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The morphology and taxonomy of Chlorococcum submarinum

(Chlorococcales) isolated from a tidal rockpool

Article  in  British Phycological Journal · June 1991

DOI: 10.1080/00071619100650101

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The morphology and taxonomy

of Chlorococcum submarinum
(Chlorococcales) isolated from a tidal
rockpool
a c b a
John R. Blackwell , Eileen J. Cox & D. James Gilmour
a
Robert Hill Institute, Department of Molecular Biology and
Biotechnology , University of Sheffield , Sheffield, S10 2TN, UK
b
Department of Animal and Plant Sciences , University of Sheffield ,
Sheffield, S10 2TN, UK
c
Department of Biological Sciences, University , College of Wales ,
Aberystwyth, Dyfed, SY23 3DA, UK
Published online: 17 Feb 2007.

To cite this article: John R. Blackwell , Eileen J. Cox & D. James Gilmour (1991) The morphology
and taxonomy of Chlorococcum submarinum (Chlorococcales) isolated from a tidal rockpool, British
Phycological Journal, 26:2, 133-139, DOI: 10.1080/00071619100650101

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 Br. phycol. J. 26:133-139
l June 1991

T h e Morphology and Taxonomy of Chlorococcum submarinum

(Chlorococcales) Isolated from a T i d a l Rockpool

By JOHN R. BLACKWELL*,EILEEN J. Cox'~ and D. JAMES GILMOUR~

Robert Hill Institute, Department of Molecular Biology and Biotechnology and tDepartment
of Animal and Plant Sciences, University of Sheffield, Sheffield SIO 2TN, UK

A unicellular green alga recently isolated from a Yorkshire (UK) tidal rockpool is shown to
be a species of Chlorococcum, very similar to the previously characterized species
C. submarinum. Although the original description of C. submarinum is incomplete and does
not meet current taxonomic specifications, we nevertheless consider our isolate conspecific
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with it. Our isolate can be differentiated from all other currently recognized species of
Chlorococcum. A full description of C. submarinum is given, with comments on the use of
standard techniques in taxonomic assessments.

In 1986, we isolated a unicellular green
alga from a tidal rockpool at Filey Brigg on
the Yorkshire (UK) coast. This organism has
non-motile vegetative cells, each with a
parietal cup-shaped chloroplast containing a
pyrenoid and walled zoospores with two
flagella of equal length. It is, therefore, a
member of the genus Chlorococcum
Meneghini (Starr, 1955). The features of our
Filey isolate are very similar to those of
Chlorococcum submarinum Alvik, which was
isolated from Norwegian oyster beds and
described by Alvik (1934). More recently,
Rees & Russell (1977) rediscovered this alga
at Hilbre Island, River Dee, Cheshire and
deposited a culture with the Culture Centre
of Algae and Protozoa, Oban, U K (strain
no. 213/10). A comparison o f Hilbre Island
isolate with the Filey isolate showed them to
be very similar if not identical. However,
since Alvik (1934) described C. submarinum,
standardized conditions for the isolation,
growth and identification of members of the
genus Chlorococcum have been prescribed in
two detailed works (Starr, 1955; Archibald &
Bold, 1970). These publications also listed
* Present address: Department of BiologicalSciences,
University College of Wales, Aberystwyth, Dyfed
SY23 3DA, UK.
++Corresponding author.
algae that had been described as members of
Chlorococcum and grouped them into
(a) algae which conformed to stipulated
taxonomic requirements and (b) those which
did not, C. submarinum was overlooked and
appeared in neither list. C. submarinum also
does not appear in the key to Chlorococcum
species drawn up by Gartner & Ettl (1988),
although it is listed in a comprehensive
review of freshwater phytoplankton
(Komarek & Fott, 1983), with reproductions
of some of Alvik's original drawings. Having
been alerted to Alvik's work, Archibald con-
sidered (pers. comm.) that the description
was too incomplete to allow it to be included
as a properly described species. In her
opinion, to be certain of its taxonomic
status, morphological observations of the
organism ought to be performed, using the
same media and conditions as Starr (1955)
and Archibald & Bold (1970). In this paper
we provide a full description of the Filey
isolate adopting, as far as possible, these
criteria.
MATERIALS AND METHODS
Isolation and selection
A shaded pool on the Ball Beds on the north
side of Filey Brigg, Yorkshire, UK (O.S. grid

