US Long-Term Ecological Research Network
A.    Chlorophyll Extraction
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 spatula to chop up the filter into small pieces. This should take approximately 1 minute.
3.       Grind filters: Grind filters using the tissue homogenizer. Place the homogenizer into the tube with the chopped filter. Turn on the homogenizer once it is in the tube and grind for at approximately one minute, moving tube up and down to ensure all chopped filter pieces are ground up. Be sure to turn off homogenizer before removing it from the tube. Rinse homogenizer with acetone after every sample.
4.    Return the sample tubes to the freezer for 24 hours: Most protocols call for storing the grinded samples at 4 degree C. However, after storing duplicate samples in the freezer and refrigerator (after grinding) there was no significant difference in the chlorophyll results. Because past samples have been stored 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 “vis light” to allow the light to warm up for 60 minutes. 
1.     Loading the Marathon 21000 Fisher Scientific centrifuge: Making sure that the caps 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. It is essential to load centrifuge buckets evenly to ensure proper balance.
2.       Running the centrifuge: Run the centrifuge at 4000 RPMs for 15 minutes. This is programmed as Program 1 on the centrifuge. If the centrifuge is on Program 1, just press start and it will go automatically.    If there is an imbalance in the centrifuge the centrifuge will shut down.   In this case, stop the centrifuge and attempt to locate the imbalance. Sometimes this requires a placement shift in the center post of the bucket holders.
3.       Unloading the centrifuge: 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 freezer until it is time to process.


C.     Running a Sample:
Ensure the spectrophotometer vis light has been on for at least 60 minutes. Exit from the small screen with the start-up results. Make sure the cuvette holder in the spectrophotometer is empty and clear of anything that could block the light waves.
1.       Select the test: Click on “Fix Wavelength”, then click on “Method” on the top of the screen, choose “LTERCHLA” (the screen will not change). Exit from this small screen.  
2.       Run a blank and check that all cuvettes read near 0: Add acetone to a 1x1 mm, chemical resistant, macro, disposable cuvette and wipe it clean with a tissue then place it in the holder inside the spectrophotometer. Left click on “Blank”. No numbers will appear, this is just a calibration. Click on “Read Sample” on the top left of the screen. All of the readings at all wavelengths should be within .001 of 0 or under 0.0004. 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.
3.       Add sample to a cuvette: Before bringing the samples into the spectrophotometer room, turn off the overhead light. 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).
4.       Check the 750 nm reading and run the sample: Insert the cuvette into the spec. making sure that the clear sides of the cuvette are facing left and right. The default reading on the spec is 750 nm. Click on “Run Sample”. Check to make sure that this reading is less than 0.010. 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.
5.       Rinse the pipette tip: Before adding another 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. Be sure that the pipette tip is firmly on the pipette (press it on the bottom of the rinse beaker).
6.       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. 
C. 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.     Record the results of each sample for every wavelength designated on spreadsheet before and after acidification. Be sure to include the lake, mL filtered, date of sample and date of analysis.
1.       Rinse thoroughly and discard all used cuvettes and tubes of processed samples. 
2.       Dilute all samples to a solution of less than 20% Acetone and disposed of down the drain. 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.


LTER Keywords
Protocol ID