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A METHOD

FOR PHYTOPLANKTON

STUDY

33. J. Ferguson Wood


Division

of Fisheries

and Oceanography,

C.S.LR.O.,

Cronulla,

Australia

ABSTBACT

A method for quantitative


and qualitative
investigations
of phytoplankton
which can be
employed at sea on fresh material is described.
Samples arc collected in van Dorn type samplers, ccntrifugcd
and examined under the
fluorcsccncc
microscope.
Phytoplankton
is counted by taking advantage of the red autofluorescence
of chlorophyll,
and total organsims by the Striigger
technique
using the
induced fluorescence
of acridine orange. Total particles
(Icptopcl)
can be counted by
ordinary light using the same cquipmcnt.
A Pctroff Hausscr bacterial counter is used as
this has a thin slide and gives maximum light intensity for fluorcscencc of the object. Qualitativc examinations are made on the same material, after sedimentation,
using a special cell
to minimize the effect of the ships motion.

mit Lund and Tallings advantage to be


fully realized.
In the authors opinion, counting has a
definite place amongst the methods of phytoplankton study, especially in areas where
particulate matter is liable to vitiate other
methods of assessing standing crop. It is
easy to distinguish
between chlorophyllbearing, non-photosynthetic
and other material, and to count the particles of each type
under the microscope at sea by the methods
described in this paper.

INTBODUCTION

Recent reviews of the measurement of


phytoplankton
(e.g., Braarud 1957; Lund
and Talling 1957; Strickland 1960) differ in
their judgment of the usefulness of the methods available. The recent trend has been
towards chemical and biochemical methods,
which have the disadvantage that they do
not take account of biological factors such as
the growth phase of the organisms or the
diversity index ( Margalef 1958) of the population.
Braarud and Strickland stress the fact that
counting does not give a mcasurc of the
standing crop unless a weighting procedure
is adopted. Lund and Talling, while admitting this, believe that counting is justified by
the three great advantages it possesses, nix.,
that the algae are observed each time a
count is made so that any changes in appearance, size, shape or aggregation of the cells
can be seen . . . that estimations can be made
of populations whose density is so small that
again at present no other method can be used
with equal accuracy and , . . that counting
enables small numbers of specific algae to
be distinguished from others and from unwanted debris; although the latter may often
limit the sensitivity of the estimation. These
authors suggest further study of methods of
estimating cell volume in conjunction with
counting.
If a rapid and easy method of estimating
cell volumes can be obtained (e.g., perhaps
by conductivity changes of organisms passing through an orifice), counting would per-

METHODS

Sampling
For samples taken on station, plastic samplers of the van Dorn type as described by
Davis ( 1957) are used from a Kelvin sounding winch or a bathythermograph
winch.
For samples taken under way, a cylindrical sampler with tapered nose and a
finned tail is used. This sampler has a tube
extending from nose to about Y2in. from the
base, and four %-in. holes just behind the
nose cone. It is towed on a bridle and holds
just over 5 L, and can collect samples from
a light winch at speeds of up to 15 knots.
Surface samples arc collected in this way
because bucket samples frequently contain
material from the ships side, e.g., Enteromorpha.
For purely qualitative studies, such as distribution studies of the larger diatoms and
dinoflagellates, a modified Hardy plankton
indicator is used. The front fins are removed,
and a grid of 120 mesh/in. is inserted in place
32

