Confusion over live/dead stainings for the detection of vital microorganisms in oral biofilms - which stain is suitable?

BMC Oral Health

Volumn 14, Issue 1, 2014, Pages

  Netuschil, Lutz ^a   Auschill, Thorsten M ^b   Sculean, Anton ^c   Arweiler, Nicole B ^a  

^a PHILIPPS UNIVERSITY MARBURG   (Germany)

^b University of Berne ^*

^c UNIVERSITY OF BERN   (Switzerland)

Author keywords

Bacterial viability kit; Biofilm; Dental plaque; Microorganisms; Mutagenicity; Viability state; Vital fluorescence

Indexed keywords

3',6'-DIACETYLFLUORESCEIN; COLORING AGENT; ETHIDIUM; FLUORESCEIN DERIVATIVE; FLUORESCENT DYE; MUTAGENIC AGENT;

BACTERIAL LOAD; BIOFILM; DIAGNOSTIC USE; FLUORESCENCE; GROWTH, DEVELOPMENT AND AGING; HUMAN; MICROBIAL VIABILITY; MICROBIOLOGICAL EXAMINATION; MICROBIOLOGY; NOMENCLATURE; TOOTH PLAQUE;

BACTERIAL LOAD; BACTERIOLOGICAL TECHNIQUES; BIOFILMS; COLORING AGENTS; DENTAL PLAQUE; ETHIDIUM; FLUORESCEINS; FLUORESCENCE; FLUORESCENT DYES; HUMANS; MICROBIAL VIABILITY; MUTAGENS; TERMINOLOGY AS TOPIC;

EID: 84892400962     PISSN: None     EISSN: 14726831     Source Type: Journal    
DOI: 10.1186/1472-6831-14-2     Document Type: Article

References (156)

