If bacterial cells are completely lysed following treatment with an antimicrobial- then washed- then PI stained- Why is concentration lower?

Based on the information you've provided, it seems likely that the lower concentration of cells observed in the PI stained samples after treatment with an antimicrobial is indeed due to cell lysis and subsequent washing steps. Let's break down the possible reasons for this:

Cell Lysis: Antimicrobial treatments can disrupt bacterial cell membranes, leading to the lysis of cells. When cells are lysed, their intracellular contents, including DNA, are released into the surrounding medium.

Washing Steps: After the antimicrobial treatment, you mentioned that the samples were washed. Washing is a standard step in many experimental protocols to remove any extracellular material, including cell debris and released intracellular content.

PI Staining: Propidium iodide (PI) is a commonly used dye to stain nucleic acids, specifically DNA. It can enter cells with damaged or compromised membranes and intercalate with DNA, resulting in fluorescence. In your experiment, the PI is likely staining the released DNA from lysed cells.

Putting it all together, after the antimicrobial treatment and washing steps, the lysed cells release their DNA into the medium. When you stain the samples with PI, it primarily stains the extracellular DNA, as it can no longer penetrate the intact membranes of viable cells. Since the PI is staining mostly the released DNA from lysed cells and not the intact intracellular DNA of viable cells, the observed concentration of PI-positive cells will be lower in the treated samples.

To verify this explanation and further interpret your results, you may want to consider the following:

Control Experiment: Include an untreated control sample without antimicrobial treatment to compare the results and determine the baseline level of PI staining due to any natural cell death or lysis during the experiment.

Time Course Analysis: Perform a time course analysis after antimicrobial treatment to observe the kinetics of cell lysis and DNA release. This will help you understand the rate at which cells are lysing and DNA is being released.

Microscopy: If possible, consider using microscopy to visually confirm cell lysis and DNA release, which can give you additional insights into the mechanism.

Quantification of Intracellular DNA: Explore methods to specifically quantify intracellular DNA in treated and untreated samples. This could provide further confirmation of the impact of antimicrobial treatment on cell lysis and DNA release.

All the best buddy

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