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2004 Fluorometric Determination of Chlorophyll a

Arctic LTER Protocol

 

References-method is adapted from EPA Method 445.0, Arar, J and G. B. Collins.  In Vitro Determination of Chl a and Pheo a in Marine and Freshwater Algae by Fluorescence.  Revision 1.9, 1997

http://www.epa.gov/nerlcwww/m445_0.pdf

 

Pre-collection Prep/materials

-         15ml graduated Vulcan Tubes or similar-note make sure they are PP and not polystyrene (Falcon# 352096 or 352196)

-         90% Buffered Acetone-in 1L volumetric add 100ml DI, 1mg MgCO3, and fill to volume

-         with 100% acetone, invert to mix

-         47mm Whatman GFF 0.7um filters

-         Amber Nalgene Bottles (PP, HDPE) 1L or 500ml

-         Cooler with ice (for extraction)

-         Forceps

-         Gelman or similar vacuum filtration tower

-         1000ml plastic graduated cylinder

-         0.1N HCL (8.5ml 12.1N HCl, fill to volume 500ml with DI)

-         100-1000uL Pipette

-         13x100mm Borosilicate Glass Tubes

-         Van Dorn or similar for field collection

 

Field Collection

     Water samples are collected using a Van Dorn or similar collection device and stored in 1L Amber Nalgene Bottles (PP or HDPE).  Most samples are filtered in the lab within 1hr of collection.  If stored for over an hour they are placed in a cooler with ice or similar dark, cool container.

 

 

Lab Filtering

Filter water from amber bottles through Gelman filtration set-up. 

1.   Using forceps place pre-combusted Whatman GF/F 47 mm (0.7um pore) glass fiber filter onto frit; attach upper reservoir to filter holder.

2.   Rinse graduated cylinder a few times with a small amount of sample water.

3.   Measure 300 ml of sample (200 ml from 500 ml bottles) in graduated cylinder.  Pour into filter reservoir.  Turn on vacuum pump, be sure not to allow pressure differential to exceed .3 ATMs, to minimize damage to delicate organisms and don’t let filter pull dry (slowly release vaccum as final volume of water is filtered).

3.      Place filter into 10ml of 90% acetone (1mg MgCO3/l) in centrifuge tubes (Vulcan Tubes).  Make sure filter is completely immersed in acetone, cap tightly and place in light tight box while processing, then move to cooler with ice for extraction. These filters will be analyzed for Chlorophyll a content after a 16-18 hour extraction period.

4.      Tubes should be inverted/mixed at least once during extraction, but allow sufficient time (>4hours) for particles to settle before reading.

 

Reading Samples on Turner Designs10-AU Fluorometer

 

      -TD-10AU should already be calibrated and ready for use, for more information regarding calibration, lamp/filter arrangements, etc. refer to users manual or Fluorometer use protocols.  Do not change settings or parameters without consulting I. Washbourne or C. Crockett.

 

*Safety Precautions, chronic exposure to acetone can cause health problems (see MSDS).  Always wear gloves, eye protection, and make sure snorkel hood is working properly.

 

1.      Verify TD fluorometer is on, if not press red button at front of unit and allow to warm up for 20 minutes.

2.      Make sure windows are covered and other light sources subdued

3.      Print out spreadsheet (see below) or make your own for recording fluorescence values

4.      Place chlorophyll solid standard in fluorometer, replace black cap, wait 2 minutes for reading to stabilize, press [*] to read average and record values for both the high and low std.  To switch between the two simply turn 180 degrees till solid slides into place.  If readings deviate from mean std. readings by more than 10%, the unit may need recalibration (see Lakes or Nutrient RA).

5.      Read and record resting fluorescence with no tube in fluorometer.  It should be around 0.328.  This will help you catch problems associated with changes in lamp intensity.  A second check is a clean glass tube which has a fluorescence around 3.32.

