I’m studying the effects of different types and doses of antibiotics on resistant and non-resistant e.coli.
I have been using the peristaltic pumps for dosing automation but have wondered if anyone has any suggestions on improving my pump priming method to keep things sterile and prevent the antibiotic from falling into my sample prematurely.
As per the priming protocol, I have to run my antibiotic through the entire length of the tubing when priming so the pump doses accurately during my experiment. As I’m dosing with such small volumes of the antibiotic (0.15 mL & 0.45 mL) I do not want to risk even the slightest amount of antibiotic falling in.
Suggestion 1: Run the pump for just long enough to reach the end of the tubing, watch it to make sure no antibiotic falls in. Needless to say, this is risky and I don’t want to do that.
Suggestion 2: The tubing is not attached to the pioreactor vial during priming, and I can let the antibiotic run through freely. Then I can either attach it to the Luer end of the vial tubing or push the tubing through the hole on the vial cap. The first one leaves some of the wire empty (defeats the point of priming), and the second causes some of the antibiotic to squeeze out of the tubing whilst inserting it into the cap (really not ideal).
Normally I have my LB getting to temperature whilst priming the pumps so that they’re ready to dose immediately after inoculation, and to prevent them from sitting around for too long between priming and inoculation to prevent them from becoming contaminated.
I think an extra cap with tubing would be my best bet so I can prime the pumps whilst connected to the cap of a waste container, before swapping it out with the one on my sample jar (on while heating so LB is not exposed). But this again risks antibiotics dropping into my sample (though it’s less likely) and also exposes my sample to potential contaminants.
Can anyone think of an accurate, sterile and logistically doable way to prime the pumps? Thanks!
This may not work exactly for what you’re looking for and is somewhat convoluted (and requires access to a biosafety cabinet or similar environment to maintain sterility) but may help. I haven’t played around with antibiotics beyond standard amounts in the media of strains I’ve used, so you may need to modify this protocol for how you use your alt-media pump.
I autoclave all components (media bottles, tubing separated from pump heads, custom GL45 lids with tubing, Pioreactor vessels, etc.) and bring into a BSC
Independently sterilize as best as possible a Pioreactor + pumps (without pump heads or tubing, so just the pump body with exposed shaft the pump head seats on) and associated pumping dovetail platform by spraying and wiping down with 70% ethanol and set up in same BSC (plug into outlet in BSC and confirm on and ready in the UI)
Do the same thing as step 2 for all pump heads (still without tubing)
Assemble pump heads with appropriate tubing for the number of pumps used for the Pioreactor you’re priming (in the BSC!)
I then connect tubing between media bottles - media pump head - Pioreactor vessel and Pioreactor vessel - output pump head - output bottle (and do the same for your antibiotic alt-media pump setup too if I’m undestanding how you’re trying to run things)
Attach 0.22 µm filters on any lines that will not be used for anything beyond air exchange (and also where possible on pumping lines that aren’t either actively being primed or already connected to their intended point)
With tubing connected, I unscrew the lid of the Pioreactor vessel and pull up the tubing of all lines except the output line about as far as I can while still being able to comfortably pull them down later as needed
I then screw this vial lid onto a different Pioreactor vessel vial that’s filled with DI water and run the output pump until the line is primed (depending on what assays you want to run on your output bottle contents you may want to screw the gl45 lid for the output bottle on a different container while pumping so things aren’t more dilute than they should be)
I then return the height of other lines on the vial lid to the intended positions and run the media pump (and similarly for your alt-media pump) with the vial lid on another extra Pioreactor vial for holding the output of this pumping
At this point, media, alt-media, or DI water theoretically should be in each line from the input bottle all the way to the output line into the Pioreactor vessel (or from the vessel to the output bottle) so I then screw the lid of the Pioreactor vessel used for priming onto the inoculated vessel (previously set up with a different vessel lid with 0.22 µm filters on each luer lock to try to prevent any contamination)
Then remove the pump heads from the “dummy” Pioreactor in the BSC and carefully carry the whole connected setup (media bottle, alt media bottle in your case, output bottle, appropriate pumps, and inoculated vessel) to where your Pioreactor array is and “install” the pump heads to their proper pumps and seat the vial in the relevant Pioreactor.
Repeat for each Pioreactor in your experiment
My thought process with this is that it minimizes contact with non-sterile environments as best as possible while allowing the pumps to be properly primed (or at least for the most part). If you put sterile filters on any of your lines that media flows through that adds an extra wrinkle of wetting the filter and then connecting it to the proper line, but in my experience the one time I tried that it worked relatively well.
Since you’re just running pumps continuously until there is visible outflow on the other end, you don’t have to worry about differences in calibrations between individual pump body + pump head combinations.
There’s obviously some downsides to this workflow (lots of wasted glassware, need to make extra media/alt-media beyond the amount needed for the number of intended dosing cycles of a given experiment, have to go in and out of the BSC often increasing risks with sterility, and so on), but this has worked successfully for me thus far to prime my pumps without introducing contamination.
The biggest drawback in my experience is that this is a quite time consuming process, so there is some drift in the culture between when makes sense to remove it from an incubator or dilute it to a target starting OD to inoculate in vessels and when you actually can start running all the Pioreactors in your array and start your dosing automations so your milage may vary on that.
I unfortunately haven’t taken pictures of this at different parts of the process, so let me know if there’s any parts that are unclear because of that.
Let me know if there’s any points of confusion on this, and this is by no means a perfect solution, it’s just what I’ve found to work for me thus far, so if you have any suggestions on how to improve it, I’m all ears!
I also work with antimicrobials. Prior to acquiring the pioreactors, we worked with our own DIY mini-bioreactors whose size and geometry made them impossible to use inside biosafety cabinets. On numerous occasions, I´ve had ruined experiments during priming of the antimicrobial tubing.
However, with the pioreactors I use a procedure similar to that of Donald C:
All the equipment is autoclaved, except for the tubing section of the peristaltic pumps (it is easy to break the pump head fittings when removing/ inserting the tubing), which is sterilised with sodium hypochlorite. The connection of the different sections of tubing is carried out inside the laminar flow cabinet to ensure asepsis. Once I’ve inoculated the vial with culture medium and cells, I cover it with a cap compatible with the pioreactor vials. On the other hand, I connect the original cap of the morbidostat to a compatible empty vial. I run the pumps until the tubes are primed. Then, I unscrew the cap from the ‘priming vial’ and connect it to the pioreactor vial.