Determination of the Rates and Optimal Temperature for Aerobic Respiration and Photosynthesis. Lab report

APPENDIX E: Use of the Oxygen Electrode and Data Analysis In Lab 4A, we will measure the rates of oxygen consumption and/or evolution as a function of temperature using an oxygen electrode (shown in Figure 1 ). At the bottom of the reaction chamber is a bare, platinum electrode covered by a porous Teflon membrane. A small polarizing current is passed through the electrode. Oxygen diffuses across the membrane and is reduced at the electrode tip to form water. The higher the concentration of oxygen in the solution being measured, the higher the rate of oxygen diffusion across the electrode membrane, and the greater the pen deflection on a chart recorder which is attached to the electrode. Conversely, as oxygen is utilized, the concentration of oxygen in the solution will decrease, resulting in a negative slope on the chart recorder. Figure 1: The reaction chamber of the oxygen electrode. In (A), the reaction chamber and controls can be observed. Note the tubes extending from the chamber, which allows for the circulation of water of a given temperature around the chamber. In (B), the inside of the chamber can be seen. The oxygen electrode is in the middle of the orange O-ring.

The following procedure describes how to properly calibrate the oxygen electrode:

1. The water bath has been set to 20 o C and a vial of tap water placed within for use during your experiment.

2. Examine the oxygen electrode apparatus. You will notice that the reaction chamber is full of water. There should be a small magnetic stir bar visible at the bottom of the chamber. The chamber has a plastic stopper loosely inserted in the top. The power switch on the blue cabinet should be "on". A B 3. Remove the plastic stopper, and suck the water out of the reaction chamber with the plastic pipette provided. Discard this water. BE CAREFUL THAT YOU DO NOT PUNCTURE THE MEMBRANE THAT IS COVERING THE ELECTRODE AT THE BOTTOM OF THE CHAMBER . Add 3-4 mls of fresh distilled water from a squeeze bottle, and then remove this water with the plastic pipette. Repeat several times to ensure that the chamber is clean, and free of any residual chemicals from any previous experiment.

4. Suck all the water out of the chamber. Retrieve the tube of water that you have suspended in the water bath, and shake it well to thoroughly aerate it. Add 2.0 ml to the reaction chamber with a pipette. Leave the top of the reaction chamber off at this point . Switch on the stirring motor, and adjust it to a low or moderate speed (about setting 5). 5. With this air-saturated mixture, the pen should be near the maximum side (the right-hand side) of the chart. Using the black “sensitivity knob” on the oxygen electrode control box , set the pen to 95 units on the chart, which will represent the maximum amount of oxygen for your experiment (See Figure 2 ).

Figure 2: Oxygen probe and chart recorder. Use the “Sensitivity” knob (shown in A) to adjust the chart to 95 units (shown in B) 6. With the reaction chamber still open , carefully bubble nitrogen gas into the solution using the tubing/pipette tip assembly which is attached to the nitrogen gas cylinder. You must place a finger over the “blow hole” in the tubing in order to get the gas to flow through the pipette tip.

The nitrogen gas will displace oxygen, and the pen will move slowly towards zero on the chart. As the pen begins to level out towards the left-hand side of the chart, adjust the pen such that the arbitrary "zero oxygen" level is at 5 units on the chart paper; the control knob to do this is marked " 0 ”, and is on the right side of the chart recorder. The system is now calibrated for all experiments performed at this temperature . See Figure 3 on the following page.

2 95 units = maximum oxygen = 100% A BUse to adjust to 95 units units Figure 3: Use the chart recorder “0” knob to adjust to 5 units after reading is stabilized following the addition of nitrogen gas to the chamber .

At this point the oxygen probe is calibrated and ready for use. Remember to turn off the stir bar prior to emptying the chamber. Add 2 mL of your culture and adjust the pen on the scale according to your procedure through the addition of “oxygen” (air) or nitrogen gas. When sealing the chamber, you must ensure that all excess air is removed. This can be ensured by observing the culture in the capillary tube of the plug (see Figure 4 ) and sealing the top appropriately with Parafilm TM . You can then perform your readings for the time indicated in your procedure. Remember to cover the chamber with the black cloth (visible in Figure 1 ) if required.

Figure 4: Inappropriate versus appropriate sealing of the reaction chamber. If the plug has not been sufficiently inserted, an air bubble will be apparent underneath (A). If the plug has been depressed sufficiently, liquid can be observed within the capillary tube of the plug (B).

35 units = minimum oxygen = 0% Use to adjust to 5 units units A BAir bubble Liquid in capillary tube You have arbitrarily set a distance of 90 chart units to represent the total amount (or 100% ) of oxygen present in the reaction chamber, at 20 o C . To convert this total pen deflection to µg oxygen/min you will use the maximum solubility values for oxygen found in Table 1 below to first calculate the amount of oxygen/unit. TABLE 1: Solubility of oxygen in pure water as a function of temperature at sea level.

Temperature ( o C) Oxygen ( µ g/mL) Temperature ( o C) Oxygen ( µ g/mL) 10 11.29 40 6.46 20 9.10 50 5.76 30 7.58 60 5.37 For example, at 60 o C the amount of oxygen/unit is:

O 2 / unit ( in 2 ml ) = (max. O 2 / ml) x 2 ml total units = (5.37 μ g/ml) x 2 ml 90 units = 0.119 μ g/unit Remember, that this will need to be determined for each temperature for your analysis. We can then determine the rate using the change in units/min obtained from the chart reader (see Figure 5 for an example). As each group has standardized their data by ensuring that their culture contained the same number of cells, we can simply average the determined units/min and use the formula shown below:

observed rate ( μ g/min/ml) = units/min x max O 2 ( μ g) /unit 2 ml Figure 5: Sample of data obtained using the oxygen electrode.