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A Comparative Study of the Bacterial Flora of the Hemolymph Of MFR PAPER 1144 R. R. Colwell is with the Depart­ ment of Microbiology, University of Maryland, College Park, MD 20742. T. C. Wicks is with the De­ partment of Biology, Georgetown University, Washington, DC 20016. A Comparative Study of the H. S. Tubiash is with the Biological Laboratory, National Marine Bacterial Flora of the Hemolymph Fisheries Service, NOAA, Oxford, of Callinectes sapidus MD 21654. The isolates from the holding tanks were obtained from animals maintained R. R. COLWELL, T. C. WICKS, and H. S. TUBIASH in the laboratory for more than I week. Crabs obtained from the fish market were held in a tank for I to 2 days before ABSTRACT-The bacterial flora of blue crabs (Callinectes sapidus) from sampling. Crabs from the Rhode River Chesapeake Bay was examined. Hemolymph of normal, healthy blue crabs was 5 in Chesapeake Bay were sampled im­ found non-sterile, with total bacterial counts ranging from <100 to >3.0 x 10 mediately after capture. Crabs from 5 on a seawater-based medium and 0 to >10 on Standard Methods Agar. Marumsco Bar were also sampled in the 3 Counts of 0-10 were observed on TCBS agar. Vibrio spp., including Vibrio field without delay after capture. The parahaemolyticus, were the major taxonomic groups found in the crab largest sample ofanimals examined was hemolymph. A comparison of crab data with that of Chesapeake Bay oysters a catch made offFranklin City, Va. The revealed a qualitative difference in that the crab hemolymph flora was almost hemolymph was sampled 12-16 h after entirely Vibrio spp., whereas the oyster flora included species of a variety of capture. other genera. Total viable counts of aerobic, heterotrophic bacteria in blue crab INTRODUCTION by various investigators (Society for hemolymph were carried out as follows. General Microbiology, 1962; Beers et After removing the fluid with a sterile The normal bacterial flora of blue a!., 1962; Silvestri eta!., 1962;Sokaland 25-gauge needle and syringe wetted crabs (Cal/inectes sapidus) from Sneath, 1963). Colwell and Liston with a sterile solution of 5 percent sodi­ Chesapeake Bay has not been exten­ (1961), in one of the earliest numerical um polyanethol sulfonate (G RO­ sively studied. Interest in the micro­ taxonomy studies, examined marine BAX)I,Z, an anticoagulant reportedly biology of shellfish, traditionally, has bacteria. Subsequently, several other effective for isolating bacteria from focused on human pathogens found in investigators examined the taxonomy of blood (Morello and Ellner, 1969), I ml commercially important species, as in marine bacteria (Floodgate and Hayes, of the hemolymph was withdrawn from the case of the blue crab. Many of the 1963; Hansen et a!., 1965; Liston et aI., each crab, either through the posterior papers published on the microflora of 1963; Pfister and Burkholder, 1965; hinge ofthe carapace or from the base of the blue crab have concerned pathogens Qualding and Colwell, 1964). Results of the coxa ofthe flippers. A dilution series rather than the normal microbial flora these studies proved helpful in identify­ was made, using a sterile diluent com­ present in the healthy animal (Couch, ing and classifying the bacterial flora posed offour salts and distilled water in 1967; Fishbein et a!., 1970; Krantz et from blue crab hemolymph. the concentration: NaCI, 24.0 g; KCI, a!., 1969; Rosen, 1967; Sawyer, 1969; 0.78 g; MgClz, 5.3 g; MgS04 • 7HzO, 7.0 Williams-Walls, 1968). In addition, in­ g; and distilled water, I liter. One-tenth formation has been gathered concerning MATERIALS AND METHODS ml of each dilution was inoculated onto the bacteriology of processing, han­ agar plates, in triplicate, and spread dling, packaging, or storage of crab­ Healthy adult blue crabs (Callinectes evenly with a sterile, bentglass rod. The meat (Loaharanu and Lopez, 1970; sapidus) weighing 150-230 g were ob­ three media used were: MSYE (Pro­ Ulmer, 1961; Ward and Tatro, 1970). tained from seafood markets or col­ teose peptone, I g; yeast extract, 1 g; This study was conducted to deter­ lected from several areas in Chesapeake agar, 15 g; four salts solution, as above; mine the total viable, aerobic, hetero­ Bay and Chincoteague Bay during the pH adjusted to 7.4-7.6 with 0.1 N trophic, bacterial flora of blue crab summer of 1970. Male and female crabs NaOH), SMA, the Standard Methods hemolymph in freshly caught, possessing hard carapaces ("soft-shell" Agar (Casein digest, 5.0 g; yeast ex­ aquarium-held, and market crabs during crabs undergoing ecdysis were not in­ tract, 2.5 glucose, 1.0 g; agar, 15 g; dis­ the summer months and to