TABLE 1.

Troubleshooting table

Problem	Possible reason	Solution
Lack of biofilm formation (at the end of Step 11)	Problems with counting the seeding inocula	Make sure the dilutions and calculations are made appropriately
	Some fungal isolates do not form biofilm	These isolates should be considered “non-biofilm” formers
		Establish “cut-off” values to identify non-biofilm forming isolates
		Consider using some other surfaces/materials for biofilm formation
Biofilms visible by the naked eye but no orange color develops (at the end of step 21)	Old XTT solution	Prepare a fresh solution of XTT
	Menadione not added	Always remember to add menadione to the XTT before adding to the wells
Microbial contamination (at the end of step 11)	Not maintaining sterility	This is a rare occurrence.
	Poor handling	Always maintain proper microbiological handling techniques
	Storage of reagents	Minimize re-opening of reagents. Discard any reagent with signs of contamination
Biofilms peel-off from bottom of wells (all washing steps, but particularly after step 11)	Aggressive washing	Be gentle during washings (follow recommendations)
		Most often this will be visible by the naked eye. Do not use these wells for final calculation
		Make sure enough replicates are performed to minimize problems
	Biofilms too old (mature)	Disaggregation should be considered normal in the biofilm life cycle as biofilms reach maturity. Use younger biofilms