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The document specifies general requirements, sample handling, apparatus and materials (including incubator conditions and test tubes for confirmation), and reagent controls such as penicillin G positive control, negative controls, and beta-lactamase confirmation. It also details assay plate preparation, including agar formulation, temperature control at 64±2°C, spore tablet inoculum steps, plate pouring volume guidance, solidification time, storage limits, and a performance check using positive and negative controls.",{"@graph":69,"@context":122},[70,84,105],{"@type":71,"itemListElement":72},"BreadcrumbList",[73,77,79,82],{"item":74,"name":75,"@type":76,"position":8},"https://docshare.wps.com","Home","ListItem",{"item":78,"name":9,"@type":76,"position":14},"https://docshare.wps.com/document/",{"item":80,"name":35,"@type":76,"position":81},"https://docshare.wps.com/document/healthcare/",3,{"item":83,"name":65,"@type":76,"position":19},"https://docshare.wps.com/document/detection-of-inhibitory-substances-in-milk-bacillus-stearothermophilus-disc-assay-bsda-charm-tablet-method-ims-9-b2-general-requirements-and-assay-plate-technique/403289/",{"url":83,"name":65,"@type":85,"image":86,"author":91,"headline":65,"publisher":94,"fileFormat":97,"inLanguage":63,"description":67,"dateModified":98,"datePublished":99,"encodingFormat":97,"isAccessibleForFree":100,"interactionStatistic":101},"DigitalDocument",{"url":87,"@type":88,"width":89,"height":90},"https://docshare.wps.com/thumbnails/detection-of-inhibitory-substances-in-milk-bacillus-stearothermophilus-disc-assay-bsda-charm-tablet-method-ims-9-b2-general-requirements-and-assay-plate-technique/403289.png","ImageObject",300,407,{"name":92,"@type":93},"aboduw","Person",{"url":74,"name":95,"@type":96},"DocShare","Organization","application/pdf","2026-11-01","2026-09-27",true,{"@type":102,"interactionType":103,"userInteractionCount":34},"InteractionCounter",{"@type":104},"ViewAction",{"@type":106,"mainEntity":107},"FAQPage",[108,114,118],{"name":109,"@type":110,"acceptedAnswer":111},"What assay method is used to detect inhibitory substances in milk?","Question",{"text":112,"@type":113},"The procedure uses Bacillus stearothermophilus Disc Assay (BsDA) with the Charm Tablet Method, applied to raw commingled cow milk, raw commingled goat milk, and NCIMS-accepted pasteurized cow milk products.","Answer",{"name":115,"@type":110,"acceptedAnswer":116},"What are the required temperature conditions during plate preparation?",{"text":117,"@type":113},"Agar is cooled promptly to 64±2°C before inoculation, and the spore preparation is incorporated into agar tempered to the same 64±2°C condition.",{"name":119,"@type":110,"acceptedAnswer":120},"How are positive and negative controls used in the procedure?",{"text":121,"@type":113},"Before any sample analysis, positive and negative controls must give appropriate zones. Out-of-range zones require corrective actions, and records must be maintained; suitability of controls is tested by adding to discs and checking expected zone behavior.","https://schema.org",{"og:url":83,"og:type":124,"og:title":65,"og:site_name":95,"og:description":67},"article",{"robots":126,"canonical":83},"index,follow",{"doc_id":128,"site_id":62},403289,1790957105,{"code":4,"msg":5,"data":131},{"doc_id":128,"user_id":132,"nickname":92,"user_avatar":133,"doc_module":4,"category_id":34,"category_name":35,"doc_title":65,"doc_description":67,"doc_content":134,"file_id":135,"file_url":136,"file_type":137,"file_size":138,"view_count":34,"is_deleted":4,"is_public":8,"is_downloadable":8,"audit_status":8,"page_count":44,"language":139,"language_code":63,"site_id":62,"html_lang":63,"table_of_contents":140,"faqs":141,"seo_title":142,"seo_description":67,"update_tm":143,"read_time":144,"preview_page_count":4},3985747862266,"https://ap-avatar.wpscdn.com/davatar_085a072bc5b1113ac321206ff7593b45","DETECTION OF INHIBITORY SUBSTANCES IN MILK  \nBacillus stearothermophilus Disc Assay (BsDA), Charm Tablet Method ( Raw Commingled Cow Milk, Raw Commingled Goat Milk, and NCIMS Accepted Pasteurized Cow Milk Products)  \nIMS\\# 9-B2  \n[Unless otherwise stated all tolerances are ±5%]  \nGENERAL REQUIREMENTS  \n1. Laboratory Requirements (see Cultural Procedure (CP), items 33 & 34), except    \na. For Appendix N testing, see Appendix N General Requirements (App. N GR),  \nitems 14 & 15    \nSAMPLES  \n2. See CP, item 33, except    \na. For Appendix N testing, see App. N GR items 9    \nAPPARATUS & MATERIALS  \n3. See CP items 1-23, except    \na. For Appendix N testing, see App. N GR items 1-8    \n4. Equipment    \na. Incubator thermostatically controlled at 64±2 °C    \nb. Heating block, water bath or other acceptable method to heat to at least  \n82±2 °C, for confirmation    \nc. Pipettor-90 µL and 500 µL (optionally 50 µL) and disposable tips (see App. N GR item 7 or CP item 6)    \nd. Forceps, Fine Points, Stainless Steel    \ne. Vernier, Dial or Digital Calipers, metal (readable to 0.1 mm)    \nf. Stirring hot plate/stirring bar (optional)    \ng. 100 mL Class A graduate cylinder    \nh. 250 mL Erlenmeyer flasks    \ni. 13 x 100 mm test tubes for beta-lactam confirmation    \nj. Timer    \nMATERIALS  \n5. See CP, items 24-32    \na. Filter Paper Discs, Blank, Unimpregnated, Non-sterile   Brand:   Lot \\#:      \n1. High absorbability, diameter 12.7±0 .1 mm    \nb. Charm PM Indicator Agar    \n1.  