A METHOD

FOR PIIYTOPLANKTON

STUDY

33

of the standard grid, This sampler can be light to confirm his diagnosis if ncccssary.
Thus, a count of the chlorophyll-bearing
towed at the surface at speeds up to 15 knots,
and can be hand-hauled. It is often used on organisms is possible. The total number of
merchant ships and other vessels not fitted _living organisms is observed by means of
with hydrological winches.
acridinc orange (l/5,000) which stains living protoplasm and fluoresces green under
Treatment of Samples
blue-violet light. Organisms without chloroplasts are then estimated by difference.
Water from the 5-L samplers is transIn fluorescence, the microscope is used
ferred to polythene bottles and taken to the
laboratory.
Routine oceanic samples are with the monocular tube, to avoid light abexamined on board ship. The whole 5 L are sorption by prisms. An Abbe condenser, 10,
20, or 40 objectives and 5 or 10X oculars are
centrifuged through a continuous Foerstnormally used as required. A mechanical
type centrifuge ( Davis 1957). Centrifugastage is very desirable, but special microtion takes 15 min at 15,000 rev/min.
The
solids which have adhered to the walls of the scopic equipment is not essential, though it
can improve the ease of counting. A bluecup are carefully suspended in the sea water
violet filter is mounted in the substage filter
remaining in the cup, carefully washed into
a graduated vessel and the volume made up holder and a yellow compensating filter is
placed over the eycpicce. Filters used are a
to 10 ml.
Material collected on the Hardy grid is Wild J3G12 with an OGl exclusion filter.
Ten fields are counted if there are less
washed into a vial, and formalin added to
.
than 5 organisms per field; 4 fields if there
give 2% final concentration.
This material
are 5 or more. The mean number of organis usually examined in a shore laboratory.
isms per field is recorded and the count is
Quantitative Examination
expressed as organisms per liter. Although
The suspension from the centrifuge is the Pctroff Hausscr counter has a grid,
which can be made visible in blue light by
carefully shaken to disperse the organisms
the use of fluorescent paint, it is more conand a drop placed on the grid of a Petroff
venient to count fields, and divide the result
Hausser bacterial counting chamber. The
by the area of the field to give results per
standard cover glass is placed on the slide
mm2 of slide, which forms a cell l/SO mm
which is examined on the stage of a monocudeep.
lar microscope. A Petroff Hausser chamber
In addition to the fluoresccncc examinais used because it has a thin slide which
allows better illumination of the object than tion, it is usual to examine the slide in white
does a thicker hcmacytomctcr slide, cspc- light, in order to count the total number of
particles (leptopel),
and to dctcrminc the
cially as it is necessary to use an immersion
ratio between larger and smaller phytocondenser to obtain fluorescence (Wood
plankton elcmcnts. If the phytoplankton
1956).
numbers arc large, differential counts can
The basis of the quantitative examination
also be made, but this is best done in the
is the autofluorescence of chlorophyll, which
chamber described below,
fluoresces bright red in blue-violet light,
followed by the green fluorescence of living
Qualitative Examination
protoplasm when stained by acridine orange
according to the method of Strtigger ( 1948).
The centrifuged samples are allowed to
Chlorophyll-bearing
organisms appear as settle for 10 min or more, or better are again
single or groups of red shapes, according to centrifuged by means of a hand centrifuge,
the shape and number of the chloroplasts.
A sample of the concentrated sediment is
Once the observer is familiar with the phytoplaced in a small cell made by cementing a
plankton organisms of the area, little diffiring of thin (1 mm) lucite to a 3 x l-in.
culty is experienced in determining whether
glass slide. The internal diameter of the ring
he is looking at a single organism or a group
is about 15 mm, and a groove is cut in one
of organisms, He can also revert to white
side of the ring to allow water to escape

34

E. J. FERGUSON

when a cover glass is put on, The groove


helps to avoid air bubbles between the cover
glass and the water, and, if the glass fits
snugly to the ring, movement of the sample
due to the ship will be greatly reduced, so
that examinations even under high power
are possible on a ship either hove to or under
way. Examinations
have been made on
board ship in winds up to 50 knots.
For qualitative examination, a binocular
tube replaces the monocular and phase-contrast can be used if required. The smaller
phytoplankton
organisms can be readily
identified.
If doubt arises as to whether a
given organism possesses chlorophyll, fluorescent light can be used to solve the point;
for example, a tintinnid was found to possess
zooxanthellae, and Ornithocercus splendidus to possess chlorophyll in the girdle lists.
In order to facilitate identifications of the
organisms at sea, an atlas of diatoms, dinoflagellates and myxophyceae of the region
has been prepared, and is supplied to each
research vessel. As new records are made,
illustrations are prepared and included in
the Atlas prior to publication elsewhere so
that the atlas may at all times be up to date.
Recording

of Information

Quantitative results for 6 depths (0,25,50,


75, 100, and 150 m ) are recorded for each
station, and later punched on IBM cards
together with the rest of the data from that
station.
Qualitative results are recorded on edgepunched cards so that station list, charts of
occurrence, salinity-temperature
relations,
seasonal fluctuations,
etc., can be easily
derived.
DISCUSSION

OF THE

METHOD

The generally accepted method for microscopic study of phytoplankton


is that of
Utermohl ( 1936), in which the phytoplankton is allowed to settle in a cylindrical vessel
and cxamincd by an inverted microscope. It
is claimed frequently that loss of organisms
by centrifugation
or filtration is avoided.
Losses by centrifugation are placed by Steemann Nielsen ( 1938) and others as about
30%, but this applies to discontinuous and
not to continuous centrifugation.
In centri-

