8

Based on the previous experiment, you've evaluated as less probable the hypothesis that rapid degradation of proton pumps initially present in the membrane of the food vacuoles causes the pH decrease process (hypothesis B).
Now you wish to perform another experiment that will further support hypothesis C: fusion of the food vacuoles with lysosomes.

Your professor suggested to add a toxin called N-ethyl maleimide (NEM) to the medium in which the Paramecia are grown. This toxin prevents membrane fusion, and will thus prevent the fusion of the food vacuoles to the lysosomes.
You recalled that addition of toxins might influence several phenomena within the cell.
After a long discussion (illustrated to the right) you decided to add NEM for a very short while, to minimize the damage.

Here is the detailed protocol you've both worked out:

Add yeast cells stained with Congo Red to the medium for 30 seconds.
Thirty seconds later (allowing a limited formation of dyed food vacuoles) add NEM for another 45 seconds.
Then examine the paramecia under the microscope at different time points and check the following parameters (monitored in previous experiments as well):

Vacuoles pH
Vacuoles size
Vacuoles migration
Yeast cells degradation

Continue.

The results are illustrated below.

**In light of these results, what can you say now about the food vacuole’s acidification?**
(Choose between right and wrong.)
A. NEM prevents the digestion of the yeast cells, thus preventing the vacuole’s acidification. B. NEM treatment does not affect any of the measured parameters. Thus, fusion plays no role in the acidification process. C. The fusion of lysosome with the food vacuole accounts for the acidification of the paramecium food vacuole. D. The NEM effect is not a result of fusion prevention but may be caused by the fact that the pH of the added NEM solution is high, thus fusion is allowed but the resulting acidification is prevented, and consequently yeast cells are not degraded.	Compare with initial findings in the absence of NEM.