When I isolate Xen41 (Pseudomonas Aeruginosa PAO1 with Luciferase system) from LB/tetracycline plates, place into saline, and read the O.D., the values are not stable and actually decrease. (e.g. 436nm t0=0.333, t5min=.294,t10=.270, t20=.246)
This makes estimating CFU difficult. Why does this happen and how can i work around it?
I have tried resuspending the bacteria in different media (e.g. HBSS, RPMI, LB) and reading at different wavelengths (436, 600, 725, 825nm) but the decrease still occurs.
Any ideas?
Isn't it just because of the sedimentation? Read the OD right after you resuspend bacteria and consider it as the "real" OD.
Don't think that is it. It does not occur with our other pseudomonas strain (no luciferase), nor with our e.coli hb101. If sedimentation were the cause, then I would also expect the OD to return to its initial value upon mixing, but it drops even more.
Does this P. aeruginosa strain also have a functional flagellum? (probably yes but I'm just asking...)
Thibault
They just lyse, this is normal for P. aeruginosa. May be some aeration (shaking) of the stock suspension (resuspended in LB) could help. They are strictly dependent on oxygen, E. coli - not.
Thibault: Yes, they have functional flagella.
Rimantas: Would it be normal for half the population of Pseudomonas to lyse within 20 minutes? I doubt this because i've plated the bacteria immediately after re-suspension, and ~40minutes later when I was finished inoculating hosts and the CFU were essentially the same.
When they lyse, they lyse immediately. P. aeruginosa are very sensitive to different kind of stress, i.e. Tris medium, EDTA, other membrane-active compounds. But it can be that in your case the cells do not lyse (if CFU stays the same), but just become swollen. This also leads to the drop in OD.
A rapid sedimentation can occur when the cells scraped from the plates, once resuspended in saline or buffer, form clots of different size, and respective velocity of sedimentation.
In order to check this possibility spot a 20 microliter drop onto a micro slide and control the cell density under the microscope (phase contrast/darkfield). If you can see aggregates of cells of different sizes, maybe this is the answer to your question.
If this is due to the bacteria swelling, how should I work around this? Resuspend the bacteria in a hypertonic solution (relative to saline)?
Regina: If the bacteria are aggregating, shouldn't the OD return to its initial value if I re-mix the bacteria with a pipette? The OD actually drops after mixing.
Patrick: I use 436 because it is a bit more sensitive. Also, when I label the bacteria with different fluorophores, I want to use a wavelength that will not be interfered with by the fluorophore.
Mixing with a (Pasteur)pipette does not sufficiently desegregates clots or clumps of the cells. Please compare results of sedimentation from cells mixed a) with a pipette, and b) after vortexing at full speed for at least 15 seconds. Compare both results under the microscope, too.
This is the beauty of working with any luciferase system in vitro. Why only Pseudomonas, virtually all luciferase producing organisms shows the same phenomenon when grown in vitro. The most probable answer is that the luciferase action needs a continuous input of high energy which we do not provide in the medium.
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