RNA/DNA ANALYSES

A second group of promising monitoring techniques includes the determination of RNA/DNA ratios, enzyme analyses, and measurements of lipid content. These analyses of eucaryotic or procaryotic populations can be streamlined, but all currently require extractions. The analyses for each of the biological materials discussed above are simple to quantify and could be adapted for use on board ship. As with ATP monitoring, the analyses would need to be correlated with actual ballast water populations for predictive purposes. These analyses would probably require some preparation on the part of crew members, but the extractions and chemical additions could be simplified to be analogous to swimming pool analyses. Specifically, safe, premeasured portions of chemical additives and small amounts of glassware could be supplied to generate a visible residual. The residue could then be readily analyzed colorimetrically on board ship.

Surrogate methods of analysis, such as ATP analysis and determination of RNA/DNA ratios, may be particularly useful if on board treatment procedures such as filtration or disinfection are used. In these cases, it would only be necessary to determine the absence or presence of biological materials such as ATP or DNA to indicate a high degree of treatment effectiveness. Such monitoring processes will probably be the least expensive to implement on board ship but will require some training to facilitate the analysis by crew members.

FLOW CYTOMETRIC TECHNIQUES

A promising technique that could eventually be applied to monitoring ballast water for unwanted biological organisms is flow cytometry. This technique uses a modification of a typical Coulter counter system and can be operated in a flow-through mode. The equipment allows for a small passage of water through an aperture of preset size (usually 2 to 20 µm). Organisms that pass through this aperture can be measured by the cytometer. The system not only quantifies the size and volume of organisms passing through the orifice, but can also detect the natural fluorescence associated with the presence of chlorophyll. Therefore, flow cytometry can be used to quantify all microorganisms without an outside excitation. This equipment is currently available commercially and has been installed and operated on research vessels where its portability, while requiring some improvement, has been proven.

A flow cytometer could also be used to identify the presence of specific organisms if either dyes or DNA/RNA probes were added to the water prior to measurement. For instance, dyes added to a sample of water before passage through the flow cytometer affect the membrane potential of the cells. This potential can be detected by the flow cytometer, and it will be different depending on whether a cell is active or inactive. Because inactive cells have no induced membrane potential, the viability of organisms passing through the cytometer can be determined.



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