..... There are just a few classic tests for the pollution of surface and ground waters. .. One of them is for FECAL CONTAMINATION, which is a matter of public health. .. The second is concerned with the dumping of so much biodegradable dissolved wastes into the water that it overloads the system such that the ravenous bacterial decomposers deplete the waters of all dissolved oxygen ("D.O.") in the course of their degrading the wastes. ..So many aquatic creatures depend upon the dissolved oxygen that if it is depleted massive kills of fish and shellfish quickly ensues with consequent great loss of ecologic and commercial value.

..... Lab #12 of the A.P. Biology Laboratory Manual dealt with the net production of dissolved oxygen under light conditions. ..This extension is an exercise using the same materials but that will show the net reduction of dissolved oxygen using
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• A control of distilled water
• Several samples taken from local ponds, streams or rivers
• A series of dilutions containing known amounts of readily biodegradable organic compounds

..... The series of known dilutions of organic material will give you an idea of just how much or how little organic matter is required to allow the resident microbes to deplete the dissolved oxygen to zero.
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1. "SERIAL DILUTIONS:" ..Make up some 1% (w/v) nutrient broth or beef boullion (dissolve 1 gram of the dry material into about 50 ml of warm water; once dissolved add water up to 100 ml)
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   1. Pour 10 ml of this 1% into another 100 ml graduated cylinder, and fill that one to 100 ml with distilled water. ..You now have a 0.1% solution.
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   2. Pour 10 ml of this 1% into another 100 ml graduated cylinder, and fill that one to 100 ml with distilled water. ..You now have a 0.1% solution.
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   3. Pour 10 ml of this 0.1% into another 100 ml graduated cylinder, and fill that one to 100 ml with distilled water. ..You now have a 0.01% solution.
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   4. Pour 10 ml of this 0.01% into another 100 ml graduated cylinder, and fill that one to 100 ml with distilled water. .. You now have a 0.001% solution.
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   5. Pour 10 ml of this 0.001% into another 100 ml graduated cylinder, and fill that one to 100 ml with distilled water. .. You now have a 0.0001% solution.
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2. You are now ready to fill your oxygen bottles. ..Before you do so oxygenate each sample fully by sloshing some of it violently in a large bottle or flask. ..Then fill the respective bottles.
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   • You have 6 bottles for the 10-fold serial dilutions of the nutrient (don't forget that your "control" is the sixth bottle! .. It is 0.0000%).
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   • And you have bottles filled from various surface waters - ponds, streams, marshes, rivers and lakes.
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3. Before capping, add one drop of pond water to each oxygen bottle to inoculate it with a wide assortment of bacteria. ..(This is especially important to do with the bottles containing the serial dilutions of the nutrient.)
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4. Now cap the bottles such that no air bubble is inside.
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5. Set the bottles in the DARK so that photosynthesizers will not add oxygen.
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6. After three days, determine the amount of dissolved oxygen remaining.
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7. Since only bright students are doing this exercise, they should be able to figure out how to determine the percentage of "D.O." that has been depleted. ..That is the B.O.D.!