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Measurement of Chlorophyll a using the Turner 10-AU Fluorometer

Chlorophyll a is the most commonly used measure of microalgal biomass. Fluorometry, owing to its superior sensitivity compared to spectrophotometry or chromatography, is the method of choice for estimating phytoplankton chlorophyll a. Fluorometry is based on the excitation of chlorophyll a molecules in extract solution by blue light and the release of red fluorescence, which is then quantified by the fluorometer.

The Turner 10-AU fluorometer uses a narrow pass filter to let in excitation light at 436 nm and another narrow pass filter to detect emitted light at 680 nm. This combination of filters, together with a light source that optimizes light emission in the 430-440 nm range, is described by Welschmeyer (1994) as optimal for quantifying chlorophyll a with negligible interference by other pigments.

The Turner 10-AU also features microprocessor-controlled calibration, blanking, and output averaging, and can dump data to a spreadsheet program in an interfaced computer.

Method

Filter an appropriate amount of water sample through clean glass fiber filters. The proper amount must be determined by practice, but the sensitivity of the fluorometer permits small sample volumes. Record filtrate volume. Filtered samples may be folded over, wrapped in foil, labeled, and frozen for later analysis, or extracted immediately by sonication for 10-15 seconds followed by a 15 minute extraction period. All extractions should be done in 10 ml (or appropriate volume) of 90% acetone. Extracts must be shielded from light. Frozen samples may be extracted in acetone in a refrigerator for 20-24 hours before analysis or sonicated. Care must be used with sonication!

The fluorometer should be warmed up for 30 minutes before use. If the spreadsheet interface is to be used to download data to the computer, follow the instructions for the software and dump your data to a labeled diskette. Never dump data to the hard (c:) drive.

Blank the fluorometer with 90% acetone in a rinsed, cleaned cuvette (13 mm cuvettes for the fluorometer have a white line on them and must never be used for any other purpose). It is best to have two or three clean cuvettes available. Insert a cuvette with 90% acetone and wait for the reading on the fluorometer to stabilize. It should give a reading of "-0.0", or at least a value much less than 1.0. If not, you have a problem. You will note that removing the cuvette cover on the machine causes it to change readings. To minimize the time the machine spends getting back to the right reading, do not leave the cuvette cover off for long between samples. Ideally you pour the next sample to be read into a cuvette, then replace the sample in the machine with the next one in one fast step.

The best way to get a good reading is to mash "*" on the console, which starts the autoaveraging program. Take the reading after the screen beeps and flashes "done". If you use the spreadsheet program, mash "9" to send the datum to the computer.

Note that the fluorometer can autorange. If you put in a sample too concentrated or too low to read in one range, it will flash at you, change sensitivity scale (low, med, or high) and then settle into a numerical reading. If it goes offscale on the high range, you must dilute and correct for that dilution (mls diluted volume/mls original volume) in your calculations.

Do not pour solvent or sample into the cuvet while it is in the machine. You should only need to turn the machine on (red button) and off. If you get out of the "home" screen (the normal display with numbers and bar graph), simply mash the "home" button. Never mess with the other screens or settings unless you have expert guidance and prior permission from LBC.

Calculate your results by taking the reading you get from the machine (care: the units on the screen readout are micrograms chlorophyll a per liter of extract; do not use these numbers as final values), multiplying by the ratio of extract volume in liters to filtrate volume in liters, and multiplying by any dilution factor necessary. The resulting values have units of micrograms of chlorophyll a per liter of water.

Reference: Welschmeyer, N.A. 1994. Fluorometric analysis of chlorophyll a in the presence of chlorophyll a and phaeopigments. Limnol. Oceanogr. 39:1985-1993.

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