Professional Documents
Culture Documents
FOR PHYTOPLANKTON
STUDY
of Fisheries
and Oceanography,
C.S.LR.O.,
Cronulla,
Australia
ABSTBACT
INTBODUCTION
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
of Information
OF THE
METHOD
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
STUDY
35