Microalgal Isolation Techniques

Serial dilution
1. Equipment

 Culture tubes, (sterile) screw-capped or steristoppered (see Note below).

 Test-tube racks, open mesh.
 Media — usually dilute e.g. f10, f20.
 Automatic dispenser (sterile) or 10 mL sterile glass pipettes.
 Glass pipettes 1 mL or pasteur (sterile), rubber or silicone teat.
 Bunsen burner or small flame.

2. Method

 Using the aseptic technique, dispense 9 mL of media into each of ten test tubes with a
sterile automatic dispenser or sterile 10 mL pipettes. Label tubes 10-1 to 10-10 indicating
dilution factor.
 Aseptically add 1 mL of enrichment sample to the first tube (10-1) and mix gently.
 Take 1 mL of this dilution and add to the next tube (10-2), mix gently.
  Repeat this procedure for the remaining tubes (10-3 to 10-10).
 Incubate test-tubes under controlled temperature and light conditions:

(a) temperature and photoperiod — as close to the natural environment as possible.

(b) light intensity — slightly lower than the natural environment.

 Examine cultures microscopically after 2—4 weeks by withdrawing a small sample

aseptically from each dilution tube. A unialgal culture may grow in one of the higher
dilution tubes e.g. 10-6 to 10-10. If tubes contain two or three different species then
micromanipulation can be used to obtain unialgal cultures.

Note: Sterilizing screw-capped culture tubes

The following procedure is required to remove toxic materials from culture tubes and screw
caps.

 Half-fill culture tubes with distilled water. Plug with steristoppers or non-absorbent
cotton wool. Autoclave.
 Take screw-caps from culture tubes and place open side down in a glass petri dish. Pour
distilled water around caps, replace the top of the petri dish, and autoclave.
 Under aseptic conditions remove steristoppers or cotton-wool plugs and pour out distilled
water. Flame tube and replace steristopper or screw-cap.

Streak plating
This is a suitable method for small species (<10 mm) or algae that grow well on a substrate.

1. Equipment

 Petri dish — sterile, disposable, 90 mm diameter

 Media — e.g. f2
 Agar — Bacto-Agar, Difco, Cat. No. 0140—01
 Wire loops — nichcrome or platinum
 Bunsen burner or small flame
 Parafilm

2. Method

 Prepare petri dishes containing growth medium solidified with 1-1.5% agar medium. The agar
should be 1/2–2/3 the depth of the dish.
 Place 1—2 drops of mixed phytoplankton sample near the periphery of the agar. Flame sterilize a
wire loop. Using aseptic technique use the sterile loop to make parallel streaks of the suspension
on the agar. Note that there are 16 streaks (4 sets of 4) to be made and the whole surface of the
agar plate is used (see fig below).
 Cover and seal plate with parafilm. Invert and incubate under low light at constant temperature.
 Select colonies that are free of other organisms for further isolation. Remove a sample using a
sterilized wire loop and place in a drop of sterile culture medium on a glass slide. Check
microscopically that the desired species has been isolated and is unialgal.
 Repeat the streaking procedure with the algal cells from a single colony and again allow colonies
to develop. This second streaking reduces the possibility of bacterial contamination and of
colonies containing more than one algal species.
 Transfer selected colonies to liquid or agar medium.

Fig Streak plating with wire loop

Micromanipulation
1. Equipment

 Inverted microscope or stereo microscope with magnification up to 200 x, although 40—100 x

should be sufficient in most cases. Phase contrast or dark field optics is an advantage
 Capillary tubes or haematocrit tubes — approx. 1 mm diameter x 100 mm long
 Bunsen burner or small flame
 Silicone tubing to fit over end of capillary tube. Length approximately 300—400 mm
 Hot plate with beaker containing distilled water
 Clean Glass slides
 Agar plates (1.5% Bacto-Agar, eg Difco Cat. No. 0140—01) made up in petri dishes (disposable,
90 mm diam.) Tissue culture plates
 Pasteur pipettes (sterile), rubber or silicon teat
 Sterile media, usually at dilute nutrient concentrations; e.g. f20, f50
 Sterile tissue culture multi-well plates or sterile disposable petri dishes (e.g. 33 or 55 mm diam or
sterile culture tubes)

