Bio5LAManual12f

Wait at least two minutes 5 apply a coverslip and

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Unformatted text preview: toplasm close to the cell wall. If the cell is healthy, the chloroplasts may be moving, but they always remain near the wall. The nucleus is often difficult to identify. It is found close to the cell wall and has a grey, shadowy appearance. 4. Once you have made the above observations, prepare another slide with a fairly large drop of 1.0 M NaCl. Then use a forceps to carefully transfer the Elodea leaf from your first slide to the drop of salt solution. Wait at least two minutes. 5. Apply a coverslip and blot off any excess salt solution. Observe the effect of this treatment upon the Elodea cells. Analysis. Describe the structural change in the Elodea cells elicited by the NaCl treatment. Explain the structural change that you observed. Osmosis in mammalian red blood cells. Red blood cells offer many advantages for this kind of study. They are readily available, easy to handle, and have a relatively simple structure characterized by a fixed size and unique shape (please see p. 911 of your text for a more detailed description of their structure). This latter feature will allow easy assessment of shape changes elicited by osmotic water movement. We will be using whole sheep blood obtained from a supply house. When handling these cells, you must keep in mind that you are dealing with a cell suspension. Thus the cells will tend to settle with time. As a result, you will need to resuspend the cells anytime they are transferred or observed. At this point, your group should obtain 5 ml of the RBC suspension* for your experiments. Your TA will dispense this into a clean test tube which you take to your lab station and place into a small beaker that is partially embedded in ice – each table will have an ice bucket for this purpose. [*Your TA will prepare this mixture by combining 6.0 ml of whole sheep blood with 40.0 ml of isotonic saline.] Biology 05LA – Fall Quarter 2012 Lab 3 – page 6 Part 1: Correlation of RBC structure in response to exposure to solutes of different tonicity. Procedure: 1. Obtain 3 small test tubes and label each with either hypo-, iso-, or hyper- (tonic). 2. In the proper tube, add 5.0 ml of hypo-, iso-, or hypertonic solution. 3. Once filled, add 0.2 ml of the RBC suspension to each of the 3 tubes -- keep in mind that you must re-suspend the RBCs by swirling the stock tube prior to dispensing. 4. Close each of these tubes with a small square of Parafilm and mix by gentle inversion. 5. Set these tubes aside for about 5 minutes before observation. 6. Students should now pair up to make the necessary microscopic observations. 7. For each of the samples, apply a drop of the RBC suspension to the center of a glass slide (remember to swirl before doing so), add a coverslip, and observe. [Caution, do not begin your observations with the hypotonic sample.] Do not dispose of your sample tubes at this time; you will need to record their appearance (either clear or cloudy) in your lab notebooks. 8. Make careful drawings in your lab notebook of a few of the RBC's from each of the samples. Note the shapes of the cells and their relative numbers for each of the samples. Record this information in your lab notebook along with the appearance of the tube (either clear or cloudy) from which they came. Analysis. Describe the appearance the cells in each of the treatments. Explain the structural change that you observed. Part 2: Estimation of the osmolarity of red blood cells. An awareness of osmolarity is essential to biologists working with isolated cells and tissues because exposure of these isolates to anything but isotonic conditions would result in unintended swelling or shrinkage. Such changes would probably affect their experimental results. Thus, there must be some way to estimate the osmolarity of their experimental subjects. Actually there are several ways to do this, some requiring special instrumentation. We will use a relatively simple approach. In this exercise, a fixed amount of the RBC suspension will be exposed to a series of solute concentrations extending from hypo- to hypertonic. Somewhere near the interface of these extremes, the cells will be isotonic with their environment. At this point, the osmolarity of the cells and their environment will be the same. That is, the rate of osmotic water movement into the cells will equal that moving out of the cells. Your task will be to approximate the location of this point. By now you should realize that the cells exposed to hypotonic conditions will swell and lyse (burst) and those exposed to hypertonic conditions will be intact. Thus, we will be seeking to identify the concentration change that results in a change from the lysed to the intact condition (this concentration should be close to isotonic). Procedure: Two different solutes will be used for this study; glucose and NaCl. For each lab table, one group of 4 will use glucose and the other will use NaCl. Thus, each group will only need to prepare one set of dilutions as given in Table 3. When this study i...
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This note was uploaded on 08/27/2013 for the course BIO BIOL05LA taught by Professor Abbottl during the Fall '12 term at UC Riverside.

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