Do Not Autoclave- (see plate preparation, item 19 below)    \nc. Charm Spore Tablets    \n1.  Bacillus stearothermophilus (Geobacillus stearothermophilus) tablets  \ncontaining 100,000,000 (±10 million) spores per tablet   Lot \\#:   Exp. Date:      \nd. Plastic Petri dish (15 x 100 mm, bottom plate inner diameter 86.1-87.0mm)    \nREAGENTS  \n6. Reagents    \na. Charm 5.0 ppb Penicillin G Standard Positive Control   Lot \\#:   Exp. Date:      \n1. Store according to label directions    \n2. Rehydrate according to label instructions    \n3. Test for suitability each time prepared, add to one (1) disc, must  \nproduce zone 16-20 mm; maintain records    \nAvg. Zone Size:      \n4. Use rehydrated standard within 48 hours if refrigerated    \nLab Prep. Date:      \n5. Or, aliquot within 24 hours and freeze at-15°C or colder in  \nnon-frost-free freezer or in an insulated foam container in a frost-free  \nfreezer, use within 2 months (Once thawed, maintain according to  \nmanufacturer’s instructions)    \nLab Prep. Date:   Lab Exp. Date:      \nb. Negative Control    \n1. Charm Zero Control Standard  \n________  \nLot \\#:   Exp. Date:      \na. Reconstitute according to label instructions    \nb. Use rehydrated negative control within 72 hours if refrigerated   Lab Prep. Date:   Lab Exp. Date:      \nc. Or, aliquot within 24 hours and freeze at-15°C or colder in non-frost-free freezer or in an insulated foam container in a frost-free freezer, use within 2 months (Once thawed, maintain  \naccording to manufacturer’s instructions)    \nLab Prep. Date:   Lab Exp. Date:      \n2. Inhibitor Free Raw Milk   Sample ID:   Date Tested:      \na. Use within 72 hours if refrigerated   Lab Prep. Date:   Lab Exp. Date:      \nb. Or, aliquot within 24 hours and freeze at-15°C or colder in non-frost-free freezer or in an insulated foam container in a frost-free freezer, use within 2 months. Once thawed, use within  \n24 hours    \nLab Prep. Date:   Lab Exp. Date:      \n3. Test for suitability, add to one (1) disc, produces no zone;  \nmaintain records    \nZone Size:      \nc. Charm Beta-lactamase tablet or liquid concentrate (not required if  \nbeta-lactamase is not used for confirmation)    \n1. Stored at-15°C or below    \n2. Do not use beyond expiration date   Lot \\#:   Exp. Date:      \n3. Reconstitute freeze dried concentrate as per manufacturer  \ninstructions    \na. Liquid concentrate stored at-15°C or below in a non  \nfrost-free freezer or in an insulated foam container in a  \nfrost-free freezer; use within 2 weeks  ","cbCain7OM2cCEOAv","https://ap.wps.com/l/cbCain7OM2cCEOAv","pdf",761651,"English","# GENERAL REQUIREMENTS\n## Laboratory Requirements\n## SAMPLES\n# APPARATUS & MATERIALS\n## Equipment\n## Materials\n# REAGENTS\n## Positive control and negative control\n## Inhibitor free raw milk and suitability testing\n## Beta-lactamase control (confirmation)\n# ASSAY PLATE\n## Preparation of Plate\n# TECHNIQUE\n## Performance Check","[{\"question\":\"What assay method is used to detect inhibitory substances in milk?\",\"answer\":\"The procedure uses Bacillus stearothermophilus Disc Assay (BsDA) with the Charm Tablet Method, applied to raw commingled cow milk, raw commingled goat milk, and NCIMS-accepted pasteurized cow milk products.\"},{\"question\":\"What are the required temperature conditions during plate preparation?\",\"answer\":\"Agar is cooled promptly to 64±2°C before inoculation, and the spore preparation is incorporated into agar tempered to the same 64±2°C condition.\"},{\"question\":\"How are positive and negative controls used in the procedure?\",\"answer\":\"Before any sample analysis, positive and negative controls must give appropriate zones. Out-of-range zones require corrective actions, and records must be maintained; suitability of controls is tested by adding to discs and checking expected zone behavior.\"}]","DETECTION OF INHIBITORY SUBSTANCES IN MILK - Bacillus stearothermophilus Disc Assay (BsDA), Charm Tablet Method - IMS# 9-B2 - General requirements and assay plate technique | PDF",1790538680,23]