{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,26]],"date-time":"2026-09-26T16:01:24Z","timestamp":1790438484560,"version":"4.1.0"},"reference-count":30,"publisher":"Wiley","issue":"9","license":[{"start":{"date-parts":[[2026,9,25]],"date-time":"2026-09-25T00:00:00Z","timestamp":1790294400000},"content-version":"vor","delay-in-days":24,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,9,1]],"date-time":"2026-09-01T00:00:00Z","timestamp":1788220800000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"funder":[{"DOI":"10.13039\/501100002322","name":"Coordena\u00e7\u00e3o de Aperfei\u00e7oamento de Pessoal de N\u00edvel Superior","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100002322","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Current Protocols"],"published-print":{"date-parts":[[2026,9]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>\n                    Obligate anaerobic bacteria are important constituents of the human microbiota and are frequently involved in opportunistic infections. However, due to their fastidious nature and oxygen sensitivity, antimicrobial susceptibility testing (AST) is often neglected, resulting in empirical treatments and an increased risk of therapeutic failure. The broth microdilution method offers a cost\u2010effective and scalable alternative for antibacterial activity evaluation but is typically restricted to certain genera due to concerns over oxygen exposure. In this study, we present a complete standardized in\u2010house protocol for determining the minimum inhibitory concentration (MIC) of natural and synthetic antimicrobial agents against obligate anaerobes using the broth microdilution technique under strictly anoxic conditions. Our method is optimized for implementation in laboratories equipped with anaerobic chambers and allows high\u2010throughput screening using minimal sample volumes, making it ideal for natural product\u2013based antibacterial discovery. The protocol is based on other guidelines with critical adaptations that ensure bacterial viability throughout the procedure. It provides reproducible results without oxygen interference, thus representing a valuable tool for researchers investigating novel antimicrobials targeting anaerobic pathogens, within a period of approximately 6 days. \u00a9 2026 The Author(s).\n                    <jats:italic>Current Protocols<\/jats:italic>\n                    published by Wiley Periodicals LLC.\n                  <\/jats:p>\n                  <jats:p>\n                    <jats:bold>Basic Protocol 1<\/jats:bold>\n                    : Preparation of working solutions\n                  <\/jats:p>\n                  <jats:p>\n                    <jats:bold>Basic Protocol 2<\/jats:bold>\n                    : Preparation of the 96\u2010well microplate\n                  <\/jats:p>\n                  <jats:p>\n                    <jats:bold>Basic Protocol 3<\/jats:bold>\n                    : Inoculum standardization\n                  <\/jats:p>\n                  <jats:p>\n                    <jats:bold>Basic Protocol 4<\/jats:bold>\n                    : Reading and interpreting the results\n                  <\/jats:p>","DOI":"10.1002\/cpz1.70474","type":"journal-article","created":{"date-parts":[[2026,9,25]],"date-time":"2026-09-25T14:16:01Z","timestamp":1790345761000},"source":"Crossref","is-referenced-by-count":0,"title":["Standardized In\u2010house Protocol for Determining the Minimum Inhibitory Concentration (MIC) via Broth Microdilution for Natural Products Against Obligate Anaerobic Bacteria"],"prefix":"10.1002","volume":"6","author":[{"given":"Daniela Silva","family":"Gon\u00e7alves","sequence":"first","affiliation":[{"name":"Laboratory of Antimicrobial Testing, Institute of Biomedical Sciences Federal University of Uberl\u00e2ndia  Uberl\u00e2ndia Minas Gerais Brazil"},{"name":"These authors contributed equally to this work"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mariana Brentini","family":"Santiago","sequence":"additional","affiliation":[{"name":"Laboratory of Antimicrobial Testing, Institute of Biomedical Sciences Federal University of Uberl\u00e2ndia  Uberl\u00e2ndia Minas Gerais Brazil"},{"name":"These authors contributed equally to this work"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lucas Andrade","family":"Sousa","sequence":"additional","affiliation":[{"name":"Laboratory of Antimicrobial Testing, Institute of Biomedical Sciences Federal University of Uberl\u00e2ndia  Uberl\u00e2ndia Minas Gerais Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Carlos Henrique Gomes","family":"Martins","sequence":"additional","affiliation":[{"name":"Laboratory