926
Notes
with ice cold medium there was a considerable loss of permease-accumulated
substrate pools. Leder also observed that this
loss was reduced if cells were cooled over a
period of 1 min rather than l-2 s. He suggested that slower cooling allowed the
membrane to maintain its integrity. In my
study, organisms were chilled over several
minutes, and the results indicate that there
was little membrane damage. Placing bottles in ice immediately after adding acid
would therefore appear to be a valuable
modification to the kinetic method of Hobbie and Crawford ( 1969).
Angela J. Ramsay
Cawthron Institute
P.O. Box 175
Nelson, New Zealand
References
ALBRIGHT, L. J., AND J. W. WENTWORTH.
1973.
Use of the heterotrophic
activity technique as
a measure of eutrophication.
Environ. Pollut.
5: 59-72.
BELLY, R. T., M. R. TANSEY, AND T. D. BROCK.
1973. Algal excretion
of 14C-labelled compounds and microbial
interactions
in Cyan&urn
caldarium mats. J. Phycol. 9: 123127.
1974.
BURNISON, B. K., AND R. Y. MORITA.
Heterotrophic
potential for amino acid uptake
in a naturally
eutrophic
lake.
Appl. Microbiol. 27: 488-495.
1971. Some methods for the staELLIOTT,
J. M.
tistical analysis of samples of benthic invertebrates.
Freshwater
Biol. Assoc. U.K. Sci.
Publ. 25.
GRIFFITH, R. P., F. J. HANUS, AND R. Y. MORITA.
1974. The effects of various water-sample
treatments on the apparent uptake of glutamic
acid by natural marine microbial populations.
Can. J. Microbial.
20: 1261-1266.
HOBBIE, J. E., AND C. C. CRAWFORD. 1969.
Respiration corrections for bacterial uptake of
dissolved organic compounds in natural waters. Limnol. Oceanogr. 14: 528532.
LEDER, I. G.
1972. Interrelated
effects of cold
shock and osmotic pressure on the permeability of the Escherichia
coli membrane to perJ. Bacterial.
mease accumulated
substrates.
111: 211-219.
MORGAN, K. C., AND J. KALFF.
1972. Bacterial
dynamics in two high-arctic lakes. Freshwater
Biol. 2: 217-228.
SNEDECOR. G. W., AND W. G. COCHRAN. 1967.
Stati&ical methods.
Iowa State.
WRIGHT, R. T. 1973. Some difficulties
in using
14C-organic solutes to measure heterotrophic
bacterial activity, p. 199-217.
In H. L. Stevenson and R. R. Colwell
[eds.], Estuarine
microbial ecology. Univ. South Carolina.
- AND J. E. HOBBIE.
1965. The uptake of
orianic solutes in lake water.
Limnol. Oceanogr. 10: 22-28.
Submitted:
Accepted:
3 November 1975
17 May 1976
Improved extraction of chlorophyll a and b
from algae using dirnethyl sulfoxide
Abtiract-Dimethyl
sulfoxide ( DMSO) and
90% acetone extracted equal amounts of chlorophyll
from diatoms and blue-green
algae,
but DMS0 was superior to 90% acetone for all
green algae tested giving 2-60 times more
chlorophyll
depending
on the species. The
absorbance spectra of pure chlorophyll
a ( and
b ) from 600 nm to 750 nm were identical
whether dissolved in 90% acetone or a mixture
of DMS0 and 90% acetone ( 1: 1 v/v).
Thus,
several equations for estimating
chlorophyll
concentration
based on extinction in 90% acetone are applicable with this solvent.
Spectrophotometric
methods for estimation of chlorophylls
(Vernon 1960; Gottschalk and Muller 1964; SCOR-UNESCO
1966) depend on a suitable method for
extraction, complete and rapid enough to
avoid formation of degradation products.