1. Mason CA, Hamer G, Bryers JD. The death and lysis of microorganisms in environmental processes. FEMS Microbiol Rev 1986, 39:373-401.
2. Roszak DB, Colwell RR. Survival strategies of bacteria in the natural environment. Microbiol Rev 1987, 51:365-379. 373117, 3312987.
3. Kaprelyants AS, Kell DB. Rapid assessment of bacterial viability and vitality using rhodamine 123 and flow cytometry. J Appl Bacteriol 1992, 72:410-422.
4. Kaprelyants AS, Gottschal JC, Kell DB. Dormancy in non-sporulating bacteria. FEMS Microbiol Rev 1993, 104:271-285.
5. Decker E-M. The ability of direct fluorescence-based, two-colour assays to detect different physiological states of oral streptococci. Letters Appl Microbiol 2001, 33:188-192.
6. Hannig C, Basche S, Burghardt T, Al-Ahmad A, Hannig M. Influence of a mouthwash containing hydroxyapatite microclusters on bacterial adherence in situ. Clin Oral Invest 2013, 17:805-814. doi:10.1007/s00784-012-0781-6.
7. Tawakoli PN, Al-Ahmad A, Hoth-Hannig W, Hannig M, Hannig C. Comparison of different live/dead stainings for detection and quantification of adherent microorganisms in the initial oral biofilm. Clin Oral Invest 2013, 17:841-850. doi:10.1007/s00784-012-0792-3.
8. Netuschil L. Der Biofilm dentale Plaque - Antibakterielle Beeinflussung, Strukturaus-sagen und Modellentwicklungen auf Basis von Vitalfluoreszenzuntersuchungen [Dental Plaque as a Biofilm - Antibacterial Measures, Structure and Models as developed due to vital fluorescence assessments]. Ph.D. thesis, Faculty of Medicine Carl Gustav Carus, TU Dresden 2004 1-274. SLUB, Saxonian State University Library, Signature 3206 400 53 001.
9. Rupf S, Idlibi AN, Marrawi FA, Hannig M, Schubert A, von Mueller L, Spitzer W, Holtmann H, Lehmann A, Rueppell A, Schindler A. Removing biofilms from microstructured titanium ex vivo: a novel approach using atmospheric plasma technology. PLoS One 2011, 6(10):e25893. doi:10.1371/journal.pone.0025893, 10.1371/journal.pone.0025893, 3189945, 22016784.
10. Davey HM. Life, death, and in-between: meanings and methods in microbiology. Appl Environ Microbiol 2011, 77:5571-5576. 10.1128/AEM.00744-11, 3165249, 21705550.
11. Pamp SJ, Sternberg C, Tolker-Nielsen T. Insight into the microbial multicellular lifestyle via flow-cell technology and confocal microscopy. Cytometry A 2009, 75A:90-103.
12. Amor KB, Breeuwer P, Verbaarschot P, Rombouts FM, Akkermans ADL, de Vos WM, Abee T. Multiparametric flow cytometry and cell sorting for the assessment of viable, injured, and dead Bifidobacterium cells during bile salt stress. Appl Environ Microbiol 2002, 68:5209-5216. 10.1128/AEM.68.11.5209-5216.2002, 129875, 12406706.
13. Shi L, Günther S, Hübschmann T, Wick LY, Harms H, Müller S. Limits of propidium iodide as a cell viability indicator for environmental bacteria. Cytometry A 2007, 71A:592-598.
14. Mailaender C, Reiling N, Engelhardt H, Bossmann S, Ehlers S, Niederweis M. The MspA porin promotes growth and increases antibiotic susceptibility of both Mycobacterium bovis BCG and Mycobacterium tuberculosis. Microbiology 2004, 150:853-864. 10.1099/mic.0.26902-0, 15073295.
15. Haugland RP. The Handbook - a guide to fluorescent probes and labeling technologies. Eugene, Molecular Probes 2005, 10, Section 15.3; sheet mp07007.
16. Zhu M, Takenaka S, Sato M, Hoshino E. Influence of starvation and biofilm formation on acid resistance of Streptococcus mutans. Oral Microbiol Immunol 2001, 16:24-27. 10.1034/j.1399-302x.2001.160104.x, 11169135.
17. Bürgers R, Witecy C, Hahnel S, Gosau M. The effect of various topical peri-implantitis antiseptics on Staphylococcus epidermidis, Candida albicans, and Streptococcus sanguinis. Arch Oral Biol 2012, 57:940-947. 10.1016/j.archoralbio.2012.01.015, 22365324.
18. Rüttermann S, Bergmann N, Beikler T, Raab WH, Janda R. Bacterial viability on surface-modified resin-based dental restorative materials. Arch Oral Biol 2012, 57:1512-1521. 10.1016/j.archoralbio.2012.05.005, 22673754.
19. Pan P, Barnett ML, Coelho J, Brogdon C, Finnegan MB. Determination of the in situ bactericidal activity of an essential oil mouthrinse using a vital stain method. J Clin Periodontol 2000, 27:256-261. 10.1034/j.1600-051x.2000.027004256.x, 10783839.
20. Hope CK, Clements D, Wilson M. Determining the spatial distribution of viable and nonviable bacteria in hydrated microcosm dental plaques by viability profiling. J Appl Microbiol 2002, 93:448-455. 10.1046/j.1365-2672.2002.01703.x, 12174043.
