Table 4.

Techniques for XTT assay troubleshooting

Problem	Possible reasons	Options
Biofilm disruption during washing	Too-vigorous washing	Use gentle washes
	Subculture conditions not optimal for biofilm formation	Consider alternative subculture conditions
	Medium not permissive for growth	Use uridine or other nutrient supplementation as required for strains
	Adhesion- or biofilm-defective strain
Susceptibility not detected	Strain resistant to antifungal concn	Shorten duration of biofilm growth
		Increase antifungal concn
		Decrease drug dosing interval
	XTT incubation too long	Shorten XTT incubation time
Resistance not detected	Immature biofilms	Increase duration of biofilm growth
	Strain susceptible to concn and dose	Decrease antifungal concn
Fluctuating results	Too-vigorous washing	Use gentle washes
	Subculture conditions not optimal for biofilm formation	Consider alternative subculture conditions
	Medium not permissive for growth	Use uridine or other nutrient supplementation as required for strains
	XTT not in solution	Filter or centrifuge XTT to remove insoluble material
		Mix XTT prior to use
	Agents exposed to light	Add assay agent in the dark
Poor biofilm formation in outer wells	Liquid in outer wells evaporates during incubation	Avoid using outer wells for experiments
		Add liquid to outer wells to prevent evaporation from inner wells
		Use Parafilm and/or foil around plate to prevent evaporation