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(ii) Specimen preparation. Enamel specimens (3 mm in diameter) were drilled from extracted, sound, human, lower permanent incisors which had been obtained from oral surgeons and sterilized by soaking in 3% buffered (neutral) formalin since the time of extraction. Each specimen was mounted on a polyacrylic rod by using methyl methacrylate resin. The specimens were first ground by using 600-grade silicon carbide paper to remove approximately 50 ?m of the surface and then polished to a high luster with Gamma Alumina (0.05 ?m) by standard methods. The specimens were then randomly assigned to test groups, with each group initially composed of 14 specimens. All specimens were sterilized with ethylene oxide gas. Two specimens from each group were randomly chosen before treatment and examined to obtain baseline confocal microscopy data. The 12 specimens that remained in each group were secured in caries-forming vessels by gluing the ends of their plexiglass rods to a round plexiglass base that fit in the bottom of the vessels.
(iii) Treatment regimen and circulating fluids. Trypticase soy broth without dextrose (Difco Laboratories) supplemented with 5% sucrose (TSBS) was used as the bacterial nutrient broth for study 1. For each caries-forming vessel there was one 1-liter bottle of TSBS that dispensed the medium (0.7 ml/min) at three different times each day, for 30 min each (2), for a total of 63 ml/day by means of a peristaltic pump (Wiz Peristaltic Pump; ISCO, Inc., Lincoln, Nebr.). Study 2 used Trypticase soy broth without dextrose (Difco) supplemented with 1% glucose (TSB) as the bacterial nutrient broth to control for sucrose-dependent colonization.

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