Arrangement of steps in Gram staining - (May/12/2008 )

What if you used the safranin stain instead of the crystal violet stain first, and used Gram stain at last instead of the safranin stain; what would be the outcome for a (i)Gram +ve strain (ii) Gram -ve strain of bacteria under such a mistake?

Thanks.

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Im not 100% sure what would happen. If you are willing to wait 24 hours from this post, I can have an answer for you. I will test E. coli (Gram negative) and Staphylococcus aureus (Gram positive).
I do the following:

1) stain with basic fuschion stain [I don't have safranin, but it does the same thing]
2) ddH20 rinse
3) Iodine
3) ddH20 rinse
4) acetone/alcohol wash
5) ddH20 rinse
6) crystal violet stain
7) ddH20 rinse, and dry slide.

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Everything comes up dark purple. (i.e both bacteria stain as if they are gram +)

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Thank you so much! And for the effort to actually do the test! I thought so too but was really unsure about it. Especially since my friend said that the alcohol would cause the cell to dyhydrate and close the peptidoglycan pores.

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Alcohol decreases CW permeability. It doesn't completely close up pores (it probably could and also completely dehydrate the cell if you expose the cell long enough) which means CV can still stain the cell after you wash out (using alcohol) safranin. Iodine isn't even necessary to stain the cell blue/purple since the iodine here is used only as a mordant.

Even if you have a dead cell (assuming the cell hasn't lysed), you'd still be able to stain it with CV. That would be like applying CV to any organic nonviable matter you can think - your cotton shirt, cooked spinach.

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