This standard specifies the test method for Listeria monocytogenes. This standard is applicable to the test for Listeria monocytogenes in food and food poisoning samples. GB/T 4789.30-2003 Food hygiene microbiological test Test for Listeria monocytogenes GB/T4789.30-2003 Standard download decompression password: www.bzxz.net

ICS07.100.30
National Standard of the People's Republic of China
GB/T4789.30-—2003
Replaces GB/T4789.30—-1994
Microbiological examination of food hygieneExamination of Listeria monocytogenes
Microbiological examination of food hygieneExamination of Listeria monocytogenesPromulgated on August 11, 2003
Ministry of Health of the People's Republic of China
Standardization Administration of the People's Republic of China
Implementation on January 1, 2004
GB/T4789.30—2003
This standard revise GB/T4789.30--1994 "Microbiological examination of food hygieneExamination of Listeria monocytogenes".
Compared with GB/T4789.30-1994, the main modifications of this standard are as follows: - The format and text of the standard text are modified in accordance with GB/T1.1-2000. - Modify and standardize the "equipment and materials" in the original standard. Content of culture medium components: A.4.1 Sodium nitride 5g is changed to 20g. - 6.8 The pathogenicity test for mice is modified. From the date of implementation of this standard, GB/T4789.30--1994 will be abolished at the same time. Appendix A of this standard is a normative appendix.
This standard is proposed and managed by the Ministry of Health of the People's Republic of China. The drafting unit of this standard: Nutrition and Food Safety Institute of Chinese Center for Disease Control and Prevention. The main drafters of this standard: Bai Jingyu, Fu Ping, Li Zhigang, Ji Rong. This standard was first issued in 1994, and this is the first revision. 240
1 Scope
Food Hygiene Microbiological Examination
Listeria Monocytogenes Examination
This standard specifies the examination method for Listeria monocytogenes. This standard applies to the examination of Listeria monocytogenes in food and food poisoning samples. 2 Normative References
GB/T4789.30—2003
The clauses in the following documents become the clauses of this standard through the reference of this standard. For any dated referenced document, all subsequent amendments (excluding errata) or revisions are not applicable to this standard. However, the parties to the agreement based on this standard are encouraged to study whether the latest versions of these documents can be used. For any undated referenced document, the latest version shall apply to this standard. GB/T4789.28—2003 Food Hygiene Microbiological Examination Staining Method, Culture Medium and Reagents 3 Equipment and Materials
3.1 Refrigerator: 4℃~-20℃.
3.2 Constant temperature incubator: 30℃±1℃, 24℃±1℃. 3.3 Constant temperature water bath: 46℃±1℃.
3.4 ​​Homogenizer or sterile mortar.
3.5 Microscope: 10×~100X.
Centrifuge: 4000r/min.
3.7 Scale plate medicine balance: 0g~500g, precision 0.5g. 3.8 Conical flask: 100mL, 500mL.
3.9 Sterile pipette: 1mL (with 0.01mL scale), 10mL (with 0.1mL scale). 3.10
Sterile flat III: diameter 90cm.
Sterile test tube: 16mm×160mm.
Centrifuge tube: 30mmX100mm.
Sterile syringe: 1mL.
Standard strain of Listeria monocytogenes. Rhodococcus equi.
5 White mice: 16g~18g.
4 Culture media and reagents
4.1 TSB-YE containing 0.6% yeast extract: see Chapter A.1. 4.2 TSA-YE containing 0.6% yeast extract: see Chapter A.2. 4.3 EB enrichment broth: see Chapter A.3.
4.4 LB enrichment broth (LB); see Chapter A.4. 4.5 TSI agar: according to 4.26 and 4.27 of GB/T4789.28-2003. 4.6 SIM motility medium: see Chapter A.5. Blood agar: 4.6 of GB/T4789.28-2003. 4.7
GB/T4789.30-2003||tt| |4.8 Modified McBride (MMA) agar: see Chapter A.5. 4.9 Nitrate culture medium: 3.17 of GB/T4789.28-2003. 4.10 Buffered glucose protein water (for MR and VP tests): 3.4 of GB/T4789.28-2003. 4.11 Sugar fermentation medium: 3.2 of GB/T4789.28-2003. 4.12 Catalase test: 4.38 of GB/T4789.28-2003. 4.131% Acriflavine HCl solution: see Chapter A.4.2.1. 4.141% Naldixic acid sodium salt solution: see Chapter A.4.2.1 Chapter 5 Test Procedure
The test procedure for Listeria monocytogenes is shown in Figure 1. Test sample 25 g (mL)
Isolation culture
Pure culture
Benzene staining
Microscopic embedding
Observation of movement
EB enrichment solution 225 ml
30℃, 48h
SIM motility medium
Selective volatile matrix (MMA)
30c.24h48h
2d~5d (umbrella)
TSA-YE medium
30,24h
Transorbital hydrogen fermentation test
LB, enrichment solution 225 mL
30c24h
LB enrichment solution 10mL
30℃.24h
TS agar
Pseudopathogenicity test
6 Operation steps
6.1 Sample collection and processing
GB/T4789.30—2003
Take 25g (mL) of sample aseptically and put it in a sterile homogenizer, add 225mL EB and LB enrichment solution, and stir thoroughly until homogenized. If it cannot be tested in time, it can be temporarily stored in a 4℃ refrigerator.
