Microalgal Isolation Techniques

¤ Antibiotics and specific cell inhibitors

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.

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 aseptic technique, dispense 9 ml of media into each of ten test tubes with 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 (1O^-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 top of 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 (<10mm) 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 ½-2/3 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

Density centrifugation

In preparation : use of silicon colloids such as Percoll

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 that 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&T=0&P=270) 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 CCLM we use final concentrations of 0.4 to 1 mg /L. As GeO₂is 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-sterilise. 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 performed in plastic culture ware.

Lewin (Phycologia 6: 1-12,1966)

Markham, J.W. & Hagmeier, E. (1982) "Observatons on the effects of germanium dixoide on the growth of macro-algae and diatoms." Phycologia 21(2):125-130.

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