LAB: Fermentation

BIOLOGY LAB
FERMENTATION

INTRODUCTION:

Have you ever seen what happens when bread rises or when apple cider "goes hard"? These actions are the result of fermentation, the process by which an organism breaks down glucose in the absence of oxygen. During this process, 2 molecules of adenosine triphosphate (ATP) are made from a glucose molecule (instead of 36 made in the presence of oxygen), and a variety of by-products are produced. Fermentation is important in making many foods, and it produces the alcohol found in beer and wine. In this lab, you will create conditions in which cabbage will become fermented, in the presence of certain bacteria, and will turn into sauerkraut.

PRE-LAB PREPARATION:

You should understand how glucose is broken down in the absence of oxygen. When no oxygen is present, nicotinamide adenine dinucleotide (NAD) donates the 2 hydrogen (H) atoms it picks up in glycolysis to pyruvic acid, the end product of glycolysis. The fate of pyruvic acid depends on the organism. Humans have an enzyme that combines pyruvic acid and 2H to form lactic acid. Some bacteria and yeast form alcohol and carbon dioxide. Others form acetic acid (vinegar) and carbon dioxide. Still others form a variety of chemicals depending on enzyme specificity.

(A) Pyruvic acid (CH₃COCOOH + 2H) ------> lactic acid (CH₃CHOHCOOH)

(B) Pyruvic acid (CH₃COCOOH + 2H) ------> ethyl alcohol (CH₃CH₂OH + CO₂)

MATERIALS:

Test tube
Test tube rack
Shredded cabbage
*2.5% NaCl (salt) solution
pH paper
**Methylene blue
Toothpicks
Balloon 7 inch round (to fit over test tube)
Microscope
Microscope slides
Coverslips

* To prepare 100 ml of a 2.5% salt solution, dissolve 2.5 grams of NaCl in 97.5 ml of water.
**This is a stain for tissue slides. You can use the blue or green tropical fish medicine as a substitute. Sometimes I even prefer it.

PROCEDURE:

1. Pack the test tube with shredded cabbage.

2. Cover with 2.5% NaCl (salt) solution until the test tube is full.

3. Cover the test tube with the balloon.Try to leave as little air as possible in the balloon. For the next 2 weeks, make sure the test tube remains full. Replenish with more NaCl solution as needed.

For day 0, record the color and texture of the cabbage, the odor, whither any gasses are produced (check for bubbles), and the pH of the solution (use pH paper). Record your data in the DATA TABLE of your report sheet.

4. Check the condition of the cabbage cells. Make a wet mount of a few cells peeled from the lower epidermis. Stain these cells using methylene blue. Examine under the microscope using middle and high power. Look carefully to see if the cell membrane has separated from the cell wall. Record in the DATA TABLE.

5. To check for the presence of bacteria, make a wet mount by gently scraping the bottom of the cabbage with a toothpick and putting it on a slide with a drop of brine. Stain with methylene blue and examine under high power. Record in the DATA TABLE.

6. Make daily observations and recordings for the next 10 days. Remember to replenish with more NaCL solution as needed. Record all data in the DATA TABLE.

7. On day 10, in addition to the other data, check the condition of the cabbage cells and the presence of bacteria as you did in steps 4 and 5. Record your observations and make a sketch of any bacteria you see.

8. Make a graph showing the pH from day 0 to day 10.

YourDATA TABLE should contain the following for days 1-10: date, color of cabbage, texture of cabbage, odor, any gasses produced, and pH. Also for days 1 and 10, condition of cabbage cells, presence of bacteria in brine, and description of bacteria in brine.

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