0007-1617/91/020133+07 $03.00/0 © 1991 British PhycologicalSociety

 134 J . R . Blackwell, E. J. Cox and D. J. Gilmour
reference TA 1381) was selected for sampling
(Blackwell, 1990). It was about 10 m beyond the
eastern end of the boulder clay cliff of Carr Naze
and about 1"5 m below the top of the reef. On
26 August 1986, a 100 cm 3 aliquot of water was
taken using a sterile bottle (autoclaved and kept
in foil until sampling). A spatula, similarly steri-
lized, was used to scrape up some of the sediment
from the bottom of the pool and to introduce
about 1 g into the sampling bottle. On return to
the laboratory, the sample was shaken vigorously
and approximately 35 em 3 was poured into a
sterile 125cm 3 conical flask with 50cm 3 of
Woods Hole MBL medium with the optional
NaHCO3, set at pH 7.5 using 20 mol m -3 tris-
(hydroxymethyl)aminoethane (Tris) buffer and
supplemented with 0.175 mol m -3 NH4HPO 4 and
1 kmol m -3 NaCI (Nichols, 1973). The sample
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was incubated at a constant 20+ 1-5°C, illumi-
nated continuously with an intensity of 50-60 laE
m -2 s -l and shaken at 115 rpm for 9 days. After
this period a dense brown community had
developed. The benthic component was partially
suspended by scraping with a sterile spatula and
vigorous shaking. A 35 cm 3 aliquot of the suspen-
sion was poured into a sterile 50 cm 3 glass centri-
fuge tube, which was then covered by sterile
aluminium foil and centrifuged at 3000 g for
10 min. The supernatant was discarded and the
pellet was resuspended, by vortex mixing, in 5 cm 3
of Woods Hole MBL medium with no added
NaCI. This suspension was poured into a fresh
sterile 125 cm 3 conical flask and the volume was
made up to 75 cm 3 with Woods Hole MBL
medium without NaCI. The osmotically shocked
culture was then incubated as above for a further
9 days. At the end of this time, the organism
which had come to dominate the culture was a
unicellular green alga. Axenic colonies of this
organism were produced by serially streaking out
colonies derived from single cells on Woods Hole
MBL medium set in 1-5% agar.
Taxonomic tests
The materials and methods employed for sub-
sequent taxonomic investigation of the organism
are as detailed in Archibald & Bold (1970), but
with three major exceptions. Firstly, as the alga
would not grow in liquid unshaken 3N Bold's
Basal Medium (BBM) unless bicarbonate was
present, liquid cultures were maintained in 3N
BBM supplemented with 5 tool rn -3 NaHCO3.
Secondly, zoospores were not observed in or on
any of the modified BBM media used by
Archibald & Bold (1970), but were produced in
abundance both in liquid and on solidified Woods
Hole MBL media. Zoospore morphology was,
therefore, described after growth on Woods Hole
MBL medium, supplemented with 0"25 kmol m -3
NaC1 (the optimal salt concentration for growth
of this strain). Thirdly, very clear nuclear staining
was achieved using the fluorochrome ethidium
bromide. For cells grown on 3N BBM agar,
colonies were picked off using a nichrome wire
loop and were dispersed in a drop of liquid 3N
BBM mixed with a drop of aqueous ethidium
bromide solution (20 ktg ml -z) on a clean micro-
scope slide. For cells grown on 0"25 kmol m -3
NaCI Woods Hole agar, 1 drop of 0"5 kmol m -3
NaCI Woods Hole medium was mixed with 1
drop of 0.25 kmol m -3 culture and 1 drop of
ethidium bromide, to maintain isotonicity. The
cells were then examined using a Leitz Ortholux 2
fluorescence microscope fitted with a pressure
mercury lamp and a four-mirror (Ploempak) filter
system. Filter 4 (Excitation Filter BP 530-560,
Absorption Filter LP 580) was used for this work.
Photographs were taken using a Leitz
Vario-Orthomat 2 automatic microscope camera
with spot lightmeter fitting. The film used was
Kodak Ektachrome 200 rated at 400 and
800 ASA.
Electron microscopy
Samples were fixed in 3% glutaraldehyde in
100 mol m -3 phosphate buffer. After half an hour
the specimens were re-centrifuged and washed in
10% sucrose in 100 mol m -3 phosphate buffer,
using three changes of half an hour followed by
an overnight incubation. The cells were post-fixed
in 2% aqueous osmium tetroxide for an hour, re-
centrifuged and placed in specimen vials. The
samples were then dehydrated using ethanol solu-
tions, the last 100% ethanol change being dried
over anhydrous copper sulphate. Embedding was
performed by transferring the cells through two
15 min changes in propylene oxide and leaving
overnight in propylene oxide:araldite resin. The
specimens were then placed in pure araldite resin
for 6-8 h before embedding in fresh resin, being
left for 48 h at 60°C to polymerize.
OBSERVATIONS
I n logarithmic growth phase, vegetative
cells vary between spherical cells 10-15 Ixm
in diameter with walls up to 1 lam, thick a n d
ellipsoidal cells 5 - 7 x 8-11 lxm with t h i n n e r
walls (up to 0.5 ~tm). I n s t a t i o n a r y phase the
cells are spherical, u p to 16 lxm in diameter
with thicker walls (up to 2 ~tm). The cells
have a distinct p y r e n o i d s u r r o u n d e d by a
biplate starch sheath a n d are u n i n u c l e a t e
 Chlorococcum from a tidal pool 135
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(a) (b)

