Procedure

 

Overview

Oysters will be placed in a circular, recirculating tank of sterile seawater.  A known amount of Escherichia coli will be added to the tank with oysters and an identical control tank with no oysters.  Water samples will be taken at set intervals from both the oyster-containing tank and the control tank.  E. coli levels in each sample will be recorded and plotted to show the rate of decline in bacterial concentrations.  The rate of decline in the oyster-containing tank will be compared to that of the control tank to determine how much bacteria the oysters are filtering out of the water column.  The first phase will be repeated three times to ensure accuracy.

 

Clean and disinfect tanks and equipment

1. Scrub down and rinse tanks and equipment with freshwater
2. Position equipment (circulation pump & air hose, heater, and particle suspension rig) in tank
3. Fill tanks with 5% bleach/water solution and let stand for at least 24 hours
4. Drain and rinse tanks
5. Fill tanks with 100 grams of vitamin C in solution to neutralize residual bleach
6. Drain and rinse tanks

 

Set up experiment

1. Fill seawater reservoir with 115 liters of fresh seawater
2. Attach 5 micron filter and UV sterilizer to reservoir output
3. Fill each tank with 50 liters of filtered, sterilized seawater
4. Scrub exterior shells of oysters to remove extraneous materials
5. Place oyster in oyster tank, positioning them in rows of 3, upright and facing the flow of water (flatter valve goes up, side opposite the hinge faces flow from circulation pump)
6. Turn on all equipment (water and air pumps, heater timer device)
7. Add 5 ml of a 24-hour student-safe Escherichia coli broth culture to each tank
8. Allow both tank systems to sit and circulate for one hour to thoroughly mix bacteria into water
9. Take water samples and record water temperatures at indicated time intervals, process all water samples as indicated in the Colilert® 18 hr sampling procedure
   • Time intervals: initial, 3 hrs, 6 hrs, 9 hrs, 12 hrs, 15 hrs, 18 hrs, 22 hrs, 26 hrs, 30 hrs, 34 hrs
   • Repeat 3 of the 22 tests to confirm accuracy of sampling procedures

 

Colilert Sampling Procedure:

 

1.     Turn on Quanti-Tray sealer

2.     Take two pre-filled, sterile, warm, 100 ml mixing bottles out of the incubator, grab two sterile empty sample bottles, and two sterile 97-well Quanti-Trays

3.     Label a sample bottle and a mixing bottle with an “O” for oyster-tank and the other sample bottle and mixing bottle with a “C” for control-tank.  Label the top of one Quanti-Tray with the sample number and a “C” and the other with the sample number and an “O”

4.     Fill each sample bottle with water from corresponding tanks

5.     Using a mechanical pipette, transfer .1 ml of water from sample bottle into the corresponding mixing, using a fresh sterile pipette tip for each transfer

6.     Tap the entire contents of one Colilert® 18-Hour reagent packet into each mixing bottle

7.     Cap bottles and shake vigorously until reagent is completely dissolved

8.     Open 97-well Quanti-Tray by squeezing top and pulling the tab out, pour the contents of each mixing bottle into the correspondingly labeled Quanti-Tray

9.     Seal each tray by placing it upside down in the rubber form and pressing the seal button

10. On the data sheet, record the sample number, source, time of sample, and temperature of each tank.

11. Place trays in incubator for 20-22 hours at 35° C

12. Read trays by counting and recording the number of yellow large wells and yellow small wells

13. Plug numbers into Idexx chart to find the MPN of E. coli concentrations.  Record on data sheet

 

Section 3 responses

a)     A student-safe strain of Escherichia coli was used in all experiments.

b)      Student-safe E. coli was purchased from Carolina Biological Supply Co..