Goals of the program are to:
- involve participants in the direct, hands-on collection of research
data using current technologies,
- include them in the collation and analysis of data samples; and,
- allow each participant to prepare oral and written presentations of
observations, interpretations and conclusions regarding the data.
The following document types are represented:
BACON, J.J., D.A. IVERSON and G.M. WORTHAM .
1993. Antibiotic sensitivity testing at hyperbaric conditions (1.62 ATA).
Proc. S.D. Acad. Sci. 72:286.
The increased and extended uses of undersea habitats as laboratories,
observatories, and living quarters will subject diving researchers to
prolonged hyperbaric and environmental conditions reported to have
significant influence on the distribution of the normal bacerial flora of
the body. Condsideration must be given to the epidemiology, pathology, and
chemotherapy of bacterial diseases augmented by an redistribution of this
This study examined the sensitivity of Escherichia coli, Pseudomona
aeruginosa, and Staphylococcus epidermidis to anitbiotics
administered in two hyperbaric microenvironments. The micorenvironments
were maintained at 1.62 ATA and varied in ventilation rates (20 and 30
scfm). The Kirby-Bauer Method of antibiotic sensitivity testing was used
to evaluate the bacterias' responses to ampicillin, erythromycin,
gentamicin, polymyxin B, tetracycline, and vancomycin.
In both hyperbaric microenvironments, E. coli, P. aeruginosa,
and S. epidermidis demonstrated antibiotic susceptibilities
congruous (p < 0.01) with the normobaric Inhibition Zone Diameter
Interpretive Standards (Difco Laboratories). Significant variations in
individual bacteria's sensitivities within the susceptibility ranges were
observed. E. coli 's sensitivities to erythromycin, gentamicin, and
tetracycline increased in a 20 scfm microenvironment (p < 0.02). P.
aeruginosa demonstrated no significant correlation between antibiotic
sensitivities and hyperbaric conditions, exclusive of its increased
sensitivities (intermediate) to tetracycline in both the 20 and 30 scfm
microenvironments (p < 0.01).
Data analyses suggest the efficacies of protein sythesis inhibiting
antibiotics (erythromycin, gentamicin, and tetracycline) may be modified
by the increased and reapportioned gas partial pressures in hyperbaric
DUBBELS, B.L., J.D. FRENCH, J.D. TRANAS,
R.T. DICKEY, D.J. DINGSOR AND W.J. SOEFFING . 1996. The effects of
hyperbaric air on the population growth rates of the common etiological
agents of otitis and the development of a buffered general growth meidum
for use in compressed air environments. Proc. S.D. Acad. Sci. submitted.
The redistribution of normal bacterial flora of the human body has been
implicated with the prevalence of auditory canal infections in saturation
divers living in and working from undersea habitats. This study examined
the influence that hyperbaric air environments have on the population
growth parameters of three bacterial species (Escherichia coli,
Pseudomonas aeruginosa and Staphylococcus epidermidis)
associated with epidemiology and pathology of otitis externa and
media. As a result of preliminary data analyses, the need for the
development of a general growth medium capable of minimizing the influence
of carbon dioxide gas absorption on the pH of experimental conditions was
identified. Subsequent experimentation into the characterization of that
medium was pursued.
Standardized bacterial subcultures were incubated at 37 degrees Celsius
in a humidified compressed air microenvironment (hyperbaric chamber).
Standard plate count methods were used to evaluate population growth as
influenced by normobaric, 151, 162, 182 and 202 kPa pressures. Ancillary
experimentation was conducted in a manned undersea habitat (MarineLab, Key
Largo, FL) pressurized at 162 kPa and population growth was evaluated as
The carbon dioxide absorption profiles of phosphate and bicarbonate
buffered and non-buffered media were indirectly evaluated by measuring the
change in pH of 50 ml samples during four hour exposures to the manned
undersea habitat environment. The buffering capacities of suitable
buffered and non-buffered general growth media were evaluated by
standardized acid-base titrations at normobaric conditions.
