You perform Gram reaction with crystal violet on pure cultures and not mixed cultures. So, please purify your cultures and apply the crystal violet to what you want. The cell culture techniques is a different protocol entirely.

Hi Maurice Ekpenyong, I'm actually performing the Gram staining on a tissue section, so wondering is there any better method if not Gram reaction to identify bacteria? Cheers

Hi Alex,

Why do not you use KOH Technique? It is very fast and I think reliable.

The following article will be useful.

Efficacy of the Ryu nonstaining KOH technique for rapidly determining gram reactions of food-borne and waterborne bacteria and yeasts.

Hi Negar Bagheri , it seems like the KOH technique is more for bacteria culture. Is it a reliable technique for bacteria detection in cell tissue? Cheers

The Crystal Violet assay is based on staining cells that are attached to cell culture plates. It relies on the detachment of adherent cells from cell culture plates during cell death. During the assay, dead detached cells are washed away. ... The cell biomass can be used to infer levels of cell viability / cytotoxicity.. https://www.abcam.com/crystal-violet-assay-kit-cell-viability-ab232855.html

Also check https://conductscience.com/lab/crystal-violet-stain/

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Maurice Ekpenyong .-. It is most common to gram stain our pure cultures after isolation to determine whether gram + or - as well as morphology. But, it is also valid to gram stain in other situations. For example, to double check to see if there is bacterial contamination in a culture (should be done to double check what you think is a pure culture). To check a pathology sample to see if the infection is polymicrobial. A sample may also be gram stained to see if there might be other bacteria present that are not culturable, or not by the methods that you are using. These and other reasons provide good reasons that we can, and often should, do gram stains on pathological, environmenmental, and laboratory cultures to see what or if other organisms are present.

Alex Peh Yes you are right, KOH is more for pure bacterial culture. In the other hand KOH also make cell tissue more visible maybe you can combine this with crystal violet staining.

Alex Peh: if you are staining tissue sections for bacteria, then first you need thin sections. You could use frozen sectioning. Then fix the section on the slide with absolute ethanol or methanol for 5 min and dry. Then apply Gram film stains, but vary the method: crystal violet 2 min, Lugol's iodine 2 min, then wash in tap water and soak in tap water for 5 min to remove all free iodine, that reacts with counterstain to form black crystals. The shake off water and apply decolorizer for a few minutes, adding more if necessary until all free stain is removed (ie no more coming out) then apply red counterstain for 3 minutes. You might have to experiment with timings to get it just right, but this should give you a nicely red-stained tissue structure and no stain deposits, and you should be able to see gram positive bacteria easily. Gram negative bacteria will still stain (red) but you need to search carefully for these. The bacteria might be intracellular.

i think Wigherts modification of Gram stain may help