GB Translated English of Chinese Standard: GB NATIONAL STANDARD OF THE

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1 Translated English of Chinese Standard: GB Buy True-PDF Auto-delivery. GB NATIONAL STANDARD OF THE PEOPLE S REPUBLIC OF CHINA GB National food safety standard - Food microbiological examination - Examination of Listeria monocytogenes 食品安全国家标准食品微生物学检验 - 单核细胞增生李斯特氏菌检验 Issued on: December 23, 2016 Implemented on: June 23, 2017 Issued by: National Health and Family Planning Commission; China Food and Drug Administration. Buy True-PDF Auto-delivery. Page 1 of 23

2 Table of Contents Foreword Scope Equipment and materials Media and reagents Testing procedures Operation procedures Test procedures Operation procedures Result count Results report Testing procedures Operation procedures Results and reports Appendix A Medium and reagents Appendix B Listeria monocytogenes most probable number (MPN) search table Buy True-PDF Auto-delivery. Page 2 of 23

3 National food safety standard - Food microbiological examination - Examination of Listeria monocytogenes 1 Scope This standard specifies the test method for Listeria monocytogenes in food. The first method of this standard is applicable to the qualitative test of Listeria monocytogenes in food; the second method is applicable to the counting of Listeria monocytogenes in foods with higher Listeria monocytogenes; the third method is applicable to the count of Listeria monocytogenes in foods with low Listeria monocytogenes (< 100 CFU/g) and high bacterial content, especially milk, water and food containing particulate matter which interferes with the counting of colonies. 2 Equipment and materials In addition to routine sterilization and culture equipment in microbiology laboratories, other equipment and materials are as follows: 2.1 Refrigerator: 2 C ~ 5 C. 2.2 Constant temperature incubator: 30 C ± 1 C, 36 C ± 1 C. 2.3 Homogenizer. 2.4 Microscope: 10 x ~ 100 x. 2.5 Electronic balance: the sensitivity is 0.1 g. 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 micropipette and tip. 2.8 Sterile plate: 90 mm in diameter. 2.9 Sterile test tube: 16 mm 160 mm Centrifuge tube: 30 mm 100 mm Sterile syringe: 1 ml. Buy True-PDF Auto-delivery. Page 4 of 23

4 HOMOGENIZE it at 8000 r/min ~ r/min for 1 min ~ 2 min. CULTURE it at 30 C ± 1 C for 24 h ± 2 h, TAKE 0.1 ml, TRANSFER it to 10 ml of LB2 monocytogenes solution, CULTURE it at 30 C ± 1 C for 24 h ± 2 h. 5.2 Separation TAKE the LB2 secondary monocytogenes solution, INOCULATE it onto the Listeria chromogenic plate and the PALCAM agar plate in a line shape, CULTURE it at 36 C ± 1 C for 24 h ~ 48 h, OBSERVE the colonies growing on each plate. Typical colonies are small round gray-green colonies on PALCAM agar plates with brown-black hydrolyzed circles around them, some colonies have black depressions; colony characteristics on Listeria colordeveloping plates are determined by reference to product specifications. 5.3 Initial screening TAKE to five typical or suspect colonies from selective agar plates, respective INOCULATE them into the xylose and rhamnose fermentation tubes, CULTURE it at 36 C ± 1 C for 24 h ± 2 h, meanwhile MARK a line on the TSA-YE plate, CULTURE it at 36 C ± 1 C for 18 h ~ 24 h, then SELECT the pure culture of xylose-negative and rhamnose-positive for further identification. 5.4 Identification (or selection of biochemical identification kits or fully automated microbial identification systems, etc.) Staining microscopy: Listeria monocytogenes is a Gram-positive Brevibacterium, the size is (0.4 μm ~ 0.5 μm) (0.5 μm ~ 2.0 μm); the bacterial suspension is made with physiological saline, which is observed in oil mirror or under the phase contrast microscope, the bacteria shows slight rotation or rolling-like motion Dynamic test: PICK a single suspicious colony to puncture semi-solid or SIM dynamic medium in pure culture, CULTURE it at 25 C ~ 30 C for 48 h. Listeria is motivated and has an umbrella shape growth above semi-solid or SIM medium. If the umbrella growth is not obvious, it can continue to culture it for 5 d, then observe the results Biochemical identification: PICK the single suspicious colony in pure culture to perform catalase test. Colonies with positive catalase reaction is continued to be subject to sugar fermentation test and MR-VP test. The main biochemical characteristics of Listeria monocytogenes are as shown in Table Hemolysis test: DIVIDE the bottom surface of fresh sheep blood agar plate into 20 ~ 25 small cells, PICK a single suspicious colony of pure culture, INOCULATE it onto the blood plate by puncture, one colony per cell, and INOCULATE the positive control bacteria by puncture (Listeria monocytogenes, Listeria ivanovii and Listeria seeligeri) and negative control bacteria (Listeria Buy True-PDF Auto-delivery. Page 7 of 23