21. Caldwell DE, Wolfaardt GM, Korber DR, Lawrence JR. Do bacterial communities transcend Darwinism?. Adv Microb Ecol 1997, 15:105-191.
22. Korber DR, Choi A, Wolfaardt GM, Ingham SC, Caldwell DE. Substratum topography influences susceptibility of Salmonella enteritidis biofilms to trisodium phosphate. Appl Environ Microbiol 1997, 63:3352-3358. 168640, 9292984.
23. Giertsen E, Guggenheim B, Thurnheer T, Gmür R. Microbiological aspects of an in situ model to study effects of antimicrobial agents on dental plaque ecology. Eur J Oral Sci 2000, 108:403-411. 10.1034/j.1600-0722.2000.108005403.x, 11037756.
24. Barcina I, González JM, Iriberri J, Egea L. Effect of visible light on progressive dormancy of Escherichia coli cells during the survival process in natural fresh water. Appl Environ Microbiol 1989, 55:246-251. 184086, 2650620.
25. Barer MR, Gribbon LT, Harwood CR, Nwoguh CE. The viable but non-culturable hypothesis and medical bacteriology. Rev Med Microbiol 1993, 4:183-191.
26. Bogosian G, Sammons LE, Morris PJL, O'Neil JP, Heitkamp MA, Weber DB. Death of the Escherichia coli K-12 strain W3110 in soil and water. Appl Environ Microbiol 1996, 62:4114-4120. 168233, 8900002.
27. Bogosian G, Morris PJL, O'Neil JP. A mixed culture recovery method indicates that enteric bacteria do not enter the viable but nonculturable state. Appl Environ Microbiol 1998, 64:1736-1742. 106224, 9572945.
28. Bowden GHW, Hamilton IR. Survival of oral bacteria. Crit Rev Oral Biol Med 1998, 9:54-85. 10.1177/10454411980090010401, 9488248.
29. Button DK, Schut F, Quang P, Martin R, Robertson BR. Viability and isolation of marine bacteria by dilution culture: theory, procedures, and initial results. Appl Environ Microbiol 1993, 59:881-891. 202203, 16348896.
30. Colwell RR. Nonculturable but still viable and potentially pathogenic. Zentralbl Bakteriol 1993, 279:154-156. 10.1016/S0934-8840(11)80392-0, 8219487.
31. Colwell RR, Brayton PR, Grimes DJ, Roszak DB, Huq SA, Palmer LM. Viable but nonculturable Vibrio cholerae and related pathogenes in the environment: implications for the release of genetically engineered microorganisms. Biotechniques 1985, 3:817-820.
32. Dawe LL, Penrose WR. " Bactericidal" property of seawater: death or debilitation?. Appl Environ Microbiol 1978, 35:829-833. 242937, 350153.
33. Duncan S, Glover LA, Killham K, Prosser JI. Luminescence-based detection of activity of starved and viable but nonculturable bacteria. Appl Environ Microbiol 1994, 60:1308-1316. 201475, 8017919.
34. González JM, Iriberri J, Egea L, Barcina I. Characterization of culturability, protistan grazing, and death of enteric bacteria in aquatic ecosystems. Appl Environ Microbiol 1992, 58:998-1004. 195368, 1575503.
35. Gribbon LT, Barer MR. Oxidative metabolism in nonculturable Helicobacter pylori and Vibrio vulnificus cells studied by substrate-enhanced tetrazolium reduction and digital image processing. Appl Environ Microbiol 1995, 61:3379-3384. 167617, 7574647.
36. Höfle M. Long-term changes in chemostat cultures of Cytophaga johnsonae. Appl Environ Microbiol 1983, 46:1045-1053. 239517, 16346411.
37. Hood MA, MacDonell MT. Distribution of ultramicrobacteria in a Gulf Coast estuary and induction of ultramicrobacteria. Microb Ecol 1987, 14:113-127. 10.1007/BF02013017, 24202640.
38. Kell DB, Ryder HM, Kaprelyants AS, Westerhoff HV. Quantifying heterogeneity: flow cytometry of bacterial cultures. Antonie Van Leeuwenhoek 1991, 60:145-158. 10.1007/BF00430362, 1725477.
39. Koch AL. What size should a bacterium be? A question of scale. Ann Rev Microbiol 1996, 50:317-348.
40. Kogure K, Simidu U, Taga N. A tentative direct microscopic method for counting living marine bacteria. Can J Microbiol 1979, 25:415-420. 10.1139/m79-063, 378340.
41. Korber DR, Choi A, Wolfaardt GM, Caldwell DE. Bacterial plasmolysis as a physical indicator of viability. Appl Environ Microbiol 1996, 62:3939-3947. 168211, 8899980.
42. McKay AM. Viable but non-culturable forms of potentially pathogenic bacteria in water. Lett Appl Microbiol 1992, 14:129-135.
43. Morgan JAW, Rhodes G, Pickup RW. Survival of nonculturable Aeromonas salmonicida in lake water. Appl Environ Microbiol 1993, 59:874-880. 202202, 8481011.
44. Nebe-von Caron G, Stephens P, Badley RA. Assessment of bacterial viability status by flow cytometry and single cell sorting. J Appl Microbiol 1998, 84:988-998. 10.1046/j.1365-2672.1998.00436.x, 9717283.
