Table 2.

Troubleshooting Guide for Dental Plaque on a Chip In Vitro Culture Model

Problem	Possible Cause	Solution
Bubbles disrupt biofilm	Air introduced through injection port, cellular respiration.	Pay careful attention to inoculation and stop injection before air is introduced. Place channel slide on an angle so that flow occurs up a slope allowing bubbles to float up away through flow channel.
Biofilm overgrown	Media too concentrated, culture time too long, or cell density too high.	Dilute media systematically in di H2O, adjust culture time, or dilute inoculation cell density.
Biofilm lacks diversity	Dental plaque obtained from volunteer contains low level of viable cells, culture time too short, or media is too dilute.	Dental plaque viability can be verified by live/dead bacterial staining and multispectral labeling. If Live/Dead staining shows a large proportion of volunteer plaque is not viable. Discard sample and obtain new sample. If Live/Dead staining shows good level of viability (i.e., ≥ 40%) then increase culture duration, frequency of re-inoculation or media concentration.
A labeled taxon is not detected in sample	Oral biofilms are heterogenous in spatial structure and therefore certain taxa may not be present in everywhere in the sample	Image across a larger field of view. Consider acquiring tile scan images to increase the statistical probability that all taxa will be captured in the imaged volume.