How to mix bacteria sample enough

Hello there,
We have a Liquid Handler from the company Copan in our Lab, the „Cyclone“.
It is a machine that can process microbiological plates automatically. It uses Hamilton Zeus pipettes.
I put 2 samples at a time on the device and while it is diluting the first sample the second sample has to wait until the first dilution series is done. My results compared to manual plating look good for the first sample but for the second sample they are not matching. It is always too high. It seems to me that the device is not mixing the sample enough again after it standing for a while and the bacteria cells settle down to the bottom. I think it then aspirates a high concentrated part of the liquid and that’s why the results are too high.
I am currently mixing a 20 ml sample 10 times 800 microliters. I already tried to use a different container and only 10 ml of sample but the results were the same. Today I tried to mix 20 times, I will have the results in a few days.
I cannot mix more volume than 1 ml, I use 0.8 ml currently because it is mixing crazy fast.

Do you have any ideas what I can try? Different mixing velocities? Do you have experience with mixing of samples on liquid handlers? I would very much appreciate any input!

Hi, could you prepare the dilution of your 2 samples in the same time ? In whitch kind of tube are your samples? Do you have the possibility to upgrade your liquid Handler to add a 5mL channel ?

Rather than using defined mix steps, I’d probably try to aspirate at a fixed height, then dispense at a different fixed height in the tube in a loop. Personally I would try aspirating from towards the bottom and then dispensing towards the top but it could be better the other way around.

Not sure what your interface looks like for controlling the pipettes, but adding a mix on those aspirate and dispenses for a couple of cycles for each of those steps can help too.

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I wouldn’t over-mix excessively because that can cause frothing with your bacterial culture. Just out of curiosity, do you eject tips after the first dilution?

Unfortunately I cannot prepare the dilution series at the same time. I also cannot upgrade the pipettor to use 5 ml Pipette tips.
My samples are in 12 ml tubes because in total I have a volume of 10 ml in these.

That sounds like a good idea. I cannot Programm these steps myself and need the vendor for this but I will suggest this to them and hope they will try.

Yes I eject the tips after each dispensing.