45. Nilsson L, Oliver JD, Kjelleberg S. Resuscitation of Vibrio vulnificus from the viable but nonculturable state. J Bacteriol 1991, 173:5054-5059. 208195, 1860818.
46. Nyström T. The trials and tribulations of growth arrest. Trends Microbiol 1995, 3:131-136. 10.1016/S0966-842X(00)88901-5, 7613753.
47. Postgate JR. Death in macrobes and microbes. In: Gray TRG, Postgate JR (Eds.): The survival of vegetative microbes. Symp Soc Gen Microbiol 1977, 26:1-19.
48. Postgate JR, Hunter JR. The survival of starved bacteria. J Gen Microbiol 1962, 29:233-263. 10.1099/00221287-29-2-233, 13985691.
49. Rose AS, Ellis AE, Munro ALS. Evidence against dormancy in the bacterial fish pathogen Aeromonas salmonicida subsp. salmonicida. FEMS Microbiol Lett 1990, 68:105-107.
50. Roszak DB, Colwell RR. Metabolic activity of bacterial cells enumerated by direct viable count. Appl Environ Microbiol 1987, 53:2889-2893. 204217, 3324968.
51. Roszak DB, Grimes DJ, Colwell RR. Viable but nonrecoverable stage of Salmonella enteritidis in aquatic systems. Can J Microbiol 1984, 30:334-338. 10.1139/m84-049, 6372975.
52. Stevenson LH. A case for bacterial dormancy in aquatic systems. Microb Ecol 1978, 4:127-133.
53. Whitesides MD, Oliver JD. Resuscitation of vibrio vulnificus from the viable but nonculturable state. Appl Environ Microbiol 1997, 63:1002-1005. 1389128, 16535534.
54. Wilson M, Lindow SE. Relationship of total viable and culturable cells in epiphytic populations of Pseudomonas syringae. Appl Environ Microbiol 1992, 58:3908-3913. 183203, 1476434.
55. Winterberg H. Zur Methodik der Bakterienzählung [Concerning methods to count bacteria]. Zeitschr Hyg 1898, 29:75-93.
56. Hehewerth FH. Die microscopische Zählungsmethode der Bakterien von Alex. Klein und einige Anwendungen derselben [The microscopic counting method of Alex. Klein and some applications thereof]. Arch Hyg 1901, 39:321-389.
57. Kelly CD, Rahn O. The growth rate of individual bacterial cells. J Bacteriol 1932, 23:147-153. 533308, 16559540.
58. Ziegler NR, Halvorson HO. Application of statistics to problems in bacteriology. IV. Experimental comparison of the dilution method, the plate count, and the direct count for the determination of bacterial populations. J Bacteriol 1935, 29:609-634. 543626, 16559814.
59. Jennison MW. Relations between plate counts and direct microscopic counts of Escherichia coli during the logarithmic growth period. J Bacteriol 1937, 33:461-477. 545160, 16560015.
60. Jannasch HW, Jones GE. Bacterial populations in seawater as determined by different methods of enumeration. Limnol Oceanogr 1959, 4:128-139.
61. Razumov AS. Mikrobiologija 1932, 1:131-146. as cited by Staley and Konopka 1985 (cf. [62, 63]).
62. Staley JT, Konopka A. Measurement of in situ activities of nonphotosynthetic micro-organisms in aquatic and terrestrial habitants. Ann Rev Microbiol 1985, 39:321-346.
63. Amann RI, Ludwig W, Schleifer K-H. Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol Rev 1995, 59:143-169. 239358, 7535888.
64. Fraser CG. The action of methylene-blue and certain other dyes on living and dead yeast. J Phys Chem 1920, 24:741-748.
65. Henrici AT. Differential counting of living and dead cells of bacteria. Proc Soc Exp Biol Med 1923, 20:293-295.
66. Rahn O, Barnes MN. An experimental comparison of different criteria of death in yeast. J Gen Physiol 1933, 16:579-592. 10.1085/jgp.16.4.579, 2141224, 19872723.
67. Gay FP, Clark AR. The differentiation of living from dead bacteria by staining reactions. J Bacteriol 1934, 27:175-189. 533612, 16559693.
68. Strugger S. Die fluoreszenzmikroskopische Unterscheidung lebender und toter Zellen mit Hilfe der Acridinorangefärbung [Fluorescence-microscopic differentiation of living and dead cells with the aid of Acridinorange staining]. Dtsch Tierärztl Wschr 1941, 49:525-527.
69. Postgate JR. Viable counts and viability. Meth Microbiol 1969, 1:611-628.
70. Azam F. Introduction, history, and overview: The 'methods' to our madness. Meth Microbiol 2001, 30:1-12.
71. Hope CK, Wilson M. Analysis of the effects of chlorhexidine and oral biofilm vitality and structure based on viability profiling and an indicator of membrane integrity. Antimicrob Agents Chemother 2004, 48:1461-1468. 10.1128/AAC.48.5.1461-1468.2004, 400577, 15105093.
72. Amann R, Springer N, Ludwig W, Görtz H-D, Schleifer K-H. Identification in situ and phylogeny of uncultured bacterial endosymbionts. Nature 1991, 351:161-163. 10.1038/351161a0, 1709451.
