John D. Buck

Effects of medium composition on the recovery of bacteria from sea water

Abstract Water samples were collected from offshore and inshore localities at various depths off the Connecticut coast over a two-year period. Spread plates for bacterial counts at 20 °C were made on a variety of complex solid media. Counts on Difco-Marine Agar were controls in all cases with counts on test media related to these in ratio form. Initially, nine media were used and represented some from the literature as well as personal formulations. Differences between inshore and offshore samples were greatest with media containing the highest peptone concentrations. Two media containing the peptones Gelysate and Trypticase showed the highest overall counts. A second phase concerned a comparative study of these peptones varying in concentration from 0.1 to 10.0 g/l in a constant basal medium. None of the media invariably gave counts greater than the control, but peptone concentrations of 10.0 and 5.0 g/l resulted in the lowest comparative counts. Considering all samples, peptone levels of 0.1 and 1.0 g/l showed the highest counts. Counts for both inshore and offshore water samples decreased as peptone concentration increased. Qualitatively, high peptone media showed large, mucoid, confluent colonies which made the counting of smaller ones difficult. Pigmented colonies were more frequent on low peptone media. Bacteria were isolated from all media and from all stations; the percentage of various groups varied with peptone concentration and source of sample. Media containing three fish peptones in varying concentrations have also been investigated. None produced overall counts greater than Difco-Marine Agar and counts decreased with increasing peptone levels: there was a trend towards higher counts in offshore waters with fish extracts. Quantitative and qualitative aspects of the work are discussed.

Clostridium perfringens as the Cause of Death of a Captive Atlantic Bottlenosed Dolphin (Tursiops truncatus)1

A previously healthy captive female bottlenosed dolphin (Tursiops truncatus) died suddenly. At necropsy, Clostridium perfringens was isolated from dorsal muscle, blood, left heart ventricle, thoracic fluid, and abdominal fluid. An identical strain was recovered from pool water. A male dolphin in the same pool had inflicted several “rake” marks on the dorsal surface of the female. Water-borne bacteria probably entered these lesions which served as the focus for anaerobe penetration and spread.

The effect of bacteria and bioturbation by Clymenella torquata on oil removal from sediment

Clymenella torquata, a representative of conveyor-belt feeding tube worms, was studied in laboratory microcosms for its effect on the removal of No. 2 fuel oil from sediment. Possible enhancement of microbial degradation of the oil was considered as well. Concentrations of the aromatic fraction of the oil were measured by synchronous fluorescence spectroscopy. Clymenella reduced the oil concentration of the surrounding sediment by 20 to 50% (p<0.05) in 10 days. Concentrations of oil in the worm tubes were 25 to 32% (p<0.001) less than the surrounding sediment. Sediment bacteria cultured in oil supplemented artificial sea water could use the oil as their sole C source, but did not significantly degrade the aromatic fraction.

Distribution and characterization of luminescent bacteria in a temperate estuary

The effect of temperature and salinity on numbers of luminescent bacteria present in waters of the Mystic (Conn.) River estuary was evaluated. Counts decreased with decreasing salinity; none were detected at freshwater stations. A population maximum of 35 per ml was noted at the highest salinity station (30±2‰). Highest counts were observed during winter and spring and lowest numbers occurred during summer and fall months. Isolates (111) were identified and compared with previously-described luminescent bacteria; i.e.,Beneckea (Vibrio) harveyi, Photobacterium (V.) fischeri, P. phosphoreum, andP. leiognathi. All species were isolated but distinct seasonal differences were noted.P. (V.) fischeri andB. (V.) harveyi represented 93% of the luminous population on an annual basis. Only the former was found during the period December through March (highest count 7 per ml) whileB. (V.) harveyi was the dominant species noted between May and October (maximum count 11 per ml).P. leiognathi andP. phosphoreum were found only during July and August as 7% of the total luminous population. All isolates grew at NaCl concentrations between 6 and 30‰; none grew below 6‰

Occurrence ofCandida albicans in fresh gull feces in temperate and subtropical areas

The occurrence ofCandida albicans in fresh gull (Larus spp.) feces was compared in temperate and subtropical locations. Of 239 fresh samples, 133 were obtained in southeastern Connecticut and 106 from different sites on the southeastern and central western coasts of Florida. Overall, 60% of all feces containedC. albicans. Of the Connecticut samples, 78% were positive, whereas 38% of the Florida samples revealed the presence of the yeast. Only 1 of 24 samples of fresh brown pelican feces containedC. albicans. Differences inC. albicans occurrence in birds in various locations was ascribed to variations in habitat and feeding behavior. Samples of water from a municipal reservoir in Connecticut were routinely positive, with an average cell density of 20/liter. Two fresh gull samples obtained on the reservoir bank containedC. albicans at an average cell concentration of 5, 200/g. The frequency ofC. albicans in gull droppings was higher than reported by others, and the yeast is common in temperate waters. These findings have important public health implications.