WOOD

fugation by the means used in this laboratory, cultures of marine bacteria ( a Bacillus
and a Pseudomonas) with 200 organisms/ml
were completely retained by the centrifuge
as shown by sterility tests on the effluent.
Further, filtered effluent from centrifuged
plankton catches collected on HA Milliporc
Filters showed less than 1% of the number of
organisms in the plankton, except in the case
of Oscillatoria ( Trichodesmium)
erythraea
which can neither be centrifuged or sedimented, when in the bloom stage.
Filtration has been compared with centrifugation by Ballantine ( 1953) who showed
the latter to be more effective. The writer
found that, by directing a quartz-mercury
lamp on fresh phytoplankton
on milliporc
filters the red fluorescence of chlorophyll
could bc observed. However, when blue
light was directed through the lens and
prism system 0E a Zetopan microscope, from
a quartz-mercury
lamp, insufficient
light
could be brought to bear on the filters, and
the fluorescence technique could not be
used.
The disadvantages of the Utcrmijhl system are that living material cannot be used,
and it is not possible to distinguish routinely
between photosynthetic and non-photosynthetic microorganisms, especially in the case
of the ultra plankton, This could lead to
false estimations of the phytoplankton, since
I have found that colorless flagellates frequently represent a large, at times a major,
portion of the total microorganisms.
In the method described in this paper, living phytoplankton is studied; thus, differentiation between photosynthetic
(i.e., chlorophyll-bearing ) and colorless organisms is
rapid and easy, qualitative examination of
the same material is easy, so direct correlations can be made. Phytoplankton
can be
studied aboard ship on and between stations.
This last is a very great advantage as programs can be varied immediately if interesting features of distribution
require it.
Further, the equipment described is relatively simple and inexpensive in comparison
with most oceanographic equipment, and is
compact, requiring about 6 ft of bench space.
It is recognized that recently dead organ-

A METHOD

FOR PHYTOPLANKTON

isms will give the chlorophyll fluorescence


and that the distinction between living and
dead organisms is not always clear when
acridine orange is used. This difficulty
occurs also when other stains arc used. Largi
chloroplasts in small organisms and diffuse.
chlorophyll can mask the green fluorescence
of acridine orange.
REFERENCE3

BALLANTINE, D. 1953. Comparison of the diffcrcnt methods of estimating nannoplankton.


J.
Mar. Biol. Assoc. U.K., 32: 129-147.
BRAARUD, T. 1957. Counting
methods for the
determination
of standing crop of phytoplankton. Rapp. Proc. Verb., Cons. Int. Explor.
Mer, 144: 17-19.
DAVIS, P. S. 1957. A method for the dctcrmination of chlorophyll
in sea water.
C.S.I.R.O.
Aust. Div. Fish Oceanogr. Rep. 7.
LUND, J. W. G., AND J. F. TALLING.
1957. Botanical limnological
methods with special refcrence to the algae.
Bot. Rev., 23: 489.

STUDY

35

MARGALEF, R. 1958. Spatial hcterogencity


and
temporal succession of phytoplankton.
Proc.
Symposium Pcrspectivcs in Mar. Biol., pp. 323349. Univ. Calif. Press.
STE~ANN
NIELSEN, E. 1933. Ober quantitative
Untcrsuchung
von marincm Plankton mit Utcrmijhls umgekchrtem
mikroskop.
J. Cons. Int.
Explor. Mcr, 8: 201-210.
-,
AND T. VON BRAND. 1934. Quantitative
Zcntrifugcn-mcthoden
zur
Planktonbcstimmung.
Rapp. Proc. Verb., Cons. Int, Explor.
Mer. 89: 87-99.
STRICKLAND, J. D. II.
1960. Measuring the production of marinc phytoplankton,
Fish, Rcs.
Bd. Canada. Bull. 122.
STR~~GGE~, S. 1948. Dcr Gegcnwartige
Stand
der Forschung auf dem Gcbiet dcr fluoreszcnzmikroskopischcn
Untcrsuchung
der Baktcrien.
Mikroskopie,
3 : 23-38.
UTEHM~IIL, H. 1936. Quantitativcn
Methoden zur
Untcrsuchung
des Nannoplanktons.
Handb.
Biochcm. Arbeitsmcthoden,
9: 1879-1937.
WOOD, E. J. F. 1956. Fluorcsccnt microscopy in
marinc microbiology.
J. Cons. Int. Explor.
Mcr, 21: 6-7.