(c)
00 (d)

I I
8/.~rn
FIG. 1. Drawings of Chlorococcum submarinum. (a) Zoospore showing stigma, nucleus, split pyrenoid and flagella
originating from papilla; (b) vegetative cell in log phase of growth; (c)aplanospores being released from
aplanosporium; (d) stationaryphase vegetativecell showingincreased thicknessof wall.

(Fig. 1). Asexual reproduction is by zoo- Sexual reproduction is by fusion of iso-

spores and aplanospores formed by succes-
sive bipartition of the protoplasm. The zoo-
spores are 3 ~tm wide by 8 ~tm long with a
median stigma, anterior nucleus and two
flagella arising from a small papilla (Fig. 1).
gametes indistinguishable from zoospores.
Figure 2 is an electron micrograph of
several algal cells showing the cup-shaped
chloroplast with a pyrenoid embedded in the
thickened basal part and a single nucleus
 136 J.R. Blackwell, E. J. Cox and D. J. Gilmour
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FIG. 2. Transmission electron micrograph of a group of Chlorococcum submarinum cells grown in 0.25 kmol m 3
NaC1 medium showing cup-shaped chloroplasts (C), a split pyrenoid (P), single nuclei (N) and lipid droplets (L).
Magnification × 13865.

within the cup. The electron-dense areas
obvious in most of the cells are large lipid
deposits. The diagnostic split pyrenoid
through which at least one thylakoid passes
is also shown in Fig. 2. Confirmation that
the ceils were uninucleate was provided by
staining the cells with the fluorochrome ethi-
dium bromide (data not shown).
A number of features were investigated in
culture as follows.
Morphology of colonies on agar. After 4
weeks' growth on 3N BBM agar, the
colonies were dull-shiny (sensu Archibald &
Bold, 1970), smooth, hemi-spherical, olive-
green mounds.
Production of secondary pigments. There was
no obvious accumulation of carotenoids or
other secondary pigments after 4 weeks'
growth on 3N BBM medium in agar.
Nitrate reduction. C. submarinum failed to
grow in the nutrient broth used to test this
ability, or on nutrient agar supplemented
with nitrate. Thus, it did not fit into any of
the previously recorded categories.
Starch digestion. C. submarinum produces no
extracellular amylases under standard test
conditions.
Gelatin hydrolysis. Cells stabbed into 3N
BBM gelled with gelatin in test tubes failed
to grow. While this is characteristic of a
number of species of Chlorococcum, it was
felt that in the case of the Filey isolate the
failure could be due to low bicarbonate
tensions since it had been shown to be sensi-
tive to these conditions. In addition to the
standard test, therefore, algae were streaked
onto the surface of the gels. The alga did not
grow in or on any of the gels and so it was
 Chlorococcum from a tidal pool
concluded that the alga is unable to use
gelatin as a sole source of nitrogen.
Tolerance of crystal violet. In the presence of
crystal violet, the Filey isolate grew well at
concentrations up to 1 x 10 4% (w/v),
poorly at 1 x 10 3%, very slightly at
2× 10 3% and growth was completely
inhibited at 4-5 x 10 3%.
DISCUSSION
Starr (1955) considered that "The genus
Chlorococcum is distinguished from other
genera in the Chlorococcaceae by its
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possession of three constant attributes:
asexual reproduction by means of
Chlamydomonas-type zoospores, vegetative
cells with a parietal, hollow spherical chro-
matophore and vegetative cells with at least
one pyrenoid". "Chlamydomonas-type"
zoospores were defined (Starr, 1955) as
walled and possessing two flagella of equal
length. There is no doubt, therefore, that the
Filey isolate is a species of Chlorococcum. To
ascertain its specific identity, comparison
was made with the key to currently recog-
nized species of Chlorococcum (Archibald,
1988). According to this key, which super-
sedes the earlier work (Archibald & Bold,
1970), the only species closely resembling the
Filey isolate is C. pamirum, which was
described by Archibald in 1988. Indeed,
C. pamirum seems to be morphologically
very similar to our isolate, but C. pamirum