identify and cluded) were sampled to determine total tilled water, I liter; pH adjusted to 7.0 classify the bacterial strains isolated, numbers and types of aerobic, hetero­ with NaOH) and TCBS, the Thio­ using the methods of numerical tax­ trophic bacteria comprising their nor­ sulfate-citrate-bile salts-sucrose agar3 onomy. Numerical taxonomy as a mal microflora during the summer method for the analysis of taxonomic months when the water temperature I Roche Diagnostics, HotTman-LaRoche, Nutley, data for bacteria was first proposed by ranged between 23°-32°C. Sources of N.J. Sneath (1957) and has been used, with crabs, bacterial isolates, and dates of 'Reference to trade names does not imply en­ dorsement by lhe National Marine Fisheries Ser­ modifications of the original methods, sampling are given in Table I. vice, NOAA. 29 Table I.-Source and date of Isolation of bacterial methyl carbinol, and urease; production Table 2.-Bacterial counts of the hemolymph of crabs strains Included In the numerics' taxonomy analysis. collected at several locations on the media employed of ammonia from peptone, NOz, and in the study. Counts given are per milliliter hemolymph Strain number Date of isolation Source' of Individual crabs. Figures given for each sample set ; N0 3 hydrolytic activity on starch, are mean values, except where otherwise indicated. 1-10 7/15f70 HT gelatin and casein; methyl red test; and 11-27 7119170 FM No. 28-33 7/21/70 RR utilization of alanine, proline, glutamic Source sampled MYSE SMA TCBS 34-39 8/22170 MB acid or methionine as sole carbon and 40 8/27/70 FC Laboratory 41 8/27170 FC nitrogen sources. holding tank 3 1.8 x lOS 3 X 10' 10 42 8/27170 FC Drug sensitivity of each pure culture Fish market 2 6 x 1()4 4 x 103 1 43 8/27/70 FC Rhode River 6 2.1 x lOS 60 0 44 8/27/70 FC was determined using penicillin, 10 Marumsco Bar 4 1.3 x lOS 2 X 105 100 45 8/27170 FC units; Chloromycetin, 30 J.1g; tetracy­ Franklin 46 8/27170 FC Cily. Va. 33 3.1 x 10' 2 X 10' 33 47 8/27170 FC cline, 30 J.1g; dihydrostreptomycin, 10 Total range 48 -:100--·3.0 x 10' (}_>105 0-10' 48 8/27/70 FC J.1g; and colimycin, 10 )J.g5. Sensitivity 49 8/27170 FC was recorded without quantification of colony type appearing on TCBS agar 'HT ~ Holding tank. Georgetown University: FM ~ Fish market. District of Columbia; MB = Marumsco Bar. Md. the diameter ofthe inhibition zone. Sen­ was yellow, 2 mm in diameter, and simi­ (Chesapeake Bay): RR = Rhode River, Md. (Chesapeake sitivity to the pteridine compound lar to Vibrio alginolyticus. Results of Bay): FC = Franklin City, Va. (Chincoteague Bay). (0/129) was also recorded (Shewan et total viable counts are given in Table 2. (yeast extract, 5.0 g; polypeptone pep­ a!., 1954). No change in counts was observed be­ tone, 10 g; sodium citrate, 10.0 g; All media, with the exception of the tween 48 hand 7 days. sodium thiosulfate, 10.0 g; oxgall, 5.0 g; blood agar, were prepared with the salt The strain clusters obtained from the sodium cholate, 3.0 g; sucrose, 20.0 g; solution, described above, as diluent. In computer analysis of the taxonomic NaCI, 10.0 g; iron citrate, 1.0 g; thymol most cases, dehydrated medium was data are shown in Figure 1. All strains blue, 0.04 g; bromthymol blue, 0.04 g; mixed directly with the salt solution as grouped atS ;;3 57 percent and the major agar, 14.0 g; distilled water, I liter; pH diluent, but some media required spe­ phenons detected are shown in Figure 8.6). The inoculated media were incu­ cial preparation. Media for the casein I, A through 1. Strains clustered in Phe­ bated at 25°C for 48 h, after which hydrolysis, methyl red, Voges­ non A were not found to be closely re­ counts were made and the media again Proskauer, and carbohydrate utilization lated, indicated by the low similarity incubated for an additional 5 days for a tests were prepared double strength in values (62-64 percent). Phenon B con­ repeat count. distilled water, and a double strength, sisted of strains more closely related to Forty-nine cultures were isolated, sterile salt solution was added (I: I each other (5 = 72-74 percent). Phenon purified, and subjected to the testing volJvol) after sterilization. C comprised the largest group, consist­ procedure used for numerical tax­ A total of 126 features used in the ing of strains 41 through II (Figure I) onomy. Colonies were selected ran­ computer analysis were scored, with clustering atS ;;3 75 percent. Three sub­ domly and were streaked three times to the code: 0 (negative), I (positive), or 3 groups (I, II, III) were observed in ensure purity of the cultures. After (not tested or not applicable). The Phenon C (Figure 1).
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