Table 3.

Critical parameters and troubleshooting.

Problem	Possible Cause	Solution
Poor or no Biofilm forms on bottom of plate	Plates are not tissue-culture treated	Use tissue-culture treated plates
	Medium may not be adjusted with optimal glucose concentration	Repeat with medium that is properly adjusted with glucose
	Bad plate lot	Try a different lot from the same manufacturer, or a different manufacturer
	Incubation period too long, biofilms falling apart	Optimize length of incubation
Biofilms robust after washing, fall apart with the addition of ethanol	Heat fixation may be required	Heat fix biofilms at 60 °C for at least 60 min
Biofilms robust after heat fixation, mostly gone after staining step	Not heat-fixed long enough	Increase time for heat fixation
	Stain incubated in wells too long	Decrease staining time
Large hole taken out of biofilms when washed with a plate washer	Aspiration tips may be too low in well, or aspiration rate too high	Raise height of aspiration tips, or decrease the rate of aspiration
		Decrease number of aspiration steps
Biofilms on edges of the plate are less dense than biofilms on the interior of the plate	Possible edge effect taking place, incubation parameters not consistent across entire plate	May require an incubation atmosphere that will allow entire plate to grow at the same rate; i.e., anaerobic conditions