73. Vollertsen J, Jahn A, Nielsen JL, Hvitved-Jacobsen T, Nielsen PH. Comparison of methods for determination of microbial biomass in wastewater. Water Res 2001, 35:1649-1658. 10.1016/S0043-1354(00)00450-4, 11329666.
74. Barer MR, Smith RJ, Cooney RP, Kimmitt PT. Relationships between culturability, activity and virulence in pathogenic bacteria. J Infect Chemother 2000, 6:108-111. 10.1007/PL00012148, 11810547.
75. Colwell RR. Viable but nonculturable bacteria: a survival strategy. J Infect Chemother 2000, 6:121-125. 10.1007/PL00012151, 11810550.
76. Kell DB, Kaprelyants AS, Weichart DH, Harwood CR, Barer MR. Viability and activity in readily culturable bacteria: a review and discussion of the practical issues. Antonie Von Leeuwenhoek 1998, 73:169-187.
77. Nystrom T. Not quite dead enough: on bacterial life, culturability, senescence, and death. Arch Microbiol 2001, 176:159-164. 10.1007/s002030100314, 11511862.
78. Yamamoto H. Viable but nonculturabel state as a general phenomenon of non-spore-forming bacteria, and its modeling. J Infect Chemother 2000, 6:112-114. 10.1007/PL00012149, 11810548.
79. Lahtinen SJ, Ahokoski H, Reinikainen JP, Gueimonde M, Nurmi J, Ouwehand AC, Salminen SJ. Degradation of 16S rRNA and attributes of viability of viable but nonculturable probiotic bacteria. Lett Appl Microbiol 2008, 46:693-698. 10.1111/j.1472-765X.2008.02374.x, 18444975.
80. Lahtinen SJ, Gueimonde M, Ouwehand A, Reinikainen JP, Salminen S. Probiotic bacteria may become dormant during storage. Appl Environ Microbiol 2005, 71:1662-1663. 10.1128/AEM.71.3.1662-1663.2005, 1065127, 15746375.
81. Molecular probes Product information LIVE/DEAD ® BacLight™ bacterial viability Kit. 2001, Molecular probes.
82. Stocks SM. Mechanism and use of the commercially available viability stain, BacLight. Cytometry A 2004, 61A:189-195.
83. Berney M, Hammes F, Bosshard F, Weilenmann H-U, Egli T. Assessment and interpretation of bacterial viability by using the LIVE/DEAD BacLight Kit in combination with flow cytometry. App Environ Microbiol 2007, 73:3283-3290.
84. Haffajee AD, Socransky SS, Feres M, Ximenez-Fyvie LA. Plaque microbiology in health and disease. Dental plaque revisited 1999, 255-282. Chippenham: BioLine Antony Rowe Ltd., Newman HN, Wilson M.
85. Otten MPT, Busscher HJ, van der Mei HC, Abbas F. Retention of antimicrobial activity in plaque and saliva following mouthrinse use in vivo. Caries Res 2010, 44:459-464. 10.1159/000320267, 20838045.
86. Lahtinen SJ, Gueimonde M, Ouwehand AC, Reinikainen JP, Salminen SJ. Comparison of four methods to enumerate probiotic bifidobacteria in a fermented food product. Food Microbiol 2006, 23:571-577. 10.1016/j.fm.2005.09.001, 16943053.
87. Bürgers R, Eidt A, Frankenberger R, Rosentritt M, Schweikl H, Handel G, Hahnel S. The anti-adherence activity and bactericidal effect of microparticulate silver additives in composite resin materials. Arch Oral Biol 2009, 54:595-601. 10.1016/j.archoralbio.2009.03.004, 19375069.
88. Gosau M, Hahnel S, Schwarz F, Gerlach T, Reichert TE, Bürgers R. Effect of six different peri-implantitis disinfection methods on in vivo human oral biofilm. Clin Oral Implants Res 2010, 21:866-872.
89. Tomás I, García-Caballero L, Cousido MC, Limeres J, Álvarez M, Diz P. Evaluation of chlorhexidine subtantivity on salivary flora by epifluorescence microscopy. Oral Dis 2009, 15:428-433. 10.1111/j.1601-0825.2009.01570.x, 19413678.
90. Cousido MC, Tomás Carmona I, Garcia-Caballero L, Limeres J, Álvarez M, Diz P. In vivo substantivity of 0.12% and 0.2% chlorhexidine mouthrinses on salivary bacteria. Clin Oral Invest 2010, 14:397-402.
91. Tomás I, Cousido MC, García-Caballero L, Rubido S, Limeres J, Diz P. Substantivity of a single chlorhexidine mouthwash on salivary flora: influence of intrinsic and extrinsic factors. J Dentistry 2010, 38:541-546.
92. Filoche SK, Coleman MJ, Angker L, Sissons CH. A fluorescence assay to determine the viable biomass of microcosm dental plaque biofilms. J Microbiol Methods 2007, 69:489-496. 10.1016/j.mimet.2007.02.015, 17408789.
93. Quéric N-V, Soltwedel T, Arntz WE. Application of a rapid direct viable count method to deep-sea sediment bacteria. J Microbiol Methods 2004, 57:351-367. 10.1016/j.mimet.2004.02.005, 15134883.
94. van der Mei HC, White DJ, Atema-Smit J, van de Belt-Gritter E, Busscher HJ. A method to study sustained antimicrobial activity of rinse and dentifrice components on biofilm viability in vivo. J Clin Periodontol 2006, 33:14-20. 10.1111/j.1600-051X.2005.00859.x, 16367850.
