# Goal/Experiment:
This experiment aims to measure 10 phenotypic traits of centric diatoms using high-throughput methodologies. This versatile assay provides detailed insights into the various characteristics of microalgae, particularly focusing on growth rates, reactive oxygen species production, photophysiological traits, and other key indicators of cellular health and activity.
## A High-Throughput Assay for Quantifying Phenotypic Traits of Microalgae
### Authors
Phoebe Argyle<sup>1,2</sup>, Jana Hinners<sup>3</sup>, Nathan G. Walworth<sup>4</sup>, Sinéad Collins<sup>5</sup>, Naomi M. Levine<sup>4</sup>, Martina A. Doblin<sup>1,6</sup>
1. Climate Change Cluster, University of Technology Sydney, Sydney, NSW, 2007, Australia
2. Ministry of Marine Resources, Cook Islands
3. Institute of Coastal Ocean Dynamics, Helmholtz-Zentrum Hereon, 21502, Geesthacht, Germany
4. Department of Biological Sciences, University of Southern California, Los Angeles, CA, 90089-0371, USA
5. Institute of Evolutionary Biology, University of Edinburgh, Edinburgh, EH9 3JF, UK
6. Sydney Institute of Marine Science, Mosman, NSW, 2088, Australia
### Abstract
This outlines a workflow for measuring 10 phenotypic traits of centric diatoms using a variety of methodologies.
### Citation
- Argyle, P. A., Hinners, J., Walworth, N. G., Collins, S., Levine, N. M., & Doblin, M. A. (2021). A high-throughput assay for quantifying phenotypic traits of microalgae. *Frontiers in microbiology*, 12, 706235.
## Protocol Steps
### Set up Experimental Cultures
1. **Initial Setup (Approx. 5 minutes)**:
- Grow experimental cultures in 12-well tissue culture plates.
- Use triplicate cultures per treatment.
- Initial cell concentration: 2000 cells/mL, adjustable based on growth expectations.
2. **Stock Preparation**:
- Add 400 µL of stock culture (at 11000 cells/mL) to 4 mL of growth media per well, achieving 4.4 mL total volume with 2000 cells/mL.
- Measure concentration of initial stock using flow cytometry:
```markdown
**Protocol:** Measuring Growth Rates of Diatom Cells in Culture
**Created By:** Phoebe Argyle
```
3. **Concentration Adjustment**:
- Use centrifugation (1000 x g, 20°C, 5 minutes) to adjust concentration if required.
4. **Sealing Plates**:
- Seal plates with breathable seal (Breathe-Easy<sup>®</sup> sealing membrane from Sigma-Aldrich, SKU: Z380059-1PAK).
### Track Growth
3. **Initial Fluorescence Measurement**:
- Take in vivo fluorescence measurement post-inoculation with a microplate reader:
```markdown
**Protocol:** Measuring Growth Rates of Diatom Cells in Culture
**Created By:** Phoebe Argyle
```
4. **Daily Monitoring**:
- Measure in vivo fluorescence daily at least 1 hour post-photoperiod onset (e.g., 9 am post 6 am light onset on a 12:12 light cycle).
- Monitor the growth phase and note exponential growth for trait measurement.
### Trait Measurements
5. **Mid-Exponential Phase**:
- Once culture reaches mid-exponential phase, begin trait measurements.
- Ensure staggered harvesting due to varying readiness of culture wells.
### Detailed Workflow (Quantitative Phenotyping Assay - QPA)
#### At Mid-Exponential Phase
6. **Split Culture (Flow Cytometry Preparation)**:
- 200 µL culture + PFA fixative for flow cytometry.
7. **Reactive Oxygen Species (ROS) Measurement**:
- 2 x 500 µL culture (1 stained H<sub>2</sub>DCFDA + 1 blank control).
- 2-hour dark incubation at growth temperature, followed by fluorescence reading.
8. **Photophysiological Traits**:
- Daily in vivo fluorescence tracking.
- Measure using Water-PAM for Rapid Light Curves.
9. **Silicification**:
- 1 mL culture + 1 mL artificial seawater into a quartz cuvette.
- Analyze post-Water-PAM measurements.
10. **Additional Flow Cytometry**:
- Use BODIPY 10-minute incubation, followed by flow cytometry.
- Traits measured: Cell Size, Granularity, Chlorophyll a, Neutral Lipids, Silicification.
### Statistical Analysis
11. **Data Analysis**:
- Perform Principal Component Analysis (PCA) to visualize multivariate trait data.
- Identify differences between strains/species and relationships between traits.
```markdown
### References
- Argyle, P., et al. (2021). High-throughput assay for quantifying phenotypic traits of microalgae. Frontiers in microbiology, 12, 706235.
- Licenses: This protocol follows the Creative Commons Attribution License.
- Protocol Status: Working, created on Aug 25, 2021.
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