Cell counting through flow cytometry
For M. alcaliphilum 20ZR - S. sp. PCC7002 pair, two
0.5 mL samples of culture broth were taken and each sample was
immediately treated with 0.25 mL of 50 mM EDTA and 0.25 mL of 4%
paraformaldehyde to fix the cells. After 10 minutes of fixation, the
samples were centrifuged at 10,000 RPM and 0.5 ml of supernatant was
removed, then each sample was treated with 0.5 mL of 0.05% Tween-20
detergent for 20 minutes (away from light) to minimize cells sticking to
each other. Next, after removing Tween-20 through centrifugation, the
samples were washed and re-suspended in DI water. For M.capsulatus – C. sorokiniana pair, the overall procedure
is similar, with the differences being that the samples were first
treated with 0.2% Tween-20 detergent for 20 minutes and then treated
with 200 mM EDTA and 4% paraformaldehyde for 20 minutes to fix the
cells. After sample preparation, 25 µL of the re-suspended sample was
counted on a Beckman Coulter Cytoflex LX cytometer with 6 active lasers
and 21 channels for fluorescence detection. FlowJo Version 10.6.1 was
used to analyze the data obtained from the flow cytometer. As both the
cyanobacteria and microalgae used in this work are green, and both
methanotrophs are white, different filter of excitation wavelengths were
used to help differentiate the cells in the coculture. For M.alcaliphilum 20ZR - S. sp. PCC7002 pair, the forward
scatter (FSC-H) was paired with the filter of excitation wavelength at
610nm (Y610-mCHERRY-H fluorochrome) to separate the two populations. ForM. capsulatus – C. sorokiniana pair, the FSC-H was
paired with the filter of excitation wavelength of 710nm (Y710-PC5.5-H)
to separate the two populations.