Culturing Algae

Culturing
Algae
CarOlina BiOlogical SUDDIU CornDanu
CONTENTS
3
TYPES OF CULTURING
5
MEDIA
7
WATER
8
STERILIZATION
8
CULTURE MAINTENANCE
9
REQUIREMENTS OF SPECIFIC ALGAE
13
ISOLATION AND PURIFICATION TECHNIQUES
19
ALGAE MEDIA
22
FURTHER READING
23
UNIALGAL CULTURES AND SUPPLIES
Additional copies of this booklet may be ordered from
CAROLINA BIOLOOICAL SUPPLY COMPANY
BURLINGTON. NORTH CAROLINA 27215
iO
C1C
1978 Carolina Biological Supply Company
Printed in U.S.A.
ISBN 089278 005 3
CULTURING ALGAE
DANIEL E. JAMES
From the Algae Department,
Carolina Biological Supply Company, Burlington, North Carolina 272/5
As with any diverse group of organisms, algae vary in their requirements.
demanding different media and different handling techniq'ues from culture to
culture. This is compounded when you want a specific culture to do a specific
thing at a specific time in the classroom. Even so, using the basic techniques
employed with algae, you will find this group of organisms one of the simplest
to handle, requiring very little sophisticated equipment, yet allowing you a
wealth of teaching material to demonstrate a wide range of biological princi·
pIes.
Culture media is the basis for establishing good quality algal teaching
materials. A search through the literature of the past few years will yield a
vast number of different media designed to fit any number of circumstances.
One important aspect of selecting an algal medium is to select one that does
what you want it to do with the least amount of work on your part.
TYPES OF eLLTLRI1\G
LONG TER\l PRESER\',-\ T 0:\
Types of cuI.' rin~ m2~ e di Idea in'o long term and short term preserva­
tion. Normally. ne \. _ ale use l ng term culturing to carry stock cultures
over extended eriod.s Q - :ime as a way of maintaining a specific culture in
the laboratory for future use. This means that you must select a med ium
that will keep a c Iture viable for periods of three months to a year without
having to continually subculture. In long term preservation the material
is not usually suitable for classroom use until subcultured in fresh medium
or in another medium for short term growth.
One of the best vehicles for long term culturing is nutrient agar. Bold's
basic agar with proteose peptone added does well for many of the green
algae, and Allen's modified blue-green agar grows a wide range of blue·
green algae. One disadvantage of using agar for long term preservation is
that the cultures must be axenic. For bacterized unialgal cultures and those
that \oviIl not grow on agar, Soil-Water Medium or one of its variants is
good. Add ing a layer of liquid (fresh water or seawater) over agar which con­
tains the nutrients also makes a good long term medium.
By choosing a good medium for long term preservation, lowering the
temperature 5 to 8 '. and decreasing illumination by about half, much of the
work of maintaining a collection of algae for teaching can be reduced. About
three or four weeks before the algae are needed for classroom use, they
4
Anabaena (15-1710)
N err;um (15-2380)
should be subcultured to the appropriate medi<;l for short term growth. About
one week before class they should be subcultured again.
SHORT TERM PRESERVATIOl\'
For actual classroom use. short term preservation of cultures is recom­
mended. Different media are best suited for particular uses of the algae and
care should be taken to assure the medium you will be using will fit your
specific needs. Media such as Soil-Water Medium with its variations and
Fishmeal Medium are good media for obtaining cultures with normal mor­
phology, but often growth in these media is slower than in other media. For
fast growing dense cultures to be used, for example, in physiology experi­
ments where morphology is not a concern, enrichment variations of Bold's
Basic Medium, Closterium Medium, or Alga-Gro® Medium will work well.
As a generalization, for normal morphology in many algae it is necessary to
use a dilute medium low in enriching organics. Slow growth is often a pre­
requisite for normal morphology.
In addition to culturing your own algae, you will be handling cultures re­
ceived from outside sources. During the past few years, delivery service
by the U. S. Postal Service has become less reliable, necessitating earlier
and earlier shipments of living materials to assure on time arrival. In some
instances this causes early arrival of cultures which must be held until time
for class use. In most cases the material can be kept in good condition for
two or three days by loosening the caps of the tubes and placing them in a
dim, cool area. If they are to be held in excess offour days, the algae should
be transferred to a weak nutrient solution such as sterile spring water (or
seawater for marine forms) or half strength Alga-Gro® Medium. This will
keep the material in prime condition for classroom use.
Many persons prefer to receive their cultures several weeks in advance
of actual use in the classroom and carry the algae through one or two sub­
cultures. This has· the advantage of material being in prime condition for
class and assures that material will be available when it is needed. It is
best when doing this to try whenever possible to use the same medium in
subculturing that the algae has been growing in. Many algae require several
subcultures in new media before they adjust physiologically to the nutri­
tional differences. This is especially true of Soil-Water Medium-a major
I,
5
fault of an otherwise excellent medium. Different soils from different
sources do not have the same chemical components, and a long lag in growth
often results from a soil change.
