1 Scope
This standard specifies the examination method for Listeria monocytogenes in food.
In this standard, Method I is applicable to the qualitative examination of Listeria monocytogenes in food; Method II is applicable to enumeration of the Listeria monocytogenes in food with high content of Listeria monocytogenes; Method III is applicable to enumeration of the Listeria monocytogenes in food with low content of Listeria monocytogenes (<100CFU/g) but high content of infectious microbe, especially the milk, water and food containing particulate matters that interfere with colony counting.
2 Apparatus and Materials
In addition to the apparatus for conventional sterilization and cultivation in microbiological laboratory, other apparatus and materials are as follows:
2.1 Refrigerator: 2℃~5℃.
2.2 Constant temperature incubator: 30℃±1℃, 36℃±1℃.
2.3 Homogenizer.
2.4 Microscope: 10x~100x.
2.5 Electronic balance: 0.1g in sensibility.
2.6 Conical flask: 100mL, 500mL.
2.7 Aseptic pipette: 1mL (scale division of 0.01mL), 10mL (scale division of 0.1mL) or micropipettor and pipette tip.
2.8 Aseptic plate: 90mm in diameter.
2.9 Aseptic test tube: 16mm×l60mm.
2.10 Centrifugal tube: 30mm×100mm.
2.11 Aseptic injector: 1mL.
2.12 Listeria monocytogenes ATCC 19111 or CMCC 54004, or any other equivalent standard bacterial strain.
2.13 Listeria innocua ATCC 33090, or any other equivalent standard bacterial strain.
2.14 Listeria ivanovii ATCC 19119, or any other equivalent standard bacterial strain.
2.15 Listeria seeligeri ATCC 35967, or any other equivalent standard bacterial strain.
2.16 Staphylococcus aureus ATCC 25923 or other β-hemolysis-ring-generated staphylococcus aureus, or any other equivalent standard bacterial strain.
2.17 Rhodococcus equi ATCC 6939 or NCTC 1621, or any other equivalent standard bacterial strain.
2.18 Mouse: ICR weight 18g~22g.
2.19 Full-automatic microbe biochemical identification system.
3 Culture Medias and Reagents
3.1 Trypticase soy broth with 0.6% yeast extract (TSB-YE): see A.1.
3.2 Trypticase soy agar with 0.6% yeast extract (TSA-YE): see A.2.
3.3 Listeria enrichment broth (LB1, LB2): see A.3.
3.4 1% acriflavine HCl solution: see A.3.2.1, A.3.2.2.
3.5 1% naladixic acid solution: see A.3.2.1, A.3.2.2.
3.6 PALCAM agar: see A.4.
3.7 Gram stain: see A.5.
3.8 SIM motility medium: see A.6.
3.9 Buffered glucose peptone water [for methyl red (MR) and V-P test]: see A.7.
3.10 5%~8% sheep blood agar: see A.8.
3.11 Sugar fermentation tube: see A.9.
3.12 Hydrogen peroxide reagent: see A.10.
3.13 Listeria chromogenic medium.
3.14 Biochemical identification kit or full-automatic microbial identification system.
3.15 Buffered peptone water (BPW): see A.11.
Method I Qualitative Examination for Listeria Monocytogenes
4 Examination Procedures
See Figure 1 for the qualitative examination procedures for Listeria monocytogenes .
Figure 1 Qualitative Examination Procedures for Listeria Monocytogenes
5 Operation Steps
5.1 Enrichment
Take 25g (mL) sample by aseptic operation and put it into a homogenizing bag that contains 225mL LB1 enrichment broth, and homogenize for 1min~2min continuously with a slap type homogenizer; or put the sample into a homogenizing cup that contains 225mL LB1 enrichment broth, and homogenize for 1 min~2 min at a speed of 8000r/min~10000r/min. Cultivate the sample for 24h±2h at 30℃±1℃, transfer 0.1mL solution into 10mL LB2 enrichment broth to cultivate for 24h~2h at 30℃±1℃.
5.2 Isolation
Take LB2 secondary enrichment broth for streak inoculation on Listeria colorplate and PALCAM agar plate; cultivate for 24h~48h at 36℃±1℃ and observe the growing colonies on the plates. On PALCAM agar plate, the typical colony is small greyish green round colony with brownish black hydrolytic circle around while some have black dent; refer to the product instructions for determination of the colony characteristics on Listeria colorplate.
5.3 Primary screening
Respectively pick 3~5 typical or suspicious colonies from the selective agar plate, and inoculate them into the xylose fermentation tube and rhamnose fermentation tube to cultivate for 24h±2h at 36℃±1℃; meanwhile, streak on TSA-YE plate to cultivate for 18h~24h at 36℃±1℃; then select xylose-negative and rhamnose-positive pure culture to continue the identification.
5.4 Identification (or selecting biochemical identification kit or full-automatic microbial identification system, etc.)
5.4.1 Gram staining microscopic examination: Listeria is gram-positive brevibacterium with a size of (0.4μm~0.5μm)×(0.5μm~2.0μm); prepare the bacterial suspension with normal saline, observe under oil immersion lens or phase contrast microscope, and this bacterium has slight movement, like rotation or rolling over.
