US Long-Term Ecological Research Network

After Field Collection

 Equipment

Scintillation vials for chlorophyll filters-glass, 20mL
Labeled caps, lake abbreviation and depth, for scintillation vials
Light impenetrable storage box-small
Forceps
 
Filter Preparation
This should occur in as little light as possible. Separate the filter holders placing the filter side face up. Then using the forceps fold the filter in half and then roll the filter around the forceps. This will allow easier movement into the scintillation vials. Place the filter in the vial and cap it with the correct labeled cap. Place the samples in the storage box and freeze for at least 24 hours but no more than 5 days.
 
 
Day 1
 
Equipment
100% methanol Optima-“good methanol”
1 Liter repipet for 100% methanol set for 10mL samples
Homogenizer
Eye and ear protection for use with homogenizer
Methyl alcohol-low acetone-“bulk” methanol
2 Glass jars
Graduated plastic centrifuge tubes with lids-labeled with lake abbreviation and depth
Light impenetrable storage box-large
 
Extraction and Homogenization
Remove the storage box from the freezer and fill the 1L repipet up with “good” methanol. Add 10mL of “good” methanol into each vial and let the samples sit for 20-30 min. in the fridge. Don’t allow any light to penetrate the samples while they sit. While this occurs find the centrifuge tubes and put them in the order that will be used throughout the next two days. Also fill two glass jars with the “bulk” methanol. Rinse the homogenizer with one of the jars; always have liquid surrounding the homogenizer while it is running. After the filters have sat for 20-30 min homogenize each filter for 30 seconds or until there is a mushy consistency, or no large pieces of filter left. Make sure to wear ear plugs and safety glasses during homogenization. By gently tapping the vial over the centrifuge tubes, the ground up filter should slide down. This will allow all of the sample to go into the tube. Between each sample rinse the homogenizer twice by dipping it in the two different jars. The homogenization should take place in the least light possible. Place the centrifuge tubes in a larger storage box and place in the refrigerator for 24 hours. Soak the scintillation vials in Milli-Ro water overnight then set them to dry.
 
 
Day 2
 
Equipment
Kontron 930 Spectrophotometer with 1 cm cuvette holder
Disposable 1cm cuvettes
Lens paper
“Bulk” methanol
“Good” methanol
Disposable pipet
N HCL pipet 25µL
1.0 N NaOH pipet 40µL
Glass stir rod
Waste jar
Styrofoam tray for centrifuge tubes/cuvettes (2 trays)
Light impenetrable storage box-large
pH paper 3.8-5.5
Chlorophyll lab sheet

Starting the Spec.
Before switching the spec on, install the 1cm cuvette holder. Turn on the Kontron 930 Spectrophotometer as soon as you arrive on station, make sure there is the amount of light in the room as will be when running the samples. Try to minimize this light by just using light from the hallway. The fan in the spec. room should be turned on. The spec. should warm up for 30 min., this allows time for a baseline to run. While waiting fill out the chlorophyll worksheet with the correct lakes and their depths. Try to place a blank in between each lake. For example (Bla= Blank, i= Blind, depth = Regular Sample):                
            Sparkling:        0     3    5    8    10    12   15    18    i10    Bla
            Trout:               0     3    5    8    10    15    20    27    31    i10
            Big Muskie      Bla 0    3    5     8     10    12    16    19    i0  
            Crystal             Bla 0    3    5     8     10    12    15    19    i15  
 
Log onto the Spec. computers and start the Ukon Spectrophotometer program. Click on Execute, then Baseline to run the baseline. After baseline is finished, under Methods choose New Method then Wavelength Scan. Now load user method LTER003. Change the number of samples running to the correct number(10 or 7). 
 
Centrifuging Samples
Darken the centrifuge room by using only the hall light with the door partially shut. Then remove the samples from the freezer and place the first two lakes in the centrifuge. Make sure that both sides are balanced. Run these for 10 min. at 3000RPM. While this occurs check the disposable cuvettes for any large scratches. If they have any throw it away. Check the centrifuge once during the 10 min. to make sure that it reached 3000RPM. After they are finished spinning place the centrifuge tubes in the large storage box and take them to the spec room.
 
Running Samples on Spec.
Place the centrifuged samples behind the cuvettes and fill the cuvettes with liquid using the disposable pipet. Be careful as to not suck up any paper. The blanks should be filled with “good” methanol. Fill an additional cuvette with “good” methanol and place in the back cuvette holder inside the spectrophotometer. This will be the “reference” blank and will remain in place throughout your readings. Make sure to watch the methanol level in this cuvette as it may need refilling about half-way through your sample runs due to evaporation. Place the first sample cuvette in the spec and click the “measure” icon. Always check each run and make sure the peak of the graph is at 665. If the line starts high and has the wrong peak it is an indication of particles in the cuvette. Except in the bogs and the deepest part of Trout Lake or Big Musky the peak should be at 665, in the bogs and those 2 lakes there could be bacteria that are producing a wavelength that is different than 665. Continue with all 10 or 7 samples. After all samples have run save the data in UVIKON c:\lterscan\04scans…   with the file name, year (2digit) day number(3digits) and 2 letter lake code, ie. 04163tr for Trout Lake on June 11, 2004. While the file is still open go to File> Export> ASCII and click OK. Then close the file and start on the second lake. Once both lakes are saved, add 25µL of 1.0N HCl to each cuvette and still with the glass rod. Wipe the glass rod between samples with a Kimwipe Let these stand for 3 min then add 30µL of 1.0N NaOH and stir, wiping the glass rod off between samples. Allow these to stand for 3 min to let the reaction occur. During this process if you forget whether you have added the acid or base test the sample on pH paper. After both acid and base have been added measure the samples with the spec. Save the files with the same file name but add an “A” at the end, i.e. 04163tra. Also, export this file with ASCII while it is still open. Pour the liquid out of the cuvettes and rinse them 3 times with bulk methanol and let dry while you start the next 2 lakes to be centrifuged. Don’t forget to check the cuvettes for scratches before adding the next sample. Remember to remove the blank “reference” cuvette from the spec after you are done.

Clean Up
Pour all the sample liquid into a glass jar and place under the fume hood to allow it to evaporate. Rinse the cuvettes 3 times with bulk methanol and put them away. Disassemble the acid and base pipettes, rinse thoroughly with Milli-Ro water and allow them to dry on the counter. Rinse the stir bar and transfer pipettes with Milli-Ro water. Put the excess liquid and ground up filters from the centrifuge tubes into a glass jar and place them under a fume hood to allow them to evaporate. Rinse the centrifuge tubes and caps 3 times with Milli-Ro water and place into a rack to dry. Transfer the ASCII files from c:\LTER\04scans to Groups O:\LTER\Chloros.
 
 
(reviewed 9/04 Anne Small)
LTER Keywords
Protocol Format
Process
Protocol ID
chlorophyll1
Protocol Type
laboratory