I’m back with another problem:
At the beginning of my workflow, I have to transfer samples from tubes to plates. The samples are lysed in an SDS-containing buffer and after bead-beating and incubation a bit of foam remains even after long centrifugation.
I know that cLLD and pLLD in combination can be used to detect foam on a liquid however, I struggle to implement it in an elegant way.
So I’m wondering if I could get any guidance regarding this topic:
- Any ideas how to get rid of the foam in the tubes (would be the easiest)
- If I want to solve this programmatically:
Use dual LLD in a first aspirate, cancel the aspirate in case of a difference larger than 2 mm, retrieve the lower value (should be the liquid surface), and perform a second aspiration at that height.
To further complicate things: I cannot redispense into the tubes, since there is a tissue pellet on the bottom that cannot be disturbed.
Any input here would be awesome,
Is there an expected volume where you could use a fixed height aspiration near well bottom (at slow speeds as to not disturb the pellet) to avoid the foam?
Similarly, is it possible to add a small volume of something like mineral oil prior to beating to prevent foam in the first place? It is likely easier to detect an oil layer rather than a foam layer at the transfer step.
Can you clarify - are you looking to detect foam or just move past the foam to aspirate the liquid in the tube? If the latter, you could just use pLLD by itself. I have used that approach oftentimes when dealing with patient samples where occasionally there are some with more bubbles/foam which triggers cLLD. Switching to just pLLD and disabling cLLD helped make the transfers more consistent.
Fixed height is not an option as the pellets will differ in size. Mineral oil might be possible, I’ll check this out.
I just need to move past the foam to aspirate the liquid. Any recommendations on the pLLD sensitivity settings? Sounds like the easiest solution to me
From tubes, probably the default or low sensitivity would work. I rarely found I have to change LLD settings from default.
Just tested it. Even with the low sensitivity the pLLD still seems to be to sensitive in this case. So I guess I have to opt for a combination of pLLD and long centrifugation. Or does anybody else have another good idea?
Would the phase aspiration step in the star pipetting tools library work for this problem?
I’m also looking to solve a somewhat similar issues, would like for robot to distinguish between two layers (water and an immiscible organic solvent) and draw from specified one but pLLD and TADM haven’t proven to be successful for me. Any suggestions would be appreciated!
Hi @jdhawkins15 ,
For @dominik.b 's issue, Phase Aspiration would not help, but for your 2 phase question that function will work.
Assuming the Organic is floating on top, you can call the PIP_PhaseAspiration function using pLLD to remove the top layer until the TADM band (lower band) has been crossed. You just need to collect TADM data for the top layer and draw the bottom band so that when it aspirates into the water layer it crosses the band. Below is an example TADM graph of 2 immiscible liquids being detected using TADM.
The errored bands are the transition points in which the new liquid is detected and the aspiration stops. Then the channels dispense back what was aspirated to prevent carry over to the target location.
I would suggest making a new topic if further discussion is needed though, to prevent the original question from being lost.
Do you know how much volume is in the tube to begin with? A back calculation of the container may be an option to determine the fixed height that would be under the foam.
Unfortunately not. I start with tube’s that contain 1 ml of a mixture of tissue / soil and ethanol. I remove the ethanol in a first step and then add a fixed volume of lysis buffer, however the total volume in the tube is then the sum of the tissue pellet plus the additional liquid.
The problem mostly occours with the negative controls since they seem to build more foam, the actual samples are mostly free of foam after 3 Minutes of centrifugation. I thought about using antifoam, but that is quite expensive. Another option would be to store the tubes for one night after lysis and then only centrifuge, however that limits my throughput.
thank you so much! Will try and if I’m still having issues will make a new thread.
Could you use TADM coupled with continuously decreasing aspiration heights to find the liquid level then start an aspiration at that height?
We know that foam/bubbles gives a super choppy pattern where a proper aspiration is predictable.
What follows is a half-baked idea:
- You start at a fixed height in the tube and aspirate with liquid following
- Your TADM curve is set per a correct aspiration in your liquid
- During aspiration with liquid following 3 things can happen. 1: You only aspirate air meaning you have not reached foam nor liquid yet and TADM throws an error. 2: You start to aspirate foam and TADM throws an error. 3: You are in the liquid and the aspiration was successful.
- In cases 1 & 2 above you will retry the aspiration moving down by a certain fixed height increment and try to aspirate again.
- Eventually you will successfully aspirate the liquid for all channels. You can then dispense in the plate.
IDK it’s an idea
Thanks for the input, this could actually work, but is really complicated. I’ll also take a look into anti-foaming reagents, they don’t seem to be expensive and would be an easy fix for my problem if they work as intended.
Is this in a plate or tubes? I would personally use an imaging platform or a more modern liquid handling tech.
It is in tubes, could you elaborate how that would help?
Vision seems useful here. Make/acquire a carrier with cutaways, take a picture, get foam height from the image. You might even be able to get away with traditional image processing rather than ML if the foam is colored differently enough from the rest of the liquid.
This shouldn’t be too complicated. The hardest part is creating the curve, but capturing the aspiration errors should be super simple!
I may be able to help in the next year if you are still struggling!
Thanks very much for the offer, I’ll come back to it if I’m still struggling with this in the upcoming year.
I’ll take a look into anti-foaming agents in the meantime. A solution with visuals would also be possible but highly complicated in my setup. The tubes are in 48-well racks and cannot be picked individually in my automation.
If you don’t know how much is in the tube, how do you know how much you are aspirating? How are you avoiding the pellet if you don’t know how large it is?
Without knowing the answers to the above questions, I think that the following might work:
- teach the tube “bottom” so that it is high enough to avoid all pellets
- start at a fixed height that is definitely going to be within the liquid
- turn liquid following to “On”
- set the aspiration mode to “Aspirate All”
It will travel down as it aspirates, until it gets to the “bottom” then it will stay at that position and continue to aspirate until it has finished aspirating the designated volume.
Another option if the foam layer is always around the same depth, set the submerge level to foam height + 2 mm