-6.2 Enrichment culture
EB enrichment solution is placed at 30℃±1℃ for 48h, 225mL LB enrichment solution is placed at 30℃±1℃ for 24h, 0.1mL is aspirated, and 10mL LB is added to enrichment solution for secondary enrichment.
6.3 Isolation and culture
Separate the EB enrichment solution and the LBz secondary enrichment solution on the selective medium MMA agar plate, culture at 30℃±1℃ for 48h, select suspicious colonies, and illuminate the plate with an incandescent lamp at a 45° angle. The colonies of Listeria are gray-blue or blue, small round colonies. 6.4 Select five or more of the above suspicious colonies and inoculate them on three sugar iron (TSI agar) and SIM power culture medium, culture at 25℃±1℃, and observe whether they are motile and grow in an umbrella or crescent shape. Generally observe for 2d to 7d, and those who are positive can proceed to the next step of identification. 6.5 Pure culture
Inoculate the above suspected cultures that are motile, umbrella-shaped, and produce acid but not hydrogen sulfide in the upper and lower layers of the three sugar iron agar medium on the trypsin casein soy agar medium (TSA-YE) for pure culture, and make the following identification. 6.6 Staining microscopy
Perform Gram staining on the above suspected pure cultures and make wet mounts Inspection: Listeria is a Gram-positive small rod with a size of (0.4μm~0.5μm)×(0.5um2.0μm); when the bacterial suspension is made with physiological saline and observed under an oil immersion lens or phase contrast microscope, the bacteria show slight rotation or tumbling movements. 6.7 Biochemical characteristics
The above-mentioned suspected bacteria were subjected to further biochemical tests. The main biochemical characteristics of Listeria monocytogenes and the differences between species are shown in Table 1.
Table 1 Differences between the biochemical characteristics of Listeria monocytogenes and related bacteria Bacterial species
Listeria monocytogenes
(L. monocytogenes)
Listeria ovis
(L.ivanovii)
Listeria innocua
(L.innocua)
Listeria welshimeri
(L, rwelshimeri)
Listeria seeligeri
(L, seeligeri)
Listeria grayi
(L.grayi)
Listeria merseyii
(L.murrayi)
Hemolysis reaction Nitrate reduction
Note: V reaction is uncertain, ten positive, one negative. Urease
Mannitol
Dianlisu
Seven leaf moss
GB/T4789.30--2003
Toxicity test on mice
Inoculate the pure culture that meets the above characteristics into TSBYE, culture at 30℃ for 24h, centrifuge, discard the supernatant, and use 0.85% sterile saline to prepare a bacterial suspension with a concentration of 101°cfu/mL. Take this bacterial suspension and inject it into the abdominal cavity of 3 to 5 mice, 0.5mL each, and observe the death of the mice. The pathogenic strain dies within 2d to 5d. Known bacteria can be used as a control in the test. Listeria monocytogenes and Listeria ovis (L.iuanovii) are pathogenic to mice. 6.9 Synergistic hemolysis test (cAMP)
Staphylococcus aureus and R.equi are inoculated in parallel on the blood plate, and the suspected Listeria is inoculated vertically between them, but do not touch them. Incubate at 30℃ for 24h~48h, and check the effect of the vertical inoculation point on the hemolysis ring in the plate. The hemolysis of Listeria monocytogenes near the Staphylococcus aureus inoculation point is enhanced, the hemolysis of Listeria seeligeri is also enhanced, and the hemolysis of L.ivanovii near R.equi is enhanced. 244
Appendix A
(Normative Appendix)
Culture medium
Tryptic soy broth containing 0.6% yeast extract (TSB-YE) A.1.1 Ingredients
Polyvalent old
Yeast extract
Sodium chloride
Dipotassium hydrogen phosphate
Glucose
Distilled water
1000mL
GB/T4789.30—2003
A.1.2 Preparation method
Heat and stir the above ingredients to dissolve, adjust to pH 7.2-7.4, divide into packages, sterilize at 121℃ for 15 min, and set aside. A.2 Tryptic soy agar containing 0.6% yeast extract (TSA-YE) A.2.1 Ingredients
Polyvalent old
Yeast extract
Sodium chloride
Dipotassium hydrogen phosphate
Glucose
Distilled water
A.2.2 Preparation method
1000mL
Heat and stir the above ingredients to dissolve, adjust the pH to 7.2~7.4, divide into packages, sterilize under high pressure at 121℃, and set aside. A.3EB enrichment solution
A.3.1 Ingredients
Multivalent aged
Yeast extract
Sodium chloride
Dipotassium hydrogen phosphate
Glucose
Distilled water
Acridinium hydrochloride
1000mL
15mg/L
GB/T4789.30—2003
Purindroic acid
A.3.2 Preparation method
40mg/L
Except nalidixic acid and nalidixic acid, heat and mix the remaining ingredients, adjust the pH to 7.2-7.4, and sterilize by high pressure at 121℃ for 15min. Before use, add 15mg/L of acridinium solution and 40mg/L of nalidixic acid solution. These two ingredients should be aseptically prepared or filtered for sterilization. A.4 Listeria enrichment broth (LB1LB,)
A.4.1 Ingredients
Polyvalent aged
Yeast extract
Sodium chloride
Potassium dihydrogen phosphate
Disodium hydrogen phosphate
Aesculin
Distilled water
A.4.2 Preparation method
1000mL
Heat and dissolve the above ingredients, adjust the pH to 7.2~7.4, divide into portions, and sterilize by high pressure at 121℃ for 15min. A.4.2.1 Add the following to 225 mL of Levin solution I (LB): 1% nalidixic acid (prepared with 0.05 mol/L sodium hydroxide solution) 0.45 mL
1% acriflavine hydrochloride (prepared with sterile steamed stuffing water) A4.2.2 Add the following to 200 mL of Levin solution II (LB): 1% nalidixic acid
1% acriflavine
Aseptically dispense into 10 mL large test tubes.
