R/V Melville Extracted Chlorophyll Methodology

Direct Questions to Sara Tanner (tanner@mlml.calstate.edu) or Jodi Brewster (jbrewster@mlml.calstate.edu)


Water samples were collected from 12 depths on the CTD Rosette and 8 
depths on the TM Rosette. The TM Rosette depths were chosen at the 100, 
45, 30, 16, 10, 5, 1, and 0.1 percent light levels (so phytoplankton 
production can be related to phytoplankton biomass) (Evans et al 1987). 
The CTD also had 2 more depths scattered between .1 and 100 percent and 
one each at 200m and 300m. The water from the CTD and TM rosettes was 
collected using opaque brown bottles in 250, 500, 1000, and 2000 ml or 
white 100 ml bottles. The differing volumes depended upon the depth of 
the sample and whether the samples were taken within the patch or not. 
Sampling from the TM Rosette was done with gloves. Each bottle was 
rinsed three times with the sample water before filling to the neck of 
the bottle.
A Whatman G/FF glass Fiber Filter, (~0.7um) Polycarbonate 5 um filter, or
Polycarbonate 20 micron filter was placed in a 25 mm diameter Gelman filter
holder. Water was pumped through the filter, being careful the vacuum
pressure did not get above 6 psi to avoid cell lyse. After filtration, the
vacuum was turned off and the filter was added with forceps to a tube
filled with 8 ml of 90% acetone. The tube was labeled and stored in a
freezer for a minimum of 24 hours.
After the minimum 24 hours extraction time, the filter was removed from 
the tube and the tube was wiped down with Chem Wipes. The fluorescence of 
the chlorophyll extracts were read on a 10AU Turner Designs fluorometer.
Two drops of 10 % HCl was added and the fluorescence was reread and
recorded again. The "before" and "after" readings were plugged into
equation chl-a = K * (Rb-Ra) * (vol ext/vol filtered)*dil to calculate
chlorophyll a values. 
A standard made from Sigma Chl-a in 90% acetone was calibrated on a
spectrophotometer and used to calibrate the fluorometer at the beginning,
mid and end of the cruise. Due to the fact the fluorometer drifted both ±
according to the solid standard, and a high correlation was found between
the low solid standard and the calibration curve, Chlorophyll-a values were
corrected using the ratio of the low solid standard.