Big Creek Reserve
Coliform Monitoring Procedures

Equipment List:
" Filter membranes: 47mm, .45 micron pore size, sterile white w/ grid
" Petri dishes: 47mm, sterile, w/pad
" Millipore m-Coliblue-24 broth, 2ml ampules
" Millipore m-fc fecal coliform broth, 2ml ampules
" Incubators
" 100ml sample bottles, sterilized*
" marker
" pencils
" sterile paper towels
" pipette, 10ml, sterilized*
" small air vacuum pump
" tweezers
" Pyrex beaker, 50-80ml, sterilized*
" Filtering flask, Pyrex, 1000ml
" >100ml Filter funnel, sterilized*
" small cooler with ice or at least 2 frozen 3"x5" gel packs
*Sterilization is by boiling in water for 5 minutes.


Turn on incubators, setting one at 44.5 deg. Celsius, the other at 35 deg. Celsius.

Collection of Samples:
Place as many empty sample bottles in the cooler, with ice/gel packs, as will fit. Take 2 pencils. Proceed to the first collection site. Snap the lid open on the first bottle, being careful to not touch the rim or inside. Holding the bottle by the rim of the lid, push the bottom of the bottle under the water at an angle to fill the bottle up to the 100ml mark. Snap the lid down tight. Erase any old pencil markings on the bottle lid and write the location and time collected. Place the bottle in the cooler. Collect samples at these locations, in this order:
1. (MA) marine sample: seawater where it can be reached as far to the south side of the creek as possible.
2. (BE) creek sample: below and downstream of the cement Highway 1 arch bridge.
3. (GH) drinking water/spring sample: Tap water from faucet between the two westernmost large pine trees in the gatehouse area. Let the tap run at least 3 minutes before collection.
4. (BC) creek sample: from the pool right above the confluence of Big Creek and Devils Creek, Big Creek fork.
5. (RW) spring sample: from the tap at the sink at Redwood Camp. Let the tap run at least 3 minutes before collection.
6. (BD) creek sample: short trail down to creek downstream of the Haida House, about ___' down the road from the creek crossing to the Haida house.
7. (AD) creek sample: just upstream of the Backlar Deck.
8. (KS) spring sample: from the mouth of the culvert where it spills into the Klanky Spring tub
9. (HC) spring sample: from the tap at Highlands Camp. Let it run at least 3 minutes before collection.
10. (WS) spring sample: turn on the pump, turn off the flow from the pump to the tank, take a sample from the outlet above the pump. Turn the water on between the pump and the tank, turn off the pump.
11. (WT) control: tap water from Whale Point.

After collecting the last sample, begin boiling the water for sterilization of the funnel, forceps, beaker, and pipette if it is needed. Boil the listed items for 5 minutes. Cool on a paper towel.
Cover work area with sterile paper towels. Label the petri dishes with either a marker (smooth top) or a pencil (rough top). Make a stack for fecal samples and a stack for total samples. If using less than 100ml of the sample water for the filtration, label the amounts on the lids of the dishes. Use 2, 5, 10, 20 or 50ml of creek water for the total coliform samples, according to the time of year and amount of recent rain. If you are unsure of the amount to use, bracket by doing 2 different amounts. Use 100ml water for the fecal samples. The WT sample is only done for total coliform. Empty the contents of one whole ampule into each dish.

Filtration Procedure:
1. Connect the pump to the filtering flask.
2. Place the filter funnel base on top of the flask.
3. Peel open a filter packet. Using the sterilized forceps, place the filter on top of the funnel base.
4. Place the filter funnel top securely on the base.
5. If the amount to be sampled is 10ml or less, pour a small amount of WP tap water into the bottom of the filter funnel.
6. Using either the pipette (2-10ml samples), the beaker (20-50ml samples), or the entire vial (100ml samples), pour the water into the filter funnel.
7. Turn on the pump.
8. When the water has disappeared, use a small amount of WP tap water to flush down the sides of the filter funnel.
9. Turn off the pump.
10. Break the vacuum suction in the flask by tilting the entire funnel unit, base included, to the side until air is sucked into the flask.
11. Remove the top of the funnel filter.
12. Using the tweezers/forceps, gently peel the filter from the base being careful to not tear it.
13. Open the appropriate petri dish. Still holding the filter with the tweezers, place it in the dish using a rolling motion, bottom edge first, being careful to not trap any air bubbles between the filter and the pad.
14. Repeat steps 2-13 for the next sample from the same water source.
15. Rinse the filter funnel top and base, the tweezers, and any measuring device used in WP tap water, flushing at least 3 times. For the pipette, draw water from the bottom to the top and pour it out the top, at least 4 times.
16. Repeat steps 2-15 for each set of samples.
17. Put the petri dishes into the incubators, turning each one upside down.

Incubate for 24 hours, +/- 2 hours.
Remove the plates from the incubator. Count the colonies on each paper, using the 10x dissecting scope. Use the hand clicker and the marked grid as a guide. Count every colony, even the tiny ones. Red and blue colonies on the total coliform plates are coliforms. Clear colonies on the total coliform plates should be marked down as "other." Blue and pale yellow colonies on the fecal coliform plates are fecal coliform. Peach/pink colonies on the fecal plates may be klebsiela. Gray/cream colonies are non-fecal colonies. Write the numbers down in the blue coliform logbook, and later update the computer files.