{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,5]],"date-time":"2026-09-05T15:54:12Z","timestamp":1788623652346,"version":"build-2803163510"},"reference-count":53,"publisher":"MDPI AG","issue":"20","license":[{"start":{"date-parts":[[2021,10,11]],"date-time":"2021-10-11T00:00:00Z","timestamp":1633910400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>(1) Background and the aim: The appropriate incubation time in the antimicrobial photodynamic therapy protocol seems to have a huge impact on the efficacy of this process. This is particularly important in relation to Candida strains, due to the size of these cells and the presence of the cell wall. The aims of this study were to determine the optimal incubation time needed for the absorption of toluidine blue by cells of C. albicans, C. glabrata, C. krusei and C. parapsilosis using direct observation by optical microscopy, and to evaluate the efficacy of TBO-mediated aPDT on planktonic cells of these strains. (2) Methods: The microscopic evaluation consisted of taking a series of images at a magnification of 600\u00d7 and counting the % of stained cells. The in vitro effect of TBO-mediated aPDT combined with a diode laser (635 nm, 400mW, 12 J\/cm2, CW) on the viability of yeast cells with different incubation times was evaluated. (3) Results: The presence of TBO within the cytoplasm was observed in all tested Candida strains and at all microscopic evaluation times. However, the highest percentages of cells were stained at 7 and 10 min. The highest % reduction of CFU\/mL after TBO-mediated aPDT against Candida was obtained for the strain C. albicans ATCC 10,231 and it was 78.55%. (4) Conclusions: TBO-mediated aPDT against Candida was effective in reducing the number of CFU\/mL at all assessed incubation times. However, the most efficient period for almost all strains was 7\u201310 min.<\/jats:p>","DOI":"10.3390\/ijms222010971","type":"journal-article","created":{"date-parts":[[2021,10,11]],"date-time":"2021-10-11T21:45:32Z","timestamp":1633988732000},"page":"10971","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":20,"title":["Influence of Incubation Time on Ortho-Toluidine Blue Mediated Antimicrobial Photodynamic Therapy Directed against Selected Candida Strains\u2014An In Vitro Study"],"prefix":"10.3390","volume":"22","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-5673-7413","authenticated-orcid":false,"given":"Rafa\u0142","family":"Wiench","sequence":"first","affiliation":[{"name":"Department of Periodontal Diseases and Oral Mucosa Diseases, Faculty of Medical Sciences in Zabrze, Medical University of Silesia, 40-055 Katowice, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Joanna","family":"Nowicka","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Faculty of Medicine, Wroclaw Medical University, 50-368 Wroclaw, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9881-8294","authenticated-orcid":false,"given":"Magdalena","family":"Paj\u0105czkowska","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Faculty of Medicine, Wroclaw Medical University, 50-368 Wroclaw, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0682-4743","authenticated-orcid":false,"given":"Piotr","family":"Kuropka","sequence":"additional","affiliation":[{"name":"Department of Histology and Embryology, Wroclaw University of Environmental and Life Sciences, 50-375 Wroclaw, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8732-251X","authenticated-orcid":false,"given":"Dariusz","family":"Skaba","sequence":"additional","affiliation":[{"name":"Department of Periodontal Diseases and Oral Mucosa Diseases, Faculty of Medical Sciences in Zabrze, Medical University of Silesia, 40-055 Katowice, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anna","family":"Kruczek-Kazibudzka","sequence":"additional","affiliation":[{"name":"Private Practice, 44-100 Gliwice, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anna","family":"Ku\u015bka-Kie\u0142bratowska","sequence":"additional","affiliation":[{"name":"Department of Periodontal Diseases and Oral Mucosa Diseases, Faculty of Medical Sciences in Zabrze, Medical University of