Results obtained form experimentation conducted in the hyperbaric
chamber were reproducible and statistically significant. E. coli
and S. epidermidis demonstrated predictable declining population
growth rates along increasing pressure gradients. Conversely, P.
aeruginosa initially demonstrated an increasing population growth rate
along the pressure gradient to 162 kPa and subsequently declined. Results
obtained from experimentation conducted in the manned undersea habitat
were inconsistent but appeared to be associated (r<-0.90) with abnormally
excessive (486 +/- 7 Pa: mean +/- sem) and fluctuating partial pressures
of atmospheric carbon dioxide (385 - 571 Pa range).
Preliminary screening of buffered and non-buffered general growth media
revealed that only phosphate buffered nutrient broth possessed the initial
pH (pH 7.09) and buffering capacity (0.007 M/pH unit) appropriate for
ensuing studies. Furthermore, the buffered media did not demonstrate the
transitory pH changes observed in the non-buffered media.
The implications of varying population growth rates along a pressure
gradient, contributing to a redistribution and reapportionment of
bacterial flora, might alter symbioses that are proportion-dependent and
promote opportunistic pathogenesis. To advance the comparison of
microenvironmental in vitro experimentation, a phosphate-buffered
general growth meidum has been developed and is being characterized.
McINTOSH,K . 1994. The effect of carbon
dioxide on the effacacies of protein sythesis inhibiting antibiotics on
bacteria cultured in hyperbaric conditions. USF Colloquium Abstract
The increased utilization and extended use of underwater habitats as
research facilities, observatories, and living quarters, will subject
divers to hyperbaric and extreme environmental conditions identified as
significantly influencing the distribution of the normal bacterial flora
of the body. This redistribution has led to the frequent occurrence of
otitis media and otitis externa in underwater inhabitants.
Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus
epidermidis are consistently found to be causal agents of external and
middle ear infections. The specific purpose of this study was to test the
effacacies of protein synthesis inhibiting antibiotics on these species of
bacteria in hyperbaric conditions and to determine the effect of carbon
dioxide gas absorption by microbiological media on these results. A
modified Kirby-Bauer method was used to predict the effectiveness of the
antibiotics on a specific bacterial sample. Carbon dioxide absorption was
evaluated indirectly through pH testing and directly by carbon dioxide
concentration testing. It is believed that the absorption of carbon
dioxide causes a decrease in pH of the microbiological growth media. This
decrease in the pH of the culture medium, due to elevated atmospheric
carbon dioxide concentrations, influences the activities of protein
sythesis inhibiting antibiotics. In the clinical application of this
studies' results, it will be important for researchers to consider the
effects that hyperbaric conditions have on in vitro experiments.
RESEARCH PROJECT REPORTS:
IVERSON, D.A. . 1990. The distribution
and preponderance of coliform, pseudomonal, and staphylococcal species in
the vicinity of a shallow water habitat. HE2RP Project Report
The qualitative knowledge of microbial biocoenoses of oceans is
insufficient (ZoBell, 1963; Odum, 1971; and , Barnes and Hughes, 1982).
Microbiological studies in the oceans are sporadic, being conducted by a
small number of scientists and lagging behind other oceanogarphical
disciplines (ZoBell, 1963). The results of these studies typically
represent bacterial distributions at isolated stations. No extensive
microbiological studies have been conducted that establish quantitative or
qualitative baseline bacterial disbributions for all marine communities.
