Background

Using optical densities (O.D.) is a common measurement technique used to quantify the growth of cyanobacteria. For cyanobacteria, cell density is measured at a wavelength of 730 nm, typically using a spectrophotometer¹. In the process of creating our biophotovoltaic cell hardware, we needed to develop a method to quantify the density of our screen printed cultures in terms of O.D.₇₃₀, which we accomplished by using image analysis and our cyanobacterial growth model. While doing so, we realized that this could also be a novel method for measuring the optical densities of liquid cyanobacterial cultures and serve as a useful tool for other teams working with cyanobacteria with limited resources.

We developed our own calibration curve using the program ImageJ that can be used to determine the O.D. of Synechocystis sp. PCC 6803 samples without the use of a spectrophotometer. While the following measurement technique is based on Synechocystis sp. PCC 6803, it has the potential to be applied to other cyanobacterial strains and E. coli.

Protocol

To serve as a standard for our calibration curve, we chose to use a KimWipe box as its shade of green is similar to that of cyanobacteria. A KimWipe box also serves as an accessible standard as it is a universally used material in all lab settings.

1. Take a clear picture of the samples you wish to measure next to a KimWipe box ensuring that there is consistent lighting on the samples (Fig. 1). Ensure that everything is in the same frame. Save the image file as a PNG or JPG/JPEG.
2. Open the image file on ImageJ and convert it to 16-bit by clicking “Edit” → “Type” → “16-bit”. This will make the image black and white.
3. Set your measurements by clicking “Analyze” → “Set Measurements” and selecting “Mean gray value.”
4. Using the rectangle selection tool, select the area of culture you wish to quantify and click “Analyze” → “Measure”. A table will pop up with measured values for mean gray value.
5. Repeat the previous step to also quantitatively measure the KimWipe box. You should now have measured average gray values for both the KimWipe box and your sample of interest.
6. We have provided a standard curve relating ImageJ average gray values to O.D.₇₃₀ of liquid cyanobacterial cultures (Fig. 2). We found our KimWipe box had an “O.D.₇₃₀” of 1.252.
7. To account for differences in your lighting, adjust the y-intercept value. You can do this by setting up your equation as 1.252 = (-0.012)(measured ImageJ value of the KimWipe box) + b and solving for “b”. This will need to be done for every picture you take.
8. Your equation to convert ImageJ average gray value to O.D.₇₃₀ is now ready for use. You can enter your measured average gray value of your sample of interest and convert to O.D.₇₃₀.

Future Applications

While our presented measurement technique is based on measuring the cell density of Synechocystis sp. PCC 6803, we believe this method can also be adapted to measure cyanobacterial health. By splitting the RGB channels of the photographs taken of samples, the ratios of mean gray values can be compared between different color channels to evaluate the health, similar to the way that a health analysis is possible by finding chlorophyll A concentration/O.D.730² . If implemented, this measurement would drastically increase the accessibility of cyanobacteria work for labs with limited resources and allow for improved and increased research regarding cyanobacteria.

References

1. Clark, R. L.; McGinley, L. L.; Purdy, H. M.; Korosh, T. C.; Reed, J. L.; Root, T. W.; Pfleger, B. F. Light-Optimized Growth of Cyanobacterial Cultures: Growth Phases and Productivity of Biomass and Secreted Molecules in Light-Limited Batch Growth. Metab. Eng. 2018, 47, 230–242. https://doi.org/10.1016/j.ymben.2018.03.017.
2. Schulze, K.; López, D. A.; Tillich, U. M.; Frohme, M. A Simple Viability Analysis for Unicellular Cyanobacteria Using a New Autofluorescence Assay, Automated Microscopy, and ImageJ. BMC Biotechnol. 2011, 11, 118. https://doi.org/10.1186/1472-6750-11-118.