A reviewer of ours (unknown; cannot be contacted) has asked us to include "in and out" staining rather than z-stack microscopy in our manuscript to demonstrate bacterial invasion. Results are backed by gentamicin protection assay already however, the reviewer asked us to provide "in and out" staining using gram stain towards our bacteria (outside the human cells) to go with our EUB338 (inside and outside the human cells). We have not been able to locate a protocol and upon our first attempt our human epithelial cells (HT29) have turned pink upon safranin staining. This is not of any help as it is now impossible to tell which bacteria are only outside of the cells as the entire human cell is pink in colour.
Is anyone aware of such a procedure?
We currently use EUB338 to stain our bacteria following 4% PFA fixation (this stains both microbes outside and inside the human cells).
Thank you in advance for your help.
Why not ask the reviewer in a reply to his/her request? But only if the additional experiment is necessary. If you don't find the experiment necessary simply refuse to do it and explain your reason(s) for the refusal to the reviewer.
The reviewer might be referring to intracellular and extracellular staining. I have read about this staining, utilising antibiotics to kill the extracellular bacteria to show the presence of bacteria inside the cells (intracellular) without lysing the cells. I have seen a lot of immunofluorescence staining done to investigate the extracellular and intracellular bacteria. However, I am not sure if Gram staining can do the trick.
Nonetheless, you can opt to decline the suggestion by the reviewer, as suggested by Martin. You can thank the reviewer for an excellent suggestion and that it can be your potential investigation in the later future.
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