of Antimicrobial Testing, Institute of Biomedical Sciences Federal University of Uberl\u00e2ndia  Uberl\u00e2ndia Minas Gerais Brazil"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2026,9,25]]},"reference":[{"key":"e_1_2_9_2_1_1","doi-asserted-by":"publisher","DOI":"10.1093\/jac\/48.suppl_1.5"},{"key":"e_1_2_9_2_2_1","volume-title":"Methods for antimicrobial susceptibility testing of anaerobic bacteria","author":"CLSI","year":"2018"},{"key":"e_1_2_9_2_3_1","doi-asserted-by":"publisher","DOI":"10.1007\/s10096\u2010023\u201004708\u20104"},{"key":"e_1_2_9_2_4_1","doi-asserted-by":"publisher","DOI":"10.3389\/fmicb.2025.1549044"},{"key":"e_1_2_9_2_5_1","unstructured":"EUCAST. (2023). EUCAST disk diffusion methodology for selected rapidly growing anaerobic bacteria on Fastidious Anaerobe Agar with defibrinated horse blood (FAA\u2010HB) (Version 2.0 January 2023) European Committee on Antimicrobial Susceptibility Testing."},{"key":"e_1_2_9_2_6_1","unstructured":"EUCAST. (2025). The European Committee on Antimicrobial Susceptibility Testing. Breakpoint tables for interpretation of MICs and zone diameters (Version 15.0) European Committee on Antimicrobial Susceptibility Testing."},{"key":"e_1_2_9_2_7_1","doi-asserted-by":"publisher","DOI":"10.1556\/1886.2020.00009"},{"key":"e_1_2_9_2_8_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.anaerobe.2008.01.001"},{"key":"e_1_2_9_2_9_1","doi-asserted-by":"publisher","DOI":"10.1590\/s0074\u201002762002000700017"},{"key":"e_1_2_9_2_10_1","doi-asserted-by":"publisher","DOI":"10.3390\/molecules28176193"},{"key":"e_1_2_9_2_11_1","doi-asserted-by":"publisher","DOI":"10.3390\/toxics9050092"},{"key":"e_1_2_9_2_12_1","doi-asserted-by":"publisher","DOI":"10.1042\/EBC20240116"},{"key":"e_1_2_9_2_13_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.anaerobe.2007.09.006"},{"key":"e_1_2_9_2_14_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.biopha.2020.110467"},{"key":"e_1_2_9_2_15_1","doi-asserted-by":"publisher","DOI":"10.1021\/acs.jnatprod.9b01285"},{"key":"e_1_2_9_2_16_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.trac.2023.117288"},{"key":"e_1_2_9_2_17_1","doi-asserted-by":"publisher","DOI":"10.3390\/molecules28135163"},{"key":"e_1_2_9_2_18_1","doi-asserted-by":"publisher","DOI":"10.3390\/microorganisms11061474"},{"key":"e_1_2_9_2_19_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jep.2005.04.025"},{"key":"e_1_2_9_2_20_1","doi-asserted-by":"publisher","DOI":"10.1093\/jambio\/lxae316"},{"key":"e_1_2_9_2_21_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.ymeth.2007.01.006"},{"key":"e_1_2_9_2_22_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.anaerobe.2022.102588"},{"key":"e_1_2_9_2_23_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41598\u2010022\u201024776\u20104"},{"key":"e_1_2_9_2_24_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.anaerobe.2022.102559"},{"key":"e_1_2_9_2_25_1","doi-asserted-by":"publisher","DOI":"10.3390\/molecules27238280"},{"key":"e_1_2_9_3_2_1","unstructured":"Moraes et\u00a0al. (2020). See above."},{"key":"e_1_2_9_3_3_1","unstructured":"Silva et\u00a0al. (2022). See above."},{"key":"e_1_2_9_3_4_1","unstructured":"Santos et\u00a0al. (2025). See above."},{"key":"e_1_2_9_3_5_1","unstructured":"Satokata et\u00a0al. (2022). See above."},{"key":"e_1_2_9_3_6_1","unstructured":"The in\u2010house protocol presented here has already been successfully published in the above\u2010mentioned research articles."}],"container-title":["Current Protocols"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/currentprotocols.onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/cpz1.70474","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/currentprotocols.onlinelibrary.wiley.com\/doi\/full-xml\/10.1002\/cpz1.70474","content-type":"application\/xml","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/currentprotocols.onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/cpz1.70474","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,26]],"date-time":"2026-09-26T15:54:52Z","timestamp":1790438092000},"score":1,"resource":{"primary":{"URL":"https:\/\/currentprotocols.onlinelibrary.wiley.com\/doi\/10.1002\/cpz1.70474"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,9]]},"references-count":30,"journal-issue":{"issue":"9","published-print":{"date-parts":[[2026,9]]}},"alternative-id":["10.1002\/cpz1.70474"],"URL":"https:\/\/doi.org\/10.1002\/cpz1.70474","archive":["Portico"],"relation":{},"ISSN":["2691-1299","2691-1299"],"issn-type":[{"value":"2691-1299","type":"print"},{"value":"2691-1299","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,9]]},"article-number":"e70474"}}