Genome Study

Nucleic Acids
Bio Informatics
Genetic Disorders

Lab Shedule
Lab 01
Lab 02
Lab 03
Lab 04
Lab 05
Lab 06
Lab 07

Cell cycle game

BIO 196 Official Website

Energetics, Fermentation, and Respiration Results

Exercise A - Part II

Under anaerobic conditions, yeast cells break down sugars, releasing carbon dioxide gas. Evidence that fermentation is taking place in a yeast culture can be provided by bubbling the gas into an indicator solution.

There are two flasks on demonstration: one contains a sugar and yeast solution and the other contains a 10% sucrose solution. Each flask has a bent U-tube from the flask into the cylinder filled with a bromothymol blue indicator solution. If carbon dioxide passes from the U-tube into the cylinder, the solution in the cylinder will turn yellow. In our case, since the reaction occurred in the blue indicator solution, the resulting color was green (yellow + blue).

Close up of Yeast + Suger and the bromothymol blue:

Close up of the 10% sucrose solution and bromothymol blue:

It's fairly easy to tell from these photos that the yeast + sugar produced carbon dioxide and changed the color of the indicator while nothing occurred in the 10% sucrose solution.

Exercise A - Part III

In this experiment, the class will study the role of yeasts as well as the food source requirements for fermentation.

During the process of glycolysis and fermentation, yeasts use sugars, but not all sugars are used at the same rate. This part of the lab helped demonstrate how four alterations to the yeast sample can produce different rates of carbon dioxide production. Remember that one sample was given glucose, another was given sucrose, one was boiled before adding sugar, and a fourth was given no sugar at all. The results can be seen in the following graph:

This demonstrates that yeast can utilize both glucose and sucrose, although it digests the latter at a slower rate. Boiled yeast doesn't produce any carbon dioxide because the organisms were killed in the boiling water. Likewise, there is no gas produced by just yeast and water; they have no source of energy (sugar).

Exercise A - Part IV

Some yeasts are more metabolically active than others and can produce more alcohol. Lugol's solution can be used to tset for metabolic activity in yeast. In this experiment, you will be looking at the fermentation reaction .

Unfortunately, there is no photo for this section of the lab but a positive test would show a smalll amount of yellow percipitate at the bottom of the test tube. This should be expected in the clear (or semi-clear) layer taken from the top of the yeast culture. Ethanol was present in this layer as a product of yeast fermentation.

Exercise B

In this exercise, you will examine one of the reactions of the Krebs cycle - the conversion of succinate to fumarate , catalyzed by the enzyme succinic dehydrogenase, using the coenzyme FAD (flavin adenine dinucleotide) .

During this section of the lab, we studied the reduction of DPIP in place of FAD as an indicator that the reaction was occurring. DPIP is originally a light blue color and as it accepts hydrogen from succinate it loses its color. Following this logic, we should see the absorbance of the solution become reduced as the reaction proceeds. If the absorbance does not decrease, we can judge that the DPIP is not being reduced and the reaction is not taking place.

Table 11B-1 in your lab book lists the ingredients of each tube. Every 2 minutes, over a timespan of 16 minutes, each tube's absorbance was measured using the spectrophotometer. The results should look similar to the following:

These results can be explained by closely examining the contents of each tube:

Tube Number	Description
1	Contained all the necessary components. Hydrogen electrons that would have been used in the synthesis of fumarate were instead used to decolorize DPIP.
2	Lacked substrate. No hydrogen electrons were produced, therefore DPIP was not decolorized.
3	Enzymes were denatured by boiling. No hydrogen electrons were produced, therefore DPIP was not decolorized.
4	The inhibitor (malonate) stopped the reaction. No electrons were produced, therefore DPIP was not decolorized.
5	Reaction rate was increased by adding twice the substrate concentration. This rate will continue to increase until the reaction is limited by enzyme concentration.
6	The inhibitor (malonate) was not as effective in stopping the reaction when three times the amount of substrate (succinate) was present. It's important to remember that malonate is a competitive inhibitor and in this case it was out competed by adding more substrate (we "out-competed" the competitive inhibitor).

Exercise D

Omitted

That's about all for this lab. As always, if you have any more questions, feel free to email or call your respective TA's. Have a good weekend!

Biology 196 Labs