Feb 25, 2025
Measurement of Auxenochlorella protothecoides (UTEX 250) Cell Diameter and Density Using the Nexcelom Bioscience Cellometer
- 1Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA
- Merchant Lab UC Berkeley
Protocol Citation: Dimitrios J. Camacho, Sabeeha S. Merchant 2025. Measurement of Auxenochlorella protothecoides (UTEX 250) Cell Diameter and Density Using the Nexcelom Bioscience Cellometer. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ldr39xg5b/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: October 30, 2024
Last Modified: February 25, 2025
Protocol Integer ID: 111259
Funders Acknowledgements:
National Institutes of Health (NIH): Molecular Basis of Cell Function T32 Training Grant
Grant ID: 5T32GM007232-44
US Department of Energy (DOE), Office of Biological and Environmental Research (BER): Systems Engineering of Auxenochlorella protothecoides: from Photosynthesis to Biofuels and Bioproducts Grant
Grant ID: DE-SC0023027
University of California, Berkeley, Chancellor’s Fellowship
Grant ID: N/A
Abstract
This protocol describes a method for quantifying the cell diameter and density of Auxenochlorella protothecoides cells in culture. Cells are loaded into Nexcelom's counting chambers and are imaged by the Auto M10 Cellometer. Optimized cell size and counting parameters are used to de-cluster and quantify individual cells and cells in clumps. This method accurately quantifies densities within a range of 5×105–2.5×107 cells/mL. Cultures of greater cell densities can be diluted in the respective culture medium and measured. Images of counted cells and histograms of cell densities are generated. Cells in acquired images can be measured retroactively using different settings on the Cellometer software.
Guidelines
The Cellometer does not accurately quantify densities under 5×105 cells/ mL.
Cell sizes and morphologies may vary due to the environment, nutrient status, or genetic mutation. For example, photototrophic cells typically have a smaller cell diameter and can be difficult to measure unless specific parameters are used.
Materials
Equipment
RAININ P1000 pipette and tips
RAININ P200 pipette and tips
RAININ P20 pipette and tips
Cellometer Auto M10
Consumables
Cellometer Cell Counting Chambers SD100, Fisherscientific (Cat. No. NC0018249)
1.5 mL microfuge tubes.
Safety warnings
Avoid touching the clear portion of the counting chamber. The oil from your fingers may obstruct the view of the cells. Do not attempt to wipe the clear portion of the counting chamber, squeezing may deform the counting chamber and result in an inaccurate measurement of cell densities.
Dispose of live cultures according to your institution's guidelines.
Loading the sample
Loading the sample
Transfer 50 µL of liquid culture to a 1.5 mL microfuge tube.
Note
Tip: UTEX 250 cultures with an optical density (OD750) of approximately 0.75 will be in the ideal range for the Cellometer.
Obtain a Cellometer counting chamber and peel the protective film off from both sides.
The counting chambers may have sharp edges and the film may be difficult to peel off.
Use a sharp object to firmly press and drag the film from a corner of the base.
Note
Once the protective film is peeled, do not touch the clear portion of the chamber. Hold the base from the sides as if you are holding a compact disk (CD) or at the edges where the logos are printed. Squeezing or bending the chamber may result in an inaccurate measurement of cell densities.
Cellometer counting chamber.
Before sampling, mix the cell suspension well as cells may settle to the bottom of the tube, even after a short amount of time.
Add 20 µL of the cell suspension to the sample introduction port of each side of the Cellometer counting chamber.
Wait 2 min for the cells to settle. While the cells settle, proceed to step 4 to turn on the instrument.
Note
This waiting period is essential for the cells to settle into the same focal plane. If a cell is out of focus, it may appear larger or smaller than other cells of the same size. While you wait, proceed to steps 4-7.
Turn the instrument on using the power switch behind the unit.
Front and rear view of the Nexcelom Cellometer AutoM10 instrument.
Launch the software after powering on the instrument.
The software needs to detect the instrument in order to launch.
Home Screen of the Cellometer AutoM10 software.
Once the software is launched, click on the "Display Image" icon on the top left of the screen to enter the live view mode.
The blue panel in the middle of the screen should turn white if the counting chamber slot is empty.
Insert the counting chamber into the Cellometer and wiggle it gently so that it is properly inserted and seated.
Acquire a reference image of cells to optimize counting parameters.
Acquire a reference image of cells to optimize counting parameters.
In this section, you will capture an image of your cells to "train" the cell recognition software. The initial settings described in step 10 are used to detect a broad range of cell sizes and types. You will then systematically adjust these settings to accurately identify the cells in the reference image and differentiate them from debris and other image artifacts.
Skip this section if you have already optimized counting parameters and have loaded the appropriate "Cell Type" in the "SETUP" box on the top left.
Once the counting chamber is inserted into the Cellometer, use the focus knob to focus on the cells.
