Water Quality

While microorganisms have a very beneficial role in preserving the quality of the ecosystem, they can also serve as water contaminants that transmit disease. The microorganisms that generally cause disease are termed pathogens. Tests have been developed to determine the quality of water and certain foods. Since water borne disease is usually spread through fecal contamination of water supplies, it is more efficient to determine if fecal contamination is present that to actually look for the presence of pathogens. In the United States, coliform bacteria serve as the indicator organisms for fecal contamination. Coliforms are gram negative, aerobic or facultative anaerobic rods that are consistently present in human feces in substantial numbers. They ferment lactose to produce gas within 48 hours of inoculation of lactose broth incubated at 35°C.

Microbiologists investigating the microbiota of an environment generally find a mixture of organisms. In order to study one organism, it must first be separated from the mixture or isolated. Microbiologists use the steak plate technique as well as special media to isolate desired organisms.

A medium (plural = media) is a material which provides nutrients needed to grow microorganisms. Some media also contain dyes or chemicals that select for a desired organism. These are called selective media. Other media, called differential media, may have chemicals added that will demonstrate a metabolic property of certain microorganisms. Some media are both selective and differential.

Eosin methylene blue (EMB) agar is used to isolate some of the organisms present in the human intestine. The dyes eosin and methylene blue inhibit the growth of gram positive organisms and select for gram negative organisms. EMB agar also contains lactose. Coliforms ferment the lactose, which causes the darkening of the colonies. Some coliforms have different appearances on EMB agar. Enterobacter aerogenes colonies have a dark center with a light border and are called "fish eye" colonies, while Escherichia coli produces a dark colony with a green metallic sheen. On EMB agar, non-lactose fermenters produce pale or colorless colonies. Many intestinal pathogens are non-lactose fermenters.

The United States Environmental Protection Agency has established guidelines for evaluating the bacteriologic quality of water for drinking. The purpose of this exercise is to determine whether or not a water sample is contaminated with coliforms using the presumptive and confirmed stages of testing.

The standard method lists gas production in lactose broth as a positive presumptive test for coliforms. Students may or may not be able to determine gas production in the polystyrene tubes; all should proceed to the confirmed test.

Materials per pair (or more) of students:

• 1 empty plastic tube in plastic bag
• 1 plastic pipet
• 1 tube of lactose broth (10ml)
• 1 Eosin Methylene Blue Agar (EMB) plate
• 1 inoculating loop
• Bacti-Cinerator
• Wax marking pencils

Protocol:

1. Each pair or group takes an empty tube and collects a few milliliters of water to be tested for the presence of fecal coliforms. (Students should be encouraged to seek a site that offers the potential for fecal contamination. Collection might be best assigned over a weekend to allow time for investigation of potential sites. Different sites could also be assigned to prevent excessive duplication.)
2. Use the plastic dropper pipet to inoculate the tube of lactose broth with 1ml of the water sample. Label the tube with the group identification and source of inoculum
3. Firmly press down on the tube lid to be sure that it is tightly sealed. Incubate the inoculated tube at 35*C for 48 hours.

Follow up:

1. Attempt to determine gas production. If gas has been produced, the tube lid will be popped up by the pressure. Record any results.
2. Streak a sample from the lactose broth onto an EMB lactose plate using a sterile inoculating loop. (Instructions for streaking a plate for isolation are provided on the next page.)
3. Label the bottom of the plate with group identification and source of inoculum. Incubate the inverted plate at 35*C for 24 hours or until the next class meeting. (These plates may take longer than 24 hrs. @30*C to show typical colonies.)
4. Examine plate. Typical coliform colonies have a dark center or a metallic sheen. Record observations and indicate whether the water sample was confirmed to contain coliforms. (In the United States, Escherichia coli is the accepted indicator organism.)

Presumptive test:

Appearance:___________________ Positive or negative (circle one)

Confirmed test:

Appearance:____________________ Positive or negative(circle one)

Would you drink this water? ________________ Why or why not?

The Quadrant Streak Plate Technique

1. Using a flame sterilized inoculation loop, spread (streak) the culture over a small area near the edge of the plate (A) using a continuous motion.
2. Flame sterilize the loop and allow it to cool.
3. Turn the plate and spread the bacteria from the end of area A across area B. (You can see the streak marks of the loop in area A.)
4. Flame sterilize the loop and allow it to cool.
5. Turn the plate in the same direction and spread the bacteria from the end of area B across area C.
6. Flame sterilize the loop and allow it to cool.
7. Turn the plate in the same direction and spread the bacteria from the edge of area C across the rest of the plate (area D).
8. Flame sterilize the loop before setting it down.