Using a micropipette or Pasteur pipette, or a glass capillary having a straight, bent, or curved tip is handy for single-cell isolation. Micropipettes enable fishing out a single cell from the sample after a series of transfers into sterile rinsing droplets, without the cell being damaged in the process. Finally, the single cell can be pipetted and transferred to the culture medium after microscopic examination. Lewin (1959) recommended placing the droplets on agar to reduce evaporation, but this depends on the size of the cells. Technical skill and expertise are important in order not to shear or damage the cell. The damage may be apparent as cessation of swimming in flagellates or a difference in light refraction due to broken frustules as in diatoms, and severe damage is evident by leakage of protoplasm. The traditional method of micropipette isolation can be successfully attempted with the use of ultra-pure sterile droplets for rinsing, as marine samples hold suspended particles.

TABLE 3.4

Common Media Used for Microalgal Strains from Diverse Aquatic Environments

TABLE 3.4 (Continued)

Common Media Used for Microalgal Strains from Diverse Aquatic Environments

Alternatively, many coccoid algae and most soil algae can be isolated on agar plates. It is the preferred isolation method because it is simple and requires no further processing. Streak or pour plating on suitable agar growth medium enables suc­cessful isolation, although few algae grow embedded in agar (Brahamsha, 1996). An improvised procedure is to make a fine or atomized spray of cells, usually a liquid cell suspension atomized with sterile air under pressure, which can then be used to inoculate or spread on agar plates. Similarly, a dilution method can be used, wherein a single cell is deposited in a test tube, flask, or well of a multiwell plate (Throndsen, 1978). Selection of the appropriate maximum dilution for plating depends on the probable cell density in natural samples. The dilutions can be effected in several ways, such as dilution with sterile culture medium, distilled water, seawater, and filtered water from the sample site, or some combination of these. Also, where neces­sary, salts of ammonium, selenium, or another element can be added as supplements to specifically isolate selected species.

When samples contain a wide variety of cells, centrifugation or settling can be foreseen. The target of concentrating the cells instead of obtaining an axenic culture can easily be achieved by gravity. Also, gravity comes in handy when the goal is to separate the larger and heavier cells from smaller algae and bacteria. Specifically for large dinoflagellates and diatoms, moderate centrifugation for a short duration is enough to pelletize them, and smaller cells can be decanted. Density gradient cen­trifugation with silica sol, Percoll™, etc. has been successfully employed to separate mixed laboratory cultures so that individual species can be separated into a sharp band (Reardon et al., 1979). Large, nonmotile algal cells can be effectively separated by settling. Hence, gravimetric settling is the choice if one aims for concentrating larger cells; however, it is not effective to obtain unialgal culture and hence suggests some combination with other procedures.