Hello,
For my 1st year of Master, I am currently in internship with the objective to determine what settings to use to follow the development of bacterial cultures (count, viability).
For now, i'm trying to discriminate population of E.coli from noise in my negative control. I'm not yet using fluorescence and I would like to be able to use the plot FSC vs SSC to count my population but, part of the noise distribution is too close to my E.coli (even with bigger bacteria like Pseudomonas).
The flow cytometer I'm using is a CyFlow Space (Sysmex-Partec) and I used deionized water or filtered osmotic water (0,2µm), as control and to dilute cultures so that something like 105 cells are analyzed.
Can anyone tell me what I am doing wrong or do suggestion on why the noise distribution overlap with my E.coli ?
Dear Julien!
Plaese You look at the guideline for flow cytomoetry:
Article Guidelines for the use of flow cytometry and cell sorting in...
(page 1701, Figure 68. Graphs B and C show the intensity of GFP fluorescence emission in granulocytes distinguished by higher granularity (SSC) and lower CD45 expression (purple-colored events in graph A) after incubation of whole blood with GFP-expressing E. coli at 37ºC (graph B) or at 4ºC (graph C).
attachment shows that noise easily can be separated by drawing threshold leval..
Shah Jahan Malik
I am not referring at the noise in bottom left, but the events in my negative control that overlap with my cells (in the gates P1 or P6 here).
In water, events in the gate can represent up to 10% of the events in the same gate when I analyze cells.
It is a first time for me so I don't know if I can discriminate these events from actual cells using only FSC vs SSC plot
To get a well-separated bacterial population, you will need to increase the gain of FSC or SSC and set the appropriate threshold before any gating. And ideally, use syto9 stained bacteria as a positive control to confirm that the threshold does not preclude your bacteria.
It could help to filter the water for the dilution, the controls and the sheath fluid with a 0.1um filter. Another possibility would be to increase the concentration of bacteria.
Hi Julien,
To begin with, I am assuming that your flow cell is perfectly aligned, fluid lines are clean and the QC is passing. I could suggest a few tips.
1. It is difficult to differentiate between bacteria and debris just on the basis of scatter. I would use a fluorescent dye that is cell permeant, for e.g. SYTO9 to stain the bacteria and look for SYTO9+ population in a plot of SSC vs. Green Fluorescence. Then back-gate the SYTO9+ fluorescent population on the FSC vs. SSC plot to see where your bacteria are. You could use other dyes as well. Just confirm that CyFlow Space is equipped with the right configuration (LASER & filter sets) to detect the fluorescence of those dyes
2. I could also use the 3µm beads (QC of CyFlow Space) as a reference in the scatter as well as fluorescent plots. While I understand that refractive indices of beads and bacteria are likely to be different from each other so the positions of beads and bacteria will not exactly correspond, it will give you a useful staring point to assess where your bacteria are likely to fall. The 3um beads fluoresce in all the channels of the Blue LASER so you can use them in the plot of SSC vs. Green fluorescence. You could mix the 3µm beads and bacteria in a single tube and run.
3. I saw the figure. I can see some events sticking on the right hand side border and the top border. I would confirm that we are not losing our bacteria in those.
4. What is the power of your Blue LASER?
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