title: A new improved protocol for in vitro intratubular dentinal bacterial contamination for antimicrobial endodontic tests: standardization and validation by confocal laser scanning microscopy.
abstract: OBJECTIVE
  To compare three methods of intratubular contamination that simulate endodontic infections using confocal laser scanning microscopy (CLSM).
  
  
  METHODS
  Two pre-existing models of dentinal contamination were used to induce intratubular infection (groups A and B). These methods were modified in an attempt to improve the model (group C). Among the modifications it may be included: specimen contamination for five days, ultrasonic bath with BHI broth after specimen sterilization, use of E. faecalisduring the exponential growth phase, greater concentration of inoculum, and two cycles of centrifugation on alternate days with changes of culture media. All specimens were longitudinally sectioned and stained with of LIVE/DEAD for 20 min. Specimens were assessed using CLSM, which provided images of the depth of viable bacterial proliferation inside the dentinal tubules. Additionally, three examiners used scores to classify the CLSM images according to the following parameters: homogeneity, density, and depth of the bacterial contamination inside the dentinal tubules. Kruskal-Wallis and Dunn's tests were used to evaluate the live and dead cells rates, and the scores obtained.
  
  
  RESULTS
  The contamination scores revealed higher contamination levels in group C when compared with groups A and B (p<0.05). No differences were observed between group A and B (p>0.05). The volume of live cells in group C was higher than in groups A and B (p<0.05).
  
  
  CONCLUSIONS
  The new protocol for intratubular infection resulted in high and uniform patterns of bacterial contamination and higher cell viability in all specimens when compared with the current methods.
authors.affiliation: Department of Operative Dentistry, Endodontics and Dental Materials, Bauru School of Dentistry,, University of São Paulo, Bauru, SP, Brazil.
authors.forename: Flaviana Bombarda de
authors.initials: FB
authors.lastname: Andrade
authors.affiliation: Department of Operative Dentistry, Endodontics and Dental Materials, Bauru School of Dentistry,, University of São Paulo, Bauru, SP, Brazil.
authors.forename: Marcela Paola Castro
authors.initials: MP
authors.lastname: Arias
authors.affiliation: Department of Operative Dentistry, Endodontics and Dental Materials, Bauru School of Dentistry,, University of São Paulo, Bauru, SP, Brazil.
authors.forename: Amanda Garcia Alves
authors.initials: AG
authors.lastname: Maliza
authors.affiliation: Department of Operative Dentistry, Endodontics and Dental Materials, Bauru School of Dentistry,, University of São Paulo, Bauru, SP, Brazil.
authors.forename: Marco Antonio Hungaro
authors.initials: MA
authors.lastname: Duarte
authors.affiliation: Integrated Research Center, Bauru School of Dentistry, University of São Paulo, Bauru, SP, Brazil.
authors.forename: Márcia Sirlene Zardin
authors.initials: MS
authors.lastname: Graeff
authors.affiliation: Department of Operative Dentistry, Endodontics and Dental Materials, Bauru School of Dentistry,, University of São Paulo, Bauru, SP, Brazil.
authors.forename: Pablo Andrés
authors.initials: PA
authors.lastname: Amoroso-Silva
authors.affiliation: Department of Operative Dentistry, Endodontics and Dental Materials, Bauru School of Dentistry,, University of São Paulo, Bauru, SP, Brazil.
authors.forename: Raquel Zanin
authors.initials: RZ
authors.lastname: Midena
authors.affiliation: Department of Operative Dentistry, Endodontics and Dental Materials, Bauru School of Dentistry,, University of São Paulo, Bauru, SP, Brazil.
authors.forename: Ivaldo Gomes de
authors.initials: IG
authors.lastname: Moraes
mesh_terms: D000818:Animals; D002417:Cattle; D002498:Centrifugation; D003470:Culture Media; D003786:Dental Pulp Cavity / Q000382:microbiology*; D003804:Dentin / Q000382:microbiology* / Q000648:ultrastructure; D004195:Disease Models, Animal; D013293:Enterococcus faecalis; D016908:Gram-Positive Bacterial Infections / Q000382:microbiology; D050296:Microbial Viability; D018613:Microscopy, Confocal; D015203:Reproducibility of Results; D013997:Time Factors
chemical_list: D003470:Culture Media