BS ISO 10932:2010 pdf free download Milk and milk products — Determination of the minimal inhibitory concentration (MIC) of antibiotics applicable to bifidobacteria and non- enterococcal lactic acid bacteria (LAB)
1Scope
This International Standard specifies a method for determining the minimal inhibitory concentration (MIC) of aseries of antibiotics applicable to bifidobacteria and non-enterococcal lactic acid bacteria(LAB).
NOTE Unlike the disk diffusion method, which is semi-quantitative, the frequently used broth microdilution methodgives quantitative MICs of the test organism in a diltution series of the antibiotics. The lowest concentration of an antibioticthat prevents visible growth of a test organism is considered to be the MlC.
This International Standard recommends the broth microdilution method as the standard method.
2Normative references
The following referenced documents are indispensable for the application of this document.For datedreferences,only the edition cited applies. For undated references,the latest edition of the referenceddocument (including any amendments)applies.
ISO 6887-5,Microbiology of food and animal feeding stuffs — Preparation of test samples, initial suspensionand decimal dilutions for microbiological examination—Part 5: Specific rules for the preparation of milk andmilk products
ISO 7218,Microbiology of food and animal feeding stufts— General requirements and guidance formicrobiological examinations
ISO/TS 11133-1,Microbiology of food and animal feeding stuffs —Guidelines on preparation and productionof culture media— Part 1 : General guidelines on quality assurance for the preparation of culture media in thelaboratory
ISO/TS 11133-2,Microbiology of food and animal feeding stuffs — Guidelines on preparation and productionof culture media— Part 2: Practical guidelines on performance testing of culture media
3Terms and definitions
For the purposes of this document, the following terms and definitions apply.3.1
minimal inhibitory concentrationMIC
lowest concentration that, under defined in vitro conditions, prevents visible growth of bacteria within a definedperiod of time
[ISO 20776-1:2006[6],2.4]
NOTE MIlC is expressed in micrograms per millilitre.
4Principle
Most individual colonies from an agar plate are picked up and suspended in sterile saline.However,Bifidobacterium spp. are suspended in pre-reduced LSM-Cys medium.
The bacterial suspension is diluted with recommended medium.
The microdilution plate is prepared with a series of twofold dilutions of antibiotic.
The diluted bacterial suspension is distributed into the wells of the plate and incubated under recommendedconditions.
The lowest concentration of an antibiotic that prevents visible growth is considered to be the MIC.
5Diluents, culture media and reagents
5.1Basic materials
Use only reagents of recognized analytical grade,unless otherwise specified,and sterile ditiled ordemineralized water or water of equivalent purity.See lSO 6887-5.
5.2Diluents
See lSO 6887-5.
5.3.1.2 Preparation Suspend the ingredients in the water. Heat the suspension to boiling with frequent agitation until complete dissolution. If needed, adjust the pH (6.7) to 6,35 ± 0,2 with dilute hydrochloric acid or dilute sodium hydroxide before autoclaving. After autoclaving, the pH range of the MRS agar medium should be 6,2 ± 0,2 at 25 °C. Distribute the medium in portions of 100 ml ± 1 ml into bottles (6.8) of capacity 150 ml or in portions of 200 ml ± 2 ml into bottles (6.8) of capacity 250 ml. Sterilize in the autoclave (6.5) maintained at 121 °C for 15 min. If the medium is to be used immediately, cool it in a water bath (6.6) to between 44 °C and 47 °C. If not used immediately, melt the MRS agar (5.3.1.1) in a boiling water bath (6.6) and mix carefully to avoid gas bubbles, then cool it in a water bath (6.6) to between 44 °C and 47 °C.
Pour 15 ml to 20 ml of prepared medium into Petri dishes (6.10). Allow the medium to cool. Solidify by placing the Petri dishes with the lids in place on a cool horizontal surface. Before use, dry the agar surface in accordance with ISO/TS 11133-1. The prepared MRS agar plates may be stored in an airtight plastic bag in the dark and held between 2 °C and 8 °C for up to 2 weeks. Test agar plates for microbial contamination in accordance with ISO/TS 11133-2. The complete MRS agar is commercially available, but the results obtained may differ significantly from one supplier to another. If used, therefore, check the commercial MRS agar against the same medium prepared in accordance with this International Standard