MEDIA
A number of the more commonly used media and their variations are of
interest as to the types of cultures they produce. Formulas and instructions
for preparing these med ia are Iisted in the Algae M ed ia Section of th is book­
let.
1. Alga-Gras Freshwater Medium is a defined, dilute medium designed in
our laboratory for short term growth of a large variety of algae for mor­
phology. The standard medium is used at a pH of7.8 for most algae, but for
some blue-green algae we adjust the pH of the medium to 6.5. This medium
was originally designed for use with spring water, but an adequate medium
can be made using distilled water.
2. Allen's Blue-G reen Medium (Modified) is a good basic medium for a wide
variety f blue-green algae. We have found that several blue-green algae
such as ArihrosfJirc an"; Glo ocapsa do best on this medium.
3. Bold's B.I. ·.'..'t· " ". :, "':-:
r emen of he old Bristol's Medium
and is good :' :-'~ ;; :e-:­ r~e,e,' ,.:ion. There have been many modifica­
tions to thi~ me~;_. -:-:. D- Bol -~ ,'r e b and some of these modifica­
tions are lis:e ' ir: :.e .~Ig,,:c " , 'j. Section.
,\1 ;I/",~;, one )f the simpliest basic salts media for which
there are many \ ariations. This medium will grow a number of the more
hardy algae and is good for long term preservation.
4. Bristor
,
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oJ
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l
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Ie.
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Gloeocapsa
(/5,I~OO)
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M erismopedia (15·1835)
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6
Chlorella pyrenoidosa (15-2070)
Chlorococcum (15-2090)
5. EREM Medium is the (b) variation listed under Bold's Basic Medium.
This medium gives good morphology and growth with such organisms as
Pandorina, Eudorina and Batrachospermum.
6. Closterium Medium yields promising results for a number of algae,
especially desmids. It gives high volume yields, but we have noticed some
abnormal morphology in some cultures.
7. Fishmeal Medium is a very dilute medium which gives good morphology
in cultures of Hydrodictyon and our large form Chlamydomonas. It will
also support growth of a number of the colonial volvocales.
8. Soil-Water Medium with its variations is excellent for long term preserva­
tion and normal morphology. At this time we have found no substitute for
Soil-Water Medium for culturing Merismopedia, Spirogyra, Bulbochaete,
colorless euglenoids, and Scytonema when normal morphology is required.
The medium will support a very wide range of algae, but one inherent dis­
advantage is finding a source of appropriate soil. One answer to the problem
of a soil source has been suggested by Dr. Richard Starr. He has found that
used soil from the Indiana University greenhouses seems to be a very stable
soil source. The Indiana University greenhouses dump all their used soil in
one out-of-doors area where it is allowed to stand for about a year before
being reused in the greenhouses. Dr. Starr found that after this soil had aged
for six months to a year it made a good Soil-Water Medium. We have checked
soil from our own greenhouses and have been very pleased with the results.
We have begun to switch to this aged greenhouse soil for our stock strains of
algae which are grown on Soil-Water Medium.
The media just discussed are primarily for freshwater algae. The following
media are for marine algae. It has only been in the last few years that cul­
tured marine algae have been available for the classroom.
9. Alga-Gro~ Seawater Medium is the same formula as used with fresh­
water algae, but with aged natural seawater substituted for spring water.
7
This medium is excellent for the larger marine algae, but gives good growth
only to a limited number of marine flagellates.
10. Erdschreiber Medium was one of the early successful marine media and
it is still good for a great many algae. Its major disadvantage is that it uses
soil-water supernatant, and as pointed out earlier, a good soil source is neces­
sary. This medium not only produces good cultures of the larger marine
algae, but also supports growth of a number of the marine flagellates.
11. Provaso/i's E. S. Medium is an exceJlent medium giving good growth for
a wide range of marine algae.
12. Provasoli's ASP 6 Medium (Modified) is a completely synthetic medium
which grows a limited number of marine algae. When culturing classroom
material, we recommend using one of the media utilizing natural seawater.
Most of the synthetic media we have tried produce cultures with some degree·
of abnormal morphology.
13. Guilliard's Medium. We prefer either our own Alga-Gro® Seawater
Medium or Provasoli's E. S. Medium for culturing, but have added this one
to the list because it is used extensively and is a very good medium.
14. Soil-Semt"Qler Medium is a variation of the Soil-Water Medium men­
tioned earlier. This medium is not particularly good for larger seaweeds but
is exceiient fur marine flagellates. It may also be used to culture brackish
water algal furm: su.::h 2~ Spirulino. by mixing with distilled water in dilu­
tions of 14. :.:. )f.3 - s:rer:g h. dependif'.g o. what is needed.
WATER
One of the important aspects of any culture medium is the water source.
We use either aged natural waters or glass-distilled water when preparing
culture media. Normal tap water, deionized water, and distilled water from
metal stills often contain enough toxic material to cause problems in cul­
turing. This is also true of natural waters which have not been aged or
treated with charcoal. We age our natural seawater for at least six months in
Derbesia gametophyte (15-2155)
Derbesia sporophyte (15-2155)
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