British Phycological Journal

ISSN: 0007-1617 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/tejp19

A simple separation technique for purifying micro-

algae

S.I. Heaney & G.H.M. Jaworski

To cite this article: S.I. Heaney & G.H.M. Jaworski (1977) A simple separation
technique for purifying micro-algae, British Phycological Journal, 12:2, 171-174, DOI:
10.1080/00071617700650191

To link to this article: http://dx.doi.org/10.1080/00071617700650191

Published online: 17 Feb 2007.

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 Br. phycol. J. 12:171-174
1 June 1977

A SIMPLE SEPARATION TECHNIQUE FOR

PURIFYING MICRO-ALGAE
By S. I. HEANEY and G. H. M. JAWORSKI
Freshwater Biological Association, The Ferry House, Ambleside, Cumbria LA22 0LP

A simple separation technique using polycarbonate membrane filters for purifying micro-
algae is described.

The mechanical separation of algal cells from other organisms is generally

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regarded as the most satisfactory method of obtaining pure cultures of algae.

Techniques based on this principle have been described by several workers (e.g.
Allen, 1952; Droop, 1969; McCurdy & Hodgson, 1974) and have included the
separation of algae and bacteria by filtration. Recent advances in membrane
filter technology have made possible the efficient separation of particles of
different sizes and enabled simple procedures for the purification of algal
cultures to be developed. This work describes the use of polycarbonate mem-
brane filters, which have advantages over conventional cellulosic membranes,
to isolate pure clonal cultures of two important planktonic algae.

METHODS
THE SEPARATION OF ALGAE FROM BACTERIA
The apparatus shown in Fig. 1 was used to separate the algae from other smaller organisms
in culture. It consists of a 25 mm diameter Nucleopore Swin-LokT M membrane holder (A)
(Nucleopore Corporation, Pleasanton, California, U.S.A.) containing an 8.0/~m maximum
pore diameter Nucleopore membrane. This pore size was chosen as it retained most of the algae
but allowed the bacteria to pass through. A glass syringe and silicon rubber plug were attached
to the cap of the membrane holder and a silicon rubber tube attached to its base. The tube was
connected to a Buchner flask and a vacuum provided by a small pump. A 13 mm diameter
membrane holder (B) containing a 0"22 tzm pore size membrane was attached to the effluent
tube via a T piece. This filter was connected to the laboratory air supply and sterilized the air
used for backwashing; the filter was raised slightly from the horizontal to prevent culture
medium entering. Two clamps (C and D) were attached to the tubing as indicated, and these
allowed the vacuum and air pressures to be varied.
Sterile culture medium was added to the syringe with the clamps C and D closed. The
vacuum pump and air supply were switched on and the clamps adjusted so that the culture
medium was drawn slowly through the membrane filter. With further careful adjustment of
these clamps, it was possible to achieve conditions where the liquid was alternately drawn
through and backwashed across the membrane. The optimal adjustments were when slightly
more medium was drawn from the syringe than was backwashed into it. When the apparatus
was working satisfactorily about 50 ml of culture medium was filtered through followed by a
few mls of algal culture (culture density c. 10 t~g chlorophyll a l-t). The algal inoculum was
then washed by adding gradually 30-100 ml of culture medium. Clamps C and D were closed
when only a few ml remained in the syringe, and the vacuum pump and air supply were
switched off. The contents of the syringe were transferred aseptically to a sterile test-tube.

ISOLATION OF ALGAL CLONES

Algal clones were isolated using the pipetting and washing techniques described by Droop
(1969). Drops of filtered culture were placed on glass slides or plates containing agar culture
medium with a micro-pipette. Single filaments or colonies of algae were then washed under a
171
 172 S. I. H E A N E Y A N D G. H. M. J A W O R S K I

[-
I

i
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D[I~

(/
FIG. 1. Filtration apparatus used to purify algal cultures. For explanation of letters
see text.

dissecting microscope through several drops of sterile liquid culture medium. The algal isolates
were transferred finally to Erlenmyer flasks containing liquid culture medium or to clean agar
plates and allowed to grow.

TESTS FOR PURITY

Algal cultures were examined for the presence of contaminating organisms before and after
purification procedures and using old dying cultures. The cultures were examined by phase
contrast and fluorescence microscopy. Samples of the cultures were treated with the fluoro-
chromes acridine orange and euchrysine using the method of Jones (1974). Purity tests were
also performed by inoculating the cultures into a variety of media including those contained in
Fogg (1942), Collins & Willoughby (1962) and MeCurdy & Hodgson (1974). The cultures were
incubated for at least 4 weeks at 8, 20 and 37°C.

