Gram-positive bacteria in histologic sections may not stain properly under certain conditions.
Situation: Gram-positive bacteria do not stain properly in a tissue Gram stain
Possible causes:
(1) over-decolorization
(2) Gram-positive bacteria with damage to cell wall (death, antibiotic therapy, etc)
(3) error in the iodine solution step
(4) stained slide left too long in alcohol solutions prior to clearing and coverslipping
Pure acetone may decolorize quickly. To prevent overdecolorization the options are:
(1) use acetone but keep step very short (may be subject to variability)
(2) use a mixture of acetone and alcohol or other weaker solvent
Confirming the proper staining of bacteria highlights the need for proper controls for all special stains and the need to treat them exactly the same as patient slides.
Iodine solutions may deteriorate with exposure to air. Iodine solutions should be used fresh and applied properly.
Once staining is complete the stain should be rapidly dehydrated and mounted.