5 without additives, HOMOGENIZE it continuously for 1 min ~ 2 min on a tapping homogenizer, or HOMOGENIZE it for 1 min ~ 2 min at 8000 r/min ~ r/min. SHAKE and MIX the liquid sample uniformly, to make it into 1:10 sample homogenate USE a 1 ml sterile pipette or micropipette to pipette 1 ml of 1:10 sample homogenate, along the tube wall, INJECT it slowly into a sterile test tube which contains 9 ml of buffered peptone or LB broth without additives (note avoiding the pipette or tip end from touching the dilution solution surface), SHAKE the test tube or USE another 1 ml sterile pipette to repeatedly mix it uniformly, to prepare it into 1:100 sample homogenate PREPARE 10 times serial dilution sample homogenate in accordance with the procedure of For each incremental dilution, replace one 1 ml sterile pipette or tip. 7.2 Inoculation of samples In accordance with the estimation of the contamination status of the sample, SELECT 2 ~ 3 sample homogenate of appropriate continuous dilution (the liquid sample may include the original solution), respectively TAKE 1 ml of each dilution of the sample homogenate, at the inoculation amount of 0.3 ml, 0.3 ml, 0.4 ml, respectively ADD it onto three Listeria chromogenic plates, USE the sterile L bar to cover it over the entire plate, PAY attention to avoiding touching the plate edge. Before use, if there is water drops on the surface of the agar plate, it can be dried in an incubator at 25 C ~ 50 C until the water drops on the surface of the plate disappear. 7.3 Culture Under normal circumstances, after coating, ALLOW the plate to stand for 10 min. If the sample solution is not easily absorbed, it may place the plate in an incubator at 36 C ± 1 C for 1 h; after the sample homogenate is absorbed, INVERT the plate, PLACE it into the incubator upside down, to culture it at 36 C ± 1 C for 24 h ~ 48 h. 7.4 Typical colony count and confirmation Colony characteristics of Listeria monocytogenes on Listeria chromogenic plates are subject to product description SELECT a plate which has typical Listeria monocytogenes colonies, and the total number of colonies in the same dilution of 3 plates between 15 CFU and 150 CFU, COUNT the number of typical colonies. In case: a) The plate colony count of only one dilution is between 15 CFU ~ 150 CFU and there is typical colony, it shall count the typical colonies on the plate Buy True-PDF Auto-delivery. Page 10 of 23

6 Appendix A Medium and reagents A.1 Tryptic soy broth (TSB-YE) containing 0.6% yeast extract A.1.1 Ingredients Tryptone 17.0 g Polyad tryptone 3.0 g Yeast extract 6.0 g Sodium chloride 5.0 g Dipotassium hydrogen phosphate 2.5 g Glucose 2.5 g Distilled water 1000 ml A.1.2 Preparation method HEAT and STIR the above components to dissolve it, ADJUST the ph to 7.2 ± 0.2, CONTAIN it separately, AUTOCLAVE it at 121 C for 15 min, PREPARE for use. A.2 Tryptic soy agar (TSA-YE) containing 0.6% yeast extract A.2.1 Ingredients Tryptone 17.0 g Polyad tryptone 3.0 g Yeast extract 6.0 g Sodium chloride 5.0 g Dipotassium hydrogen phosphate 2.5 g Glucose 2.5 g Agar 15.0 g Distilled water 1000 ml Buy True-PDF Auto-delivery. Page 15 of 23