2. Method

 With a fine flame from a bunsen burner heat and draw out (holding at both ends) the
capillary tube to form two micropipettes. The narrow end should be about twice the
diameter of the cell to be micromanipulated.
 Heat distilled water to simmering point on hot plate. This is used for sterilizing the
micropipette between each transfer.
 Place drops of sterile medium onto 1.5% agar plates with a sterile pasteur pipette.
Alternatively place three drops on a glass slide.
 With silicone tubing attached to micropipette suck up and blow out with mouth a small
amount of hot distilled water. This sterilizes the micropipette.
  Locate algal cell to be isolated in drop of enrichment sample. While observing the cell,
suck up into the micropipette.
 Transfer the cell to a drop of sterile medium on agar plate or glass slide.
 Sterilize the micropipette.
 Repeat this process to “wash” the cell. The more times a cell is washed the less likely is
bacterial contamination. However, the risk of cell damage increases with the number of
times a cell is handled. The optimum number of washes will depend on the type of algae.
 Transfer the cell to dilute medium in a tissue culture plate, petri dish or culture tube.
 Place culture vessel under low light at appropriate constant temperature. Check
microscopically for growth or wait until macroscopic growth can be detected (3—4
weeks after transfer).
 A clonal uni-algal culture should result from this method.

For large non-motile cells or when time is at a premium an alternative method is to isolate into
petridishes rather than on a slide. Multiple cells of a population are quickly transferred to the first
rinse plate and then from there progressively fewer into subsequent plates. Because relatively
less care is taken to avoid non-target cell transfer each petri dish acts as an enrichment culture
with hopefully a relatively clean culture in the final plate (or even possibly clonal). The “clean”
plates can then be used as the stock for making clonal isolations when time permits.

Antibiotics
The use of antibiotics is not recommended when isolating strains that will be used for
physiological or ecological studies as mutant clones may be produced that do not reflect
populations in the wild. However for species used in aquaculture, population genetics, molecular
biology, toxicology or bioproduct screening it may be necessary to work with axenic strains and
sometimes this may only be achieved through using antibiotics.

The choice of antibiotics is made based on their efficacy against Gram (+) and/or Gram (-)
bacteria and for their mode of action, either as inhibitors of cell wall synthesis or of cell growth
via inhibition of protein synthesis. For the cell wall inhibitors to be truly effective the bacteria
need to be actively growing therefore it is advisable to add a small amount of organic matter to
the treatment media. For this reason adding additional antibiotics that slow cell growth may be
counter productive hence a cascade treatment where several antibiotics are administered
sequentially is more effective than a cocktail treatment where a single mixed dose is given. Cell
wall inhibitors generally belong to the penicillin family, such as Penicillin G which acts
primarily against Gram (+) bacteria or the cephalosporin family of beta-lactams that disrupt the
integrity of the bacteria cell wall, making it more porous and include broad-spectrum antibiotics
such as Imipenem. Aminoglycosides such as gentamycin, kanamycin, neomycin and
streptomycin attach to the bacterial cell’s ribosomes and impair protein production. Using a B-
lactam first to increase cell wall porosity may then allow a faster passage of aminoglycoside to
then disrupt protein synthesis.

Antifungal agents include cycloheximide (= acti-dione), Nystatin, or Amphotericin B

Germanium dioxide (GeO2) was used as a media supplement by Lewin (1966) as a means to
remove diatoms at a final concentration of 10 mg/L. It acts as a cell division inhibitor rather than
a toxin. Suggested concentrations vary widely (https://listserv.heanet.ie/cgi-bin/wa?
A2=ind9706&L=ALGAE-L&P=R1150) and Markham and Hagmeier (1982) found
concentrations as low as 0.045—0.179 mg/L were sufficient to kill diatoms contaminating their
kelp cultures. At ANACC we use final concentrations of 0.4—1 mg/L. As GeO2 is difficult to
dissolve, lower initial stock concentrations improve dissolution so we make up a stock solution
of 2 mg GeO2 in 100 mL of MilliQ water and filter-sterilize. We add the stock solution at a
concentration of 1—2.5 mL per 50 mL enrichment medium. If removing diatoms is the goal then
all sources of silica should be removed from the culture environment therefore the stock solution
is stored in a polycarbonate bottle and the enrichment or isolation cultures are performed in
plastic culture ware.

Density centrifugation
Density centrifugation can be used in conjunction with other isolation and algal treatments to
improve the purification of algal strains from unwanted algal and protozoan contaminants. The
technique can also be used for the separation of subcellular particles and the isolation of algal
viruses.

