The NSS Bulletin - ISSN 1090-6924
Volume 57 Number 1: 24-30 - June 1995

A publication of the National Speleological Society

Diving Protocol for Sterile Sampling of Aquifer Bacteria in Underwater Caves
Harris W. Martin, Robin L. Brigmon, and Thomas L. Morris


Microflora of phreatic conduits are not accessible to ordinary environmental microbiology sampling techniques. Therefore, ecosystems found in underwater caves have received very little study. The combination of sterile technique for environmental sampling of microorganisms with technical cave diving procedures is required if aquifer bacteria in phreatic limestone conduits are to be sampled and studied. Groundwater bacteria in underwater caves have only recently been sampled by divers. Challenges such as sample container buoyancy, confined space, darkness, remoteness of sampling sites, and an aquatic environment were met by cave divers sampling bacterial colonies while hovering in the water column in underwater caves. A set of suggested instructions is provided for qualified cave divers to collect samples from visable natural colonies of aerobic and microaerophilic bacteria and other microorganisms in underwater cave environments while maintaining sterile technique. Sterile 50 ml tubes were used successfully to collect the bacteria. Collecting bacteria in underwater caves with sterile syringes was less successful. Bulk water samples collected from the vicinity of sampled bacteria colonies were autoclaved and used to imporve isolation and culture of some fastidious aquatic bacteria such as Thiothrix ssp. A low pressure hose and air nozzle are used to purge outside non-cave water from these water sample jugs. Safety and conservation practices are important in this type of field work. Logistical details of preparation for the dive and handling of samples during and after the dive are described. Modifications for sampling anaerobic bacteria under N2 gas are suggested. Study of underwater cave microflora may contribute substantially to a better understanding of groundwater biogeochemistry, carbonate geochemistry, speleogenesis, subsurface microbial ecology, paleo-ecology, the ecology of cave macrofauna, and global nutrient cycling.

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