Question.

I just did a B & H gram stain for the first time. All tissue stained various shades of purple against a clear background. There was no yellow or red staining at all. The protocol I used replaced all acetone differentiation steps with 95% ethanol, "to avoid over-decolorizing."

What am I doing wrong? Should I:

    1. Use acetone instead of 95% ethanol, or a combination of equal amounts?
    2. Use saturated aqueous picric acid?
    3. Use 0.1% basic fuchsin (instead of 0.01%)?

Answer.

The following modifications of Brown & Hopps give consistent differentiation of Gram negatives with reduced risk of over-differentiation. Cellosolve is used instead of acetone, and tartrazine instead of picric acid.

The crystal violet staining is as in the original method. Modifications are as follows:-

Substitute Lugol's or Jensen's iodine for Gram's to give a stronger crystal violet-iodine complex.

Use cellosolve (= ethylene glycol monoethyl ether = 2-ethoxyethanol) as decoloriser. The smell can be unpleasant, but it is slower in its action and more easily controlled.

Use 0.5% basic fuchsine, for 5 mins, to counterstain the Gram negative organisms.

After rinsing  with water apply Gallego's differentiator (1% acetic acid with 2% formalin, in water) for 5 mins. Rinse with water and flood sections with 1.5% tartrazine for 1 min.

Rinse the slides with water. Now take one slide at a time: blot with filter paper, flood with cellosolve for 6 - 10 secs, blot again, and then place slide directly in xylene, 2 or 3 changes

Coverslip and mount. Repeat with the remaining slides, one at a time.

The extra step with the cellosolve seems to remove excess fuchsine from cytoplasmic elements in the background, thereby increasing visibility of Gram-negative bacteria.

Mike Rentsch
Lab. Manager, Aust.Biostain.
(ausbio[AT]nex.com.au)