Exercise 6.3 - Pinocytosis

LEVEL I

Materials

• Actively growing culture Amoeba proteus 2
• 0.001 M Alcian blue
• 0.01 M Sodium azide
• Petroleum jelly
• Slides, coverslips
• Microscope

Procedure

1. Form a small ring of petroleum jelly onto a microscope slide.

2. Add a small drop of Amoeba suspension, and add about 10 µl of alcian blue to the slide. Note the time.
   Immediately place a coverslip onto the slide and gently press to seal the amoeba within the petroleum jelly ring.

3. Observe the Amoeba with 10 X magnification of the microscope. 3

4. Record the time for each of the following events:
   a. Surface staining
   b. Rounding up of the cell
   c. Formation of rosette (crinkling of cell)
   d. Channeling (flattened pseudopodia and invagination of membranes)
   e. Pinosome formation (small vessicles of dye pinched off from invaginated membranes).
   

5. Prepare another slide with a jelly ring, but add 10 µl of sodium azide along with the Amoeba.
   Allow the Amoeba to remain in the azide solution for about 5 minutes and then add the alcian blue and a coverslip. Repeat steps 3 and 4.

6. Compare the times for each event of step 4 for the control and for those Amoeba treated with the azide.

Notes

Membranes normally do not allow larger molecules (such as dyes) to enter a cell through simple diffusion. If a dye is presented to an amoeba, however, the dye will be incorporated as part of what is referred to as receptor mediated endocytosis. In more general terms, the cell will drink or eat the dye, i.e. pinocytosis (cell drinking) or phagocytosis (cell eating). The dye will enter into internal vacuoles, known as food vacuoles.

With some dyes, the cell will actively transport the dye back out of the cell. It requires energy for this dye exclusion, energy derived from metabolism. Energy is only produced in living cells, and the phenomenon can be monitored in what is known as the dye exclusion test. The dye exclusion test becomes a way to monitor cell viability (Chapter 12).

For now, however, it implies that we can monitor the health of an amoeba by timing the movement of dye, and we can observe alterations in membrane function by alterations in the process of dye movement. A healthy, functioning cell will not stain and will have a minimum number of dyed vacuoles. Its membrane will remain intact and there will be minimum interruption of its ligand-mediated endocytotic processes. A damaged cell, however, will undergo physiological and morphological changes as the membrane receptor sites become irreversibly bound to dye. As it loses its ability to excrete the dye, it will become stained, round up and ultimately die.

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