Baseband going down with increasing OD?

[sefrem]

Hi,
We are culturing a bacteria that constitutively produces high amounts of intracellular GFP in MOPS EZ (non-autofluorescent) media. However, we’re noticing a behavior where as the OD goes up to the target (i.e. particularly in the log phase), the FP reading goes down (i.e. lower than blank). Then, once the OD reaches a high enough point, the FP turns around and only ends up being slightly higher than the starting point. This may be in part because the baseband fluorescence appears to spike early on with increasing OD, before starting to dip again, despite the culture remaining turbid.

https://i.ibb.co/Lrmn2PP/2022-06-06-graph-MOPS-k-aerogenes.jpg

I was wondering if you had any thoughts as to what might be going on here? Should we use something other than clear for the baseband in our case?

Thank you!

[harrison]

What is happening to the culture BEFORE the experiment? Are the cells at stationary or exponential phase? If you are sampling from stationary phase then it can be that the GFP/cell at that point is already at or near maximum, hence the fluorescence/cell (which the emission/baseband measurement attempts to approximate) may already be at a very high level.

As to the long term (high OD) behaviour, it is difficult to conclude too much since when you get to high ODs there are multiple conflicting factors that determine the measurement. In particular, your excitation light (e.g. baseband) may scatter multiple times or be absorbed before leaving the test tube (i.e. to be seen by the sensor) and hence you get this decrease.

That said, most of these problems go away if you have the culture at a fixed OD – is this eventually your goal, or are you primarily interested in looking at growth curves from stationary-exponential-stationary phase?

[sefrem]

Hi Dr. Steel,

Thank you for the prompt reply! The inoculum was from a culture in its stationary phase– are you saying that for bacteria constitutively producing GFP the amount of GFP per cell might be higher at this phase? And to be clear, the culture started at a very high dilution (i.e. no detectable OD/fluorescence) so presumably we’re seeing the bacteria re-entering the exponential phase pretty quickly, are we not?

Re: the high OD behavior, are you suggesting theres a max OD (seen at around the 17 hour mark) after which the baseband signal does not increase with OD due to the degree of scatter?

Yes we will be working with a fixed OD, was just trying to understand what we were seeing prior to this! Especially considering the normalized FP signal is at some points lower than the starting (pretty much blank media) signal.. want to make sure the readout we’re seeing is in fact true fluorescence!

[harrison]

Yes – if you leave them constitutively expressing it for a long time they can produce a lot per cell, more than the equilibrium amount when they are in exponential growth. But if you dilute them a lot at the beginning much of this shoudl be diluted out.

I would say in general measurements at extremes are difficult, either zero OD (where light is just passing right through, so you are just measuring the leakiness of the excitation light into the emission filter) or very high OD (where light being both scattered and blocked). If you want to get a good idea for what you are seeing the best bet is to grow non fluorescent cells at fixed OD for a while, then either induce them (chemical inducer or whatever your system is), or dilute them with fluorescent cells. That way you will have a very clear cut difference between off and on, and be able to accurately measure the dynamic range of the system for your biology.

[Author]

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