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Subject: Chlorophyll Determinations
From: Mike Hein <[log in to unmask]>
Reply-To:Mike Hein <[log in to unmask]>
Date:Tue, 9 Dec 2003 09:11:05 -0500

text/plain (93 lines)

Several weeks ago I posted the following message to Diatom-L and Algae-L.
Several people have asked for the responses which I am including following
the message (minus responses simply asking for the responses). Bottom lines:
1. Seems to be a consensus that determining chlorophyll levels on formalin
preserved samples is not a good idea.
2. Except for the paper by Dell'Anno, et al. there seems to be little actual
data to support a conclusion.
3. Our client goofed and actually wanted phytoplankton counts done on all
the samples - not chlorophyll as indicated on the bottles.

In Section 10200H (Phytoplankton - Chlorophyll) of Standard Methods for the
Examination of Water and Wastewater we state "Concentrate sample by
centrifuging or filtering as soon as possible after collection. If
processing must be delayed, hold samples on ice or at 4 degrees C and
protect from exposure to light." Briefly that means unpreserved, chilled,
and in the dark.

We just received from a client a set of samples for chlorophyll analysis
that were preserved in error with 3 percent formalin. My questions to the
group are:
Has anyone tried to run chlorophyll analysis on formalin preserved samples?
Were the results comparable to unpreserved samples?
Does the chlorophyll even extract or is it bound up by the formalin?

Michael Hein
Water & Air Research, Inc.
6821 SW Archer Road
Gainesville, FL 32608 USA
Phone: 352 372-1500
Fax: 352 378-1500

Hi Michael, as far as I know, the results are not comparable, not only does
the formalin bleach the pigment, it interfere's with the analysis.  I have
folks do this to me all the time, it gets really frustrating, because its
lost data.

I would recommend throwing the samples away.  They are undoubtedly not going
to reflect true chlorophyll.  Formalin is acidic and undoubtedly has, at the
least knocked Mg out of the ring and degraded the chlorophyll.

I am in no means an expert in the field, as i am just an undergraduate,
however, For what it is worth, i my self preserved samples from a field
trip abroad and tried to run them, my results seemed low, and the response
from the supervising professor was that cross -linking meant that I was
unable to extract the chlorophyll directly.

I tried a solvent called 'target' often used for IHC work.

My results have not been confirmed and are of little scientific use, but i
thought id pass on my thoughts!

Sorry, I can't offer any useful answers to your query except that I
had always assumed that you had to have fresh material for
chlorophyll measurements. However, if you receive any advice about
this I would be very interested to hear about it. I was thinking of
running some trials to test this with glutaraldehyde preserved
samples, but hadn't got around to it yet.

Highly variable results for chl a will result from formalin
preservation, as cell lysis is very likely.  Even if you analyzed for
chl degradation products, loss from cell lysis would compromise these

Saw your post on the Diatom list. You cannot run chlorophyll analysis using
samples preserved in formalin. Once the sample is preserved in formalin,
chlorophyll a gets converted to phaeophytin, a degradation product which has
similar fluorescence characteristics as the actively photosynthesizing
pigment chlorophyll a. This happens when the magnesium from the porphyrin
ring structure gets removed. I do not have any data comparing the relative

I am fairly sure that most of the pigment was either lost or degraded.
Possibly useful to look at, but not for pigment work.

There is a paper in Marine Ecology Progress Series that deals with the
subject of formalin preserved samples and photopigment determination:
MEPS 191:71-77 (1999).

You can read the abstract here:

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