95. Neilands J, Petersson LG, Beighton D, Svensäter G. Fluoride-supplemented milk inhibits acid tolerance in root caries biofilms. Caries Res 2012, 46:156-160. 10.1159/000337390, 22488252.
96. Shen Y, Stojicic S, Haapasalo M. Bacterial viability in starved and revitalized biofilms: comparison of viability staining and direct culture. J Endod 2010, 36:1820-1823. 10.1016/j.joen.2010.08.029, 20951294.
97. Alakomi H-L, Mättö J, Virkajärvi I, Saarela M. Application of a microplate scale fluorochrome staining assay for the assessment of viability of probiotic preparations. J Microbiol Meth 2005, 62:25-35.
98. Welin-Neilands J, Svensäter G. Acid tolerance of biofilm cells of Streptococcus mutans. Appl Environ Microbiol 2007, 73:5633-5638. 10.1128/AEM.01049-07, 2042095, 17630302.
99. Tomás I, Cousido MC, Tomás M, Limeres J, García-Caballero L, Diz P. In vivo bactericidal effect of 0.2% chlorhexidine but not 0.12% on salivary obligate anaerobes. Arch Oral Biol 2008, 53:1186-1191. 10.1016/j.archoralbio.2008.07.009, 18783772.
100. Pan PH, Finnegan MB, Sturdivant L, Barnett ML. Comparative antimicrobial activity of an essential oil and an amine fluoride/stannous fluoride mouthrinse in vitro. J Clin Periodontol 1999, 26:474-476. 10.1034/j.1600-051X.1999.260710.x, 10412853.
101. Fine DH, Furgang D, Barnett ML. Comparative antimicrobial activities of antiseptic mouthrinses against isogenic planktonic and biofilm forms of Actinobacillus actinomycetemcomitans. J Clin Periodontol 2001, 28:697-700. 10.1034/j.1600-051x.2001.028007697.x, 11422593.
102. Davey HM, Kell DB, Weichart DH, Kaprelyants AS. Estimation of microbial viability using flow cytometry. Curr Protoc Cytom 2004, 11. 11.3.1-11.3.21.
103. Lloyd D, Hayes AJ. Vigour, vitality and viability of microorganisms. FEMS Microbiol Lett 1995, 133:1-7.
104. Nebe-von-Caron G, Stephens PJ, Hewitt CJ, Powell JR, Badley RA. Analysis of bacterial function by multi-colour fluorescence flow cytometry and single cell sorting. J Microbiol Methods 2000, 42:97-114. 10.1016/S0167-7012(00)00181-0, 11000436.
105. Auty MAE, Gardiner GE, McBrearty SJ, O'Sullivan E, Mulvihill DM, Collins JK, Fitzgerald GF, Stanton C, Ross RP. Direct in situ viability assessment of bacteria in probiotic dairy products using viability staining in conjunction with confocal scanning laser microscopy. Appl Environ Microbiol 2001, 55:420-425.
106. Brehm-Stecher BF, Johnson EA. Single-cell microbiology: tools, technology and applications. Microbiol Mol Biol Rev 2004, 68:538-559. 10.1128/MMBR.68.3.538-559.2004, 515252, 15353569.
107. Keijser BJ, Zaura E, Huse SM, van der Vossen JM, Schuren FH, Montijn RC, ten Cate JM, Crielaard W. Pyrosequencing analysis of the oral microflora of healthy adults. J Dent Res 2008, 87:1016-1020. 10.1177/154405910808701104, 18946007.
108. Weiger R, Netuschil L, Brecx M. Relationship between bacterial counts, microbial vitality and the accumulation of supragingival dental plaque in humans. J Periodont Res 1992, 27:575-580. 10.1111/j.1600-0765.1992.tb01739.x, 1460546.
109. Weiger R, Netuschil L, von Ohle C, Brecx M. Microbial vitality of supragingival dental plaque during initial stages of experimental gingivitis in humans. J Periodont Res 1995, 30:204-209. 10.1111/j.1600-0765.1995.tb01275.x, 7473004.
110. Netuschil L, Weiger R, Preisler R, Brecx M. Plaque bacteria counts and vitality during chlorhexidine, meridol and listerine mouthrinses. Eur J Oral Sci 1995, 103:355-361. 10.1111/j.1600-0722.1995.tb01857.x, 8747670.
111. Mikkelsen L. Influence of sucrose intake on saliva and number of microorganisms and acidogenic potential in early dental plaque. Microb Ecol Health Dis 1993, 6:253-264.
112. Netuschil L, Reich E, Brecx M. Direct measurement of the bactericidal effect of chlorhexidine on human dental plaque. J Clin Periodontol 1989, 16:484-488. 10.1111/j.1600-051X.1989.tb02324.x, 2476466.
113. Arweiler N, Donos N, Netuschil L, Sculean A, Reich E. Clinical and antibacterial effect of tea tree oil. Clin Oral Invest 2000, 4:70-73.
114. Weiger R, Netuschil L, Brecx M. Comparison of early human dental plaque formation on vestibular and approximal enamel surfaces in situ. J West Soc Periodontol Periodontal Abstr 1992, 40:101-104. / Erratum 41: 10.