7 Glucose 0.5 g Esculin 0.8 g Ammonium ferric citrate 0.5 g Mannitol 10.0 g Phenol red 0.1 g Lithium chloride 15.0 g Casein trypsin digest 10.0 g Heart trypsin digest 3.0 g Corn Starch 1.0 g Meat and stomach enzyme digest 5.0 g Sodium chloride 5.0 g Agar 15.0 g Distilled water 1000 ml A.4.2 Preparation method HEAT and STIR the above components to dissolve it, ADJUST the ph to 7.2 ± 0.2, CONTAIN it separately, AUTOCLAVE it at 121 C for 15 min, PREPARE for use. A PALCAM selective additives Polymyxin B 5.0 mg Acriflavine HCl 2.5 mg Ceftazidime 10.0 mg Sterile distilled water 500 ml A Preparation method DISSOLVE the PALCAM base medium, COOL it to 50 C, ADD 2 ml of PALCAM selective additive, MIX it uniformly, POUR it into a sterile plate, PREPARE for use. A.5 Gram staining solution Buy True-PDF Auto-delivery. Page 17 of 23

8 After dissolving it, ADJUST the ph to 7.0 ± 0.2, CONTAIN it in different test tubes, 1 ml per tube, AUTOCLAVE it at 121 C for 15 min, PREPARE for use. A.7.3 Methyl red (MR) test A Methyl red reagent A Ingredients Methyl red 10 mg 95% ethanol 30 ml Distilled water 20 ml A Preparation method DISSOLVE 10 mg of methyl red in 30 ml of 95% ethanol, then ADD 20 ml of distilled water. A Test method TAKE an appropriate amount of agar culture, INOCULATE it into buffered glucose protein water, CULTURE it at 36 C ± 1 C for 2 d ~ 5 d. ADD one drop of methyl red reagent, immediately OBSERVE it. Bright red is positive and yellow is negative. A.7.4 V-P test A % α-naphthol-ethanol solution Ingredients and preparation method: Take 6.0 g of α-naphthol, ADD absolute ethanol to dissolve it, MAKE its volume reach to 100 ml. A % potassium hydroxide solution Ingredients and preparation method: TAKE 40 g of potassium hydroxide, ADD distilled water to dissolve it, MAKE its volume reach to 100 ml. A Test method TAKE an appropriate amount of agar culture, INOCULATE it into buffered glucose protein water, CULTURE it at 36 C ± 1 C for 2 d ~ 4 d. ADD 0.5 ml of 6% α-naphthol-ethanol solution and 0.2 ml of a 40% potassium hydroxide solution, SHAKE the test tube completely, OBSERVE the results. If red color appears immediately or after several minutes, it is positive reaction; if it is negative reaction, it shall be cultured continuously at 36 C ± 1 C for 1 h and observed. Buy True-PDF Auto-delivery. Page 20 of 23

9 meanwhile AUTOCLAVE it. ADD 5 ml of the sugar solution into 100 ml of the medium, aseptically DISPENSE it into a small tube containing the inverted tube. Or PREPARE other sugar fermentation tubes in accordance with the preparation method of A glucose fermentation tube. A.9.3 Test methods TAKE appropriate amount of pure culture, INOCULATE it into the sugar fermentation tube, CULTURE it at 36 C ± 1 C for 24 h ~ 48 h. OBSERVE the results, the blue color indicates negative and yellow color indicates positive. A.10 Catalase test A.10.1 Reagent 3% hydrogen peroxide solution: PREPARE it at the time of use. A.10.2 Test methods USE a thin glass rod or a disposable inoculation needle to pick a single colony, PLACE it into a clean glass plate, ADD 2 drops of 3% hydrogen peroxide solution, OBSERVE the result. A.10.3 Results Those which have bubbles in half a minute are positive, and those which do not have bubbles are negative. A.11 Buffered protein water (BPW) A.11.1 Ingredients Peptone 10.0 g Sodium chloride 5.0 g Disodium hydrogen phosphate (Na2HPO4 12H2O) 9.0 g Potassium dihydrogen phosphate 1.5 g Distilled water 1000 ml A.11.2 Preparation method HEAT and STIR to dissolve it, ADJUST the ph to 7.2 ± 0.2, AUTOCLAVE it at 121 C for 15 min. Buy True-PDF Auto-delivery. Page 22 of 23