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GB 4789.30-2025: National food safety standard - Food microbiological examination - Examination of Listeria monocytogenes 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 4789.30: Historical versions
Similar standardsGB 4789.30-2025: National food safety standard - Food microbiological examination - Examination of Listeria monocytogenes---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/GB4789.30-2025 GB NATIONAL STANDARD OF THE PEOPLE’S REPUBLIC OF CHINA National food safety standard - Food microbiology test Listeria monocytogenes test Issued on. MARCH 16, 2025 Implemented on. SEPTEMBER 16, 2025 Issued by. National Health Commission of the People’s Republic of China; State Administration for Market Regulation. Table of ContentsForeword... 3 1 Scope... 4 2 Equipment and materials... 4 3 Culture medium and reagents... 5 First method -- Qualitative test of Listeria monocytogenes... 6 4 Test procedure... 6 5 Operation steps... 7 6 Results and report... 10 Second method -- Plate count method for Listeria monocytogenes... 10 7 Test procedure... 10 8 Operation steps... 10 9 Results and report... 12 Third method -- MPN count method for Listeria monocytogenes... 14 10 Test procedure... 14 11 Operation steps... 15 12 Results and report... 15 Annex A Culture medium and reagents... 16 Annex B Table of most probable number (MPN) of Listeria monocytogenes... 27ForewordThis Standard replaces GB 4789.30-2016 “National food safety standard - Food microbiological test - Listeria monocytogenes test”. Compared with GB 4789.30-2016, this Standard has the following major changes. - MODIFY the scope of application; - MODIFY the culture medium and reagents, and ADD the formula of agar Listeria according to Ottaviani and Agosti; - MODIFY the enrichment solution, selective culture medium, test procedure, identification method, etc. of the first method -- Qualitative test of Listeria monocytogenes; - MODIFY the sample inoculation, colony count and confirmation, result count, and result report of the second method -- Plate count method for Listeria monocytogenes; - MODIFY the sample inoculation of the third method -- MPN count method for Listeria monocytogenes. National food safety standards Food microbiology test Listeria monocytogenes test1 ScopeThis Standard specifies the test methods for Listeria monocytogenes in food. The first method of this Standard applies to the qualitative test of Listeria monocytogenes in food; the second method applies to the counting of Listeria monocytogenes in food with a high content of Listeria monocytogenes; the third method applies to the counting of Listeria monocytogenes in food with a low content of Listeria monocytogenes.2 Equipment and materialsIn addition to routine sterilization and culture equipment of microbiology laboratories, other equipment and materials are as follows. 2.1 Refrigerator. 2 ℃ ~ 8 ℃. 2.2 Constant temperature incubator. 30 ℃ ± 1 ℃, 36 ℃ ± 1 ℃, 25 ℃ ~ 30 ℃. 2.3 Homogenizer. 2.4 Microscope. 100× ~ 1000×. 2.5 Electronic balance. the sensitivity is 0.1 g, 0.1 mg. 2.6 Conical flask. 100 mL, 500 mL. 2.7 Sterile pipette. 1 mL (with 0.01 mL scale), 10 mL (with 0.1 mL scale) or pipette (with a scale of 0.1 mL, 1 mL, 10 mL) and sterile pipette tip. 2.8 Sterile culture dish. diameter of 90 mm. 2.9 Sterile test tube. 16 mm × 160 mm. 2.10 Centrifuge. 4000 r/min. 2.11 Sterile centrifuge tube. 30 mm × 100 mm. 2.12 Sterile syringe. 1 mL. 2.13 Oil immersion lens or phase contrast microscope. 2.14 Sterile coating stick. 2.15 Listeria monocytogenes ATCC 19111 or CMCC 54004 or other equivalent strains. 2.16 Listeria innocua ATCC 33090 or other equivalent strains. 2.17 Listeria ivanovii ATCC 19119 or other equivalent strains. 2.18 Listeria seeligeri ATCC 35967 or other equivalent strains. 2.19 Staphylococcus aureus ATCC 25923 or other equivalent strains, requiring the production of β-hemolytic ring. 2.20 Rhodococcus equi ATCC 6939 or NCTC 1621 or other equivalent strains.3 Culture medium and reagents3.1 Tryptic soy broth with 0.6 % yeast extract powder (TSB-YE). see A.1 in Annex A. 3.2 Tryptic soy agar with 0.6 % yeast extract powder (TSA-YE). see A.2. 3.3 Fraser enrichment broth (FB1, FB2). see A.3. 3.4 Agar Listeria according to Ottaviani and Agosti. see A.4. 3.5 PALCAM medium. see A.5. 3.6 Gram staining solution. see A.6. 3.7 SIM motility medium. see A.7. 3.8 Buffered glucose peptone water [for methyl red (MR) and acetyl methyl alcohol (VP) tests]. see A.8. 3.9 Sheep blood agar. see A.9. 3.10 Sterile phosphate buffer. see A.10. 3.11 Sterile normal saline. see A.11. 