Silesia, 40-055 Katowice, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5700-4577","authenticated-orcid":false,"given":"Kinga","family":"Grzech-Le\u015bniak","sequence":"additional","affiliation":[{"name":"Laser Laboratory Dental Surgery Department, Medical University of Wroclaw, 50-425 Wroclaw, Poland"},{"name":"Department of Periodontics, School of Dentistry, Virginia Commonwealth University, Richmond, VA 23284, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2021,10,11]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1098","DOI":"10.1016\/j.biopha.2018.07.049","article-title":"Photodynamic therapy\u2013mechanisms, photosensitizers and combinations","volume":"106","author":"Kwiatkowski","year":"2018","journal-title":"Biomed. Pharmacother."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"111544","DOI":"10.1016\/j.jphotobiol.2019.111544","article-title":"Photodynamic therapy for skin cancer: How to enhance drug penetration?","volume":"197","author":"Champeau","year":"2019","journal-title":"J. Photochem. Photobiol. B Biol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"508","DOI":"10.1111\/den.13353","article-title":"Role of photodynamic therapy in the treatment of esophageal cancer","volume":"31","author":"Wu","year":"2019","journal-title":"Dig. Endosc."},{"key":"ref_4","first-page":"2785","article-title":"Cancer Tissue Classification, Associated Therapeutic Implications and PDT as an Alternative","volume":"37","author":"Horne","year":"2017","journal-title":"Anticancer. Res."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"176","DOI":"10.1111\/jcpe.13236","article-title":"Adjunctive laser or antimicrobial photodynamic therapy to non-surgical mechanical instrumentation in patients with untreated periodontitis: A systematic review and meta-analysis","volume":"47","author":"Salvi","year":"2019","journal-title":"J. Clin. Periodontol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"44","DOI":"10.1016\/j.pdpdt.2019.05.028","article-title":"Clinical and microbiological effects of multiple applications of antibacterial photodynamic therapy in periodontal maintenance patients. A randomized controlled clinical study","volume":"27","author":"Gaspirc","year":"2019","journal-title":"Photodiagnosis Photodyn. Ther."},{"key":"ref_7","first-page":"129","article-title":"Effects of Nd:YAG laser irradiation on the growth of Candida albicans and Streptococcus mutans: In Vitro Study","volume":"34","author":"Nowicka","year":"2018","journal-title":"Lasers Med. Sci."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"101688","DOI":"10.1016\/j.pdpdt.2020.101688","article-title":"Antimicrobial photodynamic therapy efficacy against specific pathogenic periodontitis bacterial species","volume":"30","author":"Park","year":"2020","journal-title":"Photodiagnosis Photodyn. Ther."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"19","DOI":"10.1038\/nrmicro.2017.107","article-title":"Development and regulation of single- and multi-species Candida albicans biofilms","volume":"16","author":"Lohse","year":"2018","journal-title":"Nat. Rev. Microbiol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"12300","DOI":"10.1088\/1054-660X\/26\/12\/123001","article-title":"The application of antimicrobial photodynamic therapy (aPDT) in dentistry: A critical review","volume":"26","author":"Carrera","year":"2016","journal-title":"Laser Phys."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"279","DOI":"10.1016\/S1572-1000(05)00007-4","article-title":"Mechanisms in photodynamic therapy: Part one\u2014Photosensitizers, photo-chemistry and cellular localization","volume":"1","author":"Castano","year":"2004","journal-title":"Photodiagnosis Photodyn. Ther."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"512","DOI":"10.1111\/j.1751-1097.2012.01107.x","article-title":"Photodynamic Antifungal Chemotherapy","volume":"88","author":"Rossi","year":"2012","journal-title":"Photochem. Photobiol."