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Fecal Coliform & E. coli

Synopsis of Methods (CCEE/EERL)

 Media

NA-MUG Agar (for E Coli) (makes approx. 80 plates)

- Add 9.24g of NA-MUG to 400mL sterilized nanopure water

- Heat to boiling, while stirring, until dissolved.

- While agar is still hot, place in autoclave (250 degrees F, 18 psi for 30 minutes)

- Allow to cool to 45-50 degrees C

- Using a sterile pipet, add 5 mL to each plate and swirl to coat bottom

- Once cool, close and invert petri dishes. Store at 4 degrees C until ready for use. May be stored for 2 weeks

 

M-FC Broth (for total fecal coliform) (makes approx. 100 plates)

- 1% Rosalic Acid solution

- Dissolve 1g of rosalic acid in 100 mL of 0.2N NaOH.

- Heat rosalic acid solution in hot water bath until dissolved (do NOT boil)

- Store at 4 degrees C in the dark. Discard after 2 weeks or if color changes from dark red to muddy brown

- M-FC Broth w/ 1% Rosalic Acid (for total fecal coliform, with interfering organisms)

- Add 7.4g M-FC Broth to approximately 150mL sterilized nanopure water

- Add 2 mL 1% rosalic acid solution

- Add sterilized nanopure water to bring volume to 200mL

- Heat to near boiling, while stirring, until dissolved.

- Remove from heat and allow to cool to 45-50 degrees C

- Using a sterile pipet, add approx. 2 mL to each padded plate, enough to moisten the whole pad. Pour off excess.

- Close and invert petri dishes. Store at 4 degrees C until ready for use. May be stored up to 96 hours.

Filtration of samples

Using sterile forceps (dip in ethanol and burn off in burner flame), place the membrane filter, grid-side up, on the porous plate of the filter base. Attach the funnel to the filter unit, taking care not to damage or dislodge the filter, so that the filter is now fitted between the funnel and the base. Shake the sample container vigorously (about 25 times).

Prepare at least 3 plates for each sample, with different volumes, using the record of previous dilutions and colony counts as guidance to choose what volumes to use. The normally accepted range for colonies per plate is 20-60. If a volume less than 10 mL is used, first add approx. 10 mL sterilized nanopure water to the funnel to help the sample volume disperse evenly onto the filter. For sample volumes of 10 mL or less, use a sterilize pipet to measure the sample. For volumes of 20 mL or more, measure the sample using a sterile graduated cylinder. Mark volumes used on the bottom of the plate and on the dilution record.

Turn on the vacuum to filter the sample. Leave the vacuum on and rinse down the walls of the funnel 3 times with sterile nanopure water. Remove the funnel from the base of the filter unit. Turn off the vacuum. Holding the filter at its edge with the sterilized forceps (dipped in ethanol and burned off), lift and place the filter onto the absorbent pad of a coliform (M-FC) plate, pressing down the edges to attach it securely to the pad. Close and invert the petri dish, place into Whirl-Pak bag and incubate in water bath at 44.5 degrees C for 24 hours.

Counting Fecal Coliform Bacteria

Count only colonies that contain any amount of blue or greenish blue color. Colonies that appear grey, buff, or colorless are non-fecal and should not be counted. Count “spreaders” as one colony. Mark location of fecal colonies on the lid of the dish. If the count is over 200, record as TNTC (“too numerous to count”) and dispose of the plate in a biohazard bag. If more than one half the count is taken up by “spreaders,” record as NC (“not counted”) and dispose of the plate in a biohazard bag. If there are no colonies, record as “0” and dispose of the plate in a biohazard bag.

Transferring to E. coli plates

Mark E. coli (NA-MUG) plates with sample IDs and sample volumes. Using sterilized forceps, transfer the filter from the coliform plate to the corresponding E. coli plate, making sure there are no air pockets between the filter and the agar. Discard the lid of the E. coli plate, and use the lid from the coliform plate; line up the markings on the lid with the colonies on the filter. Dispose of coliform plates (and unused E. coli plate lids) in a biohazard bag. Once all filters are transferred, place plates in a 35 degree C oven for 4 hours.

Counting E. coli

Remove plates from the oven. Observe each plate under the ultraviolet light. For samples where TNTC, NC, or 0 was recorded on the coliform record, record NC on the E. coli record. Count colonies that were previously marked as coliform and that now fluoresce under the UV light. Do NOT count colonies that fluoresce but were not marked. Record counts.

Disposal

Place all used plates in a biohazard bag and autoclave at 250 degrees F, 18 psi for 30 minutes. Dispose of autoclaved bag.

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