{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,7]],"date-time":"2026-08-07T17:01:23Z","timestamp":1786122083186,"version":"build-2736575974"},"reference-count":29,"publisher":"MDPI AG","issue":"8","license":[{"start":{"date-parts":[[2026,8,7]],"date-time":"2026-08-07T00:00:00Z","timestamp":1786060800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000060","name":"National Institute of Allergy and Infectious Diseases","doi-asserted-by":"publisher","award":["R33AI140823"],"award-info":[{"award-number":["R33AI140823"]}],"id":[{"id":"10.13039\/100000060","id-type":"DOI","asserted-by":"publisher"}]},{"award":["R33AI140823"],"award-info":[{"award-number":["R33AI140823"]}],"id":[{"id":"https:\/\/ror.org\/043z4tv69","id-type":"ROR","asserted-by":"publisher"}]},{"name":"Margaret Batts Tobin Foundation"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["J. Fungi","JoF"],"abstract":"<jats:p>Screening assays for discovering Candida albicans biofilm inhibitors typically measure either metabolic activity or attached biomass. Although biomass staining with Crystal Violet or Safranin is affordable and easy to perform, standard protocols often require toxic solvents for biofilm fixation and dye extraction. We developed and evaluated an alternative Safranin staining method that uses heat fixation and direct absorbance measurement of stained biofilms without a dye-extraction step. Under the experimental conditions tested, the proposed method reduced solvent use and plate handling and minimized reagent use while offering assay consistency and reliability for primary screening campaigns. The assay quality was verified using positive and negative inhibition controls, with acceptable Z\u2032-factor values supporting its use for primary drug screening. The protocol was further evaluated by screening 1520 compounds from the Prestwick Chemical Library to identify inhibitors of C. albicans biofilm formation. The proposed Safranin method provides a simplified, extraction-free alternative for biofilm-inhibitor screening under the experimental conditions evaluated in this study.<\/jats:p>","DOI":"10.3390\/jof12080581","type":"journal-article","created":{"date-parts":[[2026,8,7]],"date-time":"2026-08-07T08:01:36Z","timestamp":1786089696000},"page":"581","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Development of a Cost-Effective and Low-Toxicity Safranin-Based Assay for Discovering Candida albicans Biofilm Formation Inhibitors in Large-Scale Screening Campaigns"],"prefix":"10.3390","volume":"12","author":[{"given":"Augusto","family":"Vazquez-Rodriguez","sequence":"first","affiliation":[{"name":"Department of Molecular Microbiology and Immunology and South Texas Center for Emerging Infectious Diseases, The University of Texas at San Antonio, San Antonio, TX 78259, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1992-3270","authenticated-orcid":false,"given":"Jose L.","family":"Lopez-Ribot","sequence":"additional","affiliation":[{"name":"Department of Molecular Microbiology and Immunology and South Texas Center for Emerging Infectious Diseases, The University of Texas at San Antonio, San Antonio, TX 78259, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,8,7]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1494","DOI":"10.1038\/nprot.2008.141","article-title":"A simple and reproducible 96-well plate-based method for the formation of fungal biofilms and its application to antifungal susceptibility testing","volume":"3","author":"Pierce","year":"2008","journal-title":"Nat. Protoc."},{"key":"ref_2","unstructured":"Ramage, G., and L\u00f3pez-Rib, J.L. (2005). Techniques for antifungal susceptibility testing of Candida albicans biofilms. Antifungal Agents: Methods and Protocols, Springer."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"2475","DOI":"10.1128\/AAC.45.9.2475-2479.2001","article-title":"Standardized Method for In Vitro Antifungal Susceptibility Testing of Candida albicans Biofilms","volume":"45","author":"Ramage","year":"2001","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1909","DOI":"10.1038\/nprot.2008.192","article-title":"In vitro growth and analysis of Candida biofilms","volume":"3","author":"Chandra","year":"2008","journal-title":"Nat. Protoc."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1B.1.1","DOI":"10.1002\/9780471729259.mc01b01s22","article-title":"Growing and analyzing static biofilms","volume":"22","author":"Merritt","year":"2011","journal-title":"Curr. Protoc. Microbiol."},{"key":"ref_6","first-page":"71","article-title":"Exposure to crystal violet, its toxic, genotoxic and carcinogenic effects on environment and its degradation and detoxification for environmental safety","volume":"237","author":"Mani","year":"2016","journal-title":"Rev. Environ. Contam. Toxicol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"87","DOI":"10.1016\/j.mimet.2017.08.003","article-title":"Quantification of biofilm biomass by staining: Non-toxic safranin can replace the popular crystal violet","volume":"141","author":"Ommen","year":"2017","journal-title":"J. Microbiol. Methods"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"63","DOI":"10.1016\/j.mimet.2014.03.016","article-title":"Staphylococcus epidermidis biofilm quantification: Effect of different solvents and dyes","volume":"101","author":"Wu","year":"2014","journal-title":"J. Microbiol. Methods"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"214","DOI":"10.3109\/13693786.2011.580016","article-title":"Comparative analysis of Candida biofilm quantitation assays","volume":"50","author":"Taff","year":"2012","journal-title":"Sabouraudia"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"2253","DOI":"10.1038\/s41467-018-04787-4","article-title":"Gene flow contributes to diversification of the major fungal pathogen Candida albicans","volume":"9","author":"Ropars","year":"2018","journal-title":"Nat. Commun."