Most capsule staining methods use an negatively charged dye like India ink or Congo red to stain the slide, and positively charged dyes like crystal violet to stain the cells. However, I have never had a decent result with these methods.
Anthony’s method uses Crystal violet (7min), then rinses with 20%CuSO4. The only explanation I have found for this method states that the crystal violet stains the cell and the capsule purple, and the CUSO4 decolorizes and counterstains the capsule light blue.
What the stain actually looks like does not look anything like what this explanation describes. I get a light purple background, dark purple cells, and clear capsules. Beautiful stains, easy to visualize capsules, but I need to be able to explain how this works.
Anyone have any idea why CV + CuSO4 would stain the slide and the cell?
It’s been a long time since I played a microbiologist, but it seems to me that you probably have a redox reaction going on between the crystal violet in the capsule and the CuSO[sub]4[/sub]. CuSO[sub]4[/sub], like most heavy metals, won’t pass easily through the cell membrane, so the cell remains purple. As a strong chaotrope, Cu[sup]2+[/sup] probably also messes up any pores through which the unoxidized dye might leak.
I understand why the cells stain purple. Crystal violet has a + charge, bacterial cell has a net - charge. Since this method does not use an acetone/alcohol destain like the Gram stain does, (+ probably for the reasons that you state) the cell remains purple.
The question is why the background (glass slide, + charged) stains at all.
If you’re following the Anthony’s stain protocol, you’re preparing your slide from a skim milk culture; this matrix retains the stain, providing a background against which the capsules should be clear or light blue.
Thank you! I didn’t realize that Anthony’s protocol specified a skim milk culture. I had been using a litmus milk culture to promote capsule formation, and had not considered the impact of the matrix on the stain. Ignorance fought!