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STUDENT OBJECTIVE

Students assay water samples from various sources for total coliform content using a Standard Total Coliform Membrane Filter Procedure from Standard Methods. The demand and production of oxygen in water samples under various light and dark treatments is measured using a modified Winkler test for dissolved oxygen. Buffering capability of bedrock is demonstrated.

EQUIPMENT	AMOUNT
	(Class groups of 24 with 8)
Filter unit (Millipore Sterifil System) Hot plate Incubators, 37°C Transfer hood Dissecting microscope (for count of E. coli colonies) For instructor use only pH meter Fluorescent light Incubator-shaker MATERIALS	1/group 1/group 1/lab 1/lab 1/student
Aspirator Vacuum tubing Beaker, 3 L or 3—5 qt. kettle Beaker tongs Pipettes, sterile 5 ml Graduated cylinder, sterile 100 ml Filter (Millipore) Culture pad (Millipore)* Petri plate, 60 mm sterile plastic Wax marking pencil Bunsen burner or alcohol lamp Matches Forceps Test tubes, 13 x 100 mm sterile with caps Depression plates Syringe, 10 ml Needle, 18 gauge Pipettes, 0.2 ml in 0.01 ml Pipettes, 1 ml x .01 Erlenmeyer flask, 25 ml Bottles, foil-covered, wide-mouth, 200—250 ml, screw-cap Bottles, clear, wide-mouth, 200—250 ml, screw-cap Thermometer For instructor use only (E. coli preparation) E. coli culture Flasks, 125 ml Flasks, 500 ml Pipettes, sterile 1 ml x 0.01 ml (Dissolved oxygen preparation) Aquarium Limestone chips Granite chips	1/group 2 ft/group 1/group 1/group 1/group 4/lab 1/group 1/group 1/group 4/lab 1/lab 1 box/lab 1/group 1/group 1/group 1/group 3/group 1/group 1/group 1/group 2/lab 2/lab 4/lab     1/lab 4/lab 4—6/lab 1/lab Demonstration Demonstration

*Please refer to the Appendix for name and address of supplier.

SOLUTIONS

70% ethanol
Water samples (from three different sources)
Sterile water
0.85% sterile saline (NaCl)
MF-Endo medium (Difco)*
Starch solution
Manganous sulfate, (MnSO₄—H₂O)
Alkaline-iodide-azide (KOH-NaI-NaN₃)
Phosphoric acid (H₃PO₄)
Sodium thiosulfate, 0.025M (Hach)
0.01 M H₂SO₄

PREPARATION

Two Weeks before Lab

1. Water to be tested for dissolved oxygen can be obtained from an aquatic culture. A large aquarium (10—20 gal) should be filled three-fourths full with water. Elodea, duckweed, any leftover algae can be added. The culture should be illuminated with two gro-lamps for 12 hours daily; water should be added periodically.
2. Order chemicals and standard solutions as necessary. Order filters, culture pads, and culture dishes.

One Week before Lab

1. The commercial reagent sodium thiosulfate should be refrigerated upon receipt. Shelf life: 6—12 months in refrigerator.
2. Starch solution preparation:
   In 5—7 ml of distilled water, make an emulsion of 0.5 g starch. Add 83 ml of boiling distilled water and stir for several minutes. Let the resulting solution sit covered for eight hours or overnight. Decant the clear supernatant and stir in 0.104 g salicylic acid (preservative); store in refrigerator until needed.
3. Manganous sulfate solution preparation:
   To remove oxygen, boil 250 ml of distilled water. Cool; stir in 91 g manganous sulfate (MnSO₄—H₂O). Fill student dropper bottles and store the remaining solution in an airtight container. (Do not use a glass stoppered container.)
4. Alkaline-iodide-azide preparation:
   To 250 ml of boiled distilled water, add 175 g KOH and 33.75 g NaI. Stir until salts are dissolved. Be careful that solution does not boil due to heat of solution. In a fume hood, stir in 2.5 g NaN₃ previously dissolved in 10ml distilled water. CAUTION: Avoid breathing the NaN₃ dust. It is a respiratory poison with effects similar to cyanide poisoning. Fill student dropper bottles and store remaining solution in a plastic bottle.
5. Empty test tubes with caps should be sterilized for 15 minutes at 15 lb pressure. MF-Endo medium will be added later.
6. Sterile water preparation:
   Several 500 ml flasks should be filled two-thirds with water and sealed with foil. Autoclave for 15 minutes at 15 psi. This will be used to dilute aliquots of the water samples.
7. Nutrient broth preparation:

   2 g nutrient broth/250 ml distilled water

   Mix powder into water and heat until dissolved. Pour into four 125 ml flasks. Seal with foil and autoclave for 15 minutes.