cells are recorded as being bi- to multi-
nucleate whereas those of the Filey isolate
are uninucleate. Although isolated from
different habitats, there is little morphologi-
cal evidence on which to separate the strains,
except that they differ consistently in the
number of nuclei per cell. However, different
m~hodologies for nuclear staining have
been employed so that artefactual effects
cannot be excluded.
In his 1955 monograph, Starr recom-
mended that the algae should be fixed in
Schaudinn's fixative, mordanted with iron
alum (after dehydration), stained with
haematoxylin and then differentiated with
137
iron alum. Mercuric chloride-containing
fixatives such as Schaudinn's are recognized
as being excellent for the fixation of chroma-
tin (Boon & Drijver, 1986). Haematoxylin
has a long history as a nuclear dye (Bohmer,
1865; Conn, 1961; Gurr, 1965; Boon &
Drijver, 1986) and is the most frequently
used natural dye in microtechnique. Also,
iron alum is the most commonly used
mordant with haematoxylin (Boon &
Drijver, 1986). Thus, the nuclear staining
procedure followed by Starr is one thor-
oughly recommended by a number of auth-
orities. Archibald & Bold (1970) however,
for reasons which were not stated, rejected
this procedure, and used dilute potassium
iodide to stain the cells, and to locate the
nuclei. Gurr (1965) includes this technique as
a method for staining nuclei, but states that
it also stains a number of other cell com-
ponents, while Nultsche & Grahle (1979)
warn that, as a nuclear stain, it generates
artefacts. Using Starr's method to fix cells
and stain the nuclei, however, it was difficult
to properly differentiate cells of the Filey
isolate; if young cells were clearly differen-
tiated, the older cells with thicker walls and
denser cytoplasm were homogeneously black
and, if the latter were differentiated the
former had all the colour washed out of
them. Furthermore, the cells accumulate
lipid droplets, which tend to reflect or refract
light in such a way as to be confusingly like
nuclei when out of focus. Dilute potassium
iodide was tried on unfixed cells and, as
Archibald found in C. pcimirum and a
number of other species (Archibald & Bold,
1970; Archibald, 1988) the cells appeared to
have several to many nuclei. Some of these
"nuclei" were, however, located in the
chloroplast or even in the cell wall and so
were clearly artefacts. Of the others, it was
unclear how many were genuine nuclei. This
method seemed, therefore, to be highly un-
satisfactory. A number of other fixative and
staining techniques for use with bright field
light microscopy were tested for their ability
to stain C. submarinum cells. Fixatives used
included Fleming's (weak and strong),
ethanol:acetic acid (3:1) and 3% glutaral-
 138 J . R . Blackwell, E. J. Cox and D. J. Giimour
dehyde, both in phosphate buffer, pH 6-8
and in 3N BBM and Woods Hole MBL
media. The stains used included aceto-
carmine, carmalum and basic fuchsin in 40%
acetic acid. All these techniques gave unclear
or variable results, often with several
apparent "nuclei" per cell but these were
obviously artefacts since they were located in
the chloroplast or the cell wall. All these
methods were therefore rejected. However,
excellent results were achieved using fluores-
cence microscopy and the vital fluorochrome
ethidium bromide (Klut, Stockner &
Bisalputra, 1989). This dye intercalates in
double strands of nucleic acids (Jarnagin &
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Luchsinger, 1980) and its use showed very
clearly that each cell has a single nucleus
located in the space surrounded by the "cup'"
of the chloroplast (Fig. 2 and data not
shown).
A further similarity shared by the strains is
that secondary carotenoid production is slow
and not pronounced. However, although
C. pamirum grows better on solidified
medium than in liquid medium,
C. submarinum only grew on solidified
medium and showed n o growth at all in
liquid 3N BBM under standardized condi-
tions unless, as discussed above, it was
supplemented with bicarbonate ions. Also,
