Table 2.

Characteristics of appliance grown oral biofilm (AGOB) following exposure to chemical agents.

Author. Yeara	Chemical used	Methods	Study Endpoint	Methods of Analysis	Study Findings
Giertsen et al. 2000 [19]	3% glucose, 3% sucrose, 70 nM NaCl, 70 nM KCL, 2 mM MgCl	Double-blind cross over split mouth study design; Each subject tested 2 treatments for each test period in 2 treatment cycle.; by dipping the device 2 x daily for I min into randomly assigned test solution	Viability, CFU, Composition of specific bacteria	Culture, Immunofluorescence	NaF, zinc acetate and fluoride plus zinc acetate significantly reduced individual taxa but similar bacterial viability and total bacterial numbers were observed. However, chlorhexidine significantly reduced viability, total cell number and abundance of most of the enumerated taxa.
Auschill et al. 2005 [21]	Chlorhexidine, Amine fluoride/stannous fluoride	Observer-blind, controlled, cross-over study, rinse 2 x daily with device in the mouth with 10 ml for 1 min, morning and evening for 48 hrs	Thickness, Viability at different layers	CLSM	Both antimicrobials reduced thickness and viability significantly compared with control. However, differences between the two active solutions were not statistically different.
Gu et al. 2012 [24]	ZnCl (2.5, 5,10 and 20 mM)	Rinse twice daily for 2 mins with 10 ml of the solution with device intact for 48 hrs.	Thickness, Viability,	CLSM	Plaque index, biofilm thickness and biofilm viability treated with various conc of ZnCl reduced sig compared with control. 2.5 nM ZnCl was the lowest conc to inhibit bacteria in the outer layers, 5 mM was the lowest conc to inhibit middle layer and none could inhibit bacteria in the inner layer.
Quintas et al. 2015 [40]	Essential oil, 0.2% Chlorhexidine	Randomized, observer-masked, crossover study. Wear device for 4 days continuously. Rinse 2 x daily with device intact (20 ml for 30 sec)	Viability, Thickness, Covering grade	CLSM	Essential oil (EO) and 0.2% chlorhexidine (CHX) significantly more effective than sterile water at reducing bacterial viability, thickness and covering grade of biofilm. No sig diff bet EO and 0.2% CHX at reducing bacterial vitality. O.2% CHX showed more reduction than EO in reducing thickness and covering grade.
Dige et al. 2016 [36]	4% sucrose	Test 1: Wear device- after 30 mins. Immerse left flange in sucrose free solution (sucrose free group) for 2 mins and the right side in sucrose solution (sucrose group) for 2 mins every hour during the day for 2 days. Test 2: Same procedure repeated but exchange the immersion side	Extracellular pH of biofilm	CLSM	pH drop pattern did not differ between biofilms exposed to sucrose-free and sucrose -rich environment. Extracellular pH dropped rapidly in most sites after addition of glucose. Data suggest that pH drops in young (48 hrs) dental biofilms are independent of the sucrose supply during the growth.
Quintas et at. 2017 [37]	Essential oil with and without alcohol	Randomized, double blind crossover study. Test 1: Wear device for 48 hrs. Rinse (20 ml for 30 sec.) with device intact. Sample collection at 0, 30sec, 1, 3 5 and 7 hrs. from distal to mesial at each time point. Test 2: Rinse2 x daily (20 ml 30 sec) with device intact for 96 hrs.	Viability, Thickness	CLSM	Both antiseptics showed very high immediate antibacterial activity and substantivity in situ on 2-day biofilm. After 4 days both demonstrated very good antiplaque effect, but alcohol free performed better at reducing thickness and covering grade.
Xue et al. 2017 [33]	Toothpaste with and without arginine	Randomized controlled crossover study. Brushing 2 xd aily for 3 mins for 2 weeks.	Lactic acid production, MTT assay, Biomass, Vitality	SEM, CLSM	Arginine- containing toothpaste showed significant reduction of lactic acid production in both high caries and non-high caries group, but did not decrease metabolic activity, total biomass and vitality in either group.

^aStudies arranged in chronological order.