eVOLVER Forum

Using nylon caps to determine culture volumes

EDIT: Using syringe needles is recommended now (Recommendations for Septas and Needles?)

Summary
In this tutorial, I hope to teach how to build your own Smart Sleeve vials and determine culture volume when running continuous culture.

Why this is important
Precise culture volume is very important in continuous culture. This determines your target flow rate in chemostat mode and how much media should be added in turbidostat mode. In our experience, the best and simplest way to set the culture volume is via the efflux straw length. In any dilution event, the efflux pump is programmed to run for longer than the influx pumps and thus pumps the media level down to whatever height the straw is set at. This is so that if there’s ever a small mismatch in pump rates, media would not accumulate in the vessel (causing an over flow).

What are considerations with choosing materials to use?
Generally, a rule of thumb for material choice is to use autoclavable, alcohol/bleach resistance, rigid material for the efflux straw. The difficulty is that a lot of these materials don’t come in small diameters (1/32") or have the potential to rust. We tend to avoid using metal straws/needles, if possible. Of course, there are use cases where a septa is useful and one would want to use needles. In this tutorial, we show how to use PEEK tubing to fix the media level.

What you will need in this tutorial

1. Cut the tubing to the appropriate size, according to how much culture volume desired

2. Cut a small piece of Silicone Tubing and thread the PEEK tubing through this piece

This silicone tubing will keep the straw in place and fix the distance from the bottom of the nylon cap. The location of where the silicone tubing is placed is important, determining how far the PEEK tubing goes down into the vessel.

3. Slot in one of the four inserts on the nylon cap

3. Insert 1mm tubing on the other end of the PEEK tubing

4. Attach a longer silicone piece to the other side of the PEEK tubing to secure it on the nylon caps

5. Verify height and stay consistent across all vessels in the same experiment

6. Repeat same procedure for influx straw
The height for the influx straw is not as critical. It is important for the flux straw to be pointed at the walls of the vessel, to prevent bubbles from forming in the vessel, skewing the OD readings.

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Seems like batch variation in silicone tubing might cause issues for some tubing causing it to leak a bit. I would recommend using smaller tubing. Post edited accordingly.

https://www.evolver.bio/t/tubes-leaking-at-vial/95

VWR site says 60985-708 has been discontinued. did you mean to say a different part # ?

Sorry I grabbed it straight from the publication. I’ll make a new tutorial today and update it.

@Max_Schubert @langevin.am Just updated the post. The main difference is that a 1 mm silicone tubing is slotted on the PEEK tubing before the larger tubing. This is to make sure the seal between the PEEK and the 1/16" tubing is good.

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Note for others: Use a syringe filled with air or DI water to check that the straws (especially the PEEK tubing) are not obstructed by bends or nylon dust before first use (or even before autoclaving for an upcoming experiment). Even if the straws are only slightly obstructed, they can cause significant pressure build up in the lines!

Additional tips:
(1) Make sure the PEEK tubing is cut as cleanly as possible, otherwise it may curve inward, limiting flow as well.
(2) If you are concerned about nylon dust in caps, use a spare piece of PEEK tubing to clear out the channel.

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