C. pamirum readily produces amylase, traces
of gelatinase and nitrate reductase, and
grows in nutrient broth, none of which is
characteristic of C. submarinum. Therefore it
seems probable that these are two separate
species, although it is very difficult to
distinguish between them on purely morpho-
logical grounds.
Despite the limitations of his description,
Alvik's (1934) specimens were also marine in
origin and very similar to our isolate. The
only differences are (a)that the zoospores
were 2-5-3.0×4.3-8.0 pm (compared to
3.0×8.0 gm for zoospores of the Filey
isolate) and (b)that Alvik perceived the
pyrenoid as being either divided into two
segments or whole, whereas electron micro-
scopy shows that the Filey isolate always has
a divided pyrenoid. However, from certain
angles with light microscopy alone, a split
pyrenoid can appear as an entire pyrenoid,
especially in cells with dense cytoplasm. The
difference in zoospore dimensions may also
be due to viewing from an oblique angle.
Alvik (1934) mentioned that no starch had
ever been observed in the alga he was study-
ing, in contrast to the Filey isolate, which
does have starch. If starch was never found,
Alvik's taxon cannot be a member of the
genus Chlorococcum, or even of the
Chlorophyceae, but it is possible that cells in
his cultures, grown on rich, chemically unde-
fined "Schreiber's medium" produced such
dense cytoplasm that the starch was hidden.
Since he also stated that the cells had large
oil reserves, we feel it is more likely that
starch was present but obscured.
Alvik's description also implied that the
release of zoospores from C. submarinum
occurred by dissolution of the whole wall,
whereas the usual mode of release in
Chlorococcum is that the outer wall layers
first split and only later dissolve. This was
observed in our material.
In the absence of holotype material or
cultures from Alvik's collection we cannot
prove unequivocally that the Filey isolate is
identical with Alvik's taxon, but we consider
it highly likely. Thus, we are in agreement
with Rees & Russell (1977) that their Hilbre
Island isolate, with which our Filey isolate
agrees, belongs to the species C. submarinum
Alvik. It should be pointed out, however,
that had the prescribed standard isolation
and culture procedures (Starr, 1955;
Archibald & Bold, 1970) alone been
followed, C. submarinum could not have
been placed in Chlorococcum where it clearly
belongs. There are inherent dangers in
assuming, a priori, that all members of a
taxon will respond to particular growth
conditions, especially when habitats which
are potentially favourable to the genus have
not been investigated. Thus, while we agree
that, as far as possible, comparisons should
be made under identical conditions, it must
be recognized that this is not always feasible.
Growth under standardized conditions
 Chlorococcum from a tidal pool
should be regarded as a recommendation
rather than an absolute requirement for allo-
cation to a taxon.
Amended description of Chlorococcum
submarinum
Spherical vegetative cells 10-15 ~tm in log
phase, with walls 1 pm thick or less, ellip-
soidal vegetative cells 5-7 × 8-11 lam in log
phase culture with thin walls (0.5 I~m or less);
in stationary phase cells up to 16 ~tm, their
wall thickening up to 2 ~tm; distinct pyrenoid
surrounded by a biplate starch sheath; cells
uninucleate.
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Asexual reproduction by zoospores and
aplanospores formed by successive biparti-
tion of the protoplasm, the former oval,
3 ~tm w i d e × 8 ~tm long with a median
stigma, anterior nucleus, flagella arising from
a small papilla. Sexual reproduction by
fusion of isogametes indistinguishable from
zoospores.
Source: Norwegian oyster beds.
ACKNOWLEDGEMENTS
J. R. Blackwell was the recipient of a research
studentship from the Natural Environment
Research Council. We wish to thank John Procter
of the Sheffield University Electron Microscope
Unit for preparing the micrographs and Mick
Turton for producing the final photographs.
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