115. Weiger R, Friedrich C, Netuschil L, Schlagenhauf U. Effect of a chlorhexidine-containing varnish (Cervitec®) on microbial vitality and accumulation of supragingival dental plaque in humans. Caries Res 1994, 28:267-271. 10.1159/000261984, 8069883.
116. Guilbault GG, Kramer DN. Fluorometric determination of lipase, acylase, alpha- and gamma-chymotrypsin and inhibitors of these enzymes. Anal Chem 1964, 36:409-412.
117. Medzon EL, Brady ML. Direct measurement of acetylesterase in living protist cells. J Bacteriol 1969, 97:402-415. 249621, 4974398.
118. Netuschil L. Vitalfärbung von Plaque-Mikroorganismen mit Fluoresceindiacetat und Ethidiumbromid [Staining of plaque microorganisms with Fluorescein diacetate and Ethidium bromide]. Dtsch zahnärztl Z 1983, 38:914-917.
119. Netuschil L, Reich E, Unteregger G, Sculean A, Brecx M. A pilot study of confocal laser scanning microscopy for the assessment of undisturbed dental plaque vitality and topography. Arch Oral Biol 1998, 43:277-285. 10.1016/S0003-9969(97)00121-0, 9839703.
120. Brecx M, Netuschil L, Reichert B, Schreil G. Efficacy of Listerine, Meridol and Chlorhexidine mouthrinses on plaque, gingivitis and plaque bacteria vitality. J Clin Periodontol 1990, 17:292-297. 10.1111/j.1600-051X.1990.tb01092.x, 2355095.
121. Hahn R, Weiger R, Netuschil L, Brüch M. Microbial accumulation and vitality on different restorative materials. Dent Mater 1993, 9:312-316. 10.1016/0109-5641(93)90049-V, 7995483.
122. Gehlen I, Netuschil L, Georg T, Reich E, Berg R, Katsaros C. The influence of a 0.2% chlorhexidin mouthrinse on plaque regrowth in orthodontic patients. A randomized prospective study. Part 2: bacteriological parameters. J Orofac Orthop 2000, 61:138-148. 10.1007/BF01300355, 10783565.
123. Schlagenhauf U, Horlacher V, Netuschil L, Brecx M. Repeated subgingival oxygen irrigations in untreated periodontal patients. J Clin Periodontol 1994, 21:48-50. 10.1111/j.1600-051X.1994.tb00276.x, 8126244.
124. Auschill TM, Arweiler NB, Brecx M, Reich E, Sculean A, Netuschil L. The effect of dental restorative materials on dental biofilm. Eur J Oral Sci 2002, 110:48-53. 10.1046/j.0909-8836.2001.101160.x, 11878760.
125. Brecx M, Brownstone E, MacDonald L, Gelskey S, Cheang M. Efficacy of Listerine®, Meridol® and chlorhexidine mouthrinses as supplements to regular tooth-cleaning measures. J Clin Periodontol 1992, 19:202-207. 10.1111/j.1600-051X.1992.tb00640.x, 1556249.
126. Brecx M, MacDonald LL, Legary K, Cheang M, Forgay MGE. Long-term effects of Meridol® and chlorhexidine mouthrinses on plaque, gingivitis, staining, and bacterial vitality. J Dent Res 1993, 72:1194-1197. 10.1177/00220345930720080601, 8360362.
127. Gelskey S, Brecx M, Netuschil L, MacDonald L, Brownstone E, Stoddart M. Vital fluorescence: a new measure of periodontal treatment effect. J Can Dent Assoc 1993, 59:615-618.
128. von Ohle C, Weiger R, Decker E, Schlagenhauf U, Brecx M. The efficacy of a single pocket irrigation on subgingival microbial vitality. Clin Oral Invest 1998, 2:84-90.
129. Arweiler NB, Netuschil L, Reich E. Alcohol-free mouthrinse solutions to reduce supra-gingival plaque regrowth and vitality. A controlled clinical study. J Clin Periodontol 2001, 28:168-174. 10.1034/j.1600-051x.2001.028002168.x, 11168742.
130. Sculean A, Auschill TM, Donos N, Brecx M, Arweiler NB. Effect of an enamel matrix protein derivative (Emdogain®) on ex vivo dental plaque vitality. J Clin Periodontol 2001, 28:1074-1078. 10.1034/j.1600-051X.2001.281113.x, 11686830.
131. Auschill TM, Arweiler NB, Netuschil L, Brecx M, Reich E, Sculean A. Spatial distribution of vital and dead microorganisms in dental biofilms. Arch Oral Biol 2001, 46:471-476. 10.1016/S0003-9969(00)00136-9, 11286812.
132. Arweiler NB, Auschill TM, Donos N, Sculean A. Antibacterial effect of an enamel matrix derivative on in vivo dental biofilm vitality. Clin Oral Invest 2002, 6:205-209.
133. Arweiler NB, Auschill TM, Baguley N, Netuschil L, Sculean A. Efficacy of an amine fluoride-triclosan mouthrinse as compared to the individual active ingredients. J Clin Periodontol 2003, 30:192-196. 10.1034/j.1600-051X.2003.10250.x, 12631176.
134. Auschill TM, Hellwig E, Sculean A, Hein N, Arweiler NB. Impact of the intraoral location on the rate of biofilm growth. Clin Oral Invest 2004, 8:97-101.
135. Arweiler NB, Hellwig E, Sculean A, Hein N, Auschill TM. Individual vitality pattern of in situ dental biofilms at different locations in the oral cavity. Caries Res 2004, 38:442-447. 10.1159/000079625, 15316188.
