Drinking_Water

	COLIFORM IN DRINKING WATER ©David B. Fankhauser, Ph.D., Professor of Biology and Chemistry University of Cincinnati Clermont College Batavia OH 45103
Membrane Filtration	This page has been accessed times since 13 August 2001.	Applying membrane to plate

7/20/89, rvsd 6 August 1993, 23 July '95, 22 July '97, 19 July 98, 30 June 99, 17 July 00, 9 Aug 00

Modified from: Standard Methods for the Examination of Water and Wastewater, 14th Ed, (1975). pages 928-935.

Students can test the drinking water from their homes, school, etc. One student in 2001 tested her tap water before and after filtration through a water purification device. The image shows two plates, the one on the left is tap water, the plate on the right is water filtered through the filter. Clearly the filter is contributing bacterial load to the water. These devices must be regularly changed and maintained in order to prevent such contamination.

EQUIPMENT AND SUPPLIES:	millipore filtering apparatus:
Sterile 250 mL capped bottles (1/student) sterile 47 mm petri dishes (1 per student) sterile 47 mm memb. filters, 0.45 µm pores sterile 47 mm millipore pads vacuum pump 3 vacuum hoses joined with "T" joint 2 strong hose clamps 1000 mL side arm filter flask m-Endo Broth MF powder sterilized repipet in 250 mL bottle	sintered glass platform in #8 stopper glass cylinder, 300 mL capacity 800 mL beaker with 400 mL EtOH 150 mL beaker with 100 mL EtOH triangle-tipped Tongs forceps with bent tipped blade Bunsen burner protective eye wear

MAKE THESE TWO ILLUSTRATIONS WITH LISTED FEATURES LABELED:

Vacuum Filtering Apparatus:	Plate Ready for Incubation:
vacuum pump on/off power switch main vacuum line main vacuum line clamp T joint relief valve clamp filter flask #8 stopper screen platform membrane filter glass cylinder, 300 mL cylinder clamp	(exploded view) 50 mm petri dish top 47 mm membrane filter 47 mm pad with 2 mL m-Endo MF 50 mm petri dish bottom

I. COLLECTION OF WATER (Collect the same day as performance of assay):

Determine the precise name of your water district, record it in your notebook.

Run tap water until it is cold (to clear out pipes, at least a minute or so).

Fill sterile 250 mL capped bottle with water, rinse several times, finally fill to neck, cap securely, maintaining sterility.

II. PREPARATION OF 100 mL of MEDIUM:

For up to 20 determinations, weight out 2.4 g m-Endo Broth MF powder into 150 mL beaker.
Add 49 mL dH₂0 and 1 mL EtOH.
Bring to boil, remove from heat immediately.

Using sterile technique, pour into sterilized repipet vessel. Securely screw down repipet to container. Clamp to ring stand for stability.

III. PREPARATION OF PAD:

Label 47 mm top of sterile petri dish with initials, seat number, date & source.
Flame off EtOH from bent blade-tipped forceps, pick up sterile pad, place in bottom of sterile petri dish, replace cover.

Repipet 2.0 mL m-Endo Broth sterilely onto pad, replace lid, keep bottom down.

IV. SET UP PLATE: Note: Wear safety goggles, tie back hair, keep flammable materials away from flame.

PRELIMINARY: Check for adequate head space in the filter flask to contain the water you are about to filter. If inadequate, empty out the flask into the sink, and replace the support platform. If necessary, re-sterilize surface of support platform with EtOH, turn on vacuum briefly to dry, then tightly clamp the main vacuum line.

SET UP MEMBRANE FILTER: Flame off EtOH from blade forceps, pick up sterile membrane filter, center it on screen platform. (Discard blue separating disc.) Open main vacuum line to hold membrane in place (turn on pump if not on yet).

THE DANGEROUS STEP: STERILIZING AND FLAMING THE CYLINDER

STERILIZE THE CYLINDER (Dangerous step): Ensure that all surfaces of the glass cylinder are immersed in 95% EtOH, pick up with fingers (touch outside of the cylinder only), invert and allow excess EtOH to drip into 800 mL beaker, touching off the last drops on a paper towel. Grasp cylinder upside down with triangle-tipped tongs (Ekco, for instance), and CAREFULLY flame off the EtOH away from EtOH beakers and other students). It will flame up fairly high, but should burn off in a few seconds. Pass quickly through the flame once more to ensure that all of the EtOH has been removed.

SET UP FILTER APPARATUS: Grasp the outside of the cylinder with your fingers (it should not be too hot if you touched off the EtOH before flaming in step 3) and place it over the membrane filter and hold it centered in place on the support platform. Clamp in place with spring clamp, vacuum still on.

FILTERING THE WATER AND APPLYING MEMBRANE TO PREPARED PAD:

FILTER WATER THROUGH: Pour your 250 mL water sample into the cylinder, monitoring that it is not leaking at the clamped joint. When all of the water has been drawn through, unclamp and remove the cylinder, place it carefully back in the EtOH.

RELEASE VACUUM ON MEMBRANE FILTER: Clamp the main vacuum line shut and open the relief valve to release the vacuum in the flask. With sterile, EtOH-free blade forceps, gently lift the edge of the membrane filter and remove from the screen platform. (Caution: the membrane filter is brittle.)

TRANSFER MEMBRANE FILTER TO PREPARED PAD, avoiding bubbles by lowering from one side first, centering it on the pad. (Hint: Rest it on far edge of petri dish, slowly pull it across the edge down toward you until it drops down onto the pad.)

INCUBATE the plate without inverting (pad-side down) at 35C for 24 hours.
COUNT THE COLONIES: Record the total number of colonies, and the number of coliform (red colonies). Divide by 2.5 to yield the number of bacteria per 100 mL. According to national health standards for drinking water, the number of coliform/100 mL should not exceed 5.
Enter your data in this sequence and format into your notebook, and then into the spreadsheet for the class table in the computer

Desk No. Initials Source in detail (including water district) Coliform/100 mL

COLIFORM IN DRINKING WATER