Metabolic activity in most live organisms produces known waste products. The chemical composition of these waste products can be qualitatively determined through the use of specific indicators. One of these gaseous by-products of metabolism produces carbonic acid when dissolved in a water solution. This same substance can cause a precipitate to form if it is bubbled through limewater. The gas that produces these reactions is CO₂ and it will be released by the live organisms and will cause the indicators to reveal its presence.

Methods and Materials

1. Set up 7 small test tubes in a rack and add 5 drops of phenol red solution to each. Tilt each test tube slightly and gently slide a small brass screw to the bottom, point first. Now, add the following materials to the tubes:

Tube 1. Nothing

Tube 2. A small, rolled piece of paper towel moistened with yeast-sugar solution

Tube 3. A small, rolled piece of paper towel moistened with boiled yeast-sugar solution

Tube 4. 5-10 small dry seeds (radish, sunflower, or melon)

Tube 5. Same number of sprouted seeds

Tube 6. A small live insect

Tube 7. A dead insect of the same type

Stopper the tubes only after all seven have been prepared. Watch from changes in the phenol red and record the approximate time required for the change to take place.

2. In the following series of tests the characteristics of two indicators will be discovered that will help in the interpretation of the results obtained in step 1.Set up 6 standard-sized test tubes in a rack an label them 8, 9, 10, 11, 12, 13.

In tubes 8, 9, and 10 place 10-12 drops of phenol red. Fill tubes 11, 12, and 13 about one-quarter full of limewater. Record the changes that occur in the indicators as the following substances are added.

Tube 8.1-5 drops of a dilute acid ( hydrochloric, acetic, sulfuric, etc.)

Tube 9. 5-10 drops of carbonated water (water with carbon dioxide added)

Tube 10. Breathe through a straw for 10 to 30 seconds into the phenol red solution

Tube 11. 15-20 drops of an acid

Tube 12. 5-10 drops of carbonated water

Tube 13. Breathe through a straw into the limewater for 10 to 30 seconds

The hypothesis was correct in supposing that CO₂ would be produced as a by-product of living organisms. The indicator phenol red was used to identify the acid nature of CO₂ in water and the limewater identified the gas CO₂ by producing a precipitate of calcium carbonate. The source of the CO₂ in tubes 2, 5, 6, 10 and 13 were living organisms.

The brass screw in each of the tubes is there to keep the biological material from being immersed in the indicator. Tubes 8 through 13 define the nature of the indicators phenol red and limewater. Phenol red is an acid/base indicator that changes to a yellow color in the presence of an acid and red in the presence of a base. Limewater is a solution of calcium hydroxide Ca(OH)₂ that will form a precipitate of calcium carbonate (CaCO₃) when it is mixed with CO₂.

Tubes 1, 3, 4 and 7 are controls demonstrating that non-metabolizing organisms do not release any CO₂ by-products. The phenol red did not react to their presence in the tubes.

Having identified the type of reactions that phenol red and limewater produce, the results in tubes 1 through 7 are explained. Tubes 2, 5 and 6 contain live yeast, live sprouted seeds and a live insect. These organisms use carbon-based molecules as respiratory molecules and produce CO₂ as a waste product. The phenol red indicator shows the presence of an acid and the limewater test demonstrates that CO₂ is the by-product.