3.12 Sugar fermentation tube. see A.12.4 Test procedureThe qualitative test procedure for Listeria monocytogenes is shown in Figure 1.5 Operation steps5.1 Enrichment Take 25 g (mL) of sample by aseptic operation, place it in a sterile homogenizing cup containing 225 mL of FB1 enrichment broth, and homogenize at 8000 r/min ~ 10000 r/min for 1 min ~ 2 min, or place it in a sterile homogenizing bag containing 225 mL of FB1 enrichment broth, and beat it with a slapping homogenizer for 1 min ~ 2 min to make a 1.10 sample homogenate. If the sample is in liquid form, it can also be mixed by oscillation or stirring. Culture at 30 ℃ ± 1 ℃ for 24 h ± 2 h. Mix well, pipette 0.1 mL of FB1 enrichment broth, and inoculate in 10 mL of FB2 enrichment broth. Culture at 30 ℃ ± 1 ℃ for 24 h ± 2 h. 5.2 Separation Take the mixed FB2 enrichment broth and inoculate on agar Listeria according to Ottaviani and Agosti (or other equivalent Listeria chromogenic medium) plates and PALCAM medium plates respectively, culture at 36 ℃ ± 1 ℃ for 24 h ~ 48 h, and observe the colonies grown on each plate. Typical colonies form round blue-green colonies with a diameter of 1 mm ~ 3 mm on agar Listeria according to Ottaviani and Agosti plates, surrounded by opaque halos. Typical colonies form round gray-green colonies with a diameter of 1 mm ~ 3 mm on PALCAM medium plates, surrounded by brown-black hydrolysis circles. After 48 h of culture, some colonies form black spots and depressions in the center. The characteristics of colonies on other equivalent Listeria chromogenic medium plates shall be determined according to the product instructions. NOTE 1.Some Listeria monocytogenes colonies on agar Listeria according to Ottaviani and Agosti have weak or no halos around them. There are also some Listeria monocytogenes colonies on agar Listeria according to Ottaviani and Agosti have halos that appear slowly, sometimes taking more than 4 days to appear. NOTE 2.The colony morphology of Listeria ivanovii on agar Listeria according to Ottaviani and Agosti is similar to that of Listeria monocytogenes. 5.3 Pure culture Pick 3 to 5 typical or suspicious colonies (select all if less than 3) from each plate (plate that meet the requirements of 5.2), streak on TSA-YE plates or sheep blood plates, and culture at 36 ℃ ± 1 ℃ for 18 h ~ 24 h. Listeria monocytogenes on TSA-YE plates or sheep blood plates are grayish white, translucent, with neat edges, dew drop-shaped colonies, and a diameter of 1 mm ~ 2 mm. 5.4 Preliminary identification Pick an individual colony on TSA-YE plates or sheep blood plates, inoculate in xylose and rhamnose fermentation tubes, and culture at 36 ℃ ± 1 ℃ for 24 h ± 2 h; at the same time, streak on TSA-YE plates or sheep blood plates, and culture at 36 ℃ ± 1 ℃ for 18 h ~ 24 h to obtain pure culture for the next step of identification. Then select the pure culture that is xylose negative and rhamnose positive to continue identification. 5.5 Identification NOTE. It can first pick one strain from each plate (plate that meets the requirements of 5.2) for identification test. If it is identified as Listeria monocytogenes, the result of “detected” can be directly reported according to the provisions of “6 Results and reports”; the result of “not detected” can be reported only after 3 to 5 typical or suspicious colonies (select all if less than 3) selected according to the requirements of 5.3 are identified as non- Listeria monocytogenes. 5.5.1 Microscopic examination. Pick an individual colony of pure culture for 18 h ~ 24 h for Gram staining microscopic examination. Listeria is a Gram-positive brevibacterium with a size of (0.4 μm ~ 0.5 μm) × (0.5 μm ~ 2.0 μm). Use normal saline to make a bacterial suspension, observe under an oil immersion lens or phase contrast microscope, the bacteria show slight rotation or tumbling motion. 5.5.4 Hemolysis test. Divide the bottom of a fresh sheep blood agar plate into 20 ~ 25 small grids, pick an individual colony of pure culture for 18 h ~ 24 h and inoculate on the blood plate, inoculate one colony per grid, and inoculate positive control bacteria (Listeria monocytogenes, Listeria ivanovii and Listeria seeligeri) and negative control bacteria (Listeria innocua). When puncturing, try to get as close to the bottom as possible, but do not touch the bottom surface, and avoid agar rupture. Culture at 36 ℃ ± 1 ℃ for 24 h ~ 48 h, and observe in a bright place. 