I am working with marine biofilms, grown in the field on microscope slides for two weeks. I would like to remove the biofilm (keeping cells intact) so that the collected samples can be used in a DNA extraction kit. I had planned to use sonication (with a 40 kHz/70W ultrasonic bath), however, after some trials, I am find that a large amount of biofilm still remains on the slide after 30 minutes sonication time (checked by DAPI staining). I am wondering if I am missing a key step - such as, should I be adding something to the sonicaiton buffer instead of just using sterile seawater?
I have found that sonication baths do not work as efficiently as the sonication probes. If a probe is not available, I might suggest trying to increase the power and/or duration. Using a probe, I have successfully removed marine biofilms from glass at 26-30W for 2 mins (while the sample was on ice to prevent warming).
Hi Kristina, Thanks for your response. Yes, we have a probe and I had considered switching to use it instead of the bath, however, I was concerned that a probe would lyse cells - I am trying to avoid cells lysis.
The frequency applied is of extreme importance for efficient matrix removal without undesired cell breakage and/or cell wall extraction. The frequency often needs to be optimized for each boifilm/sonication system. But it is critical to keep the suspension cold (on ice) all the time. But some systems can be sonicated for up to 5 mins at 20KHz without breaking cells.
Our analytical experience confirms what Kristina supports even if our analyzes were performed on solid matrices.
Hi Kristina, The probe did remove the biofilm (Thanks!). I wanted to ask if you know have a method of measuring if cell breakage occurs. It is important for me to minimise breakage because of later steps in the experiment.
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