The hemolymph of 290 freshly collected blue crabs from Chincoteague Bay, Va., was sampled over a 15-month period from August 1968 through November 1969 and most probable numbers of bacteria were determined by tube dilution. The hemolymph of 18% of all crabs sampled was found to be sterile, with 16% sterility in summer and 23% in winter samples. Despite individual variations, male crabs as a group had a higher bacterial hemolymph burden than females, and among both sexes summer counts were higher than winter. The hemolymph of crabs with missing appendages had significantly higher counts than uninjured crabs. The annual mean hemolymph most probable numbers per ml was 2,756 for males, 1,300 for females, and 1,876 for both sexes. The higher bacterial levels found in the hemolymph of male crabs may, in part, be explained by the fact that males, which predominated in the summer samples, had a higher incidence of injury and missing appendages than did females.

Fluorescence-labeled wheat germ agglutinin binds specffically to N-acetylglucosamine in the outer peptidoglycan layer of gram-positive bacteria. The peptidoglycan layer of gram-negative bacteria is covered by a membrane and is not labeled by the lectin. By exploiting this phenomenon, an alternative Gram staining technique has been developed.The Gram stain is the most widely used taxonomic test of bacteria (5). The technique is relatively simple and, in experienced hands, gives reproducible results. The existing technique does, however, have its limitations (4). Most modifications of the Gram stain, such as that of Hucker (6), require at least four solutions and four staining steps. Furthermore, the stains used in the technique, particularly the primary stain (crystal violet), are concentrated and can be messy. The staining procedure is estimated to take approximately 3 min (2).We have developed an alternative Gram staining technique which is simpler and faster, requires fewer reagents, is easier to interpret, and, in our hands, is less susceptible to errors. This technique takes advantage of the selective binding of a lectin, wheat germ agglutinin, to N-acetylglucosamine (1,8). This molecule is a prominent component of the peptidoglycan layer found in all eubacteria except Mycoplasma spp. In gram-positive bacteria, the peptidoglycan layer is the outer portion of the cell wall. The exterior layer of gram-negative bacteria is a membrane which covers the peptidoglycan layer (9). Thus, a large molecule such as a lectin should be able to attach to the peptidoglycan layer of gram-positive bacteria but should not be able to penetrate the outer membrane and thus could not attach to the peptidoglycan of gram-negative bacteria. A total of 92 bacterial strains were tested. Gram-positive bacteria were as follows: Bacillus sp. (n = 8), Bacillus megaterium (n = 2), Corynebacterium sp. (n = 1), Lactobacillus acidophilus (n = 1), Lactobacillus lactis (n = 1), Micrococcus sp. (n = 3), Micrococcus luteus (n = 2), Mycobacterium smegmatis (n = 1), Sporosarcina ureae (n = 1), Staphylococcus aureus (n = 9), Staphylococcus epidermidis (n = 7), Staphylococcus saprophyticus (n = 1), Streptococcus faecalis (n = 4), Streptococcus mitis (n = 2), and Streptococcus pyogenes (n = 4). Gram-negative bacteria were as follows: Acinetobacter calcoaceticus (n = 2), Alcaligenes faecalis (n = 2), Cytophaga sp. (n = 1), Enterobacter aerogenes (n = 1), Enterobacter cloacae (n = 5), Escherichia coli (n = 8), Klebsiella pneumoniae (n = 4), Morganella morganii (n = 1), Proteus mirabilis (n = 2), Proteus vulgaris (n = 4), Pseudomonas sp. (n = 1), Pseudomonas aeruginosa (n = 4), Pseudomonasfluorescens (n = 1), Pseudomonas stutzeri (n = 1), Rhodospirillum rubrum (n = 1), Salmonella typhimurium (n = 1), Serratia liquefaciens (n = 1), Serratia marcescens (n = 4), and Shigella sonnei (n = 1). All strains were initially streaked to ensure purity and * Corresponding author. then were maintained at 35°C with periodic transfer. Wheat germ agglutinin labeled with flu...

Bacteria were readily isolated from the hemolymph of a majority (88%) of the blue crabs collected from Galveston Bay, Texas. The hemolymph of most crabs contained moderate (>103 bacteria/ml) to heavy (>105 bacteria/ml) infections. Large variances were observed in the bacterial number associated with individual crabs, but no significant difference was observed between the mean bacterial levels in the hemolymph of crabs collected during different seasons of the sampling year. Vibrio spp. were the predominant bacterial types in the hemolymph of infected crabs and increased in number significantly during the summer season. Warmer water temperatures were thought to be responsible for this increase. Bacterial numbers and the percentage of Vibrio spp. were highest in the interior of the crab bodies, especially in the digestive tract. The exterior of the crabs did not appear to be the source of the hemolymph's bacterial flora. Bacteria taxonomically identical to Vibrio cholerae, V. vulnificus, and V. parahaemolyticus were routinely isolated from the crab hemolymph and external carapace. V. parahaemolyticus was the most prevalent of the pathogenic Vibrio spp. and was isolated from 23% of the hemolymph samples. V. vulnificus (7%) and V. cholerae (2%) occurred less commonly in the hemolymph. The incidences of V. parahaemolyticus and V. vulnificus were related and increased in the summer months. Both organisms were frequently isolated from the same crab.

