Feb 25, 2023
Measuring growth rates of diatom cells in culture
- 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. Measuring growth rates of diatom cells in culture. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzynnxlx1/v1
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: December 13, 2021
Last Modified: February 25, 2023
Protocol Integer ID: 55902
Funders Acknowledgement:
Gordon and Betty Moore Foundation Marine Microbes Initiative
Grant ID: MMI 7397
Abstract
A method for measuring growth rates in diatoms, based on work with Thalassiosira spp. Used in:
Argyle, P. A., Walworth, N. G., Hinners, J., Collins, S., Levine, N. M., & Doblin, M. A. (2021). Multivariate trait analysis reveals diatom plasticity constrained to a reduced set of biological axes. ISME Communications, 1(1), 59.
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 materials
Paraformaldehyde 8% EM Grade aqueous solutionsEmgridCatalog #157-8
Step 2
Using In vivo fluorescence measurements
Using In vivo fluorescence measurements
Measure the in vivo chlorophyll fluorescence of the culture using a fluorometer (using a test tube) or a plate reader (for cultures grown in tissue culture plates).
Equipment
10AU Solid secondary standards
NAME
Turner Designs Digital Fluorometer
TYPE
Turner designs
BRAND
10-AU-904
SKU
Equipment
Infinite® M1000 Pro
NAME
Microplate reader
TYPE
Tecan
BRAND
n/a
SKU
LINK
Note M1000 Pro no longer in production, newer models such as the Spark are available and will serve the same purpose.
SPECIFICATIONS
For chlorophyll measurements in a plate reader, a 455/680 nm excitation/emission filter is used.
Using manual cell counts
Using manual cell counts
Take an aliquot of culture:
Take daily aliquots of culture and take measurements immediately or fix using Paraformaldehyde 8% EM Grade aqueous solutionsEmgridCatalog #157-8 .
The size of the aliquot depends on a number of things:
-Total volume of culture
-Approximate concentration
As multiple aliquots will be taken, it is important to not remove too much volume or biomass during the experiment, so as to not alter the conditions of the experiment too much. It is recommended to not remove more than 50% of the volume throughout the experiment, however less is preferable.
In order to accurately estimate concentration, a minimum of 200 cells should be counted. Therefore if a culture was initially inoculated at 2000 cells/mL, aliquots for growth estimation should be at least 100 µL . The more cells counted, the greater the accuracy of the estimate.
If cells are clumping in the culture, gently agitate before taking the aliquot, take the aliquot from the center of the culture (rather than surface or bottom).
If the culture is slow growing, aliquots may be taken every second or third day, at the discretion of the researcher. However it is preferable to capture multiple data points during the exponential growth phase.
Measure the cell concentration.
Cell concentration from the aliquot may be estimated using a haemocytometer or or flow cytometry.
Equipment
Neubauer Improved Haemocytometer Counting Chamber
NAME
Counting Chamber
TYPE
Hawksley
BRAND
AC1000
SKU
LINK
Void depth: 0.1 mm
Counting Area: 1mm²
SPECIFICATIONS
For larger cells, a Sedgewick Rafter Counting chamber may be appropriate.
Equipment
Sedgewick Rafter Counting Chamber
NAME
Microscopy counting chamber
TYPE
Graticules Optics
BRAND
02B00417
SKU
LINK
Count the cells
Count the cells
Calculate the concentration of cells in the aliquot (remembering to account for the volume of the fixative if using preserved cells) and thus the concentration of the initial culture in cells per mL.
I.e. Cells counted per volume counted.
Calculate growth rates on two points
Calculate growth rates on two points
The classic equation for calculating microalgae growth rates based on two points is:
where F2 is the fluorescence measurement at time 2 (t2) in days and F1 is the fluorescence at time 1 (t1) in days if using in vivo fluorescence measurements.
Where cell counts are used, F2 and F1 are replaced with cell concentration at both times (C2 and C1).
Calculate growth rates for multiple time points
Calculate growth rates for multiple time points
Calculating growth rates for multiple time points, a linear regression is used of the ln(Fluorescence) or ln(cell concentration) plotted against time. A linear regression fitted during the exponential growth phase gives the growth rate (the slope).
Natural log of in vivo fluorescence over time for a Thalassiosira pseudonana culture. A linear regression is fitted across the exponential phase, with the slope giving the growth rate of the culture.