Problems with the Gram stain:
(1) Gram-positive bacteria appear Gram-negative
(2) Gram-negative bacteria appear Gram-positive
(3) mottled or irregular staining
(4) excessive background staining
Risk factors that would not affect a concurrent control smear or other slides:
(1) interfering substances such as mucus or topical agents
(2) smear made too thick
(3) deterioration during transport or handling
(4) poor fixation
(5) over-heating during fixation
(6) antibiotic effect
(7) old culture or old abscess with “sickly” bacteria
Risk factors that would also affect a concurrent control smear and other smears:
(1) over-decolorization (Gram-positive bacteria appear Gram-negative) or under-decolorization (Gram-negative bacteria appear Gram-positive)
(2) operator error or inexperience
(3) deterioration in a reagent
(4) failure to filter a reagent (with precipitate)
(5) failure to use a reagent (leaving out a step)
(6) reader error
When in doubt it is always a good idea to repeat making the smear and performing the stain, taking care with each step.
