PURPOSE
How can we check for the presence of contaminating bacteria in water supplies?  In this lab you will culture coliform bacteria found in water samples, count the colonies produced, and compare that data to permitted levels.

INTRODUCTION
    To insure that the water we use is acceptably free of contaminants, a test for coliform bacteria is commonly done. Escherichia coli, (E. coli), is a common inhabitant of the large intestines of humans and other vertebrates.  If tests show that E. coli are present in amounts larger than trace quantities, then the water is being contaminated with fecal wastes.

EQUIPMENT
    Millipore filtration units

SUPPLIES (per group)
50 mL  sample of water to be tested  (it may need to be diluted if you think that it has a high concentration of organisms or particulate matter), 1 Millipore filter petri dish, 2 mL  endobroth growth media, 1 medium absorbing pad, 1 Millipore grid filter paper, 600 mL  beaker of ethanol, 10 mL  pipette, 10 mL  pipette pump

PROCEDURE

Day One
1. Assemble the Millipore Filtration Apparatus as shown in Fig. 1 on page 12.

2. Sterilize the tip of a pair of forceps in alcohol, then use the forceps to place one grid filter paper on the filter support, grid side up.

3. Gently screw the funnel to the filter support.  Attach the funnel & filter to the receiving flask.   Be sure the syringe pump mechanism is securely attached to the filter.  Add 50 mL of water sample to be tested into the funnel (the funnel is graduated).  Cap the funnel with the cover making sure three of the openings are covered with gum rubber caps and the fourth by the air filter assembly if needed.

4. Draw the water sample into the receiver by pumping the plunger until the water sample has been completely drawn through the grid printed filter.

5. Now prepare your petri dish by sterilizing the forceps again and using them to place a sterile white absorbent pad in the bottom half of the petri dish, the half that fits inside the other.  Place your team's identification on the outside surface of this half of the petri dish.

6. Pipette 2 mL of the endobroth media onto the absorbent pad. (Caution: The stain in the media could be permanent in clothing.)

7. Carefully unscrew the funnel.  Re-sterilize the forceps and use them to move the grid filter paper from the filter support base and place on the endobroth pad in the petri dish that you have just prepared.  Be sure that the grid printed surface is on top. Immediately put the top back onto the dish and invert, so that your identification is on top. Incubate at 37 C for 24 hours or at room temperature for 48 hours.

8. Unscrew the receiver flask from the filter support and funnel assembly and pour the water down the sink.  Return the apparatus so that the next team may start their filtering.

9. If instructed by your teacher, sterilize the filtration system in alcohol as follows:  submerge the funnel and filter support into a beaker of 95% alcohol (ETHANOL).  The receiver flask does not need to be dipped in alcohol.  Wipe alcohol off with a paper towel.

10. Wash hands thoroughly with soap and water.

Day Two
11. When your petri dish is removed from the incubator or has set for 48 hours, view the bacterial colonies.  You may want to wipe the moisture from the top and reassemble the dish. If there are any coliform bacteria in your sample, they will appear as shiny colonies with metallic appearing surfaces.  Non-coliform bacterial colonies will appear purple, without metallic appearing surfaces.  If available, you may want to view your petri dish under a stereoscope.

12. Count the number of coliform and non-coliform colonies on your plate and record the information.  Collect data for all water samples.

The acceptable amount of coliform bacteria in the water depends on where the water is and what the water is to be used for.  The following table shows acceptable limits.
 

1. Describe, not just name, the different water sample sources used by class members.  Variables that you might consider in your descriptions: running, standing, closed, open, filter, treated, use, oxygen content.

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2. It is possible to test for all coliform, mammalian coliform, or for specifically human coliform.  Which test would be the most

practical for recreational water? _____________________________________________________________________

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3. Which test would be the most reasonable for drinking water?

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