},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Mylona, V., Anagnostaki, E., Parker, S., Cronshaw, M., Lynch, E., and Grootveld, M. (2020). Laser-Assisted aPDT Protocols in Randomized Controlled Clinical Trials in Dentistry: A Systematic Review. Dent. J., 8.","DOI":"10.3390\/dj8030107"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"581","DOI":"10.1088\/0965-0393\/14\/4\/003","article-title":"Toluidine blue: Aggregation properties and structural aspects","volume":"14","author":"Martinelli","year":"2006","journal-title":"Model. Simul. Mater. Sci. Eng."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"347","DOI":"10.1042\/BJ20150942","article-title":"New photosensitizers for photodynamic therapy","volume":"473","author":"Abrahamse","year":"2016","journal-title":"Biochem. J."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"436","DOI":"10.1039\/b311900a","article-title":"Photodynamic therapy: A new antimicrobial approach to infectious disease?","volume":"3","author":"Hamblin","year":"2004","journal-title":"Photochem. Photobiol. Sci."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"2515","DOI":"10.2174\/0929867325666180601102333","article-title":"Promising Alternative Therapeutics for Oral Candidiasis","volume":"26","author":"Rodrigues","year":"2019","journal-title":"Curr. Med. Chem."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"2993","DOI":"10.3389\/fmicb.2019.02993","article-title":"The Fungal Cell Wall: Candida, Cryptococcus, and Aspergillus Species","volume":"10","author":"Rivera","year":"2020","journal-title":"Front. Microbiol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"495","DOI":"10.1128\/MMBR.00032-07","article-title":"Candida albicans Cell Wall Proteins","volume":"72","author":"Chaffin","year":"2008","journal-title":"Microbiol. Mol. Biol. Rev."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"33","DOI":"10.1016\/B978-0-12-407677-8.00002-6","article-title":"Fungal Cell Wall Organization and Biosynthesis","volume":"81","author":"Free","year":"2013","journal-title":"Adv. Genet."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"752","DOI":"10.1590\/0037-8682-0190-2013","article-title":"Fluconazole and amphotericin-B resistance are associated with increased catalase and superoxide dismutase activity in Candida albicans and Candida dubliniensis","volume":"46","author":"Linares","year":"2013","journal-title":"Rev. Soc. Bras. Med. Trop."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"340","DOI":"10.1016\/j.mam.2005.07.006","article-title":"Superoxide dismutases and their impact upon human health","volume":"26","author":"Johnson","year":"2005","journal-title":"Mol. Asp. Med."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"456","DOI":"10.1091\/mbc.e03-03-0179","article-title":"Superoxide Dismutases inCandida albicans: Transcriptional Regulation and Functional Characterization of the Hyphal-inducedSOD5Gene","volume":"15","author":"Martchenko","year":"2004","journal-title":"Mol. Biol. Cell"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"7445","DOI":"10.3390\/ijms14047445","article-title":"The Use of Chitosan to Enhance Photodynamic Inactivation against Candida albicans and Its Drug-Resistant Clinical Isolates","volume":"14","author":"Chien","year":"2013","journal-title":"Int. J. Mol. Sci."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"385","DOI":"10.1007\/s10103-009-0706-z","article-title":"Comparison of the photodynamic fungicidal efficacy of methylene blue, toluidine blue, malachite green and low-power laser irradiation alone against Candida albicans","volume":"25","author":"Souza","year":"2009","journal-title":"Lasers Med. Sci."},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Wiench, R., Skaba, D., Matys, J., and Grzech-Le\u015bniak, K. (2021). Efficacy of Toluidine Blue\u2014Mediated Antimicrobial Photodynamic Therapy on Candida spp. A Systematic Review. Antibiotics, 10.","DOI":"10.3390\/antibiotics10040349"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"274","DOI":"10.1046\/j.1365-2133.2001.04013.x","article-title":"Killing of cutaneous microbial species by photodynamic therapy","volume":"144","author":"Zeina","year":"2001","journal-title":"Br. J. Dermatol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"568","DOI":"10.1046\/j.1365-2133.2002.04623.x","article-title":"Cytotoxic effects of antimicrobial photodynamic therapy on keratinocytes in vitro","volume":"146","author":"Zeina","year":"2002","journal-title":"Br. J. Dermatol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"2329","DOI":"10.1128\/AAC.49.6.2329-2335.2005","article-title":"Effect of Cell-Photosensitizer Binding and Cell Density on Microbial Photoinactivation","volume":"49","author":"Demidova","year":"2005","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.micres.2007.08.001","article-title":"Antifungal photodynamic therapy","volume":"163","author":"Donnelly","year":"2008","journal-title":"Microbiol. Res."