3.6.1 Transfer Techniques

The accomplishment of bioprospecting rests with the successful long-term mainte­nance of the algal strains. The most common method used to preserve microalgal cultures is perpetual maintenance under a controlled environment. Periodic serial sub-culturing of the mother culture onto agar slants is done to maintain the strains (Day, 1999; Warren et al., 2002; Richmond, 2004). This provides metabolically active cultures that retain a vigorous, morphologically, physiologically, and geneti­cally representative population. A crucial factor to consider is that different dura­tions of sub-cultures may provide different stages of the life cycle. Proper labeling and careful checking are required before starting a serial transfer. Manipulations and transfer should be carried out under aseptic conditions. Rigorous microbiologi­cal methods, following standard guidelines for aseptic techniques and maintenance procedures, are crucial (Isaac and Jennings, 1995). The revival of preserved cultures can be successfully accomplished with 1% to 10% (v/v) of the original culture, but some dinoflagellates, Synechococcus and Prochlorococcus, may require a higher inocula level of up to 25% (v/v). Another issue with agar cultures is that some benthic diatom colonies may stick rather firmly to the agar surface or that some filamentous cyanobacteria may even grow into the agar. These can be transferred by removal of agar along with algal material. If older agar cultures are to be revived, the slant may be over-layered with fresh liquid medium for several hours prior to transfer. Another issue that needs bearing in mind is that not all species form appreciable colonies on agar, specifically many flagellates and other planktonic species; likewise, few edaphic and aquatic benthic microalgae grow well in liquid medium. Regardless of that, slant cultures are preferred because of easy and minimal handling during transfer and hence a lower risk of contamination. Mixing the algal liquid culture is customary during transfer to fresh medium or plates. However, uncontrolled mix­ing bears the risk of damage to delicate coccoid green algae, cyanobacteria, and some fragile diatoms such as Thalassiosira and Rhizosolenia. But mixing may be mandatory in some instances or, as in case of Polytoma, where resting cells settle to the bottom, the cell transfer should effect from the bottom of the culture vessel (Anderson, 2005). In contrast, certain colonial flagellate and coccoid green algae (Eudorina, Pediastrum) need agitation and aeration to obtain typical morphology.