A note on the experimental uptake and clearance of Candida albicans in a young captive gull (Larus sp.)

An immature gull (Larus sp.) was placed in captivity after having been kept as a pet for several weeks and allowed to roam freely. On day one of captivity, bird feces showed the presence of Candida albicans but the yeast was absent for the next 16 days. The gull was fed only yeast-free water and fish. On day 17 only, the bird was fed fish containing C. albicans which had been isolated from a wild gull. The yeast was present in all fecal samples (2–4 per day) for the next 13 days. Beginning on day 26 and irregularly thereafter the bird was fed fish containing 100–200 mg of ketoconazole. Feces continued to show the presence of C. albicans but only sporadically because the bird continued to reinfect itself, probably by contaminating the water supply via feet or feces. After protecting the water, yeast presence in feces decreased. The gull was released on day 57; feces that day were negative for C. albicans. During the experimental period the bird displayed no clinical symptoms of candidiasis. The observations indicated that one exposure to C. albicans was sufficient to establish the carrier state and that the possibility exists for shedding a potentially dangerous microorganism over a large geographical area.

Microbiological Observations on Two Stranded Live Whales1

coons may be of increasing importance as reservoirs for RV particularly in the southeastern USA (Kappus et al., 1970, op. cit.). Absence of clinical disease and RV antibody suggests that rabies does not frequently occur in this population. Rabies virus antibody develops relatively soon in animals after exposure to the virus and is readily detectable by the IFAT (Coe and Bell, 1977, Infect. Immun. 16: 915919). The significance of low antibody titers found in a few animals to CPV and CAV1 is not clear. The former is associated with severe disease in dogs but antibody titers in convalescent dogs are usually high (Potgieter et al., 1981, op. cit.). Low antibody levels can be observed very early after infection (Potgieter et al., 1981, op. cit.) and conceivably after an interval of several months or years after infection. Since the IFAT does not discriminate between strains of CPV or distinguish this virus from feline panleukopenia virus (Potgieten et al., 1981, op. cit.), our results suggested that the latter was not prevalent in this population. Studies of CAV1 in dogs and ranch foxes have been extensive but little is known about this virus in other wildlife (Cabasso, 1970, op. cit.). However, serologic surveys suggest that this virus occurs naturally in raccoons (Jamison et al., 1973, J. Wildl. Dis. 9: 2-3; and Parker et al., 1961, J. Am. Vet. Med. Assoc. 138: 437-440). Apparently the raccoons in Cades Cove are not exposed frequently to CAV1. The low prevalence and titers of antibodies suggested that the viruses which were surveyed were probably not endemic in this raccoon population. Thus, this raccoon population did not appear to serve as an important reservoir for these viruses at the time studied. The authors would like to thank T. A. Webb-Martin for her invaluable assistance in this investigation. This study was financed by Mclntire-Stennis Project Funds of the Agricultural Experiment Station and the Department of Forestry, Wildlife, and Fisheries, University of Tennessee, Knoxville.