Percoll, a silica colloid, is diluted to a range of concentrations to produce a density gradient. An

algal culture is then added before centrifugation. Centrifuging at low speeds, cells of varying
densities will settle to the density band to match their individual density while the Percoll
gradient remains stable. Percoll has a low osmolality and therefore doesn’t produce a
 significant osmolality gradient itself. However, care is needed to prepare isopycnic
gradients, particularly for stepwise gradient preparation, as variations in osmolality can
cause cells to shrink or swell. Density gradients can be produced either by the stepwise
addition of pre-prepared gradient mixtures to the centrifuge tube or by the self-generation of
gradient through the high-speed centrifugation of a uniform Percoll mixture. Due to the
complexities in the formation of self-generating density gradients from the physics of
centrifugation with effects from rotor angles, the non-recommended use of swinging bucket
rotors and the variation of Percoll viscosity with osmolality, only stepwise density gradients will
be covered in this method.
NOTES ABOUT HANDLING PERCOLL:
Refrigerate after opening.
To resterilise, autoclave at 120°C for 30min.
Prepared gradients should remain stable for several weeks.
Centrifuging at <10,000 x g will quickly cause the Percoll to pelletise at the bottom of the
tube.
Not suitable for acidic media types as at pH < 5.5 the Percoll may begin to gel.

1. Equipment

 Percoll
 15 mL centrifuge tubes and rack
 Sterile media/stock solutions
 Centrifuge
 Sterile 1 mL pipettes and micropipettes
 Bunsen burner or small flame
 Density marker beads (optional)
NOTE: Density marker beads can be used to define density bands within the gradient.
These added as a 1 mL mix in a separate but identical gradient to the sample and can be
used to counter-balance the centrifuge. Small variations in density can occur with marker
beads where the ionic strength of the media varies from the calibration solutions and
thus this should be considered in any interpretation. They can also help with method
development.

2. Method – Marine cultures

 A Stock Isotonic Percoll (SIP) solution that is 50% Percoll is prepared by mixing an equal part of Percoll
to an equal part of double strength evaporated media or seawater.
 Using the 50% SIP solution, prepare stepwise Percoll gradients for 10%, 20%, 30%, 40% and 50%
Percoll.
 Starting with the most concentrated gradient and working to the most dilute, carefully pipette 1 mL of
each layer into a 15-mL centrifuge tube.
 Add 1 mL of culture on the surface.
 Centrifuge at 1500 rpm for 45min
 Use a micropipette to gently remove the target algal cells from the interface that they have collected at.
 The Percoll is washed from the cells at a ratio of 5 part media/seawater to 1 part Percoll/cell mix by
centrifuging at 2500 rpm for 2 min and collecting the pellet of cells at the bottom of the tube.
 Repeat the rinse 2-3 times.
 The Percoll gradient separation may need to be repeated 3 times to effectively remove contamination.

3. Method – Freshwater cyanobacteria cultures

 As cyanobacteria tend to float, a Stock Isotonic Percoll (SIP) solution that is 90% Percoll is prepared by
mixing 9 parts of Percoll to 1 part of 10x concentrate of media.
 Using the 90% SIP solution, prepare stepwise Percoll gradients for 0%, 20%, 50%, and 80%-90% Percoll.
 Starting with the most concentrated gradient and working to the most dilute, carefully pipette the
gradients following quantities: 2 mL of 80%-90%, 5 mL of 50%, 1 mL of 20% and 1 mL of 0% into a
15-mL centrifuge tube.
 Add 1 mL of culture on the surface.
 Centrifuge at 1500 rpm for 45min
 Use a micropipette to gently remove the target algal cells from the interface that they have collected at.
 The Percoll is washed from the cells at a ratio of 5 part media to 1 part Percoll/cell mix by centrifuging at
2500 rpm for 2 min and collecting the pellet of cells at the bottom of the tube.
 Repeat the rinse 2-3 times.
 The Percoll gradient separation may need to be repeated 3 times to effectively remove contamination.

References

GE Healthcare (2007) Cell Separation Media: Methodology and applications, Sweden 80pgs

Cho J.-Y., Choi J.-S., Kong I.-S., Park S.-I., Kerr R. G. and Hong Y.-K. (2002) A procedure for
axenic isolation of the marine microalga Isochrysis galbana from heavily contaminated mass
cultures, J. App. Phycol. 14: 385-390

Lavoie, A., Mouget J.-L. and de la Noue J. (1986) Measurement of freshwater micro-algal cell
density with Percoll density gradients, J. Microbiol. Meth. 4: 251-259

Price C. A. and Reardon E. M. (1978) Collection of dinoflagellates and other marine microalgae
by centrifugation in density gradients of modified silica sol. Limnol. Oceanog. 23: 548-553

Whitelam G. C., Lanaras T. and Codd G. A. (1983) Rapid separation of microalgae by density
gradient centrifugation in Percoll, Br. Phycol. J. 18: 23-28