US Long-Term Ecological Research Network
EQUIPMENT
 
Kontron 930 Spectrophotometer with 1cm cuvette holder
Disposable 1cm cuvettes
Homogenizer
Scintillation vials for chlorophyll filters (glass, 20 mL)
Labeled caps for scintillation vial (Lake abbreviation and depth)
Holder for scintillation vials
Graduated plastic centrifuge tubes with lids (tubes labeled with lake abbreviation and depth)
Black storage boxes - 2 sizes (light impenetrable)
Square edged metal forceps (for removing filters from holders)
Curved necked metal forceps (for extracting ground filter from homogenizer)
100% methanol Optima
1 liter repipet for 100% methanol, set for 10 mL samples
Methyl Alcohol -low acetone (for homogenizer rinsing and washing)
1.0 N HCl syringe (1.5mL)
1.0 N NaOH syringe (0.6mL)
1 repipetter
2 rinse jars for homogenizer
Waste jar
Styrofoam tube/cuvette holders (3-4 trays)
Ear protection for use with homogenizer (plugs or muffs)
Eye protection for use with homogenizer
Chlorophyll lab sheet
Kontron spec. use booklet

FILTER PREPARATION
 
After returning from the field, the chlorophyll filters must be prepared for chlorophyll analysis. Filter preparation should be done in a dark room with as little light as possible. Separate the filter holder into its two halves with input side of filter facing up. Touching only the white ring around the edge of the filter, lift the filter from the holder with the square edged forceps and place input side up (the side that is discolored) on a paper towel to remove excess water. Do not squeeze filter as this may remove chlorophyll concentrated water. Place entire filter including any fragments in glass scintillation vial and cap with appropriately labeled lid. Place sample vials in a dark box and freeze for at least 24 hours but no more than 5 days.
 
LAKE AND SAMPLE SELECTION
 
If only one-half of the samples are to be processed, lakes should be divided into two groups. Extraction and homogenizing can be done to one group on Day 1 and then that group is centrifuged and read on Day 2. Group 2 is usually extracted and homogenized on Day 2 and centrifuged and read on Day 3. 
 
EXTRACTION AND HOMOGENIZATION
 
Remove frozen filters from freezer and add 10 mL of 100% Optima brand methanol to each sample vial, submersing the filter in solvent. Allow to soak for 20-30 minutes. Homogenize samples for approximately 45 seconds or until the filter-solvent mixture has a fine consistency. Make sure that there are no large pieces of filter remaining. Use forceps to remove any pieces of filter that remain attached to end of homogenizer, and return them to the sample vial. Transfer slurry to a pre-labeled centrifuge tube. Between each sample, rinse homogenizer and forceps twice by dipping in methyl alcohol wash jars. Do not operate homogenizer without it being immersed in a liquid or being very wet. Place centrifuge tubes into a dark box and refrigerate for 24 hr. Soak vials in Milli-Ro water, scrub with a nylon brush, rinse in Milli-Ro water then Milli-Q and allow to dry.
 
STARTING THE KONTRON SPECTROPHOTOMETER (also see step protocol)
 
Before switching the power on, install the 1cm-cuvette holder in the Kontron spec. Always start the Kontron Spec. and computer system in the same level of room light that the chlorophyll samples will be handled (low light). The computer will automatically run a baseline when first started. Allow at least 30 minutes of warm-up time for the Kontron spec before starting any computer set-up procedures. While waiting for the spec to warm-up, you can proceed to the next steps, ORGANIZING SAMPLE ORDER and CENTRIFUGING SAMPLES.
 
ORGANIZING SAMPLE ORDER
 
Ten samples are the maximum that can be run at one time on the Kontron, so organize your sample set accordingly. Use the CHLOROPHYLL datasheet and the field sheets. Ideally, a blank sample should be run between lake sets and be included as part of every sample set. Replicate samples from each lake should be run one after another.
 
CENTRIFUGING SAMPLES
 
First darken the centrifuge room as much as possible. Remove samples from refrigeration. Place grouped samples (usually 2 lakes at a time or as determined above) in centrifuge making sure they are balanced. Run for ten minutes at 3000 RPM. The centrifuge becomes warm after repeated use, therefore, minimize sample exposure and only run samples directly out of refrigeration. A spare centrifuge tube with equal volume of plain water or methanol can be used to balance loads. Once samples are spinning, proceed to the next step, BASELINE AND BASELINE CHECK WITH BLANKS. Transfer samples to spec. room in a dark box.
 
BASELINE AND BASELINE CHECK WITH BLANKS
 
After the Kontron Spec has warmed up, and before putting any cuvettes into the sample holder, re-run the baseline. To do this press 1 on the main menu for Uvikon start. Next highlight NEW SAMPLE BASELINE and then press ENTER/RETURN. This will take about 5 minutes. To check the baseline run methanol blanks. Highlight USER METHODS and then press ENTER/RETURN, then highlight the user method (LTER003) and press ENTER/RETURN. On the first parameter page, check the sample number. If not set to 1, highlight SAMPLE NUMBER, press ENTER, and enter a value of 1. Press ENTER to accept. Place cuvettes containing pure methanol in both the sample and reference positions and close cover. Double check that the compartment cover closes properly. Press MEASURE. The resulting scan is a reference to the baseline and the similarity of the cuvettes. Your scan should be nearly a straight line through zero. The minimum and maximum absorbency values should have values of + 0.005. If the baseline check gives you problems, try another NEW SAMPLE BASELINE after removing the cuvettes. After every NEW SAMPLE BASELINE, examine the outcome until it is acceptable. These data may be discarded once acceptable.
TROUBLE SHOOTING Things that can make an irregular baseline: (1) Error in initial baseline--make sure cuvettes have been removed, cuvette holder is properly seated, and run another baseline. (2) Reference cuvette is running low on solvent--change solvent. (3) Either sample or reference cuvette have marks on light sensing surface--replace cuvette.
*NOTE: Any time you wish to cancel an attempted baseline, before pressing MEASURE, press BREAK instead.
 
