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.