Spectrophotometer:A. Chlorophyll Extraction (using tissue grinder at DNR Research Station) 1. Dim the lights and keep the sample tubes in the freezer: Because chlorophyll degrades when exposed to light and heat, this procedure and all others associated with analyzing chlorophyll should be carried out in dim light conditions. Only one sample tube should be out of the freezer at any one time while the pre-grinding or grinding procedure is occurring. Return each tube to the freezer as soon as its filter has been ground. 2. Pre-grind filters: Use the sharpened stainless steel probe to chop up the filter into small pieces. This should take approximately 2 minutes. 3. Grind filters: The teflon tip on the tissue grinder should be sanded after grinding approximately 5 filters. Grind each filter for 2 minutes. Do not lift the teflon tip out of the test tube while the grinder is rotating. Grind the filters by attempting to keep the teflon tip in the acetone solution and pressing the tip against the filter and the tube. 4. Return the sample tubes to the freezer for 24 hours: Most protocols call for extracting the samples in the refrigerator (at 4 degrees C). However, after extracting duplicate samples in the freezer and refrigerator (after grinding) there was no significant difference in the chlorophyll results. Because past samples have been extracted in the freezer, this is the current procedure being used. B. Centrifuging the Samples: The samples should be centrifuged as close as possible to 24 hours after extraction. Before centrifuging the samples, turn on the spectrophotometer and enter the correct program number to be sure that it is working properly. Perform the procedures below in dim light. 1. Checking acetone volume: In dim light, use an identical tube as those used for the samples but with mL marked on it, to measure the volume of the acetone in the samples. Measure to the nearest 0.5 mL. If the sample has any other volume than 5 mL, write the volume on the sample label and remember to enter the volume later into the spreadsheet. 2. Loading the centrifuge: Making sure that the rubber stoppers are on tight, put tubes with equal acetone volumes opposite each other in the centrifuge. If there is an odd tube remaining or a tube with a different volume, put a spare tube opposite the sample with the same volume of water to counterbalance the centrifuge. 4. Running the centrifuge: Turn the speed dial below 40. Turn the timer past 15 minutes. Slowly turn up the speed allowing time for the centrifuge to increase in speed. If there is an imbalance in the centrifuge (or any other problem), the centrifuge will run much louder than normal. In this case, stop the centrifuge and attempt to locate the imbalance. If the centrifuge is running smoothly, set the speed at 90 and the timer at 15 minutes. Previously, the numbers on the dial were believed to correspond to revolutions per second; however, this is not the case, for the centrifuge will only reach rpms of approximately 2500. 5. Unloading the centrifuge: Allow the centrifuge to come to a stop on its own. Carefully take each sample tube out of the centrifuge with minimal mixing. If the filter paper is mixed with the liquid, it will be necessary to re-centrifuge the sample. Transport the samples to the spectrophotometer in a rack that has tinfoil on the sides in order to block out the light. C. Running a Sample: 1. Select the test: Allow the spectrophotometer to warm up for at least 15 minutes. Select the proper program by pressing the test number followed by Select. 2. Rinse the cuvettes 3 times with acetone. It is most efficient to rotate 4 matching 1 cm cuvettes. Try to touch the cuvettes only on the opaque sides avoiding touching the clear sides especially on the lower half of the cuvette. 3. Run a blank and check that all cuvettes read near 0: Add acetone to the 4 matching cuvettes (at least half full), wipe them clean with a tissue, and insert them into the spectrophotometer with the labeled sides all facing the same direction (always put the tops on the cuvettes when they are in the spec). Press Run and the spec. will ask for a blank. Use one of the cuvettes filled with acetone as the blank. Once the blank is run, run all of the cuvettes (the cuvette position is changed by pulling out the metal rod to the next notched position). All of the readings at all wavelengths should be within .001 of 0. If this is not the case, remove the suspect cuvette and rinse, wipe, add acetone, and rerun it. Make sure that the correct program is being run by checking the wavelengths. The LTER samples should be run at 750, 665, 664, 647, and 630 nm. 4. Rinse the pipette tip: Before adding sample to a cuvette, the pipette tip should be rinsed with acetone. You should have 2 different sized beakers, one for waste and one for acetone rinse. Set the 10-1000uL pipette to 1000 uL (1 mL) and pipette 1mL of acetone from the rinse beaker and dispose of it in the waste beaker. Be sure that the pipette tip is firmly on the pipette (press it on the bottom of the rinse beaker). 5. Add sample to a cuvette: Before bringing the samples into the spectrophotometer room, turn off the overhead light and turn on the desk light in the corner. Carefully remove a sample from the rack and pipette approximately 2 mL of sample into a cuvette. Use caution not to suck up any filter paper into the pipette; tilt the sample to the side and submerge the pipette tip only just below the fluid level. If the pipette tip is getting close to the filter paper when removing the second mL of sample, stop pipetting and add the partial mL to the cuvette (it is possible to read approximately 1.5 mL of sample). 6. Check the 750 nm reading and run the sample: Insert the cuvette into the spec. (making sure that the labeled side is always facing in the same direction). The default reading on the spec is 750 nm. Check to make sure that this reading is less than 0.010 A. If the reading is higher, remove the cuvette and re-wipe it with a tissue. If the reading is still high, pour the sample back into the tube and re-centrifuge it. To run the sample press Run. 7. Acidify the sample: Once the sample has been run, remove it from the spec and add 60 uL of 0.1 N HCl (30 uL per 1 mL of sample). Gently shake the sample and wait 90 seconds to run it. 8. Check the acidification ratio: The before acidorafter acid ratio of the LTER samples is usually between 1.3 and 1.7. Compare the two readings to make sure the ratio fits in this range. If the ratio is higher than 1.7, re-acidify the sample and run it again (the acid probably did not make contact with the sample). 9. Rinse the cuvette: After checking the acidification ratio, dispose of the sample in the waste beaker and rinse the cuvette 3 times with acetone. Be sure to fill the cuvette to the top with acetone during each rinse to be sure that there is not any trace of acid left. Running Multiple Samples: 1. It may be more efficient to run 2 samples before acidification and then run them both after acidification. If this is done, take caution to add the correct sample to the correct cuvette and not to mix up the samples when they are removed from the spec. for acidification. Recording the Results: 1. Write the spec. id number located on the left of the printout onto the label of the corresponding sample. Each sample should have a before and an after acidification spec. id number written on its label. After all of the samples have been run, enter the date of analysis onto the spec. printout. This date will be used to identify the spec. printout when the data is proofread (after which proofed from spec. printout should be written on the spreadsheet). Clean-up: 1. Rinse the cuvettes 3 times with acetone, allow them to dry for several minutes in the cuvette rack, and return them to their box. 2. Solutions of less than 20percent Acetone can be disposed of down the drain followed by at least 10 volumes of water. Fill the waste beaker with water and pour the waste down the sink with the water running. Leave the water running for several minutes 3. Rinse the beakers and pipette tips 3 times with tap water followed by 3 rinses with distilled water. Hang the beakers on the drying rack.