The maintenance of a stable environment for red blood cells is of vital importance. Physiological saline is a solution used to re-hydrate patients in hospital. It is perfused by an intravenous drip directly into a blood vessel.

A. Exposing the blood cells to different solutions of sodium chloride (NaCl)

MATERIALS

METHOD

1. Pipette 10cm3 of physiological saline into test tube number 1. Put a bung on it.
Set up the next 8 test tubes with bungs using 10cm³ of the prepared solutions: 0%, 0.1%, 0.3%, 0.5%, 0.7%, 0.9%, 1% and 2 % NaCl.

2. Collect a few cm³ of blood in the fat bottomed flask. Using the 1cm³ pipette add 0.1cm3 of blood to each test tubes. Replace the bungs and shake the tubes gently to mix them.

3. Set up 9 labelled microscope slides on the bench. Using a Pasteur pipette, take one drop of the each of the dilutions on the appropriate slide. Make sure that you rinse the pipette thoroughly with the new dilution before taking the drop.

4. Observe the shape of the red blood cells under high power until no further change in appearance is seen. Record your results.

5. After having left the test tubes undisturbed for at least 1 hour, observe the appearance of the tubes and note down your observations.

6. Transfer the various blood dilutions to centrifuge tubes and spin for 1 minute. Record your observations on the sediment (solid at the bottom of the tube) and the supernatant (liquid layer above the sediment).

7. Clear away all the materials apart from the centrifuge tubes of blood in the different NaCl solutions.

B. A colorimetric analysis of the supernatant

Starting the DataMate Program and calibrating the sensor

1. Use the following steps to start the DataMate program on your calculator:
   Press (for TI-73, 82 and 83 press ), then press the calculator key for the number that precedes DATAMATE. Press . An introductory screen will appear, followed by the main screen.

2. Plug the colorimeter probe into channel CH 1 on the CBL2 interface.

Start the DataMate program. Press to reset the program. DataMate will detect the sensor and display the current sensor reading. If the colorimeter is connected to the CBL2 via a DIN plug you will need to specify it. Press : setup. Channel 1 should show nothing so press . You will find the SELECT SENSOR menu. If the list of sensors does not include Colorimeter press : MORE. Another list will appear. Press the number corresponding to the colorimeter sensor. Press : OK and you will return to the setup screen.
To calibrate the colorimeter set up a blank tube containing distilled water. Press : CALIBRATE, to open the calibration menu. Press : CALIBRATE NOW .
Turn the colorimeter to 0% T (zero transmission, the lamp is off). Press and record 0% for POINT 1. Turn to an appropriate filter (remember blood is red) and record 100% for POINT 2. Then : OK to return to the set up screen.
Using the cursor buttons,  or (be patient it’s a bit sluggish!) select MODE, press (scroll up to get to the last item on the menu).
In the SELECT MODE menu press : EVENTS WITH ENTRY.  Press : OK to return to the main screen.

Collecting data

1. Suck up supernatant from a centrifuge tube using a fresh Pasteur pipette, without disturbing the sediment, and fill a colorimeter cuvette until it is 1cm from the top. Wash the pipette, take a sample from the next tube and fill a fresh cuvette. When labelling the cuvettes mark one side only near the top. Handle the cuvettes near the top too.

Set the cuvette with the first sample in the colorimeter, take care not to touch the sides where the light will pass. Select : START to begin data collection. Press to record your measurement. When you are asked to enter value, enter the NaCl concentration of your first sample. Press again. Place the next cuvette in the colorimeter and press . Then type in the next temperature. Press again and you will find the calculator producing an autoscaled scattergram of the measurements. Continue to take measurements of all your samples. You may stop data collection at any time by pressing the key. When you stop data collection  you will see the complete auto-scaled graph.

To store your data, if you are satisfied with it, return to the main screen by pressing .
Press : TOOLS, then select : STORE LATEST DATA RUN. This stores the data in lists. In this case “temperature” in L1 and the colorimeter “absorbance” in L2 with a copy of it in L3.
To check this press : QUIT, then , then and, finally, select : EDIT… You will see a spread sheet with your data in it in L1, L2 (and L3).

Process your data appropriately and discuss and evaluate the results.

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© Paul Billiet 2010