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Instructor: Chris Gillen 310 Higley Hall PBX 5399 email: GILLENC This lab is partly based upon a lab done in Biol 332 at the Department
of Zoology at the University of British Columbia. Reading:
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Biol 244 - Experimental Animal Physiology - Fall 2005
Instructor: Chris Gillen
310 Higley Hall
PBX 5399
email: GILLENCThis lab is partly based upon a lab done in Biol 332 at the Department of Zoology at the University of British Columbia.
http://www.zoology.ubc.ca/courses/bio332/Labs/OSMO.HTMReading:
Hill Wyse Anderson
- 67-74 (Concentration and Diffusion)
- 82-88 (Colligative Properties and Osmosis)
- 686-691 (Animals in Freshwater)
Introduction:
Imagine the water balance problems that a Paramecium faces. It lives in fresh water with a very low osmolarity, yet it needs to have many dissolved solutes and thus has a modestly high internal osmolarity. This creates a gradient for osmotic water movement into the Paramecium. The rate of water movement will depend upon the permeability of the Paramecium to water influx and also the magnitude of the gradient.
To prevent swelling and eventual bursting, Paramecium need to have a mechanism for excreting water. This mechanism is the contractile vacuole, which fills with water, then contracts, expelling water out of the Paramecium. We can get an estimate of the rate of water extrusion by determining the rate of contractile vacuole contration. In this lab, we will attempt to determine the iso-osmotic concentration of sucrose for Paramecium. The iso-osmotic concentration is the concentration of sucrose that causes no osmotic stress. In other words, it is the concentration of sucrose that does not cause osmotic water influx or efflux. We will determine this parameter by measuring contractile vacuole contraction rate at various sucrose concentrations, then extrapolating to the sucrose concentration where contraction rate would be zero.
Procedure:
Be sure you know how to use the microsope before beginning.
1) Place a very small drop of culture on a microscope slide and put a cover slip loosely on top. Observe the Paramecium under low power (40X).
2) In order to count contractile vacuole contractions, we will need to go to a higher power (100X), and also to remove most of the fluid from between the cover slip and slide. Removing the water will slow down the Paramecium, enabling us to view contractile vacuole contraction.
3) To remove water, place a piece of ripped paper towel on each side of the cover slip. It should begin to soak up the excess water. You should check the slide continuously to determine whether the cells have slowed. The best time to view the cells is when they have slowed down, but are not yet immobilized. At this point, you should be able to follow the cells by moving the stage at 100X while you count contractions. If you take away too much water, the cells will be crushed. Generally, cells that are completely immobilized will not provide accurate data. If you do not take away enough water, the Paramecium will continue to glide around, preventing analysis.
4) Practice counting contractions of the contractile vacuole. There are two contractile vacuoles, one anterior and one posterior. Do you see any difference in their rate of contraction? Count contractions for 2-3 minutes and report them as contractions per minute.
5) Now, we will place the cells in different sucrose concentrations. You will be assigned a few sucrose concentrations between 0 and 80 millimolar, and provided with a 100 mM sucrose stock solution. First, make a solution with the correct concentration of sucrose. If you need help making these solutions, click here. Then, add 200-500 ul of that solution to a tube of concentrated Paramecium . Analyze quickly for contractile vacuole contraction rate. Try to obtain rates from 2-4 cells per concentration of sucrose. Repeat with a different concentration - each group should obtain measurements for 3 different concentrations.
6) Save your data in p/class/biology/biol244/paramecium.xlsAnalysis:
1) Perform a regression analysis on the collected class data by plotting contractile rate vs. sucrose concentration. The iso-osmotic concentration of sucrose will be the x-intercept (the sucrose concentration at which contraction rate is extrapolated to be zero).
2) Bring a copy of your figure and your calculated iso-osmotic point to lab for next week.