Quantitative or qualitative?
A Sedgwick-Rafter counting cell (as described in detail below) can be used for highly quantitative veliger detection. It's limitation is that only 1 ml of sample can be examined at a time. For qualitative (presence/absence) information, placing a larger volume of the sample in a glass petri dish and examining with a dissection scope equipped with polarizing filters is very effective.
Equipment for quantitative analysis:
Dissecting microscope with magnification ³40X, Sedgwick-Rafter counting cell, disposable Pasteur pipettes, rubber bulbs, dissecting tools, [optional: polarizing filters and Hensen-Stempel pipette]
Sedgewick/Rafter counting cell
1. Remove a 1-ml aliquot from a well-mixed sample using either a Pasteur or Hensen-Stempel pipette. Dispense the aliquot into a Sedgwick-Rafter counting cell. Carefully place the cover slip onto the counting cell perpendicular to the long axis of the slide. Slowly swing the cover glass so that it completely covers the sample well. Careful alignment of the cover glass will prevent air bubbles from being introduced into the sample and will ensure that the sample holds its complete volume (1-ml).
2. Place the filled Sedgwick-Rafter cell under a dissecting microscope. Examination of the counting cell is simplified if the cell is placed over a grid. A general examination of the contents of the cell will provide a feel for the density of organisms. If plankton densities are too high, it may be hard to see and identify veligers. If densities are too low, time may be wasted looking for veligers. Dilute or concentrate the sample as needed, taking care to record any dilutions or concentrations.
3. Once an appropriate concentration has been reached, examine the contents of the cell and record veliger densities. If cross-polarized light is available, align the filters appropriately and begin counting veligers. Regardless of the light source, count each cell until the same number of veligers is reached twice. Using a grid system or an equivalent scanning technique will reduce the amount of time spent counting and ensure that the complete cell contents are counted.
4. If live samples were used in the analysis, dump the contents of the cell into a designated vessel that will eventually be sanitized per zebra mussel containment protocols (see Protocol for Responsible Monitoring Procedures). The counting cell, cover glass, and any pipettes should be carefully rinsed. The rinse water should be put into the containment vessel as well.
5. For each sample collected, examine at least five Sedgwick-Rafter cells for veligers, count the densities, and record the data. To calculate the veliger densities in the water sampled, see the Calculating Veliger Densities from Sedgwick-Rafter Cell Countssection.
6. To prevent cross-contamination of samples (i.e., false positives), it is recommended (Cameron Lange, personal communication) that Sedgwick-Rafter cells and all glassware be kept in a vinegar bath, which dissolves the calcium of the veliger shell.
Veliger Analysis Techniques