After a 60-minute incubation in saliva, the samples weresubjected to an acid challenge by immersion in grapefruit juice for five minutes without agitation, rinsed with water, and then reimmersed in fresh saliva. In the afternoon (2:30 p.m.± 30 minutes) the brushing and acid treatment were repeated. At the end of eachday, following a minimum of 60 minutes of incubation in saliva, one set of samples was removed for scanning electron microscopy (SEM) analysis and grading. The remaining samples were stored overnight in a moist pot at 5°C for treatment the following day. This protocol follows a previously published design.12
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