Presumptive marine Vibrio spp. were collected from an operational oil field and control site located in the northwestern Gulf of Mexico. Of 440 isolates analyzed for the presence of extrachromosomal deoxyribonucleic acid elements or plasmids by using the cleared lysate and agarose gel techniques, 31% showed distinct plasmid bands on agarose gels. A majority of the plasmids detected were estimated to have molecular masses of 10 × 10 6 or less. Multiple plasmids were observed in approximately half of the plasmid-containing strains. A number of isolates contained plasmids with similar banding and mobility patterns. The oil field area had noticeably more plasmid-containing strains (35 versus 23% in the control site) and a greater number of plasmids per plasmid-containing strain (an average of 2.5 plasmids, versus 1.5 in the control site). Oil field discharges might have resulted in increased plasmid incidence and diversity.

Antibiotic-resistant bacteria were isolated from seawater samples collected in the Atlantic Ocean off the southeastern coast of the United States. Large numbers of antibiotic-resistant bacterial strains were found to be present in harbor and inshore waters; however, the percentage of resistant strains was higher for several seawater samples collected offshore than for those collected near shore. Bacteria resistant to tetracycline, chloramphenicol, and streptomycin were found in nearly all samples collected, including samples from 200 miles (about 522 km) offshore and at depths to 8,200 m. Sediment samples, in general, were found to contain smaller populations of resistant strains as compared with the seawater samples examined. Antibiotic-resistant bacteria exhibiting phenetic characteristics common to autochthonous marine bacterial species were examined in detail, and several of the isolates exhibited unstable antibiotic resistance, which was transferable to recipientEscherichia coli cells. Deoxyribonucleic acid preparations from 10 strains examined by ethidium bromide-cesium chloride density sedimentation revealed that 6 of the strains contained covalently closed circular plasmid deoxyribonucleic acid.

Bioluminescent bacteria were found in the water column, sediment, shrimp, and gastrointestinal tract of marine fishes from the semitropical estuarine environment of the East Lagoon, Galveston Island, Tex. Populations in the water column decreased during cold weather while sedimentary populations persisted. The highest percentages of luminous organisms were isolated from the gastrointestinal tract of marine fishes, where they persisted during 5 days of starvation. The presence of chitin temporarily increased intestinal populations. All isolates were Beneckea harveyi, whose natural habitat appears to be the gut of fishes and whose free-living reservoir appears to be marine sediments. Luminescent bacteria are found saprophytically and parasitically on marine animals and live symbiotically in specialized organs in certain marine fish and cephalopods (2). Although the biochemistry, physiology and, most recently, the from the East Lagoon for 12 months (June 1977 to June 1978). Water samples were collected in sterilized milk dilution bottles, capped, and returned to the laboratory within 1 h for processing. Sediment samples were collected in a polypropylene centrifuge tube with a hole drilled in the bottom. The 928

Aims: The aim of this work was to isolate bacteriocins from the environment that would be effective in neutralizing Vibrio vulnificus in seafood. Methods and Results: Water samples from Wilmington (NC, USA) were plated to determine total viable counts and to isolate presumptive Vibrio spp. Isolates containing plasmids were checked for antimicrobial activity which was not due to lytic bacteriophage or small, non-specific molecules. Three bacteriocin producers were detected and their inhibitory spectra determined: IW1 inhibited few strains of V. vulnificus; BC1 inhibited several strains of V. vulnificus, V. cholerae and V. parahaemolyticus and BC2 inhibited all tested Vibrio spp., Plesiomonas shigelloides and Escherichia coli. Loss of inhibitory activity coincided with loss of the bacteriocinogenic plasmid. The bacteriocins were found to be between 1AE3 and 9AE0 kDa. IW1 was heat labile, while BC1 was moderately stable except at extreme temperatures. BC2 was very stable and maintained its activity when frozen, autoclaved or exposed to extreme pH values. Conclusions: Bacteriocins have been isolated from environmental isolates of V. vulnificus and V. cholerae. BC2, with its broad spectrum and stability, may be useful in neutralizing V. vulnificus. Significance and Impact of the Study: The results have significance in relation to reducing the occurrence of food poisoning caused by V. vulnificus.

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