Make sure that the cell suspension is not too dense. Cells will overlap if the suspension is too dense. Dilute if necessary and record the dilution factor used.
Create a new cell type by clicking on the "Cell Type" tab and selecting "New" on the top left of the screen.
A window should appear. See image below.
Rename the cell type.
Use the following settings:
Minimum cell diameter: 0.1 microns
Maximum cell diameter: 20 microns
Roundness: 0.1
Contrast Enhancement: 0.4
Do not test cell viability: Default Setting for "SETUP/Test Viability": No Default
Decluster Edge factor: 0.5
Decluster Th Factor: 1.0
Cell type settings window with initial settings. In the software, hover your mouse over each setting to get an explanation of how each setting affects the automated cell counting and size measurement performance.
Save the new cell type.
Click on the "Display Image" icon.
Refocus the image following instructions from step 9.
Input the sample ID
Adjust the dilution factor.
Click on the "Count" icon.
It will take a few seconds for the program to count all cells. If there are too many cells, the program will take longer and may not be able to complete the count.
A quick display of the results will be shown to the right side of the screen.
Check the "Cell Count" box to ensure that 500-5000 cells are counted.
The program acquires four replicate images (images A-D).
Each image is split into 4 quadrants (1-4).
Use the drop down to select image A.
Clicking on the icon "1" will show a zoomed in section of image A (quadrant 1).
Click on the checkbox "counted"
A green outline of each cell will be displayed.
If you have suitable cell counting parameters, each cell should have one clear green outline.
Click on the "Zoom In" check box to inspect the cells closer.
Click on the histogram icon on the top left.
View the histogram to ensure that you observe a complete distribution of cell sizes.
If the distribution is not normal and appears to be cut (see image below), you will have to adjust the minimum or maximum cell size.
The histogram below displays an incomplete left-censored distribution because cells smaller than 2 microns were not counted. A normal distribution of cell sizes is unlikely if fewer than 500 cells are counted.
Close the histogram and click on the Excel icon to export the data.
A folder with the raw images and images of counted cells will be exported in addition to an Excel sheet.
The Excel sheet will contain the cell diameter of every cell counted.
It will also contain metadata describing the cell counting parameters used.
To optimize the cell counting parameters, use a single reference image (which you just acquired and saved).
You may need to create multiple cell types with different parameters to test on the saved image.
Load the new cell type.
Import the image into the Cellometer software by clicking on the "file" tab.
Click on the option, "import photo for counting"
Select the photo that you saved in step 14.
The software will automatically start counting.
View the histogram to ensure a complete distribution (see step 14).
View quadrants 1-4 of images A-D with the counted option checked.
Visually inspect that each cell is properly counted. Each counted cell should have a green outline.
If a high proportion of cells do not have a clear green outline, repeat steps 17-18 and adjust the parameters.
If you are satisfied with your optimized counting parameters and the performance of the cell counting program, save the settings as a new "Cell Type".
Counting cells using the Auto M10 Cellometer
Counting cells using the Auto M10 Cellometer
Follow steps in the section "Loading the sample" to load a new sample.
Click on the "Display Image" icon.
Input the proper dilution factor and sample name.
Ensure that you have selected the correct "Cell Type".
Click on the "Count" icon.
Make sure that 500–5000 cells are counted.
Check the histogram to ensure a normal distribution of cell sizes.
Check if both tails exist and do not appear truncated (see step 14)
Visually inspect the images A-D and quadrants 1-4 with the "Counted" option checked.
Make sure that each cell has a green outline.
If the cell density exceeds 5×107 cells/mL, dilute the cell suspension in a sterile form of the culture medium used. Cells may overlap in the image if the sample is too dense. The software is more likely to make counting and cell size measurement errors if the cells are touching one another.
If debris is detected and counted as a cell, click on the object in the display within the green outline.
A menu should appear. Click on "Change to Debris". See the image below.
The cell count will automatically update.
Record the object ID and make sure that it is removed from the exported Excel sheet and excluded from downstream data analysis.
If cells are clumped and the declustering algorithm is unable to decluster the cells, change the clumped cells to debris.
Then add the number of cells changed to the count manually using the Manual Adjust option on the bottom right of the screen. You do not need to count all the cells in the field of view. If you Click on the "Enter" icon to incorporate the manual adjustment into the cell concentration calculation.
Export the raw images, images of counted cells, cell size distribution data, and counting metadata using the Excel sheet icon on the top right of the screen. The images can be reprocessed using different parameters at a later time (see steps 16-19).
Remove the counting chamber from the Cellometer.
Close the program before shutting off the instrument. Shutting down the instrument before closing the program will cause a software error and make the program unresponsive.
Switch off the Cellometer using the power switch in the rear.
Acknowledgements
We thank Lauren Saucedo, Dr. Maria Clara Avendaño Monsalve, and Dr. Yang-Tsung (Jon) Lin for testing the protocol and providing feedback.