Down the Drain

Charlotte Freeman
1991 Woodrow Wilson Biology Institute


  • To know what problems might occur if oxygen decreases in a body of water.
  • To model what happens in a situation.where decomposition is occurring.
  • To obtain, organize, graph, and interpret information.
  • To interpret results in terms of variables.


If water supplies contain materials which rot or decompose, oxygen is used. Decay and decomposition require vast supplies of oxygen. The use of oxygen for decay of wastes may be a life or death occurrence for organisms such as protozoa or fish. They need oxygen to live.

In this investigation, methylene blue indicator is used to follow the depletion of oxygen in a water supply.

methylene blue (oxidized)
------> methylene blue (reduced)

When oxygen is present and methylene blue is in its oxidized state, it is blue in color. When sufficient oxygen is no longer available, methylene blue becomes reduced it and is colorless. This redox reaction enables the investigator to determine when oxygen decreases below a critical concentration. Yeast is a decomposer in this lab. Milk is the "waste" which decays.

Part I. Procedure

Materials needed per team:

  • milk or cream
  • 0.01% methylene blue in dropper bottle
  • distilled water
  • yeast suspension
  • 12 test tubes
  • stop watch
  • 3-5 ml pipettes or 10 ml graduate cylinders
  • test tube rack

Step 1.

Pipette 5 ml of yeast suspension into each of 6 test tubes. (Be sure to swirl or shake the suspension before drawing yeast into tube. This will help to keep yeast evenly suspended)

Step 2.

Obtain and label six test tubes. Prepare 5 ml of milk/water solutions at the following concentrations and place in the corresponding tube: (The first two amounts have been done for you. You calculate the remaining ones.)

concentration milk (ml) H2O(ml)




Step 3

Using the pipette, add 2 ml of methylene blue into each of the milk test tubes.

Step 4.

Assign one team member to be the timer. Another should make the actual transfer of yeast to the milk solution. The timer should signal the add point by a countdown (i.e.-5-4-3-2-1-add). at the time intervals indicated in the table below. Keep the stopwatch going and record the time at which each tube shows a color change. Calculate the elapsed time for each reaction. (Subtract starting time from end time).

Time for decolorization
milk conc (%) Start
End Elapsed time






Step 5.

Plot the time on the Y axis and concentration of milk on the X axis. Interpret the graph. What is the relationship between the concentration of milk and the rate of yeast oxygen consumption?

Part II. Further Analysis

Good experimental design is necessary to insure that an investigator is testing the intended variable. The following tests were performed as they are stated. all other variables were held constant.

Oxygen Depletion Tests
Expt # Concentration of
Methylene Blue
of Yeast
of Milk
1.1 g/L2 g/40 ml100%
2.1 g/L4 g/40 ml100%
3.2 g/L2 g/40 ml100%
4.1 g/L2 g/40 ml50%
5.2 g/L4 g/40 ml50%

  1. Which of the above tests would be needed to tell the scientist the effects of waste concentration on oxygen use?
  2. Why would experiments 1 and 5 not answer this question?
  3. Which two experiments would show the effects decomposer concentrations have upon depletion of oxygen supplies?
  4. Which experiments would show the relationship between methylene blue concentration and the time needed for decolorization?
  5. Would you predict a longer or shorter time for decolorization with greater concentrations of methylene blue? Why?
  6. If a scientist performed experiments 4 and 5, what conclusions could he make? Why?

Teacher and Preparator's Guide


This lab, as written, is directed toward novice students unfamiliar with this type of data collection. Directions for more advanced students should be modified to contain less explicit steps and allow them more latitude for determining the individual procedures.

Relate this exercise to water pollution, waste water treatment, eutrophication, or other oxygen consuming problems associated with human activities.

Preparation of solutions:

  • yeast suspension: 1 package of dry yeast added to 500 ml of distilled water
  • methylene blue: .01 gm/ 100 ml
  • milk: Purchase whole milk, cream, and 1 or 2% milk. Different groups should study different kinds of milk.

Adapted from a laboratory exercise by Katherine Eidson at Woodrow Wilson Institute, Summer 1991.

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