Beginning in the 1980’s, Ballast
Organic Biofilm (BOB) units and flow cells [3
] were developed and deployed in ships visiting a total at least 16 consecutive world ports in the Atlantic, Pacific, and Mediterranean Seas. The carried coupons were examined for dominant species of microbes and particles as they were built up from station-to-station and in total [5
]. Biofilms were examined by light and scanning electron microscopy, and by fluorescence light microscopy using special stains developed for five “consensus” species that always appeared as benchmark microbes [7
], and by multiple attenuated internal reflection infrared (MAIR-IR) spectroscopy [8
] for qualitative and quantitative analyses of biofilm constituents. The five “benchmark” bacteria were originally identified and isolated from naturally formed biofilms on materials from the Atlantic Ocean and stored in Atlantic Ocean water aquaria [7
]. The bacteria were noted to be quite cosmopolitan in regard to the volumetric habitats they normally thrived in: water (fresh and salt), food, animal sources, soil, sewage, and air. Each species was a known opportunistic human pathogen, and so presents the concern that biofilm-induced invasion by these and more serious pathogenic species might someday occur. Specifically, Vibrio alginolyticus
has been isolated from infections of the ear (otitis externa), eye (conjunctiva), gastro-intestinal system, and wounds [9
]. Pseudomonas putrefaciens
) has been isolated from human skin, soft tissue, and intra-abdominal infections, and may cause sepsis, bacteremia, and meningitis. Vibrio alginolyticus
and Pseudomonas putrefaciens
), members of normal fish micro-fauna, are also bacteria that cause spoilage of harvested fish-flesh and, thereby, are microbes of health concern to the food industry [10
With approval of the university’s Animal Care and Use Committee, antibodies against the bacteria were developed by inoculations of New Zealand White Rabbits and were deliberately of the polyclonal
type. Polyclonal antiserum raised against any individual antigen consists of an assortment of antibodies of a variety of classes binding to different epitopes on the antigen with a diverse range of affinities, the proportion varying from animal to animal and within the one animal from bleed to bleed [11
]. The chances of having cross-reactions with other non-targeted bacteria that might be found in natural biofilms, using polyclonal antibodies, are – of course -- greater than if monoclonal antibodies were utilized. In monoclonal antibody (mAb) production, the cell (B-lymphocyte) producing a single specific antibody to a specific epitope is isolated and propagated in large amounts [11
]. Monoclonal antibodies decreased the chances of cross-reactions with non-targeted bacteria. On the other hand, the problem with monoclonal antibodies was that they may only have targeted a certain strain of a species, thereby being too selective for general surveys. The genus-specific Ab preparation resulting from polyclonal Abs was judged to be a more useful tool for the general survey studies reported here. Antigenicity or reactivity of the antibodies used was — in every case -- relatively weak, with titers ranging from 13:1 to 30:1, where antibodies with strong antigenicity would be in the range of 100’s:1.
The examination of immunofluorescent
-stained bacteria associated with biofilms on surfaces by the technique of fluorescence microscopy utilized incident lighting for observation and enumeration of the biofilm-sequestered bacteria formed on flat test coupons within the ballast tanks. The technique also allowed for detection of immuno-stained bacteria associated with biofilms on sediments and other particles re-suspended within those tanks. If only transmission lighting had been used, the opaque properties of the sediments or particles would not have uniformly allowed for detection of stained bacterial biofilms on these opaque, irregular bits of matter.
Scanning Electron Microscopy – Energy Dispersive X-ray Analysis:
General scanning electron microscopic and coordinated energy-dispersive X-ray surveys at low magnifications were used to confirm silicon as the major element of the biofilms’ trapped particles, mostly silica as in beach sand. Corresponding MAIR-IR spectroscopic analysis also confirmed the presence of silica as found in clay and other soil minerals of usually re-suspended harbor sediments.
Sterile cotton swabs were utilized to sample the biofilms from the coupons deployed in the flow cells and BOB samplers. The material removed from each coupon was transferred to a germanium prism for analysis by MAIR-IR spectroscopy. Spectra were evaluated for the presence of carbonates and silica-based inorganic particles associated with the organic constituents of biofilms from the transglobal ships.
Normal transmission light microscopy was used to reveal larger particles associated with the biofilms and was useful in visual comparisons among different samples. Light microscopy also was useful in observing protists and occasional small crustaceans in-situ, grazing on the primary biofilms (see video at www.wings.buffalo.edu/iucb/video.html ).