A wide variety of water miscible solvents
such as acetone, methanol, ethanol, pyridine, and acetone plus ethyl acetate have
been used (Strain and Svec 1966); however, none of these solvents remove the unaltered chlorophylls rapidly and quantitatively from all freshwater algae. Ninety
percent acetone, widely
used (SCORUNESCO 1966; Slack et al. 1973; Weber
1973), extracts the pigments readily from
diatoms and blue-green algae but is relatively inefficient
with the coccoid green
algae. Although extraction may be im-
Notes
I
Chlorophyl
I a
Acetone
DMSO/Acetone
---_
-600
650
700
750
WAVELENGTH,
600
IN NANOMETERS
Fig. 1. Absorption spectra of chlorophylls
a and b in 90% acetone
v/v).
Absorption spectra are identical for chlorophyll
a.
proved by extending the time and by
grinding with fine sand or glass, this may
result in the formation
of degradation
products and the extraction is still frequently incomplete.
Because we found
that dimethyl s&oxide
( DMS0 ) was an
efficient extractant for adenosine triphosphate (ATP) from algae, we also examined its use as a chlorophyll
extractant.
Seeley et al. (1972) found DMS0 to be
an efficient extractant of chlorophyll
c
from brown algae; they also tested it on
two genera of green algae but published
no data. There are no published spectra
for chlorophyll a or b in DMSO, nor any
comparison of spectral properties in acetone and DMSO.
We made the extractions by filtering
logarithmically
growing algae through a
0.45-pm membrane filter which was solu-
650
and DMSO:
90% acetone
( 1: 1
ble in both acetone and DMSO. The filter was ground in a glass grinding vessel
with 34 ml of spectrophotometric
grade
acetone (90%) or DMS0 with a motordriven Teflon pestle for 3 min at room
temperature.
After grinding, the volume
was doubled with 90% acetone, mixed, and
centrifuged for 10 min at about 5,000 X g.
The absorbance of the supernatant was determined at the appropriate wavelength.
Highly purified chlorophyll
a ( and b )
produced the same absorption spectrum between 600 and 750 nm in 96% acetone as
in a 1 : 1 (v/v)
mixture of DMS0 and
90% acetone (Fig. 1 ), so that equations
such as those recommended by SCORUNESCO (1966) for the estimation of
chlorophylls in 90% acetone are also valid
for DMSO/acetone.
The SCOR-UNESCO
(1966) equations were used to calculate
928
Notes
Table 1. Comparison
using dimethyl sulfoxide
of chlorophyll
extraction
and 90% acetone.
Chl a*
(mg/&)
Fvtractant
Diatoms
Litsschia
-__
(Chrysophyta)
sp. (01157406)+
Cyclotelln
____.
Greens
-IChlorelln
$1,.
(011.5740’)+
(Chlorophyta)
prenoldosa
Tetrnedron
--~
(Yo.
hitrldens
(Uo.
Selenastrum
capricornutum+
Scenedesmus
ql~adricnuda
Oocystis
marssonii
Ankistrodesmus
Blue-greens
Anac~
Anabaena
~_____
120)
(No.
(No.
braunii
Green flagellate
Chlamydomonas
395)s
76)
287)
(No.
245)
(Chlorophyta)
reinhardtii
(h’o.89)
(Cyanophyta)
nidulans
(No.
625)
flo?-aquae+
-Frcmvella
* Concentration
recommended
*losim
__
_ (No.
481)
in mg/k calculated
SCOR-UNESCO
(1966).
+ These
algae
here obtained
from
Research
Program,
fnvironmental
Corvallis,
Oregon.
