Feb 25, 2023
A High-Throughput Assay for Quantifying Phenotypic Traits of Microalgae
- Phoebe Argyle1,2,
- Jana Hinners3,
- Nathan G. Walworth4,
- Sinéad Collins5,
- Naomi M. Levine4,
- Martina A. Doblin1,6
- 1Climate Change Cluster, University of Technology Sydney, Sydney, NSW, 2007, Australia;
- 2Ministry of Marine Resources, Cook Islands;
- 3Institute of Coastal Ocean Dynamics, Helmholtz-Zentrum Hereon, 21502, Geesthacht, Germany;
- 4Department of Biological Sciences, University of Southern California, Los Angeles, CA, 90089-0371, USA;
- 5Institute of Evolutionary Biology, University of Edinburgh, Edinburgh, EH9 3JF, UK;
- 6Sydney Institute of Marine Science, Mosman, NSW, 2088, Australia
Protocol Citation: Phoebe Argyle, Jana Hinners, Nathan G. Walworth, Sinéad Collins, Naomi M. Levine, Martina A. Doblin 2023. A High-Throughput Assay for Quantifying Phenotypic Traits of Microalgae. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l24j33g1y/v1
Manuscript 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(2910).
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 25, 2021
Last Modified: February 25, 2023
Protocol Integer ID: 52691
Funders Acknowledgement:
Gordon and Betty Moore Foundation Marine Microbes Initiative
Grant ID: MMI 7397
Abstract
This outlines a workflow for measuring 10 phenotypic traits of centric diatoms using a variety of methodologies. This method is described in:
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.
Image Attribution
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(2910).
Set up experimental cultures
Set up experimental cultures
Experimental cultures are grown in 12-well tissue culture plates. Triplicate cultures per treatment are recommended.
The initial cell concentration should be 2000 cells/mL, but may be altered depending on the anticipated growth.
During developing, 400 µL of stock culture at a concentration of 11000 cells/mL was added to 4 mL of growth media in each well, resulting in a final volume of 4.4 mL at a concentration of 2000 cells/mL.
The concentration of the initial stock culture was measured using flow cytometry as outlined in:
Protocol
NAME
Measuring growth rates of diatom cells in cultureCREATED BY
Phoebe Argyle
The stock was then diluted or concentrated using centrifugation 1000 x g, 20°C, 00:05:00 to achieve the final concentration of 11000 cells/mL.
NB: The initial concentration of the cultures may be altered depending on anticipated growth, or the species of microalgae being used.
Cultures should be randomised within growth plates. Analysis during method development showed negligible variance due to 'plate' effects, however we recommend position plates randomly within growth incubators and changing their positions daily to minimize potential effects.
5m
Seal plates with breathable seal. These can act in place of the plastic lids of desired.
Equipment
Breathe-Easy® sealing membrane
NAME
Plate seal
TYPE
Breathe-Easy®
BRAND
Z380059-1PAK
SKU
LINK
Track growth
Track growth
After inoculation, take an initial in vivo fluorescence measurement of each plate using a microplate reader as outlined in :
Protocol
NAME
Measuring growth rates of diatom cells in cultureCREATED BY
Phoebe Argyle
Return plates to their experimental incubators.
Each day, track the in vivo fluorescence at least one hour after the onset of the photoperiod. During development this would be at 9am, after 'lights on' at 6am, following a 12:12 light cycle.
Light settings may vary depending on the nature of the experiment being conducted.
Track the in vivo fluorescence over time, and note the growth phase of the cultures.
Trait measurements are conducted during mid-exponential growth, so some discernment is required to estimate this stage.
For example, in this experiment, the cultures shown in blue are in exponential growth between days 2 and 7, whereas the orange cultures have a short exponential phase between days 2 and 5.
Once the experiment is harvested growth measures can no longer be taken, so if in doubt of the growth phase, an experiment of just the growth may be prudent to anticipate the best time to harvest for trait measurements.
Growth of Thalassiosira spp. cultures as measured by in vivo fluorescence over time. The blue represent 3 biological replicates of T. rotula and the orange are three biological replicates of T. pseudonana, all grown at 30°C in f/2 media.
Trait measurements
Trait measurements
Once a culture has reached mid-exponential phase, trait measurements begin according to the workflow. Note not all culture wells will be ready to harvest on any one day, creating a staggered approach.
The workflow of the Quantitative Phenotyping Assay (QPA) outlining the sequence of actions, measurements, and data outcomes. (from Argyle et al. 2021).
Flow cytometry
Flow cytometry
Taken an aliquot and fix for flow cytometry, according to the protocol:
Protocol
NAME
Flow cytometry trait measurements (size, granularity and chlorophyll-a) of diatomsCREATED BY
Phoebe Argyle
Reactive oxygen species
Reactive oxygen species
Initiate the Reactive oxygen species assay according to the protocol:
Protocol
NAME
Quantifying Reactive Oxygen Species in diatomsCREATED BY
Phoebe Argyle
Silicification via PDMPO
Silicification via PDMPO
Initiate the silicification assay according to the protocol:
LINK TO PDMPO assay to insert after publication
On the following day, when harvesting the next days' cultures, take the aliquots from the previous days' incubation and analyse via flow cytometry (according to the protocol). As this is done in plate-mode, these samples can be analysed while step 10 is completed.
Photophysiolgical traits
Photophysiolgical traits
Measure the photophysiological traits according to the protocol:
Protocol
NAME
Measuring photophysiological traits of diatoms from Rapid Light Curves using a Water-PAM CREATED BY
Phoebe Argyle
Not that during this time, it may be necessary to return to the ROS assay and take the final measurement.
Flow cytometry traits
Flow cytometry traits
Conduct flow cytometry analysis of the fixed samples collected in the morning according to the protocol:
Protocol
NAME
Flow cytometry trait measurements (size, granularity and chlorophyll-a) of diatomsCREATED BY
Phoebe Argyle
Measure neutral lipids according to the protocol:
Protocol
NAME
Measuring neutral lipids in fixed diatom cells using BODIPY 505/515CREATED BY
Phoebe Argyle
Statistical analysis
Statistical analysis
Multivariate trait data can be analysed using Principal Component Analysis to generate a multivariate trait-scape, in which differences between strains or species, as well as relationships between traits, can be visualised.
Figure 2A from Argyle et al. 2021. A trait-scape of Thalassiosira spp. strains grown in multi-well plates and assayed using the QPA. Shapes represent different growth plates, each point is a biological replicate.