Gram stain question posted in description below

Question

distilled water was acidentally used to decolorize your specimen instead of alcohol during the Gram-staining technique.

 

1.  What is the color of your Gram negative bacterium after the staining procedure is complete?

 

2.  What is the color of your Gram positive bacterium after the staining procedure is complete?

Answer 1

Gram positive bacteria have a thick peptidoglycan cell wall which binds lots of crystal violet (CV) and Iodine in the first step of a gram stain- making them purple.

Gram negative bacteria bacteria have a much thinner peptidoglycan layer that also binds CV and Iodine.  In addition they have an outer cell membrane, which initially serves to trap the purple CV and iodine inside the gram negative bacteria (and raise its concentration) and make the MUCH thinner peptidoglycan layer purple.

So at first, both gram negative and positive bacteria stain purple.

When you add alcohol/acetone, the outer membrane is dissolved (since alcohol/acetone dissolves lipids, but not peptidoglycan).  

Now the CV and I that was previously held in the gram negative bacteria can no longer accumulate there because the outer membrane is no longer helping that.  So that proportion of the purple dye in gram negatives washes away.

In addition, the alcohol/acetone can dehydrate both peptidoglycan layers (thick and thin), and cause CV and I to be squeezed out.

You are supposed to "destain" only as long as the purple is coming off you slide: i.e.: as long as the easily-removed purple is leeching out because of the dissolving of outer membrane.

 

Thus the role of the alcohol is to remove the outer membrane, and thus makes the initially purple gram negative bacteria turn colorless (and then pink after the saffrin stain).

 

So if you leave out the alcohol/acetone.  The gram negative bacteria will retain their outer cell membrane, and stay purple when your staining is done. Thus both gram positive and negative bacteria will stain purple without the alcohol/acetone step.

 

 

Answer 2

The decolorization step is key in distinguishing between gram negative and positive bacteria. When alcohol is added during the decolorization step, it causes the outer liposaccharide membrane on gram negative bacteria to be lost. During the decolorization step, the gram positive bacteria become dehydrated, trapping the crystal violet-iodine complexes within the multi-layered peptoglycan wall. If you skip this step, gram negative bacteria retain the purple dye.

 

If you decolorize properly, the saffrin dye will dye the gram negative bacteria pink.

 

Gram stains are really quick and easy to do. If you think you made a mistake, just repeat the stain with a fresh sample. Just make sure you don't leave the alcohol on too long.