GB 4789.10-2016 PDF EnglishUS$85.00 · In stock · Download in 9 seconds
GB 4789.10-2016: National food safety standard - Food microbiological examination - Staphylococcus aureus Delivery: 9 seconds. True-PDF full-copy in English & invoice will be downloaded + auto-delivered via email. See step-by-step procedure Status: Valid GB 4789.10: Historical versions
Similar standardsGB 4789.10-2016: National food safety standard - Food microbiological examination - Staphylococcus aureus---This is an excerpt. Full copy of true-PDF in English version (including equations, symbols, images, flow-chart, tables, and figures etc.), auto-downloaded/delivered in 9 seconds, can be purchased online: https://www.ChineseStandard.net/PDF.aspx/GB4789.10-2016 GB NATIONAL STANDARD OF THE PEOPLE’S REPUBLIC OF CHINA National food safety standard - Food microbiological examination - Staphylococcus aureus Issued on. DECEMBER 23, 2016 Implemented on. JUNE 23, 2017 Issued by. National Health and Family Planning Commission of the People’s Republic of China; China Food and Drug Administration. Table of ContentsForeword... 3 1 Scope... 4 2 Equipment and materials... 4 3 Medium and reagents... 5 4 Examination procedure... 5 5 Operating procedure... 6 6 Result and report... 8 7 Examination procedure... 8 8 Operating procedure... 8 9 Result calculation... 10 10 Report... 11 11 Examination procedure... 12 12 Operating procedure... 12 13 Result and report... 13 Annex A Medium and reagents... 14 Annex B Staphylococcal enterotoxin detection... 19 Annex C Retrieval table for most probable number (MPN) of staphylococcus aureus... 24ForewordThis Standard replaces GB 4789.10-2010 “National food safety standard - Food microbiological examination. Staphylococcus aureus”, SN/T 0172-2010 “Determination of Staphylococcus aureus in foods for import and export”, SN/T 2154- 2008 “Determination of coagulase-positive staphylococci in import and export food - Technique using rabbit plasma fibrinogen agar medium”. Compared with GB 4789.10-2010, the main changes of this standard are as follows. - The enrichment fluid for test is modified to 7.5 % sodium chloride broth. National food safety standard - Food microbiological examination – Staphylococcus aureus1 ScopeThis Standard specifies the examination method for Staphylococcus aureus in foods. Method I of this Standard applies to the qualitative examination of Staphylococcus aureus in foods; Method II applies to the counting of Staphylococcus aureus in foods with high Staphylococcus aureus content; Method III applies to the counting of Staphylococcus aureus in foods with low Staphylococcus aureus content.2 Equipment and materialsIn addition to routine sterilization and culture equipment for microbiological laboratories, other equipment and materials are as follows. 2.1 Constant temperature incubator. 36 °C ± 1 °C. 2.2 Refrigerator. 2 °C ~ 5 °C. 2.3 Constant temperature water bath. 36 °C ~ 56 °C. 2.4 Balance. with division of 0.1 g. 2.5 Homogenizer. 2.6 Oscillator. 2.7 Sterile pipette. 1 mL (with 0.01 mL scale), 10 mL (with 0.1 mL scale) or micro pipette and tip. 2.8 Sterile Erlenmeyer flask. with capacity of 100 mL and 500 mL. 2.9 Sterile Petri dish. with diameter of 90 mm. 2.10 Spreader. 2.11 pH meter or pH colorimetric tube or precision pH test paper.3 Medium and reagents3.1 7.5 % sodium chloride broth. see A.1. 3.2 Blood agar plate. see A.2. 3.3 Baird-Parker agar plate. see A.3. 3.4 Brain and heart infusion broth (BHI). see A.4. 3.5 Rabbit plasma. see A.5. 3.6 Diluent. phosphate buffer. see A.6. 3.7 Nutrient agar slant. see A.7. 3.8 Gram’s stain. see A.8. 3.9 Sterile saline. see A.9. Method I Qualitative examination of Staphylococcus aureus4 Examination procedureThe qualitative examination procedure of Staphylococcus aureus is shown in Figure 1.5 Operating procedure5.1 Sample treatment WEIGH 25 g of sample to a sterile homogeneous cup containing 225 mL of 7.5 % sodium chloride broth, HOMOGENIZE for 1 min to 2 min at 8000 r/min to 10000 r/min; or PLACE in a sterile homogeneous bag containing 225 mL of 7.5 % sodium chlorine broth, BEAT with a bag mixer for 1 min ~ 2 min. If the sample is liquid, pipette 25 mL of sample to a sterile Erlenmeyer flask (it may preset an appropriate number of sterile glass beads in the flask) containing 225 mL of 7.5 % sodium chloride broth. 5.2 Enrichment PLACE the homogenized sample above at 36 °C ± 1 °C to culture for 18 h ~ 24 h. Staphylococcus aureus is growing turbidly in 7.5 % sodium chloride broth. 5.3 Separation The enriched culture is inoculated into Baird-Parker plate and blood plate. The blood plate shall be cultured at 36 °C ± 1 °C for 18 h ~ 24 h. The Baird-Parker plate shall be cultured at 36 °C ± 1 °C for 24 h ~ 48 h. 5.4 Preliminary identification Staphylococcus aureus on the Baird-Parker plate is round, smooth-surface, raised, moist, with colony diameter of 2 mm ~ 3 mm, color of gray black to black color, shiny, and usually light (non-white) edge, around the opaque circle (precipitation) and a clear band outside. When the inoculating needle touches the colony, there is a butter-like sticky feeling. 