5.5.5 Cooperative hemolysis test CAMP (optional). Streak Staphylococcus aureus and Rhodococcus equi in parallel lines on sheep blood agar plates. Pick an individual colony of pure culture for 18 h ~ 24 h and streak vertically between the parallel lines, the two ends of the vertical line shall not touch the parallel lines and the distance is 1 mm ~ 2 mm. At the same time, inoculate Listeria monocytogenes, Listeria innocua, Listeria ivanovii and Listeria seeligeri and culture at 36 ℃ ± 1 ℃ for 24 h ~ 48 h. 5.6 Mouse toxicity test (optional item) Inoculate the pure culture that meets the above characteristics into TSB-YE, culture at 36 ℃ ± 1 ℃ for 24 h, centrifuge at 4000 r/min for 5 min, discard the supernatant, and use sterile normal saline to prepare a bacterial suspension with a concentration of 1010 CFU/mL. Take this bacterial suspension and inject it intraperitoneally into 3 ~ 5 mice, 0.5 mL per mouse, and observe the death of the mice at the same time. If the mice die within 2 d ~ 5 d, it is a pathogenic strain. In the test, set up a pathogenic strain of Listeria monocytogenes and a sterile normal saline control group. Listeria monocytogenes and Listeria ivanovii are pathogenic to mice.6 Results and reportCombining the identification results of 5.4 and 5.5, report whether Listeria monocytogenes is detected or not detected in 25 g (mL) of sample. If Listeria monocytogenes is not detected in 25 g (mL) of sample, it can also be reported as 0/25 g (mL).7 Test procedureThe plate count procedure for Listeria monocytogenes is shown in Figure 2.8 Operation steps8.1 Sample dilution 8.2 Sample inoculation 8.2.1 Based on the estimation of the sample contamination status, select 2 to 3 sample homogenates with appropriate serial dilutions (liquid samples may include stock solution), pipette 0.1 mL of sample homogenate for each dilution, and inoculate on 1 agar Listeria according to Ottaviani and Agosti (or other equivalent Listeria chromogenic medium) plate. Use a sterile coating stick to coat the entire plate, being careful not to touch the edge of the plate. Before use, if there are water droplets on the surface of the agar plate, place it in an incubator at 25 ℃ ~ 50 ℃ to dry until the water droplets on the surface of the plate disappear. 8.2.2 For food samples with low content of Listeria monocytogenes, pipette 1 mL of the sample homogenate with lowest dilution and coat it on 3 agar Listeria according to Ottaviani and Agosti (or other equivalent Listeria chromogenic medium) plates with inoculation volumes of 0.3 mL, 0.3 mL, and 0.4 mL respectively. The coating method is the same as 8.2.1. 8.3 Culture 8.3.1 Under normal circumstances, after coating, place the plate upright on a horizontal table for 10 min ~ 20 min, then turn the plate over and place it in an incubator to culture at 36 ℃ ± 1 ℃ for 24 h ~ 48 h. 8.4 Count and confirmation of typical colony 8.4.1 Select plates with typical or suspicious Listeria monocytogenes colonies. If.9 Results and report9.1 Count method 9.1.1 Formula (1) 9.2 Result report 9.2.1 When the number of colonies is less than 100 CFU, it shall be rounded off according to the principle of “rounding off” and reported as an integer. 9.2.2 When the number of colonies is greater than or equal to 100 CFU, the third digit shall be rounded off according to the principle of “rounding off”, and the first two digits shall be taken, and the digits behind shall be replaced by 0.It can also be expressed in the form of 10 exponentials, and two significant digits shall be retained after rounding off according to the principle of “rounding off”. 9.2.3 Report the number of colonies of Listeria monocytogenes per g (mL) of sample, expressed as CFU/g (mL). If the T value is 0, the method using the 0.1 mL inoculum volume shall be reported as less than 10 multiplied by the lowest dilution factor. The method using the 0.3 mL, 0.3 mL, 0.4 mL inoculum volumes shall be reported as less than 1 multiplied by the lowest dilution factor.10 Test procedureThe test procedure for MPN count method for Listeria monocytogenes is shown in11 Operation steps11.1 Sample dilution Phosphate buffer is used as sample diluent. The sample dilution method is the same as 8.1. 11.2 Inoculation and culture 11.2.1 Based on the estimation of sample contamination status, select 3 sample homogenates (liquid samples may include stock solution) with appropriate serial dilutions and inoculate them in 10 mL of FB1 enrichment broth. Inoculate 3 tubes for each dilution, and inoculate 1 mL in each tube. If the inoculation volume is 10 mL, inoculate it in 10 mL of double-mix FB1 enrichment broth. Culture at 30 ℃ ± 1 ℃ for 24 h ± 2 h. Pipette 0.1 mL from each tube, inoculate to 10 mL of FB2 enrichment broth, and culture at 30 ℃ ± 1 ℃ for 24 h ± 2 h. 11.2.2 Use inoculation loops to transfer 1 loop from each tube of FB2 enrichment broth, inoculate on agar Listeria according to Ottaviani and Agosti (or other equivalent Listeria chromogenic medium) plates, and culture at 36 ℃ ± 1 ℃ for 24 h ~ 48 h. 11.3 Confirmation test Pick 3 ~ 5 typical or suspicious colonies (select all if less than 3) from each plate (plate that meets the requirements of 5.2) and identify them according to 5.3, 5.4, and 5.5.12 Results and reportBased on the number of test tubes confirmed to be positive for Listeria monocytogenes, lookup the MPN retrieval table (see Annex B) and report the most probable number of Listeria monocytogenes per gram (ml) of sample, expressed as 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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