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"1525","DOI":"10.1093\/jac\/dkr160","article-title":"Influence of multidrug efflux systems on methylene blue-mediated photodynamic inactivation of Candida albicans","volume":"66","author":"Prates","year":"2011","journal-title":"J. Antimicrob. Chemother."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"150","DOI":"10.1007\/s101030050037","article-title":"Killing of the Yeast and Hyphal Forms of Candida albicans Using a Light-Activated Antimicrobial Agent","volume":"14","author":"Jackson","year":"1999","journal-title":"Lasers Med. Sci."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"59","DOI":"10.1016\/j.jphotobiol.2006.07.011","article-title":"Potential of photodynamic therapy in treatment of fungal infections of the mouth. Design and characterisation of a mucoadhesive patch containing toluidine blue O","volume":"86","author":"Donnelly","year":"2007","journal-title":"J. Photochem. Photobiol. B Biol."},{"key":"ref_34","first-page":"1567","article-title":"Genetic regulatory mechanisms of Candida albicans biofilm formation","volume":"33","author":"Guo","year":"2017","journal-title":"Sheng Wu Gong Cheng Xue Bao = Chin. J. Biotechnol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"182","DOI":"10.1016\/j.pdpdt.2017.12.001","article-title":"Photodynamic Antimicrobial Chemotherapy (PACT), using Toluidine blue O inhibits the viability of biofilm produced by Candida albicans at different stages of development","volume":"21","author":"Pinto","year":"2018","journal-title":"Photodiagn. Photodyn. Ther."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"983","DOI":"10.1007\/s10103-017-2344-1","article-title":"Photodynamic antimicrobial chemotherapy (PACT) using toluidine blue inhibits both growth and biofilm formation by Candida krusei","volume":"33","author":"Carvalho","year":"2018","journal-title":"Lasers Med. Sci."},{"key":"ref_37","doi-asserted-by":"crossref","unstructured":"Huang, M.-C., Shen, M., Huang, Y.-J., Lin, H.-C., and Chen, C.-T. (2018). Photodynamic Inactivation Potentiates the Susceptibility of Antifungal Agents against the Planktonic and Biofilm Cells of Candida albicans. Int. J. Mol. Sci., 19.","DOI":"10.3390\/ijms19020434"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"619","DOI":"10.1007\/s12275-009-0059-0","article-title":"The photodynamic effect of methylene blue and toluidine blue on Candida albicans is dependent on medium conditions","volume":"47","author":"Carvalho","year":"2009","journal-title":"J. Microbiol."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"106080","DOI":"10.1016\/j.mimet.2020.106080","article-title":"Regimen and different surfaces interfere with photodynamic therapy on Candida albicans biofilms","volume":"178","author":"Garcia","year":"2020","journal-title":"J. Microbiol. Methods"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"65","DOI":"10.1016\/j.jphotobiol.2008.07.013","article-title":"In vitro photodynamic inactivation of Candida spp. growth and adhesion to buccal epithelial cells","volume":"94","author":"Soares","year":"2009","journal-title":"J. Photochem. Photobiol. B Biol."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"180","DOI":"10.5978\/islsm.28_19-OR-14","article-title":"Antimicrobial effect on Candida albicans biofilm by application of different wavelengths and dyes and the synthetic killer decapeptide KP","volume":"28","author":"Merigo","year":"2019","journal-title":"Laser Ther."