Biological Analyses

• Bacteria - Bacteria abundance in the ballast tanks is being quantified by flow cytometry (Figure 4). Coupled with fluorometric detection and assuming reasonable distinction of particle attributes, this instrument rapidly produces particle distributions that allow differentiation and enumeration of populations within a given sample. For example based on size and chlorophyll autofluorescence, populations of bacteria and pico-eukaryotes can be distinguished from each other and from larger cells within a sample, thereby facilitating rapid enumeration of each group. Fluorometric stains that bind with nucleic acids, enzymes or other cellular constituents can also help differente biotic particles and indicate cellular viability in unpreserved samples. Molecular techniques are being used to screen for a suite of harmful bacteria: PCR is run to produce bacterial amplicon products; the bacterial amplicons have each been linked to a vector; and we are producing E. coli cell lines capable of incorporating the vector DNA product from each. Based on the data from these cell lines, we search nucleotide databases for a sequence match, and if it is not available, we determine the phylogeny. From the sequence data and species-specific PCR assays, we are identifying pathogens of interest.

• Phytoplankton - Chlorophyll a concentrations are measured in the ballast tanks as an indicator of total phytoplankton biomass. Phytoplankton taxa analyses are conducted using phase contrast light microscopy (200-600X) (Figure 5), and Utermohl chambers for quantification. Scanning electron microscopy and molecular screens are also utilized as needed to aid in identifications.

• Zooplankton - Small zooplankton (maximum dimension < 80 µm) are being identified and quantified using light microscopy. In addition to the microscopic analyses of preserved samples, live samples are collected for culture in media designed to simulate discharge into a receiving system where environmental conditions might permit establishment. The cultures are being used to assist in identification of some taxa. In addition, some of the cultured species are being tested in heat treatment experiments to assess temperature regimes that kill harmful organisms capable of surviving transport (Figure 6). Heat treatment has been considered as an effective way to treat ballast water in some ships, depending on the ship design.