6.      Read and record fluorescence of two acetone blanks (90% acetone w/MgCO3) in 13x100 glass tubes

7.      Reading samples-Carefully transfer extract from Vulcan tube to a clean 13x100mm borosilicate tube (leave last 0.5ml of acetone, filter, and any particles in the Vulcan tube).

8.      Wipe exterior of glass tube with kimwipe/tissue to remove any fingerprints or residual acetone.

9.      Place tube into fluorometer, replace black cap

10.  Allow reading to stabilize (about 5-10 seconds), press [*] and read average fluorescence value, record preacidification value (Rb).

11.  Using calibrated pipette, transfer 300ul 0.1N HCL into the sample you just read, mix by inversion (place Saran wrap over end, hold with thumb) or by using disposable glass pipette to “mix”

12.  Let stand for 2 minutes (its easiest to work out a system where you read a row unacidified, then acidify and let stand while reading 2nd row, acidify, etc, etc)

13.  Repeat steps 7-10, record post acidification fluorescence (Ra)

14.  After recording both readings, dump contents of glass tube into acetone waste container, rinse with small amount of DI

15.  Tubes are usually disposed of in broken glass container box.  You could double rinse and then dry for reuse, but trace HCL left on tubes could contaminate the next run.

16.  When finished with samples rerun and record low and high chl. solid standard values (let readings stabilize about 2 minutes)

17.  Make sure all glass tubes are rinsed and disposed of properly, place cap on acetone waste bottle, clean up kimwipes, pipettes, acid, etc.  Leave fluorometer on for next user

18.  Remove filters from Vulcan tubes dispose into filter bin, rinse tubes with DI, refill with 10ml 90% acetone, place back in dark box until next chlorophyll run.

19.  See equations below to determine chlorophyll a and pheophytin a concentrations

 

 

Chlorophyll a

 

C E, C  = Chlorophyll a concentration in the extract (usually 10ml)

 

C E, C = Fs(r/r-1)(Rb-Ra)

 

Where:

 C E, C  = corrected chlorophyll a concentration (ug/L) in the extract solution analyzed

Fs = response factor for the sensitivity setting S

r = the before to after acidification ration of a pure chl a solution (Rb/Ra)

Rb = fluorescence of sample extract before acidification

Ra = fluorescence of sample extract after acidification

 

 C S, C = corrected chl a concentration (ug/L) in the whole water sample

 

C S, C ={(C E, C x extract volume  in L x Dilution Factor)/(sample volume

in L)}

 

 

Pheophytin a

 

PE = pheophytin a concentration (ug/L) in the sample extract

 

PE = Fs (r/r-1) (rRa - Rb)

 

PS = pheophytin a concentration (ug/L) in the whole water sample

 

PS = (PE x extract volume in L x dilution factor)/(sample volume in L)

 

Note: if no dilution took place, then DF equals 1

 

 

Helpful Websites/Notes

 

Turner Designs Fluorometer 10-AU (see links at bottom of this page for PDFs of FAQ, background info, scope of applications, etc_

http://www.turnerdesigns.com/t2/instruments/10au.html

 

Quick Start Manual for 10-AU

http://www.turnerdesigns.com/t2/doc/manuals/10au_manual.pdf

Frequently asked questions about Fluorometry (Good Background Info)


 

Chlorophyll a Determination by Fluorescence-Arctic Lakes Spreadsheet

 

Name___________________             Date___________

 

Resting Fluorescence (no tube) ______ start of run     ______ end of run

 

Chlorophyll Solid Standard-mean early season 2004 High 49.11, Low 7.95

_______high start of run     ______low start of run        ______high end of run     _____low end of run

 

Blanks (90% acetone) _____ start of run       _____ start of run        _____ end of run     _____ end of run

 

Sample Name

Rb, Fluorescence before acid

Ra, Florescence after acid

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

                       

 

 

 

 

 

 

 

 

 

Please contact arc_im@mbl.edu with questions, comments, or for technical assistance regarding this web site.