However, these isolated studies have indicated large bacterial
populations in the vicinity of watersheds, coral reefs, plankton blooms,
convergences, or areas of high organic matter concentrations. In the
littoral zone, where most observations have been made, biological activity
is the greatest. ZoBell (1963) reports the presence of 100 to 1000
bacteria per millilitre of water; with extremes ranging from <1 to
100,000,000 bacteria per millilitre. Bacterial populations ranging from
<10 to 100,000,000 per gram of wet sediment have been detected in benthic
samples (mean values ranging from 100 to 100,000 bacteria per gram of
Generally, bacteria are most abundant in the topmost layers of bottom
sediments under shallow organic-rich waters (ZoBell, 1963; Odum, 1971, and
APHA, 1985). The abundance of bacteria decreases with sediment depth (ZoBell,
1963; and , APHA, 1985). Bacteria are scarcest in pelagic areas
particularly at depths exceeding 1000 meters (ZoBell, 1963). Wood (1967)
attributed the distribution of baceria to the selective pressures of the
marine environment. Jones (1963) reported the sea water varies in its
ability to support the growth of bacteria, depending upon where and when
it is collected, and the species concerned. Liston and Colwell (1963)
identified specific bacterial types (based on metabolic, thermal, and
biochemical requirements) associated with particular geographical
locations. Obviously, envirionmental variability necessitated an
investigation into the distribution of bacteria in the research locale.
This study set out to examine the distribution and preponderance of
Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus
epidermidis in the vicinity of a shallow water habitat. Since these
three species have been associated with the etiology of external ear
infections, it was necessary to examine the potential for environmental
contributions to the occurrence of these species in the divers' external
Methods and Procedures: Two water samples were collected at each of two
locations within the immediate vicinity of the undersea habitat.
Collection Station #1 included that area circumscribed by the habitat's
hatch at a depth of 10-20 cm below the air-water interface (approximately
21 ft. of water depth). Station #2 was immediately beneath a suspended
oyster culture bed and 20-30 cm above the bottom sediments (approximately
17 ft. of water depth). Water samples were collected aseptically with 60
ml presterilized syringes. Precautions were taken against bacterial
contamination or cross-contamination of the syringes to insure the
integrity of the collected sample. Water sampling coincided with each
Water samples were processed using positive-pressure, presterilized,
0.22 micron ultrafiltration sytems. The ultrafiltration system was
assembled aseptically and affixed via a Lur-Lock (TM) attachment to the
sample syringe. Three 25 ml samples were filtered from each collection
station, one sample (filter disc) for each media type.
The filter discs were aseptically transferred, organism side up, to a
prepared medium which would select and/or differentiate the sample's
bacteria (mannitol salt agar--Staphylococcus spp., M-Pseudomonas
Agar--Pseudomonas spp., and Eosin Methylene Blue Agar--coliforms).
The media plates containing the filter discs were incubated for 16 to 20
hours in a 35 degree celcius incubator at 1.62 ATA.
Results and Discussion: Enumeration and identification of colonies were
intially unsuccessful. The ambient humidity of the habitat and the media
moisture content impeded the production of discrete colonies. This
situation dictated a modification in the filtration method. A brief
exposure of the filter disc to a forced filtered-air drying produced
superior results to that previously performed.
Upon examining the first set of incubated samples, it was found that
the colorimetric indicators of the Eosin Methylene Blue Agar had not
responded under hyperbaric conditions according to the normobaric
standards. Further investigation revealed the methylene blue indicator had
been reduced by the increased partial pressure of the oxygen associated
with the hyperbaric environment of the habitat. The selective
characteristics of the media appreared to be unaffected by the hyperbaric
environment. Subsequent collaborative studies, conducted under normobaric
conditions, confirmed that the selection, but not differentiation, had
occurred. For that reason, the identification of specific coliform species
could not be made.
Data, collected after the modification of methods, indicated an
increased preponderance of Staphylococcus spp. in the vicinity of
the undersea habitat and an association of coliforms with the oyster bed.
These findings are based on an extremely small sample size and suggest
With the development of an effective forced ultrafiltration technique
and as future research is planned, it will be propitious to continue water
sampling to establish a spatial and temporal population distribution as
influenced by the research locale's environmental and seasonal variables.