RESULTS
PURIFICATION OF Aphanizomenon flos-aquae f. gracile (Lemm.) Elenk., and
ASTERIONELLA FORMOSA Hass.
This method has been used to isolate several pure clones of the blue-green
alga Aphanizomenonflos-aquae from Lough Neagh, Ireland, and the diatom
 A techniquefor purifyingmicro-algae 173
Asterionella formosa from Rydal Water, a lake in the Windermere drainage
basin. All the tests for purity failed to demonstrate the presence of other
organisms, even after numerous subcultures, and the cultures were considered
pure. On one occasion nine filaments of Aphanizomenon from a membrane
filtered culture were isolated on to agar culture medium and 7 days later these
had grown into visible colonies. Four of these colonies were without any
apparent contamination and after transfer to liquid culture were found to be pure.
Aphanizomenon was grown in ASM-1 medium (Gorham et al., 1964), modified
by the addition of 200 mg 1-1 N-2-hydroxyethylpiperazine-N'-ethanesulphonic
acid (HEPES); the pH was adjusted to 7.55, the pKa of this buffer, using 0.1 M
sodium hydroxide. Agar medium was prepared using 1-5 % m/V Oxoid bacterio-
logical agar. The medium for growing Asterionella was solution 10 of Chu (1942)
modified as described in Lund et al. (1975). Cultures of both algae were grown
at an irradiance between 1000-2000 lx [12.5-25/~E (400-700 nm) m -2 s -1 ] pro-
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vided by daylight fluorescent lamps. Aphanizomenon was grown at 20+ I°C and
Asterionella at 16+ I°C.
Both algae grew well in liquid culture media with maximum exponential
growth rates similar to those obtained with cultures containing bacteria. The
optimal doubling time of Asterionella in pure culture was just less than 12 h, the
same as reported by Lund (1949) for cultures with bacteria. The optimal doubling
time of Aphanizomenon, determined from optical density readings at 436 nm,
was 21 h and was not significantly different from the value found by Smith &
Foy (1974) who used a non-axenic culture of the same algal isolate. Clonal
cultures of these algae are maintained at the Ferry House Laboratory of the
Freshwater Biological Association with the strain designations, Asterionella
formosa Hass. L226A and B, and Aphanizomenon flos-aquae f. graeile (Lemm.)
Elenk. L1, L2 and L3. A pure culture of one of the Aphanizomenon clones, strain
designation L1401/1, is also kept by the Culture Centre of Algae and Protozoa,
36 Storey's Way, Cambridge CB3 0DT, England.

DISCUSSION
Nucleopore membranes were used because bacteria pass through the
cylindrical pores of these thin (lO #m) polycarbonate membranes more easily
than across the complicated pore systems of thicker cellulosic filters. The back-
washing enabled bacteria and algae which had settled on the membrane between
the pores to be resuspended in the liquid culture. This gave a more efficient
filtration of bacteria and lowered the bacteria-algal ratio. Several manufactures
of membrane filters now produce filtration cells which incorporate a magnetic
stirrer above the membrane to increase their efficiency. These simpler systems
may provide a more convenient, though more expensive, filtration apparatus
than the one described here.
Both the Aphanizomenon and Asterionella cultures used here had cells with
little or no mucilage, and with the exception of the heterocysts of Aphani-
zomenon, epiphytic bacteria were not observed. Because bacteria were often
found attached to the heterocysts, filaments with these cells were not isolated.
Many species of algae, however, have large mucilagenous sheaths containing
bacteria which would not be removed by filtration alone. This difficulty has
 174 S. I. HEANEY AND G. H. M. JAWORSKI

been overcome for certain mucilagenous blue-green algae by M c C u r d y &

H o d g s o n (1974) who used glass bead dispersion to separate c o n t a m i n a t i n g
bacteria from the algal mucilage. The procedure of isolating filaments on to
agar culture m e d i u m has the advantage that the growth of individual filaments
or cells m a y be followed, a n d bacterial c o n t a m i n a t i o n observed more readily.
Several workers have tried to purify Asterionella b u t have failed to m a i n t a i n
pure clones for long periods (Provasoli, 1958; Hughes & L u n d , 1962). Hughes
& L u n d (t962) f o u n d that growth in their pure culture was erratic c o m p a r e d to
growth in cultures with bacteria present. These difficulties have n o t been
experienced with the clones o b t a i n e d in this work a n d may be due to the
improved culture medium.

ACKNOWLEDGEMENTS
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We wish to thank A. Foster and P. Webster for technical assistance and G. Hall who
performed the bacteriological tests with the Asterionella culture. We also thank Dr J. W. G.
Lund, F.R.S. for his comments and criticisms of the manuscript.
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