PT  - JOURNAL ARTICLE
AU  - Nielsen, Regitze Lund
AU  - Bjarnsholt, Thomas
AU  - Jakobsen, Tim Holm
AU  - Lichtenberg, Mads
TI  - A method for separating bacterial cultures into single cells and defined size fractions of biofilm aggregates
AID  - 10.1101/2024.01.25.577188
DP  - 2024 Jan 01
TA  - bioRxiv
PG  - 2024.01.25.577188
4099  - http://biorxiv.org/content/early/2024/01/25/2024.01.25.577188.short
4100  - http://biorxiv.org/content/early/2024/01/25/2024.01.25.577188.full
AB  - In vitro microbiological experiments that aim to describe differences between planktonic and biofilm aggregate populations are challenging since liquid batch cultures contain a mix of both. Here, we present a simple method for fractioning a bacterial liquid batch culture into aggregates and single cells.Stackable cell strainers with mesh sizes of 30 μm and 10 μm were used to filtrate 6 day old batch cultures of Pseudomonas aeruginosa to produce size fractions of 0-10μm and >30μm. By confocal laser scanning microscopy measurements, we show that 95.5% of the total biomass was <10 μm in the “0-10μm size fraction” and that 92.5% of the total biomass was >30μm in the “>30μm size fraction”.Furthermore, the adjustment of bacterial concentration using CFU/ml was validated by quantifying the total DNA of viable bacteria in the two size fractions after DNase treatment to deplete eDNA and DNA from dead bacteria. Surprisingly, this showed that adjusting the bacterial concentration using CFU/ml was a valid method with no significant differences in total DNA from viable bacteria.Competing Interest StatementThe authors have declared no competing interest.