REGISTERING CUVETTE SETS.
 
Before running any sets of cuvettes or samples be sure to keep track of every run and the order it was ran. Later you will go back and rename according to this order.
Before every spec run, cuvettes should be run with optima methanol and absorbances saved. If necessary, highlight USER METHODS and ENTER. Select LTER003. Go to the first parameter page and set sample number: highlight SAMPLE NUMBER, press ENTER, and enter a value (6,7,or 10 as determined BY CUVETTE NUMBER) press ENTER to accept. Go to the second parameter screen and set it to autosave: highlight AUTOSAVE press ENTER, enter 0=off and 1=on press ENTER to accept it. Measure an absorbance scan on all cuvettes in numerical order. Each cuvette should contain blank optima methanol that can be poured from cuvette to cuvette so as to limit solvent use.
 
RUNNING SAMPLES ON THE KONTRON SPEC.
 
If necessary, highlight USER METHODS and ENTER. Select LTER003. Go to the first parameter page and set sample number: highlight SAMPLE NUMBER, press ENTER, and enter a value (6,7,or 10 as determined above) press ENTER to accept. Go to the second parameter screen and set it to autosave: highlight AUTOSAVE press ENTER, enter 0=off and 1= On press ENTER to accept it.
Carefully transfer samples from tubes to the cuvettes using a pipette. To avoid disturbing filter in bottom of centrifuge tube, do not squirt sample back into the tube. Using the MEASURE key, run all samples in current set. Next attach the 1.5mL syringe to the repipetter, set the dial to 1 and push the button to dispense 25ul of 1.0 N HCl into each cuvette. Mix the HCl into the samples using a glass stir rod. After all samples have been acidified, let the samples react for about 3 minutes. Next attach the 0.6mL syringe to the repipetter, set the dial to 4 and push the button to dispense 40ul of 1.0 N NaOH into each cuvette. Mix the NaOH into the cuvettes using a glass stir rod. After all the samples have been neutralized, let the samples react for about 1 minute .
Run all samples in current set again. Rinse cuvettes with bulk methanol between sets.
 
CLEAN UP
 
** Before discarding samples from centrifuge tubes, be sure all files are accounted for. If not refrigerate samples in a dark container as soon as possible to prevent degradation of chlorophyll.
Remove filters from centrifuge tubes under the evaporation hood. Place ground, wet filters in a waste container under the hood and allow to evaporate. Discard dry filters in the trash later. Soak tubes in Milli-Ro water, scrub with a nylon brush, rinse in Milli-Ro water then Milli-Q and allow to dry. Soak cuvettes in Milli-Ro water, scrub lightly - do not scratch, rinse in Milli-Ro water than Milli-Q and allow to dry.
 
RENAMING AND CONVERTING FILES
 
The data stored in the Kontron must be renamed and then converted to ASCII format before it can be entered as a text file into another computer or program. To do so, press methods, Highlight DISK AND FILE UTILITIES and press ENTER. The monitor will display the Kontron Main Menu. Select 9 to enter the DOS mode. Type "cd windows" and then "win" to open windows. Open file manager and then the work folder. Sort the files by name. Highlight the most recent file ran on the spec. Press ALT-F and then N to rename the samples one at a time. To rename the samples use BCMyear-run# (ex. BCMCHL04-1, BCMCHL04-2, etc.) and an "A" for a post acid run (BCMCHL04-1A). After renaming, highlight all the files. Rename all by typing " *.scn" to the end of all the files. Exit windows and at dos prompt type "9310". Select 3 FILE FORMAT CONVERSION UTILITY on the Uvikon main menu. Next select 1, ASCII FORMAT, then 4 for WAVELENGTH SCAN DATA. Highlight all files by pressing ENTER and then press EXECUTE to run the conversion. To exit press 7, then 3 then 9 to exit to dos. At C:\> type "novell". It will ask you to login. To do so type your user name and then (when asked) your password. Then select WINDOWS <A> and then LOCAL WINDOWS <B>. Open FILE MANAGER and then open O:/ Biocomplexity\Cross Lake Comparison\Data\Year\Biocom year\Chem year\Chloro. Next open the C directory-work folder and highlight all the .asc files. Drag the file icon to the O directory. Delete all the .asc files in the C directory. Next open C:/lter_chloro/1998 (or most recent). Return to C:/work and highlight the .scn files. Drag the file icon to the 1998 folder. Now you are done. Exit FILE MANAGER and then exit WINDOWS. Press ESC to the opening menu and then press K to log out of the network. At the dos prompt type "9310" to return to the main menu of the Uvikon. Turn off the spec.
 
(Updated July 1998)

 

LTER Keywords
Protocol Format
Process
Protocol ID
biocom_chlorophyll1
Protocol Type
laboratory