	ASSAY FOR COLIFORM CONTAMINATION IN AMBIENT WATER ©David B. Fankhauser, Ph.D., Professor of Biology and Chemistry University of Cincinnati Clermont College, Batavia OH 45103
Sewage Treatment Plant Outfall	This page has been accessed times since 1 August 2001. rvsd 27 July 1993, 26 July 1994, 31 July 1995, 19 July 98, 2 Aug 00, 15 July 01	Colonies from "treated" sewage

Man has long recognized the dangers of drinking water which is contaminated with fecal waste. Major diseases which are spread in this manner include cholera, salmonellosis, dysentery, shigellosis, polio and many others. Mammalian fecal wastes carry large numbers of gram negative rod-shaped bacteria which are capable of using lactose (milk sugar) as a carbon source. Bacteria with these properties are collectively termed coliform bacteria. The presence of coliform bacteria is widely used as an indicator of potential fecal contamination. Escherichia coli is the most famous member of the coliform group.

A selective and differential medium which can be used to enumerate the number of coliform bacteria is Levine EMB Agar. It contains nutrients, 1% lactose, agar and two dyes, E osin and Methylene Blue. These dyes serve two purposes: first, as a selective medium, they inhibit the growth of gram positive bacteria, and second, as a differential medium, they cause colonies which ferment the lactose ("lac+") to turn purple (E. coli typically has a green sheen ) while the "lac" colonies will be pink or uncolored.

A second medium which may be used to enumerate coliform is MacConkey's Agar which contains 1% Lactose, 0.15% Bile Salts, and the dyes Neutral Red and Crystal Violet. These dyes act much the same as the dyes in EMB agar, but lac + appear red, while lac - appear white .

By spreading a known quantity of aqueous sample (often 0.1 mL) on one of these media and incubating until colonies have formed, the number coliform will be equal to the number of lac + colonies, and indicates the degree of potential fecal contamination. A sub set of coliform, termed fecal coliform, are defined as those coliform which grow at the elevated temperature of 44.5 C instead of the usual 35 C.

RELATED PROTOCOLS:
    Commonly Used Media
    Sterile Delivery of Liquids by Pipet
    Plate Spreading Technique

EQUIPMENT LIST:

    sterile screw-capped culture tubes
    Levine-EMB and/or MacConkey Agar plates
    sterile pipets, 0.1, 0.2 and/or 1.0 mL
    spreader, turntable
    flame
    35C incubator

COLLECT SAMPLE: Collect sample as close to plating time as possible, or keep refrigerated until plating. Use sterile screw-capped culture tubes (10 or 20 mL), fill 2/3 full from midstream of a flowing stream . Do not contaminate with sediment from stream bottom. Cap immediately . Write in pencil on the tube's frosted area: location of collection point, date and your initials. Samples from above and below sewage treatment plant outfalls can be interesting, demonstrating whether raw or improperly treated sewage is contaminating the river.
PLATE OUT ON THE DIFFERENTIAL AGARS: Label plate with seat number, initials, source and aliquot volume. Using sterile technique as outlined in Plate Spreading Technique , spread 0.1 mL of sample on EMB and/or MacConkey agar. Plate 0.2 mL if the sample is thought to be unpolluted. If you believe the sample to be highly polluted, pipet 0.1 mL of sterile dH2O on the plate, then deliver 0.05, 0.01 or even 0.005 mL to the plate and spread. Incubate at 35^o C for 48 hrs.
SCORE THE PLATES: Count and record the total colonies and also the number of coliform colonies/plate. Those which are lactose fermenting are purple. Calculate the number of these categories/100 ml of sample, enter into your notebook.
ENTER INTO THE CLASS DATA SHEET. Enter your data in the spreadsheet on the computer. After class data have been collected, mount the table in your note book.

RESULTS:

We have found some interesting (and disturbing) concentrations of bacteria, and coliform in particular in samples over the past few years.

ICE TEA:
We demonstrated in 1994 that restaurant ice tea often contained extremely high numbers of coliform. Here are some plates of restaurant ice tea (0.1 mL each) one from July 2001 , and another from July 2000 . These contain so many bacteria that they could not be accurately counted, but we estimate that they contain between 500,000 and 1 million bacteria per 100 mL, most of which are coliform. The standard for drinking water is 5 coliform per 100 mL, and for recreational waters, 5,000/100 mL. It would be illegal to swim in these samples of ice tea.

STREAMS AND RIVERS:
Here is a picture of an EMB lactose plate on which was plated out only 0.05 mL of "treated" sewage from the Nine Mile Sewage Treatment plant outfall . Here is a MaConkey Agar plate containing 0.2 mL of Ohio River water taken on the same day. Notice that the bacterial count is MUCH lower than that of ice tea...