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"e01521-23","DOI":"10.1128\/mbio.01521-23","article-title":"The Candida albicans reference strain SC5314 contains a rare, dominant allele of the transcription factor Rob1 that modulates filamentation, biofilm formation, and oral commensalism","volume":"14","author":"Glazier","year":"2023","journal-title":"mBio"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"247","DOI":"10.1177\/1087057105285610","article-title":"A comparison of assay performance measures in screening assays: Signal window, Z\u2019factor, and assay variability ratio","volume":"11","author":"Iversen","year":"2006","journal-title":"J. Biomol. Screen."},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Schwab, M. (2017). Z-Factor. Encyclopedia of Cancer, Springer.","DOI":"10.1007\/978-3-662-46875-3"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"67","DOI":"10.1177\/108705719900400206","article-title":"A simple statistical parameter for use in evaluation and validation of high throughput screening assays","volume":"4","author":"Zhang","year":"1999","journal-title":"J. Biomol. Screen."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"e00205-25","DOI":"10.1128\/mbio.00205-25","article-title":"Discovery of novel antifungal drugs via screening repurposing libraries against Coccidioides posadasii spherule initials","volume":"16","author":"Saeger","year":"2025","journal-title":"mBio"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"11559","DOI":"10.1038\/s41598-018-29973-8","article-title":"A phenotypic small-molecule screen identifies halogenated salicylanilides as inhibitors of fungal morphogenesis, biofilm formation and host cell invasion","volume":"8","author":"Garcia","year":"2018","journal-title":"Sci. Rep."},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Sutar, Y., Nabeela, S., Singh, S., Alqarihi, A., Solis, N., Ghebremariam, T., Filler, S., Ibrahim, A.S., Date, A., and Uppuluri, P. (2022). Niclosamide-loaded nanoparticles disrupt Candida biofilms and protect mice from mucosal candidiasis. PLoS Biol., 20.","DOI":"10.1371\/journal.pbio.3001762"},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"Roy, A. (2022). High-Throughput screening (HTS) technology. Encyclopedia of Molecular Pharmacology, Springer.","DOI":"10.1007\/978-3-030-57401-7_73"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"e00539-18","DOI":"10.1128\/mSphere.00539-18","article-title":"Alexidine dihydrochloride has broad-spectrum activities against diverse fungal pathogens","volume":"3","author":"Mamouei","year":"2018","journal-title":"mSphere"},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Okoli, I., Coleman, J.J., Tempakakis, E., An, W.F., Holson, E., Wagner, F., Conery, A.L., Larkins-Ford, J., Wu, G., and Stern, A. (2009). Identification of antifungal compounds active against Candida albicans using an improved high-throughput Caenorhabditis elegans assay. PLoS ONE, 4.","DOI":"10.1371\/annotation\/76699885-a47e-426c-accd-35576696c4f2"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"e00263-19","DOI":"10.1128\/AAC.00263-19","article-title":"Repurposing of ribavirin as an adjunct therapy against invasive Candida strains in an in vitro study","volume":"63","author":"Yousfi","year":"2019","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Ajetunmobi, O.H., Wall, G., Vidal Bonifacio, B., Martinez Delgado, L.A., Chaturvedi, A.K., Najvar, L.K., Wormley, F.L., Patterson, H.P., Wiederhold, N.P., and Patterson, T.F. (2023). High-throughput screening of the repurposing hub library to identify drugs with novel inhibitory activity against Candida albicans and Candida auris biofilms. J. Fungi, 9.","DOI":"10.3390\/jof9090879"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"3681","DOI":"10.1128\/AAC.00680-13","article-title":"High-throughput screening of a collection of known pharmacologically active small compounds for identification of Candida albicans biofilm inhibitors","volume":"57","author":"Siles","year":"2013","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"618","DOI":"10.1016\/S1734-1140(11)70574-6","article-title":"Role of vinpocetine in cerebrovascular diseases","volume":"63","author":"Patyar","year":"2011","journal-title":"Pharmacol. Rep."},{"key":"ref_25","first-page":"211","article-title":"Updates of recent vinpocetine research in treating cardiovascular diseases","volume":"2","author":"Zhang","year":"2020","journal-title":"J. Cell. Immunol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"107988","DOI":"10.1016\/j.yebeh.2021.107988","article-title":"Vinpocetine, cognition, and epilepsy","volume":"119","author":"Meador","year":"2021","journal-title":"Epilepsy Behav."},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Vizi, E.S., and Kiss, B. (2026). Vinpocetine\u2014An \u201cOld\u201d Drug with a New Face: Moving Toward a Better Understanding of Its Neuroprotective Mechanism of Action. Biomolecules, 16.","DOI":"10.3390\/biom16030454"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"30","DOI":"10.1016\/j.ejphar.2017.11.041","article-title":"An update on vinpocetine: New discoveries and clinical implications","volume":"819","author":"Zhang","year":"2018","journal-title":"Eur. J. Pharmacol."},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Kumar, S., and Singh, B. (2026). Clinical Trials and Toxicological Evaluations. Catharanthus in Drug Discovery: From Molecules to Medicine, Springer.","DOI":"10.1007\/978-981-95-5048-7"}],"container-title":["Journal of Fungi"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2309-608X\/12\/8\/581\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,8,7]],"date-time":"2026-08-07T08:36:22Z","timestamp":1786091782000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2309-608X\/12\/8\/581"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,8,7]]},"references-count":29,"journal-issue":{"issue":"8","published-online":{"date-parts":[[2026,8]]}},"alternative-id":["jof12080581"],"URL":"https:\/\/doi.org\/10.3390\/jof12080581","relation":{},"ISSN":["2309-608X"],"issn-type":[{"value":"2309-608X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,8,7]]}}}