8. Saline preparation:

24 Hours before Lab

1. Sample preparation for dissolved oxygen using the Winkler test: From the previously established lab culture of Elodea, algae, duckweed, and such, fill two foil-covered and two clear glass bottles with equal amounts of vegetation. With the bottle fully under water, fill the bottles to the brim and tighten screw cap (permit no air over water). Provide 24 hours continuous strong light for the uncovered glass containers. Place the foil-covered containers in the dark for 24 hours. Maintain the same temperature in all the containers.
2. Inoculate the sterile nutrient broth with E. coli and put on incubator-shaker at 37^oC.

8 to 16 Hours before Lab

1. Water samples to be tested for total coliform count should be collected and held in refrigerator until needed.
2. MF-Endo medium preparation:

Morning of Lab

1. In a water bath, warm MF-Endo medium until the red color returns. (MF-Endo medium is best prepared the morning of lab.)
2. Fill dropper bottles with starch solution.
3. All water samples should come to room temperature before the exercise is begun.
4. Label the water samples and suggest the approximate aliquot of liquid that should be filtered. Use the following chart as a guideline:

   Swimming pool 300 ml
   Lake 100 ml
   River 50 ml
   Pond 5—30 ml

   Sterile water should be available to add to the water samples. Whenever the membrane filter system is used, a minimum of 100 ml of solution must pass through the filter to get adequate mixing and dispersal of sample.

5. A 0.001-0.01 ml amount of the E. coli culture should be added to 100 ml of 0.85% sterile saline and filtered to obtain the known E. coli colonies. A dilution series should be performed to determine the exact amount of culture to be used.
6. After students have added media, pads, and filters to petri dishes, tape the dishes together and incubate 24 hours at 37^oC right side up. (To maintain humidity, place a pan of water at the bottom of the incubator.)
7. Add 0.01 M H₂SO₄ to 500 ml beakers half filled with limestone or granite chips at beginning of lab.

24 Hours after Lab

1. Remove the petri dishes and record the number of colonies. If time does not permit examination, invert the dishes and store at 4^oC until the next lab class. If longer than two days’ storage is required, the plates may be frozen.
2. After obtaining the coliform count, the dishes should be sterilized and discarded.

NOTES

1. If microburets are not available for the titration in the dissolved oxygen test, a good substitute is a 3 ml syringe attached to a 1 ml disposable pipette with 2 cm piece of tubing. This "buret" can be filled and held by one student while another swirls the beaker during the titration. A small, square, white paper placed under the beaker during the titration will assist the student in seeing the end point.
2. Water quality varies according to the environment and location. Although a guideline is offered for the aliquot of water sample to be filtered, prior testing of the sample is the preferred way to determine the exact amount a student should take. Ideal count of coliform colonies on a test plate is 30—50.
3. O-rings for the membrane filter system wear out quickly. It is a good idea to have several on hand.

CLASSROOM SUGGESTIONS

1. In the membrane filter apparatus, there are many parts, and assembling them can be confusing to students. A demonstration will help the students reassemble the parts quickly without contamination.
2. If available, a dissolved oxygen meter can be demonstrated. After calibration, it could be used to check the dissolved oxygen of the water samples that the students test in the lab.
3. The use of sterile technique is confusing to beginning students. Demonstrating how to make the transfers of equipment and solutions can help the student prevent contamination.
4. The concept of water quality and its impact on the environment is a relatively new area of study for the student. The exercise can be the beginning of an investigative report on local water quality.
5. Laboratory investigations can be completed in 2—2 1/2 hours.
6. Check out the links for this lab topic at http://auth.mhhe.com/biosci/genbio/dolphin/ You will find useful materials for developing your lab introduction or summary, and in some cases, you may want to tell students to connect to a particular site for further information.

ANSWERS TO CRITICAL THINKING QUESTIONS

1. Besides pathogenic bacteria, viruses and protozoa associated with human fecal contaminations, there are a variety of pathogens that are water-borne and are independent of humans. For example, the protozoa, Giardia lamblia (causing prolonged diarrhea), may be shed into waterways by beaver, cats, dogs, and sheep; Cryptospiridium (a protozoan causing acute enterocolitis) has many species of domestic and wild animals as its reservoir; Legionella pneumophila (causative agent of Legionnaire’s disease) is free living and found in cooling towers, condensers and other water sources.

SUPPLEMENTAL MATERIALS