Methods of Enumeration of Phytoplankton and Periphyton Collected for the Algae Taste and Odor Project.

All counts are natural unit counts not total cell counts and are reported as cells or colonies/mL or cm2.

Phytoplankton

Samples are collected in 500 mL Nalgene bottles approximately 15 cm below the surface and approximately 70 - 90 cm from the sides of the canals. These are kept in a cooler for transport to the lab (ERL) where they are then placed in a walk-in cooler.

Enumeration is performed by homogenizing the 500 mL sample and extracting 10 mL with a syringe. This 10 mL sample is filtered through a 0.45 micron (or smaller) filter. This filter is placed onto a microscope slide where 1 drop of immersion oil is used to clear the filter and a 22mm X 22mm coverslip is placed on top of the filter.

The eyepiece of the microscope is fitted with an ocular reticule. The area of the entire reticule is 10 mm2 (1 cm2). This is broken into a grid of 100 squares. Each square is 1mm on a side (1mm2). The volume under 81 of these squares is 1/10,000 of a mL @ 100X or 1/40,000 of a mL @ 200X (calibrated with a Neubauer Improved Hemacytometer).

Results are an average of 6 separate counts performed on any 1 filtered sample. For example, if the average after 6 counts is 1.83 then this is multiplied by 4000 instead of 40,000 (@200X) since we are concentrating the sample by a factor of 10 during filtration. Therefore, 1.83 X 4000 = 7320 cells or colonies/mL.

Periphyton

Samples are collected by scraping 5 mL from the sides of the canals at an approximate depth of 15 cm. This is measured using a 10 mL syringe. This 5 mL sample is diluted with 50 mL of de-ionized water. The approximate area of the scrape is determined using a standard ruler and reported in cm2. This is put into a cooler for transport back to the lab (ERL) where it is placed in a walk-in cooler.

Enumeration is performed using the ocular reticule described above. The area under the entire reticule equals 0.55 mm2 @ 200X (calibrated with a 0.01 mm stage micrometer). All counts are performed at this magnification with other powers used for identification purposes only. For example, if the average after 6 counts is16.17 then 16.17 X 1.8181818 = 29.4. Since this is a dilution by a factor of 10 then, 29.4 X 10 = 294.00 cells or colonies/mm2. Converting to cm2 we multiply by 10 again and get 2940 cells or colonies/cm2. If, for instance, we scraped an area of 39 cm2 to obtain our 5 mL sample we divide 2940 cells or colonies/cm2 by 39 cm2 and we get 62.82 cells or colonies/cm2.

Artificial Substrates

Glass slides are attached to bricks with silicone and tied to the sides of the canals at various sample sites. These bricks are placed at a depth of approximately 70 - 90 cm. Slides are examined every 2 or 4 weeks. Due to the difficulty in being able to keep bricks and slides in some areas due to removal by maintenance personnel or vandalism, some of the data for various times may be missing.

Enumeration is performed by placing a 22mm X 22mm coverslip at a random area on the slide and counted using the ocular reticule described above. Results are reported in cells or colonies/cm2