High-Throughput Antimicrobial Efficacy Tests on a 384PillarPlate

High-Throughput Antimicrobial Efficacy Tests on a 384PillarPlate | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article High-Throughput Antimicrobial Efficacy Tests on a 384PillarPlate Pranav Joshi, Prabha Acharya, Manav Vanga, Oluwadara Fadeyibi, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7643728/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 27 Feb, 2026 Read the published version in World Journal of Microbiology and Biotechnology → Version 1 posted 9 You are reading this latest preprint version Abstract Preservatives, including antimicrobial compounds, are routinely added to home and personal care products such as dishwashing detergents to prevent microbial contamination during repeated consumer use. Traditional antimicrobial efficacy testing (T-AET) relies on colony counting methods, where microbial cocktails are inoculated into preservative-containing products and plated on agar to measure microbial survival. While considered a gold standard, T-AET is time-consuming, labor-intensive, costly, and prone to variability and error due to manual handling. To address these limitations, we developed a high-throughput, miniature, ATP-based on-chip AET (O-AET) using a 384PillarPlate platform. Pseudomonas aeruginosa , Enterobacter cloacae , and Staphylococcus aureus were encapsulated in 3% agarose and exposed to test compounds and detergent formulations. ATP levels in viable microbes were quantified using the BacTiter-Glo™ luminescence assay to generate dose–response curves, IC 50 values, and log reduction measurements. Two industrial antimicrobials, Acticide LG and Acticide MBR2, were evaluated, showing CV values below 8%. Nine detergent formulations from Colgate-Palmolive, including fresh and 4-week aged samples, were tested. Log reduction data from O-AET showed strong correlation with T-AET. Using a 3-log reduction cutoff, O-AET achieved 98% sensitivity, 44% specificity, and 71% overall predictivity. The lower specificity was partly attributed to limited true negative samples and nutrient deficiencies. The O-AET enables significant reduction in reagent volume, rapid turnaround, and compatibility with standard plate readers, making it cost-effective and scalable. This ATP bioluminescence-based platform provides a robust, reproducible, and high-throughput alternative to T-AET, with high potential to streamline product development and preservative screening in consumer care applications. 384PillarPlate high-throughput microbial assay preservative antimicrobial efficacy consumer product testing Full Text Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 27 Feb, 2026 Read the published version in World Journal of Microbiology and Biotechnology → Version 1 posted Editorial decision: Revision requested 17 Nov, 2025 Reviews received at journal 15 Nov, 2025 Reviewers agreed at journal 11 Nov, 2025 Reviews received at journal 20 Oct, 2025 Reviewers agreed at journal 15 Oct, 2025 Reviewers invited by journal 19 Sep, 2025 Editor assigned by journal 18 Sep, 2025 Submission checks completed at journal 18 Sep, 2025 First submitted to journal 17 Sep, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7643728","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":522481635,"identity":"022ba867-4adf-41fc-a06f-f6659c2020f1","order_by":0,"name":"Pranav Joshi","email":"","orcid":"","institution":"Bioprinting Laboratories Inc.","correspondingAuthor":false,"prefix":"","firstName":"Pranav","middleName":"","lastName":"Joshi","suffix":""},{"id":522481636,"identity":"ba121151-5be4-4426-9052-7b64c621f0a0","order_by":1,"name":"Prabha Acharya","email":"","orcid":"","institution":"University of North 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384PillarPlate","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"world-journal-of-microbiology-and-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"wibi","sideBox":"Learn more about [World Journal of Microbiology and Biotechnology](https://www.springer.com/journal/11274)","snPcode":"11274","submissionUrl":"https://submission.nature.com/new-submission/11274/3","title":"World Journal of Microbiology and Biotechnology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"384PillarPlate, high-throughput microbial assay, preservative, antimicrobial efficacy, consumer product testing","lastPublishedDoi":"10.21203/rs.3.rs-7643728/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7643728/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePreservatives, including antimicrobial compounds, are routinely added to home and personal care products such as dishwashing detergents to prevent microbial contamination during repeated consumer use. Traditional antimicrobial efficacy testing (T-AET) relies on colony counting methods, where microbial cocktails are inoculated into preservative-containing products and plated on agar to measure microbial survival. While considered a gold standard, T-AET is time-consuming, labor-intensive, costly, and prone to variability and error due to manual handling. To address these limitations, we developed a high-throughput, miniature, ATP-based on-chip AET (O-AET) using a 384PillarPlate platform. \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e, \u003cem\u003eEnterobacter cloacae\u003c/em\u003e, and \u003cem\u003eStaphylococcus aureus\u003c/em\u003e were encapsulated in 3% agarose and exposed to test compounds and detergent formulations. ATP levels in viable microbes were quantified using the BacTiter-Glo™ luminescence assay to generate dose–response curves, IC\u003csub\u003e50\u003c/sub\u003e values, and log reduction measurements. Two industrial antimicrobials, Acticide LG and Acticide MBR2, were evaluated, showing CV values below 8%. Nine detergent formulations from Colgate-Palmolive, including fresh and 4-week aged samples, were tested. Log reduction data from O-AET showed strong correlation with T-AET. Using a 3-log reduction cutoff, O-AET achieved 98% sensitivity, 44% specificity, and 71% overall predictivity. The lower specificity was partly attributed to limited true negative samples and nutrient deficiencies. The O-AET enables significant reduction in reagent volume, rapid turnaround, and compatibility with standard plate readers, making it cost-effective and scalable. This ATP bioluminescence-based platform provides a robust, reproducible, and high-throughput alternative to T-AET, with high potential to streamline product development and preservative screening in consumer care applications.\u003c/p\u003e","manuscriptTitle":"High-Throughput Antimicrobial Efficacy Tests on a 384PillarPlate","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-30 17:14:48","doi":"10.21203/rs.3.rs-7643728/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-11-17T08:01:08+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-15T17:57:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"330606706945625624202151450286758901537","date":"2025-11-11T21:08:16+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-21T00:03:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"291032093327116160314509958628810051903","date":"2025-10-15T18:09:52+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-19T08:47:37+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-18T22:27:40+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-18T07:35:58+00:00","index":"","fulltext":""},{"type":"submitted","content":"World Journal of Microbiology and Biotechnology","date":"2025-09-17T22:24:25+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"world-journal-of-microbiology-and-biotechnology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"wibi","sideBox":"Learn more about [World Journal of Microbiology and Biotechnology](https://www.springer.com/journal/11274)","snPcode":"11274","submissionUrl":"https://submission.nature.com/new-submission/11274/3","title":"World Journal of Microbiology and Biotechnology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"cd25f06f-3307-48d8-9d10-7433f51c9905","owner":[],"postedDate":"September 30th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-03-02T16:01:03+00:00","versionOfRecord":{"articleIdentity":"rs-7643728","link":"https://doi.org/10.1007/s11274-026-04805-4","journal":{"identity":"world-journal-of-microbiology-and-biotechnology","isVorOnly":false,"title":"World Journal of Microbiology and Biotechnology"},"publishedOn":"2026-02-27 15:57:49","publishedOnDateReadable":"February 27th, 2026"},"versionCreatedAt":"2025-09-30 17:14:48","video":"","vorDoi":"10.1007/s11274-026-04805-4","vorDoiUrl":"https://doi.org/10.1007/s11274-026-04805-4","workflowStages":[]},"version":"v1","identity":"rs-7643728","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7643728","identity":"rs-7643728","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}