DETECTION OF INHIBITORY SUBSTANCES IN MILK - Bacillus stearothermophilus Disc Assay (BsDA), Charm Tablet Method - IMS# 9-B2 - General requirements and assay plate technique

Sep 27, 2026

Uploaded by aboduw

DETECTION OF INHIBITORY SUBSTANCES IN MILK defines a laboratory workflow using Bacillus stearothermophilus disc assay (BsDA) with a Charm tablet method for testing raw commingled cow milk, raw commingled goat milk, and NCIMS-accepted pasteurized cow milk products. 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.

LanguageEnglish

CategoryHealthcare

FormatPDF

Pages9

Reading Time23 min

AccessFree to download

GENERAL REQUIREMENTS

Laboratory Requirements

SAMPLES

APPARATUS & MATERIALS

Equipment

Materials

REAGENTS

Positive control and negative control

Inhibitor free raw milk and suitability testing

Beta-lactamase control (confirmation)

ASSAY PLATE

Preparation of Plate

TECHNIQUE

Performance Check

DETECTION OF INHIBITORY SUBSTANCES IN MILK Bacillus stearothermophilus Disc Assay (BsDA), Charm Tablet Method ( Raw Commingled Cow Milk, Raw Commingled Goat Milk, and NCIMS Accepted Pasteurized Cow Milk Products) IMS\# 9-B2 [Unless otherwise stated all tolerances are ±5%] GENERAL REQUIREMENTS 1. Laboratory Requirements (see Cultural Procedure (CP), items 33 & 34), except a. For Appendix N testing, see Appendix N General Requirements (App. N GR), items 14 & 15 SAMPLES 2. See CP, item 33, except a. For Appendix N testing, see App. N GR items 9 APPARATUS & MATERIALS 3. See CP items 1-23, except a. For Appendix N testing, see App. N GR items 1-8 4. Equipment a. Incubator thermostatically controlled at 64±2 °C b. Heating block, water bath or other acceptable method to heat to at least 82±2 °C, for confirmation c. Pipettor-90 µL and 500 µL (optionally 50 µL) and disposable tips (see App. N GR item 7 or CP item 6) d. Forceps, Fine Points, Stainless Steel e. Vernier, Dial or Digital Calipers, metal (readable to 0.1 mm) f. Stirring hot plate/stirring bar (optional) g. 100 mL Class A graduate cylinder h. 250 mL Erlenmeyer flasks i. 13 x 100 mm test tubes for beta-lactam confirmation j. Timer MATERIALS 5. See CP, items 24-32 a. Filter Paper Discs, Blank, Unimpregnated, Non-sterile Brand: Lot \#: 1. High absorbability, diameter 12.7±0 .1 mm b. Charm PM Indicator Agar 1. Do Not Autoclave- (see plate preparation, item 19 below) c. Charm Spore Tablets 1. Bacillus stearothermophilus (Geobacillus stearothermophilus) tablets containing 100,000,000 (±10 million) spores per tablet Lot \#: Exp. Date: d. Plastic Petri dish (15 x 100 mm, bottom plate inner diameter 86.1-87.0mm) REAGENTS 6. Reagents a. Charm 5.0 ppb Penicillin G Standard Positive Control Lot \#: Exp. Date: 1. Store according to label directions 2. Rehydrate according to label instructions 3. Test for suitability each time prepared, add to one (1) disc, must produce zone 16-20 mm; maintain records Avg. Zone Size: 4. Use rehydrated standard within 48 hours if refrigerated Lab Prep. Date: 5. 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 according to manufacturer’s instructions) Lab Prep. Date: Lab Exp. Date: b. Negative Control 1. Charm Zero Control Standard ________ Lot \#: Exp. Date: a. Reconstitute according to label instructions b. Use rehydrated negative control within 72 hours if refrigerated Lab Prep. Date: Lab Exp. Date: c. 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 according to manufacturer’s instructions) Lab Prep. Date: Lab Exp. Date: 2. Inhibitor Free Raw Milk Sample ID: Date Tested: a. Use within 72 hours if refrigerated Lab Prep. Date: Lab Exp. Date: b. 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 24 hours Lab Prep. Date: Lab Exp. Date: 3. Test for suitability, add to one (1) disc, produces no zone; maintain records Zone Size: c. Charm Beta-lactamase tablet or liquid concentrate (not required if beta-lactamase is not used for confirmation) 1. Stored at-15°C or below 2. Do not use beyond expiration date Lot \#: Exp. Date: 3. Reconstitute freeze dried concentrate as per manufacturer instructions a. Liquid concentrate stored at-15°C or below in a non frost-free freezer or in an insulated foam container in a frost-free freezer; use within 2 weeks

FAQs

What assay method is used to detect inhibitory substances in milk?

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.

What are the required temperature conditions during plate preparation?

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.

How are positive and negative controls used in the procedure?

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.

Sep 27, 2026

Uploaded by aboduw