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.

Keywords: Chlamydomonas reinhardtii; Chlamydomonas reinhardtii microplate-based culture; Chlamydomonas reinhardtii toxicology; Chlamydomonas reinhardtii viability; high-throughput screening of Chlamydomonas; in vivo photosynthetic assays; nanoparticles; photosynthetic pigment analysis.

FIGURE 1

Comparing C. reinhardtii growth in flasks and microplate wells. CC-124 cultured with TAP medium under (A) 16:8 h day:night or (B) continuous photoperiods in either flasks or microplates at the indication shaker speeds. Cell concentration was determined at the indicated time points by hemocytometer (N = 3).

FIGURE 2

Calculating C. reinhardtii cell concentration based on optical density. (A) In Vivo absorbance spectrum of CC-124 cultured in liquid TAP in 96-well microplates at 150 rpm under continuous lighting for 48 h. An absorbance minimum at ≈550 nm is highlighted by the gray circle (dashed). (B) 48 h cultures were diluted in a series, then measured for absorbance at 550 nm and their actual cell count determined by hemocytometer (gray circles). Cell concentration is reproducibly correlated (R² = 0.94) (N = 4) to optical density (OD) at 550 nm (white circles) with a third order polynomial: Cell Concentration = (216944) + (8483581^∗(OD₅₅₀)) + (46233132^∗(OD₅₅₀²)) + (–36516574^∗(OD₅₅₀³)).

FIGURE 3

Effects of media type on C. reinhardtii growth. CC-124 was cultured with different medias in either a flask (gray; 100 rpm) or microplate (white; 150 rpm) under (A) 16:8 h day:night or (B) continuous light cycles. Cell concentrations were measured at 24 and 48 h (N = 4). A Welch’s ANOVA and Dunnett’s t-test were used to determine statistical significance compared to Flask TAP controls at ^∗p ≤ 0.05 or ^∗∗p ≤ 0.03.

FIGURE 4

Effects of DMSO on C. reinhardtii growth. cc-124 cultures were grown in microplates under continuous lighting at 150 rpm over a 48 h period in the indicated concentrations of DMSO (N = 4). Samples were evaluated for culture growth at 24 and 48 h intervals. R statsmod curve comparison tool was used to indicate statistical significance at p ≤ 0.05 (indicated by ‘^∗’) relative to untreated (–DMSO) controls.

FIGURE 5

Evaluating C. reinhardtii viability assays. CC-124 cultures were grown under continuous lighting in liquid TAP for 48 h with any indicated supplements (DMSO or 200 μM CuSO₄) at 150 rpm. Samples were then either directly used in viability assays (LIVE) or heat treated (DEAD) for ≈45 min at 90°C. Results were normalized to LIVE controls (A) FDA (N = 3), H₂DCFDA (N = 8), and RT-GLO (N = 5). (B) CellTox (N = 5).

FIGURE 6

Measuring C. reinhardtii photopigment concentrations in vivo. CC-124 was cultured with liquid TAP medium in microplates at 150 rpm under continuous lighting for 48 h. (Top) Photopigment measurements of uncorrected in vivo values (dark circles), predicted in vivo values (gray; equations used for predictions indicated for each pigment) and extraction values (open circles). An R² correlation (R² ≈ 0.99 for Chlorophyll A, B, and carotenoids) was established through regression analysis between actual and predicted values (Top). (Bottom) Predicted in vivo photopigment measures were normalized to extraction values (predicted/extraction) for Chlorophyll A (white), Chlorophyll B (light gray), total carotenoids (dark gray), and total chlorophyll (dotted) (N = 8). A Welch’s ANOVA was used to determine statistically significant results between actual and predicted at a threshold of p ≤ 0.05 (Bottom) indicated by ‘^∗’.

FIGURE 7

Comparing silver cation and silver nanoparticle toxicity in C. reinhardtii –C. reinhardtii cultures were grown in microplate wells in liquid TAP and the indicated concentration of either ‘free’ silver (AgNO₃) or silver nanoparticles (Ag-NPs) for 48 h (N = 5) under continuous lighting at 150 rpm. Samples were then evaluated for effects on growth (A), total chlorophyll (C), and total carotenoids (D) relative to untreated controls through a multi-wavelength absorbance assay. H₂DCF-DA was then added to evaluate for changes in ROS production as described above. (B) Statistically different results (p < 0.03) relative to untreated controls indicated by ‘^∗’.