Robust Microplate-Based Methods for Culturing and in Vivo Phenotypic Screening of Chlamydomonas reinhardtii

Abstract

Chlamydomonas reinhardtii (Cr), a unicellular alga, is routinely utilized to study photosynthetic biochemistry, ciliary motility, and cellular reproduction. Its minimal culture requirements, unicellular morphology, and ease of transformation have made it a popular model system. Despite its relatively slow doubling time, compared with many bacteria, it is an ideal eukaryotic system for microplate-based studies utilizing either, or both, absorbance as well as fluorescence assays. Such microplate assays are powerful tools for researchers in the areas of toxicology, pharmacology, chemical genetics, biotechnology, and more. However, while microplate-based assays are valuable tools for screening biological systems, these methodologies can significantly alter the conditions in which the organisms are cultured and their subsequent physiology or morphology. Herein we describe a novel method for the microplate culture and in vivo phenotypic analysis of growth, viability, and photosynthetic pigments of C. reinhardtii. We evaluated the utility of our assay by screening silver nanoparticles for their effects on growth and viability. These methods are amenable to a wide assortment of studies and present a significant advancement in the methodologies available for research involving this model organism.

Computer-Assisted Tracking of Chlamydomonas Species

“…We propose that this rate increase facilitates the search for new nitrogen sources under nitrogen-limited conditions. When coupled to the microplate-based methods we have already developed for investigating the growth and viability of this microorganism, we are now prepared to thoroughly explore and characterize the impacts of changing environmental conditions and/or mutation on multiple aspects of Chlamydomonas biology (Haire et al, 2018). Indeed, our ability to measure swimming speeds in C. moewusii strongly supports the utility of this approach for understanding motility in this important genus of over 300 species.…”

“…Individual lines were maintained by streaking cultures on plates of Tris-acetatephosphate (TAP) media with agar. Liquid cultures were prepared by growing lines individually in 25 ml of TAP media for 72 h under a 16:8-h day/night cycle (~13,800 W/m 2 ) at 23°C on a shaker (~150 rpm) (Haire et al, 2018). Minimal media studies were performed by limiting the amount of available nitrogen by restricting the amount of NH 4 Cl, either to: low (50%, 400 mg/l) or NH 4 -free TAP media.…”

Nanocosm: a well plate photobioreactor for environmental and biotechnological studies

“…33 Moreover, fluorescent probe-based assays can be used to measure enzyme activity, 40 determine the lipid content, 15,17,41 measure levels of oxidative stress 42,43 and perform multiple toxicity tests. 37,44 All these assays are validated and are easy to perform. Most of the commercially available flow cytometers and FACS are suited for microplate application and can be used to analyze environmental samples, isolate strains of interest, 45 obtain information about growth and fitness and perform stain-based experiments.…”

Effect of tobacco smoke on the growth, biochemical composition, and biodiesel characteristics of two Chlamydomonas strains

“…Monitoring photosynthetic pigments such as chlorophyll and carotenoids offers a valuable understanding of cell stress, metabolic ux, and photosynthetic potential [31]. Changes induced by various stressors are initially detectable in photosynthetic pigments changes before the recognition of any changes in the growth [32]. Therefore, pigment analysis is broadly applied to understand photosynthetic organism's responses towards stress.…”