A reemphasis-germ tubes diagnostic for Candida albicans have no constrictions

Descriptive and illustrative material in several recent diagnostic manuals for medical mycology are unclear with respect to proper designation of germ tubes formed by Candida albicans. Because of the increasing significance of this and other yeast species in human disease, mycologists should be aware that germ tubes, unlike buds or pseudohyphae, do not have constrictions at the point of origin. Light and scanning electron micrographs are presented to emphasize this diagnostic characteristic.The pathogenicity of the yeast Candida albicans for humans is well-known (9, 19). However, several other yeast species are opportunistic pathogens whose occurrence in humans has been noted with increasing frequency in recent years (2, 11, 17). Consequently, the clinical microbiologist has the task of accurately identifying yeast isolates as they assume added significance in medicine.Perhaps the most used diagnostic procedure for separating C. albicans, the more common and serious yeast pathogen, from other species is the germ tube test. Most descriptions of the technique stress that germ tube walls exhibit no constriction at their points of origin (1, 4, 8, 20). A recent paper which reported germ tube-positive isolates of C. tropicalis (16) received further discussion (14). Comments underscored the need to distinguish clearly between pseudohyphae with basal constrictions in both C. tropicalis and C. albicans and true germ tubes in C. albicans which have no constrictions and which confers specificity to the test. This morphological distinction has been noted previously (5) with photomicrographs although the title of the article did not emphasize this aspect.One of us (JDB) received a letter recently in response to inquiries regarding the eventual standardization of a selective C. albicans medium (6). The comment was made ‘... (the author) ... states that in C. albicans there is no constriction of the germ tube at its point of origin. This is not true. Although most C. albicans do not show constrictions, it certainly does sometimes occur as shown ... (ref. 10).’ In fact, the photo referred to shows only one yeast cell with a cellular extension which could possibly be interpreted as a germ tube although there is an obvious constriction at the base. Without conducting an extensive literature search, we did find another laboratory manual (12) which has a photo showing one cell having a structure identified as a germ tube. This too has a quite obvious constriction. In this publication, a germ tube is defined as an appendage one-half the width of and three to four times the length of the cell from which it arises. No mention is made of constriction or lack thereof. Another clinical manual (15) shows a photo of about ten C. albicans cells with cellular extensions, only one of which can clearly be discerned as a germ tube and it is poorly marked with an arrow since one other cell with a constricted pseudohypha is positioned over the germ tube. Again, germ tubes are defined only as ‘short lateral hyphal filaments.’We are concerned that the differentiation of germ tubes from buds, pseudohyphae, and perhaps other cell extensions is becoming clearly less defined in the recent literature. We are disturbed that an eventual consequence may be that clinical workers now being trained will eventually produce inaccurate diagnoses of yeast infections which are increasing in medical importance.To illustrate the matter of constrictions in C. albicans germ tubes, we used a culture recently isolated on mCA medium (6) from raw sewage collected at the University of Connecticut treatment facility. Cells were transferred from Sabouraud dextrose agar to commercially available calf serum and incubated at 37 C for 3 hours. Figs. 1A and 1B show, under phase microscopy, obviously constricted buds and/or pseudohyphae which closely resemble structures cited above (10, 12, 15) as being germ tubes. Further examination of the same culture revealed unconstricted cell extensions (Figs. 1C and 1D) which were in fact germ tubes.

Comparison ofin Situ andin Vitro survival ofCandida albicans in seawater.

The survival in seawater of several laboratory and field isolates ofCandida albicans was investigated. Initial studies were madein vitro (flasks) to confirm previous reports. Frequent sampling of viable cells showed that flask experiments, even repeated, produced varied patterns of survival in this closed system. As an alternative, multiple experiments were run in untreated seawater in dialysis bags and plexiglas chambers at ambient temperature (17‡ to 22‡C) in flowing seawater. Die-off rates of all cultures tested in dialysis bags were very rapid in the first day and may have been related to high levels of dissolved organic carbon in the tubing. Distilled water-or acid-washed bags did not yield significantly higher survival rates in all cases. When plexiglas chambers closed with Nuclepore membranes were used, survival rates decreased to 5% to 15% of the original population after 6 days. Chamber data were more uniform and represented approximately a twofold increase in survival over that shown previously inin vitro (flask) studies. Some evidence was obtained in all three test systems for the greater survival rate of a field isolate ofC. albicans compared with that noted for a laboratory (ATCC) strain. The results are considered to more accurately depict the survival ofC. albicans in summer temperate recreational waters.

Distribution of aquatic yeasts — Effect of incubation temperature and chloramphenicol concentration on isolation

Fresh (river), estuarine, and marine waters in and along the coastline of Connecticut were cultured by the membrane filter technique at 20 and 37°C on a complex medium containing 0–1000 mg/L of chloramphenicol. Using counts on medium with 500 mg/L antibiotic as a base, ratios of total and pink yeast counts were recorded for other chloramphenicol concentrations at both temperatures for the waters sampled. Variable results were obtained; in general, both total and pink yeast counts decreased with increasing antibiotic levels, being most apparent at > 400 mg/L chloramphenicol. Medium without antibiotic and with 100 mg/L always produced baterial overgrowth. A total of 209 white yeasts were isolated from all platings; the genera Torulopsis, particularly T. Candida, and Candida were dominant with lesser numbers of Cryptococcus, Trichosporon, sporogenous genera, and Kloeckera. Most species isolated were found on media at all chloramphenicol levels. Comparisons were made of yeast distributions in these temperate waters with reports from other areas.