Hello, I’ve had a persistent problem with small particles of unknown material being introduced into vials through the pump system, which interferes with OD readings and potentially contaminates our cultures. These particles show up even when only purified water is being pumped through the lines, and no amount of flushing with any common solvents (bleach, ethanol, water) seems to remove them. After having eliminated all other possible sources of contamination, my conclusion is that either the tubing itself or the luer lock components at the ends of the tubing must be the source of these particles. Are there any suggestions about how best to mitigate this? Thanks, Eli
Hi Eli,
Mind sharing pictures of your fluidic lines and if possible the particles? It’s hard to diagnose without that.
Hi Nate, thanks for responding. It is a bit difficult to get pictures since we have our setup inside an anaerobic chamber, but I have done my best to show the full setup. I also have a picture of the particles in a vial of LB media that was pumped directly from a clean container.
I also found that there are no particles present when the luer lock component is removed and media is dispensed from the tubing itself, so I suspect that this part is the culprit. Do you have any suggestions on how to clean the luer lock components? Are they autoclave safe?
Thanks, Eli
Those are some small particles! I wouldn’t expect them to interfere with OD readings unless there were a whole lot of them.
Considering the luer locks are all nylon (hard inert plastic), we’ve never had an issue with particulates coming off of them. They should be fine to autoclave, though this would only sterlize them and not remove particulates.
While unlikely, it’s possible that the batch of luer locks you got has these particulates. Perhaps try unplugging them and swirling them around in bleach to dislodge? Also pumping for an extended amount of time should eventually fix your problem right?
Best of luck!
Thanks for your input. I’ve been troubleshooting a bit more and have come to some new conclusions about the nature of these particles.
First, the particles do not appear to decrease no matter how long I pump for, and no amount of rinsing the luer lock components with water or bleach appears to help either. This leads me to believe that the particles are being introduced somewhere upstream and then accumulating in the luer lock components. To test this, I took raw OD sensor readings of purified water for five vials and calculated the standard deviation of the readings across 10 minutes. The control vial was filled using a sterile pipette, completely bypassing the pump system. where the other four had every possible combination of the luer lock plug and vial caps on or off.
As you can see below, the amount of particles introduced (as measured indirectly by noise level, and confirmed visually) is much higher when the media is pumped through either the luer lock plugs or the vial caps alone, and is highest when both are used. There also appears to be a small amount of particles introduced through the pump system itself, though I would have to do repeat testing to verify if effect on noise levels is significant.
In any case, this is certainly impacting the accuracy of our OD readings, so any further advice you could give on how to mitigate this issue would be greatly appreciated.
Sleeves 0 - 2 dont actually look that noisy to me. They’re slowly changing over time as one would expect to see if they are still coming to temperature (temperature directly effects OD). I would assume that you recently pumped room temperature water into those vials and thus changed their temperature from whatever your set point is. If you were to wait longer for them to equilibrate (30 minutes) and take that data it would be more accurate.
It’s definitely not a temperature effect. I let the vials equilibrate for an hour before taking measurements this time, and confirmed that temperature stayed constant the whole time. The media was completely clear except for the particles. Here is the “growth curve” I got for that:
I was able to isolate the particles and image them under a microscope. Clearly there there is some sort of contamination, unsure what exactly but possibly fungus. I will try to decontaminate the lines with bleach and report back.
At this point yeah I think you’re working with contamination in your lines. @danhart will be more versed in dealing with something like this
Was your ‘growth curve’ done with media or water in the vial? I know working with bleach can be tricky in the anaerobic chamber because you don’t want to poison the palladium catalyst, but are you letting the 10% bleach sit for at least 30 minutes when you run it through the lines? I’ve found that autoclaving the vials with caps on and only removing the caps right before connection in the chamber is helpful to avoid contamination that way. Other things you can try are only using sterile beakers and water for mixing bleach, and adding 5g/L alconox (a strong detergent) to your bleach mixture (use a stir plate to dissolve). The last, most drastic step is to take your tubing out of the pump heads and autoclave everything, which is time consuming but might be easier than tracing down contamination from other sources.
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