Gram staining?

Question

In gram staining, can we inverse the crystal violet and safranin dye? Do any problem will be created due to this?

Answer 1

No, no, no, a thousand times no. Due to the composition of the bacterial cell wall, it is essential that crystal violet be used first, flushed with water, then Gram's Iodine, then decolorized with acetone-alcohol, then counterstained.

Wikipedia has a more extensive, yet easily understandable explanation.

Answer 2

good question!!!!!!! y didnt i think of that while performing gram staining!!!!!!i'll definitely ask this ques to my teacher..lets see wt answer she has................

Answer 3

yes there will be problems...

it has to be in the correct order:

1. crystl violet

2. iodine

3. 40% acetone in alcohol

4. safranin

crystal violet added... the cell wall absorbs it n becomes purple...

acetone in alcohol added to decolourise the effect c.violet has n the cell wall... that is.... this has to be done for only 3-5 secs or else all the c.violet will be decolourised....

safranin is then taken up by those cell membranes that have been decolourised...

therfore.... those with thick cell walls (purple) are gram +ve because they have not been completely decolourised, and those which have thin cell walls (pink) are gram -ve because the decolourised cell wall was able to absorb the safranin....

Answer 4

It is important for the crystal violet to be added first as it must form a complex with the iodine. The alcohol wash removes any crystal violet that has not complexed hence in Gram neg cell wall. This will be replaced with the counterstain (in this case safranin). If they were switched you'd never get a result as both dyes would be washed away!!!

Answer 5

No! Do it the right way round and all will be well. You can substitute another dye for the saffranin if you want,but the crystal violet staining and alcohol wash must come first.