eVOLVER Forum

Recommendations for Septas and Needles?

Question:

Does anyone have any recommendations for extra long needles and septa for applications requiring gas exchange, using the typical 40mL chemglass borosilicate vessel?

Answer:

We’ve recently been 3D printing nylon mounts that enable the vessel to interface with more liquid/air inputs that have been generally useful for use in eVOLVER:

These mounts are designed to screw onto the typical 40 mL glass vessel. You can either use them with a silicone septa or without.

Without a Septa
Running the system without a septa is more convenient and sterility hasn’t been an issue in our hands (even with the sampling port on the top). I would recommend using 1/16" PEEK tubing to be threaded through the holes. PEEK tubing is polymer based, very resilient, rigid, and inert. This is a great choice for defining the volume of the culture vessels (efflux length) and also has the added advantage of not rusting (whereas needles might rust over time).

PEEK Tubing

With a Septa
Adding a septa helps in conditions where sterility is more of a concern (mammalian cell culture) and gas control is important. However, this makes sampling more difficult and you would need to use needles to interface with the culture vessel.

Silicone Septa
I typically cut a small piece off the septa with a razor blade where the sampling port is to make it easier to sample with a pipette tip. Additionally, this helps vent the vessel and prevent pressure build up.

Influx Needles
Input needles/ Venting Port

Efflux Needles
You can choose what length best fits your application

Extra long Needles

Premium Veterinary Hypodermic Needles, Sterile, Air-Tite, Length=3 (76 mm), Gauge=16

I measured out the volumes for each port and different syringe needle lengths. It’s hard to find 3.5’’ needles, but putting 0.5’’ tape on the 4’’ needle works listed above works.

Volumes are in mL.

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Thanks Zack! This is great. One more detail is to watch out for the needle gauge. Small needles have high resistances. This may cause bubbles if used during influx lines (caused by high velocity ejection rate) or overflow from slow update rate if used in efflux lines.

We recommend you to test it under your experimental conditions (depends on pumps, etc) but typically 16 gauge works well for us.

If we plan on using the silicone septa, does that require different vial caps? Like the ones shown here:

No they should be compatible with the ones that came with the eVOLVER. They should fit right under the 3D printed nylon caps.

are the 3D printed nylon caps autoclavable? I noticed on the Jove paper you autoclaved with the caps- were those the 3D printed nylon ones?

Yes they are autoclavable

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Are STL files for the nylon mounts available somewhere?

Thanks! I had some confusion over what the post was saying and thought the second image was indicating there are additional mounts that go on the nylon caps ports for holding the septa that were not on the repo.

We had tried using needles and septa for a while, but it seemed to take a disproportionate amount of work to get the septa cut and into the ports given it’d have to be done each time as the vast majority of the septa came out with the needle.

Is there a specific way that works well for interfacing the septa and vial caps? I had been cutting small “pyramids” and inserting them into the ports on the pointed end and twisting.

I dont have specific guidance there, as for most of our work we used silicone tubing and that was very annoying to get the volumes consistent. This was implemented toward the tail end of a lot of my experimental work. Maybe someone else will have more guidance on this? Ultimately, do whatever works best for your experiments!

I usually just push the needles through the septa. I sometimes see some tiny septa particles in the culture from the larger gauge needles but it’s never caused a problem in my experiments. You could use a smaller gauge needle, but the flow rates might be affected.

What is the minimum culture volume that you would feel comfortable running? As in there’s no interference on the OD readings, even with a high stirring rate.

I typically l like running mine at 25 mL, but a simple experiment can determine whats best for your system.

Brandon

Just to add to that, I’m pretty sure the LED/PD are around the 5 mL level. I’ve run experiments down to 10 ml with slower stir rates and it seemed to work, but you should test on your setup before setting up anything. I never did further testing as my experimental needs changed. Running at 25 mL as Brandon suggested is definitely the safest way to go though.

Zack

Hi,
So we have gone another way actually, with the same caps. We are using standard needles of the appropriate length and various gauge…and then we “fix” them on the caps with a small bit of heat-shrinkable tubing. This way, we can just put the whole thing in the autoclave after washing the tubes, without removing the needles. Between experiments, we soak the caps+needles in EtOH, then water, and then we assemble the vials with the caps and put everything in the autoclave. We have done this over many many cycles without issues. This saves us for re-assembling the caps and needles every time. We have had no issues with using metal needles several times (we have run experiments over 3 months in a row so…)

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Thanks for the tip @dcoudreuse. Just did a test of this! Works well without the septa and is pretty snug.

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