136. Auschill TM, Hein N, Hellwig E, Follo M, Sculean A, Arweiler NB. Effect of two antimicrobial agents on early in situ biofilm formation. J Clin Periodontol 2005, 32:147-152. 10.1111/j.1600-051X.2005.00650.x, 15691343.
137. Arweiler NB, Böhnke N, Sculean A, Hellwig E, Auschill TM. Differences in efficacy of two commercial 0.2% chlorhexidine mouthrinse solutions: a 4-day plaque re-growth study. J Clin Periodontol 2006, 33:334-339. 10.1111/j.1600-051X.2006.00917.x, 16634954.
138. Auschill TM, Deimling D, Hellwig E, Arweiler NB. Antibacterial effect of two toothpastes following a single brushing. Oral Health Prev Dent 2007, 5:25-32.
139. Al-Ahmad A, Wiedmann-Al-Ahmad M, Auschill TM, Follo M, Braun G, Hellwig E, Arweiler NB. Effects of commonly used food preservatives on biofilm formation of Streptococcus mutans in vitro. Arch Oral Biol 2008, 53:765-772. 10.1016/j.archoralbio.2008.02.014, 18395697.
140. Arweiler NB, Lenz R, Sculean A, Al-Ahmad A, Hellwig E, Auschill TM. Effect of food preservatives on in situ biofilm formation. Clin Oral Invest 2008, 12:203-208.
141. Arweiler NB, Pergola G, Kuenz J, Hellwig E, Sculean A, Auschill TM. Clinical and antibacterial effect of an anti-inflammatory toothpaste formulation with Scutellaria baicalensis extract on experimental gingivitis. Clin Oral Invest 2011, 15:909-913.
142. Arweiler NB, Auschill TM, Sculean A. Antibacterial effect of taurolodine (2%) on established dental plaque biofilm. Clin Oral Invest 2012, 16:499-504.
143. Arweiler NB, Netuschil L, Beier D, Grunert S, Heumann C, Altenburger MJ, Sculean A, Nagy K, Al-Ahmad A, Auschill TM. Action of food preservatives on 14-days dental biofilm formation, biofilm vitality, and biofilm-derived enamel demineralisation in situ. Clin Oral Invest 2013, [Epub ahead of print] doi:10.1007/s00784-013-1053-9.
144. Botzenhart K, Heizmann W, Sedaghat S, Heeg P, Hahn T. Bacterial colonization and occurrence of legionella pneumophila in warm and cold water, in faucet aerators, and in drains of hospitals. Zbl Bakt Hyg 1986, B183:79-85.
145. Rundegren J, Hvid E, Johansson M, Aström M. Effect of 4 days of mouth rinsing with delmopinol or chlorhexidine on the vitality of plaque bacteria. J Clin Periodontol 1992, 19:322-325. 10.1111/j.1600-051X.1992.tb00653.x, 1517477.
146. Herles S, Olsen S, Afflitto J, Gaffar A. Chemostat flow cell system: an in vitro model for the evaluation of antiplaque agents. J Dent Res 1994, 73:1748-1755.
147. Kourkouta S, Walsh TF, Davis LG. The effect of porcelain laminate veneers on gingival health and bacterial plaque characteristics. J Clin Periodontol 1994, 21:638-640. 10.1111/j.1600-051X.1994.tb00756.x, 7806682.
148. Gaffar A, Afflitto J, Nabi N, Herles S, Kruger I, Olsen S. Recent advances in plaque, gingivitis, tartar and caries prevention technology. Intern Dent J 1994, 44:63-70.
149. Gaffar A, Afflitto J, Nabi N. Chemical agents for the control of plaque and plaque microflora: an overview. Eur J Oral Sci 1997, 105:502-507. 10.1111/j.1600-0722.1997.tb00237.x, 9395116.
150. Mullally BH, James JA, Coulter WA, Linden GJ. The efficacy of a herbal-based toothpaste on the control of plaque and gingivitis. J Clin Periodontol 1995, 22:686-689. 10.1111/j.1600-051X.1995.tb00827.x, 7593698.
151. Walsh TF, Ünsal E, Davis LG, Yilmaz T. The effect of irrigation with chlorhexidine or saline on plaque vitality. J Clin Periodontol 1995, 22:262-264.
152. Walsh TF, Ünsal E, Varella-Centelles P. Comparison of digitised and visual plaque vitality measurement. J Clin Periodontol 1995, 22:653-654. 10.1111/j.1600-051X.1995.tb00820.x, 8583024.
153. Zaura-Arite E, van Marle J, ten Cate JM. Confocal microscopy study of undisturbed and chlorhexidine-treated dental biofilm. J Dent Res 2001, 80:1436-1440. 10.1177/00220345010800051001, 11437215.
154. König J, Storcks V, Kocher T, Bössmann K, Plagmann H-C. Anti-plaque effect of tempered 0.2% chlorhexidine rinse: an in vivo study. J Clin Periodontol 2002, 29:207-210. 10.1034/j.1600-051x.2002.290304.x, 11940138.
155. Hannig C, Hannig M, Rehmer O, Braun G, Hellwig E, Al-Ahmad A. Fluorescence microscopic visualization and quantification of initial bacterial colonization on enamel in situ. Arch oral Biol 2007, 52:1048-1056. 10.1016/j.archoralbio.2007.05.006, 17603998.
156. Wojcik K, Dobrucki JW. Interaction of a DNA intercalator DRAQ5, and a minor groove binder SYTO17, with chromatin in live cells - influence of chromatin organization and histone-DNA interactions. Cytometry A 2008, 73:555-562.