Cryopreservation of Chlamydomonas reinhardtii (Chlorophyta)

Abstract

Cryopreserved Chlamydomonas reinhardtii cultures remained viable when frozen by cooling slowly to −55°C, then plunging into liquid nitrogen for at least 1 day of storage. High viability (>40%) was retained when cultures contained 2–10% (v/v) methanol as a cryoprotective agent prior to freezing, while dimethyl sulphoxide was ineffective. However, methanol was lethal to cells in the presence of light. Frozen cultures became non-viable within 24 h when stored at −80°C, whereas those stored below −130°C remained viable for at least several months. Highest viability was attained in cultures that were frozen and stored at a low cell density. High viability also required that frozen cultures be warmed rapidly and the cryoprotective agent removed immediately thereafter in preparation for culturing in liquid or on solid medium. Individual cell viability was determined by measurements of colony counts after cell plating and by the penetration of Evans blue dye into non-viable cells. Viability in bulk culture was conveniently measured by comparing the rates of photosynthetic oxygen evolution, corrected for dark respiration, in previously frozen cultures with the corresponding rates in unfrozen controls. Cultures that had lost viability as measured by a decline in whole-cell photosynthetic oxygen evolution after freezing and thawing retained functional Photosystem II activity for some time thereafter, indicating that the loss in photosynthetic activity was due to some process other than photoinhibition of Photosystem II.