Although single-celled, colonial, or filamentous algae growing on the agar surface can be isolated by streak plate or spraying, any flagellates as well as other types of algae require the use of advanced techniques. A unialgal culture would contain only one kind of alga, usually a clonal population, but may contain other life forms such as bacteria, fungi, or protozoa. Alternatively, cultures may be axenic, in that they contain only one species of alga. Unialgal cultures are best isolated by targeting the isolation of the zoospores immediately after release from parent cell walls as those cells that begin attaching to surfaces are likely to add contaminants. The algal isola­tion techniques involving cell separation pose limitations with highly heterogeneous samples or when the cells are suspended in a solution of different chemicals, biomol­ecules, and cells. This can be overcome by employing a micromanipulator, which successfully permits the separation of a single cell from a liquid culture. The single cell can be easily separated from an enriched environmental sample and grown in liquid medium as monoculture or in agar plates, thus facilitating a significant time saving over the conventional plating technique. The micromanipulator is the ideal tool for algal screening and isolation, provided the person handling it has acquired skill in handling the equipment (Kacka and Donmez, 2008; Moreno-Garrido, 2008). Using micromanipulation techniques requires expertise and skill. It requires the handling of an inverted microscope or stereo zoom microscope with a magnification up to 200x. Phase contrast or dark-field microscopy offers advantages. Capillary tubes or hematocrit tubes of approximately 1 mm diameter x 100 mm long are used for picking individual cells (Godhe et al., 2002; Knuckey et al., 2002).

High-throughput cell sorting is possible when coupled with flow cytometry, which facilitates the rapid and efficient screening of microalgal strains. Microalgae possess different photosynthetic pigments, emitting various auto-fluorescence, which can be applied in flow cytometry to identify algae (Davey and Kell, 1996). Literature on the isolation of microalgae from natural waters employing flow cytometric cell sort­ing is available (Reckermann, 2000; Crosbie et al., 2003). Chlorophyll is used as a fluorescent probe to distinguish different strains of microalgae. Reckermann (2000) and Sensen et al. (1993) used the chlorophyll auto-fluorescence (CAF) properties of eukaryotic phytoplankton, diatoms, and pico-autotrophic cells for isolation of axenic cultures, whereas Crosbie et al. (2003) used both red and orange auto-fluorescence to differentiate species of algae. Similarly, green auto-fluorescence (GAF), which is common in both autotrophic and heterotrophic dinoflagellates, is also a valuable taxonomic consideration (Tang and Dobbs, 2007). Hence, flow cytometry, coupled with cell sorting, can signify a vital tool for screening and exploiting microalgal strains for specific drives, including biodiesel feedstock development. As compared to fluorescence microscopy, flow cytometry helps the investigator perform rapid and quantitative experimentation. Fluorescence-activated cell sorting (FACS) permits cells with a specific characteristic—or indeed a combination of characteristics—to be separated from the sample. Sinigalliano et al. (2009) compared electronic cell sorting and conventional methods of micropipette cell isolation with dinoflagellates and other marine eukaryotic phytoplankton. Fragile dinoflagellates such as Karenia brevis (Dinophyceae) were distressed upon conventional micropipette procedures while cells were viable on electronic sorting. However, electronic single-cell sorting combined with automated techniques for growth screening has the possibility of screening novel algal strains (Sinigalliano et al., 2009). The benefits and shortcom­ings of the microalgal isolation and purification protocols described in this section are summarized in Table 3.5.

In addition, several immunological and nonimmunological methods to isolate desired unicellular algal cells exist. The immunologic reaction of a specific inte­grated protein on the membrane decides the protocol for cell separation. Large-scale commercialized cell separation involves techniques such as FACS (Takahashi et al., 2004), magnetic-activated cell sorting (Han and Frazier, 2005), and affinity-based cell sorting (Chang et al., 2005), all of which are highly specific and selective. But the limitation is that the immunologically isolated cells may undergo trauma and the inclusive separation system involves high cost. Further, immunoreactions and follow-up elution with capturing antibodies are quite complicated processes. Alternatively, nonimmunological techniques such as dielectrophoresis (Doh and Cho, 2005), hydrodynamic separation (Shevkoplyas et al., 2005), aqueous two-phase system (Yamada et al., 2002), and ultrasound separation (Petersson et al., 2004) have also been employed. These methods work based on the interactive physico-chemical property of a cell with that of the surrounding media, and lack specificity.