Ocean, was carried out to evaluate effects of wastes on the ecology of the microflora of surface waters of the dump site. In addition to bacteriological monitoring of the waste plume created by the release of wastes from the disposal barge, stations along a series of transects, extending north from coastal waters through and beyond the dump site, were sampled. Largest numbers of culturable bacteria on marine agar were found at stations closest to shore and in the vicinity of the dump site. Bacteria recovered on marine agar were predominantly Vibrio and Aeromonas spp., with the relative abundance of these organisms decreasing as gram-positive organisms (staphylococci, micrococci, and bacilli) became dominant in areas immediately affected by waste dumping. Total numbers of bacteria (determined by acridine orange direct counts [AODC]), which were relatively stable throughout the region, and a direct estimate of viable cells (DVC), i.e., those cells responsive to additions of yeast extract and nalidixic acid, were determined by acridine orange staining and epifluorescence microscopy. Heterotrophic bacterial activity, measured by the uptake (V(max)) of C-labeled amino acids, declined relative to distance from land. Increases in specific activity indices (DVC/AODC and V(max)/AODC) were observed near the dump site. The composite results of this study, i.e., increased specific activities (determined by two methods), increased numbers of culturable marine bacteria, and marked alteration of the taxonomic composition of the culturable bacterial community in waters within and surrounding the Puerto Rico dump site, indicate demonstrable changes in the marine microbial community in the region used for waste disposal.

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The contributions of different sources of error in sampling mixed and unmixed bacterial microcosms were evaluated by using analysis of variance. Culturable heterotrophic bacteria from a turbid freshwater impoundment were sampled from 9-liter tanks that were unagitated or mixed with magnetic stirrers or pumps and from dilution bottles that were unagitated or agitated with a mechanical shaker. Axenic cultures of Enterobacter aerogenes were also sampled from manually shaken test tubes. In both agitated and unagitated tanks and in unagitated dilution bottles, dilutions made from the same sampling pipette were significantly different, showing a clumping of bacteria on the scale of millimeters. Also, microcosms within a single experiment differed from one another by a large margin. Dilution mean squares and tank or bottle mean squares were homogeneous for all types of tanks and unagitated bottles, indicating that the gentle mixing provided by pumps and stir bars did not reduce either millimeter scale or intermicrocosm variability over what prevailed in unagitated microcosms. By contrast, the vigorously shaken bottles and test tubes showed no millimeter scale variability. Intermicrocosm variability was undetectable in test tubes and two orders of magnitude less in shaken bottles than in unshaken bottles. When these facts are coupled with the inherent statistical advantage of replicating large rather than small experimental units, it is concluded that sampling error in the enumeration of aquatic bacteria in microcosms will be reduced by using numerous, small, violently agitated microcosms with a minimum of subsampling per microcosm.

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