5.5 Confirmation identification 5.5.1 Stain microscopic examination. Staphylococcus aureus is Gram’s positive cocci, arranged in grape-like, no spores, no capsule, with the diameter of about 0.5 μm ~ 1 μm. 5.5.2 Plasma coagulase test. PICK at least 5 suspicious colonies (select all when there are less than 5) on Baird-Parker plate or blood plate; INOCULATE them to 5 mL of BHI and nutrient agar slants respectively; CULTURE at 36 °C ± 1 °C for 18 h ~ 24 h. 5.6 Staphylococcal enterotoxin examination (optional) For identification of suspicious food poisoning samples or Staphylococcus aureus strains producing staphylococcal enterotoxin, Staphylococcal enterotoxin shall be tested according to Annex B.6 Result and report6.1 Result determination. when complying with 5.4, 5.5, it can be determined as Staphylococcus aureus. 6.2 Result report. Staphylococcus aureus is detected or not detected in 25 g (mL) of sample.7 Examination procedureThe examination procedure of Staphylococcus aureus plate counting method is shown8 Operating procedure8.1 Sample dilution 8.1.1 Solid and semi-solid sample. WEIGH 25 g of sample in a sterile homogeneous cup containing 225 mL of phosphate buffer or saline, HOMOGENIZE at 8000 r/min ~ 10000 r/min for 1 min ~ 2 min; or PLACE in a sterile homogeneous bag containing 225 mL of dilution, BEAT with a bag mixer for 1 min ~ 2 min to make into 1.10 sample homogenate. 8.1.2 Liquid sample. PIPETTE 25 mL of sample with a sterile pipette in a sterile Erlenmeyer flask containing 225 mL of phosphate buffer or saline (an appropriate number of sterile glass beads shall be preset in the flask), MIX well, to make into 1.10 sample homogenate. 8.1.3 PIPETTE 1 mL of 1.10 sample homogenate with a 1 mL sterile pipette or micro pipette and slowly INJECT it along the tube wall to a sterile test tube containing 9 mL of phosphate buffer or saline (note that the pipette or tip shall not touch the surface of the diluent), SHAKE the test tube or USE a 1mL sterile straw to repeatedly blow to make it evenly mixed, to make into 1.100 sample homogenate. 8.2 Sample inoculation According to the estimation of the contamination status of the sample, select sample homogenates of 2 ~ 3 appropriate dilutions (for liquid sample, it can include the original solution). 8.3 Culture Under normal circumstances, after spreading, the plate stands for 10 min; if the sample homogenate is not easy to absorb, the plate can be placed in an incubator at 36 °C ± 1 °C to culture for 1 h. After the sample homogenate is absorbed, inverse the plate, and culture at 36°C ± 1 °C for 24 h ~ 48 h. 8.4 Counting and confirmation of typical colony 8.4.1 Staphylococcus aureus on the Baird-Parker plate is round, smooth-surface, raised, moist, with colony diameter of 2 mm ~ 3 mm, color of gray black to black color, shiny, and usually light (non-white) edge, around the opaque circle (precipitation) and a clear band outside. When the inoculating needle touches the colony, there is a butter- like sticky feeling. 8.4.2 SELECT the plate with typical Staphylococcus aureus colonies, and the total number of colonies on all three plates of the same dilution is between 20 CFU ~ 200 CFU, to count the number of typical colonies. 8.4.3 SELECT at least 5 suspected colonies (select all when there are less than 5) from typical colonies for identification test.9 Result calculation9.1 If the number of typical colonies on only one dilution plate is between 20 CFF and 200 CFU, the typical colonies on the dilution plate shall be counted and calculated as equation (1). 9.2 If the number of typical colonies on the lowest dilution plate is less than 20 CFU, the typical colonies on the dilution plate shall be counted and calculated as equation (1). 9.3 If the number of typical colonies on a certain dilution plate is greater than 200 CFU, and there is not typical colony on the next dilution plate, the typical colonies on the dilution plate shall be counted and calculated as equation (1). 9.4 If the number of typical colonies on a certain dilution plate is greater than 200 CFU, and the number of typical colonies on the next dilution plate is within the range of 20 CFU ~ 200 CFU, the typical colonies on the dilution plate shall be counted and calculated as equation (1). 9.5 If the number of typical colonies of 2 consecutive dilution plates is between 20 CFU ~ 200 CFU, calculated as equation (2). 9.6 Calculation equation10 ReportAccording to the calculation results of the equations in Clause 9, report the number of Staphylococcus aureus in per g (mL) of sample, expressed as CFU/g (mL); if the T value is 0, report the lowest dilution factor multiplies by less than 1. Method III Staphylococcus aureus MPN counting11 Examination procedureThe examination procedure of Staphylococcus aureus MPN counting is shown in Figure 3.12 Operating procedure12.1 Sample dilution CARRY out according to 8.1. 12.2 Inoculation and culture 12.2.1 According to the estimation of the contamination status of the sample, select sample homogenates of 3 appropriate dilutions (for liquid sample, 12.2.2 USE inoculating loop to take a loop of inoculum from the cultured 7.5 % sodium chloride broth, and transplant it on Baird-Parker plate and culture at 36 °C ± 1 °C for 24 h ~ 48 h. 12.3 Confirmation of typical colony CARRY out according to 8.4.1, 8.4.3.13 Result and reportAccording to the number of test tubes with Staphylococcus aureus confirmed to be positive, refer to the MPN retrieve table (see Annex C) and report the most probable number of Staphylococcus aureus in per g (mL) of sample, expressed in MPN/g (mL). ......Source: Above contents are excerpted from the full-copy PDF -- translated/reviewed by: www.ChineseStandard.net / Wayne Zheng et al. 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