©David B. Fankhauser, Ph.D.,
Professor of Biology and Chemistry
University of Cincinnati Clermont College,
Batavia OH 45103
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
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.
Commonly Used Media
Sterile Delivery of Liquids by Pipet
Plate Spreading Technique
Levine-EMB and/or MacConkey Agar plates
sterile pipets, 0.1, 0.2 and/or 1.0 mL
We have found some interesting (and disturbing) concentrations of bacteria, and coliform in particular in samples over the past few years.
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...