:
Chl b*
(w/e)
nm0
Acetone
1.57
1.71
O.OD
@.OO
nvso
Acetone
(I.86
0.86
0. on
0.00
DNX
Acetone
2.06
il.64
0.64
0.11
DVSO
Acetone
1.81
0.76
0.6.5
0.23
DblSO
Acetone
1.18
0.02
0.30
0.00
DMS0
Acetone
1. is
0.60
O.bb
0.71
DMS0
Acetone
1.55
0.07
0.36
0.00
DVSO
Acetone
1.05
0.21
0.59
0.14
DMS0
Acetone
0.89
0.89
0.47
0.48
nMso
Acetone
0.40
0.40
0.00
0.00
nKi0
Acetone
2.45
2.45
0.00
0.00
DMS0
Acetone
3.28
3.16
0.00
0.00
using
the
the National
Protection
equations
Eutrophication
Agency,
The algae
kith
a two or three
digit
number
after
the name
were obtained
from the culture
collection
of algae
at
Indiana
University
(Starr
1964).
chlorophyll a and b concentrations (Table
1). The ratio of absorbance at 663 nm
(for chlorophyll a) and 645 nm (for chlorophyll b ) before and after acidification
(with hydrochloric acid) was the same in
90% acetone as in DMSO/acetone
for the
pure chlorophylls.
The ratio of absorbance
at 663 nm before and after acidification
was the same in either solvent with the algal extracts.
DMS0 was superior to acetone for the
extraction of chlorophyll from green algae.
This extraction appeared to be complete
in that further extraction produced no additional chlorophyll
nor was the centrifuged precipitate green. DMS0 was as
effective as 90% acetone for the diatoms
and blue-green algae tested, and the turbidity which often accompanies acetone extracts (Strain and Svec 1966) was not observed. Table 1 compares extractions for
12 species using acetone and DMS0 acetone mixture. With all green algae tested,
DMS0 was far superior.
Note: The toxicity of dimethyl sulfoxide
is relatively low. However, because of its
skin penetration
properties
( similar to
those of ethanol, toluene, benzene, carbon
tetrachloride,
and dimethyl
formamide )
any such solvent should be used with care.
A discussion of the chemistry and toxicology of DMS0 can be found in Jacob et
al. ( 1971).
W. Thomas Shoaf
Bruce W. Lium
U.S. Geological Survey
6481 Peachtree Industrial Blvd.
Doraville, Georgia 30340
References
GOTTSCHALK, W., AND F. MULLER.
1964. Quantitative Pigmentuntersuchungen
an strahleninduzierten Chlorophyllmutanten
von Pisum satiuum.
Planta 61: 259-282.
JACOB, S. W., E. E. ROSENBAUM, AND D. C. WOOD.
1971. Dimethyl
sulfoxide, v. 1. Basic concepts of DMSO.
Dekker.
SCOR-UNESCO.
1966. Determination
of photosynthetic
pigments in sea-water.
Monogr.
Oceanogr. Methodol.
1. 69 p.
SEELEY, G. R., M. J. DUNCAN, AND W. E. VIDAVER.
1972. Preparative
and analytical
extraction
of pigments from brown algae with dimethyl
sulfoxide.
Mar. Biol. 12: 184-188.
SLACK, K. V., R. C. AVERETT, P. E. GREESON, AND
R. G. LIPSCOMB. 1973. Methods for collection and analysis of aquatic biological
and
microbiological
samples.
U.S. Geol. Surv.
Tech. Water Resour. Invest. Bk. 5.
STARR, R. C. 1964. The culture collection
of
algae at Indiana University.
Am. J. Bot. 51:
1013-1044.
STRAIN, H. H., AND W. A. SVEC. 1966. Extraction, separation,
estimation
and isolation
of
chlorophylls,
p. 21-66.
In L. P. Vernon and
G. R. Seeley [eds.], The chlorophylls.
Academic.
VERNON, L. P. 1960. Spectrophotometric
determination of chlorophylls
and phaeophytins
in
plant extracts.
Anal. Chem. 32: 1144-1150.
WEBER, C. I. 1973. Biological
field and laboratory methods for measuring the quality of
U.S. EPA-670/
surface waters and effluents.
4-73-001.
Environ.
Monit. Ser. 164 p.
Submitted: 14 November 1975
Accented: 27 Anri 1976