},{"key":"ref_42","doi-asserted-by":"crossref","unstructured":"Sherwani, M.A., Tufail, S., Khan, A.A., and Owais, M. (2015). Gold Nanoparticle-Photosensitizer Conjugate Based Photodynamic Inactivation of Biofilm Producing Cells: Potential for Treatment of C. albicans Infection in BALB\/c Mice. PLoS ONE, 10.","DOI":"10.1371\/journal.pone.0131684"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"5710","DOI":"10.1128\/AAC.05404-11","article-title":"Blue Dye and Red Light, a Dynamic Combination for Prophylaxis and Treatment of Cutaneous Candida albicans Infections in Mice","volume":"55","author":"Dai","year":"2011","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref_44","doi-asserted-by":"crossref","unstructured":"Lin, C.-H., Chien, H.-F., Lin, M.-H., Chen, C.-P., Shen, M., and Chen, C.-T. (2018). Chitosan Inhibits the Rehabilitation of Damaged Microbes Induced by Photodynamic Inactivation. Int. J. Mol. Sci., 19.","DOI":"10.20944\/preprints201805.0453.v1"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"141","DOI":"10.1016\/j.pdpdt.2012.11.004","article-title":"In vitro photodynamic inactivation of Candida species and mouse fibroblasts with phenothiazinium photosensitisers and red light","volume":"10","author":"Rodrigues","year":"2013","journal-title":"Photodiagnosis Photodyn. Ther."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"241","DOI":"10.1016\/j.pdpdt.2019.06.007","article-title":"Assessment of sensitivity of selected Candida strains on antimicrobial photodynamic therapy using diode laser 635 nm and toluidine blue\u2013In vitro research","volume":"27","author":"Wiench","year":"2019","journal-title":"Photodiagnosis Photodyn. Ther."},{"key":"ref_47","doi-asserted-by":"crossref","unstructured":"Nielsen, H.K., Garcia, J., V\u00e6th, M., and Schlafer, S. (2015). Comparison of Riboflavin and Toluidine Blue O as Photosensitizers for Photoactivated Disinfection on Endodontic and Periodontal Pathogens In Vitro. PLoS ONE, 10.","DOI":"10.1371\/journal.pone.0140720"},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"1059","DOI":"10.1007\/s10103-013-1473-4","article-title":"Photodynamic antimicrobial chemotherapy (PACT) inhibits biofilm formation by Candida albicans, increasing both ROS production and membrane permeability","volume":"29","author":"Rosseti","year":"2014","journal-title":"Lasers Med. Sci."},{"key":"ref_49","first-page":"907","article-title":"Photodynamic inactivation of Candida albicans mediated by a low density of light energy","volume":"29","author":"Silva","year":"2013","journal-title":"Lasers Med. Sci."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"4436","DOI":"10.1128\/AEM.02945-05","article-title":"Cellulose Acetate Containing Toluidine Blue and Rose Bengal Is an Effective Antimicrobial Coating when Exposed to White Light","volume":"72","author":"Decraene","year":"2006","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"726","DOI":"10.1039\/c3pp50426c","article-title":"Treatment of oral fungal infections using antimicrobial photodynamic therapy: A systematic review of currently available evidence","volume":"13","author":"Javed","year":"2014","journal-title":"Photochem. Photobiol. Sci."},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"103575","DOI":"10.1016\/j.micpath.2019.103575","article-title":"Characterization of oral candidiasis and the Candida species profile in patients with oral mucosal diseases","volume":"134","author":"Hu","year":"2019","journal-title":"Microb. Pathog."},{"key":"ref_53","unstructured":"Rasband, W.S. (2021, March 14). ImageJ.US National Institutes of Health, Bethesda, Maryland, USA, 1997\u20132018, Available online: https:\/\/imagej.nih.gov\/ij\/."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/22\/20\/10971\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T07:11:46Z","timestamp":1760166706000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/22\/20\/10971"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,10,11]]},"references-count":53,"journal-issue":{"issue":"20","published-online":{"date-parts":[[2021,10]]}},"alternative-id":["ijms222010971"],"URL":"https:\/\/doi.org\/10.3390\/ijms222010971","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,10,11]]}}}