I am trying to isolate bacteria with and without bound IgA from oral washes using anti-IgA APC antibodies and anti-APC beads plus MACS columns. Both populations when run through the flow cytometer have a lot of debris/cells and the positive population looks like it has a lot of 'negative' bacteria as well.
Is there a way to clean up the bacteria population? The reason is we want to do single cell sequencing on the bacteria so need a really clean population.
Dear Dr.Sophie Irwin,
You may perform streak plating.
It is a technique wherein the oral wash sample, which likely contains a mixture of bacteria, debris, and other cells, is initially streaked onto an agar plate. The sample is then repeatedly streaked across the plate, with each streak covering a smaller area than the previous one. This creates a dilution gradient where the number of bacteria gradually decreases.
By the time the last streak is made, individual bacterial cells are hopefully far enough apart that they can grow into separate colonies when the plate is incubated. Each colony represents a single bacterial cell that has multiplied and grown into a visible cluster on the agar plate.
This technique effectively separates bacteria from debris and other cells in an oral wash sample, and it works by diluting the sample and creating areas where individual bacterial cells can grow into isolated colonies. These isolated colonies can then be further used for various purposes, such as identification or culture.
Regards,
Malcolm Nobre
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