A.5 Modified McBride Agar (MMA)
A.5.1 Ingredients
Beef extract
Glucose
Sodium chloride
Disodium hydrogen phosphate
Phenylethanol
Anhydrous glycine
Lithium chloride
Distilled water
A.5.2 Preparation method
1000mL
Heat and stir the above ingredients to mix well, adjust the pH to 7.2-7.4, divide into portions, sterilize under high pressure at 121℃ for 15 min, and set aside. 246
A.6, SIM power medium
A.6.1 Ingredients
Polyvalent ferric sulfate
Ammonium ferric sulfate
Sodium thiosulfate
Distilled water
A.6.2 Preparation method
1000mL
Heat and mix the above ingredients, adjust pH to 7.2, divide into small test tubes, and sterilize by high pressure at 121℃ for 15min. GB/T4789.30—20031 Ingredients
Multivalent aged
Yeast extract
Sodium chloride
Potassium dihydrogen phosphate
Disodium hydrogen phosphate
Aesculin
Distilled water
A.4.2 Preparation method
1000mL
Heat and dissolve the above ingredients, adjust the pH to 7.2-7.4, divide into portions, and sterilize by high pressure at 121℃ for 15 min. A.4.2.1 Add the following to 225 mL of Levin solution I (LB): 1% nalidixic acid (prepared with 0.05 mol/L sodium hydroxide solution) 0.45 mL
1% acriflavine hydrochloride (prepared with sterile steamed stuffing water) A4.2.2 Add the following to 200 mL of Levin solution II (LB): 1% nalidixic acid
1% acriflavine
Aseptically dispense into 10 mL large test tubes.
A.5 Modified McBride Agar (MMA)
A.5.1 Ingredients
Beef extract
Glucose
Sodium chloridebzxz.net
Disodium hydrogen phosphate
Phenylethanol
Anhydrous glycine
Lithium chloride
Distilled water
A.5.2 Preparation method
1000mL
Heat and stir the above ingredients to mix well, adjust the pH to 7.2-7.4, divide into portions, sterilize under high pressure at 121℃ for 15 min, and set aside. 246
A.6, SIM power medium
A.6.1 Ingredients
Polyvalent ferric sulfate
Ammonium ferric sulfate
Sodium thiosulfate
Distilled water
A.6.2 Preparation method
1000mL
Heat and mix the above ingredients, adjust pH to 7.2, divide into small test tubes, and sterilize by high pressure at 121℃ for 15min. GB/T4789.30—20031 Ingredients
Multivalent aged
Yeast extract
Sodium chloride
Potassium dihydrogen phosphate
Disodium hydrogen phosphate
Aesculin
Distilled water
A.4.2 Preparation method
1000mL
Heat and dissolve the above ingredients, adjust the pH to 7.2-7.4, divide into portions, and sterilize by high pressure at 121℃ for 15 min. A.4.2.1 Add the following to 225 mL of Levin solution I (LB): 1% nalidixic acid (prepared with 0.05 mol/L sodium hydroxide solution) 0.45 mL
1% acriflavine hydrochloride (prepared with sterile steamed stuffing water) A4.2.2 Add the following to 200 mL of Levin solution II (LB): 1% nalidixic acid
1% acriflavine
Aseptically dispense into 10 mL large test tubes.
A.5 Modified McBride Agar (MMA)
A.5.1 Ingredients
Beef extract
Glucose
Sodium chloride
Disodium hydrogen phosphate
Phenylethanol
Anhydrous glycine
Lithium chloride
Distilled water
A.5.2 Preparation method
1000mL
Heat and stir the above ingredients to mix well, adjust the pH to 7.2-7.4, divide into portions, sterilize under high pressure at 121℃ for 15 min, and set aside. 246
A.6, SIM power medium
A.6.1 Ingredients
Polyvalent ferric sulfate
Ammonium ferric sulfate
Sodium thiosulfate
Distilled water
A.6.2 Preparation method
1000mL
Heat and mix the above ingredients, adjust pH to 7.2, divide into small test tubes, and sterilize by high pressure at 121℃ for 15min. GB/T4789.30—2003
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