5.4.2 Motility test: pick purely-cultivated single suspicious colony and penetrate it into semi-solid or SIM motility medium to cultivate for 48h at 25℃~30℃; Listeria has motility and it can grow in umbrella shape; if the umbrella-shaped growth is not apparent, cultivate for another 5d and then observe the result.
5.4.3 Biochemical identification: pick purely-cultivated single suspicious colony to conduct the catalase test; the catalase-positive colony shall be subjected to further sugar fermentation test and MR-VP test. The major biochemical characteristics of Listeria monocytogenes are detailed in Table 1.
5.4.4 Hemolytic test: divide the bottom surface of fresh sheep blood agar plate into 20~25 grids, pick purely-cultivated single suspicious colony and inoculate one colony in each grid of the blood plate by stabbing, and also inoculate the positive contrast bacteria (Listeria monocytogenes, Listeria ivanovii and Listeria seeligeri) and negative contrast bacteria (Listeria innocua) by stabbing. Approach to the bottom as much as possible when stabbing but not touch the bottom, meanwhile, avoid the agar cracking, cultivate for 24h~48h at 36℃±1℃ and observe at bright place. Listeria monocytogenes shall present narrow, clear and bright hemolytic circle; Listeria seeligeri shall present weak transparent hemolytic circle around the stabbing point; Listeria innocua shall not have hemolytic circle and Listeria ivanovii shall present wide β-hemolysis area with clear boundary. If the result is inapparent, place the plate in 4℃ refrigerator for another 24h~48h.
Note: streak inoculation is also applicable.
5.4.5 cAMP test (optional): inoculate staphylococcus aureus and Rhodococcus equi on sheep blood agar plate by parallel streaking, and pick purely-cultivated single suspicious colony to inoculate between the parallel lines by perpendicular streaking, with the perpendicular line keeping 1mm~2mm away from the parallel line. Meanwhile, inoculate Listeria monocytogenes, Listeria innocua, Listeria ivanovii and Listeria seeligeri to cultivate them for 24h~48h at 36℃±1℃. Listeria monocytogenes shall present approx. 2mm β-hemolysis enhancement area near the staphylococcus aureus; Listeria seeligeri shall also present weak hemolysis enhancement area; Listeria ivanovii shall present approx. 5mm~10mm "arrow-like" β-hemolysis enhancement area near the Rhodococcus equi; Listeria innocua shall not present hemolysis. If the result is inapparent, place the plate in 4℃ refrigerator for another 24h~48h.
Note: 5%~8% Listeria monocytogenes show enhanced hemolysis on the end of Rhodococcus equi.
Table 1 Difference in Biochemical Characteristics
between Listeria Monocytogenes and Other Listeria Bacteria
Bacteria Hemolytic reaction Glucose Maltose MR-VP Mannitol Rhamnose Xylose Esculin
L.monocytogenes + + + +/+ - + - +
L.grayi - + + +/+ + - - +
L.seeligeri + + + +/+ - - + +
L.welshimeri - + + +/+ - V + +
L.ivanovii + + + +/+ - - + +
L.innocua - + + +/+ - V - +
Note: + means positive; - means negative; V means uncertain reaction.
5.5 Mouse virulence test (optional)
Inoculate the pure culture with the aforesaid characteristics in TSB-YE to cultivate for 24h at 36℃±1℃, then centrifuge for 5min at 4000r/min; abandon the supernatant and prepare the remaining solution to 1010CFU/mL bacterium suspension with stroke-physiological saline solution; inject 0.5mL of the bacterium suspension into the abdominal cavity of 3~5 mice respectively, and observe the death of mice. The mice inoculated with pathogenic strain die in 2d~5d. Listeria monocytogenes pathogenic strain and aseptic normal saline control are set for the test. Listeria monocytogenes and Listeria ivanovii are pathogenic to mice.
5.6 Result and report
The detection of Listeria monocytogenes in 25g (mL) sample shall be reported by combining the above biochemical tests and hemolytic test results.
Contents
Foreword i
1 Scope
2 Apparatus and Materials
3 Culture Medias and Reagents
4 Examination Procedures
5 Operation Steps
6 Examination Procedures
7 Operation Steps
8 Result Enumeration
9 Result Report
10 Examination Procedures
11 Operation Steps
12 Result and Report
Appendix A Media and Reagent
Appendix B Most Probable Number (MPN) of Listeria Monocytogenes
1 Scope
This standard specifies the examination method for Listeria monocytogenes in food.
In this standard, Method I is applicable to the qualitative examination of Listeria monocytogenes in food; Method II is applicable to enumeration of the Listeria monocytogenes in food with high content of Listeria monocytogenes; Method III is applicable to enumeration of the Listeria monocytogenes in food with low content of Listeria monocytogenes (<100CFU/g) but high content of infectious microbe, especially the milk, water and food containing particulate matters that interfere with colony counting.
2 Apparatus and Materials
In addition to the apparatus for conventional sterilization and cultivation in microbiological laboratory, other apparatus and materials are as follows:
2.1 Refrigerator: 2℃~5℃.
2.2 Constant temperature incubator: 30℃±1℃, 36℃±1℃.
2.3 Homogenizer.
2.4 Microscope: 10x~100x.
2.5 Electronic balance: 0.1g in sensibility.
2.6 Conical flask: 100mL, 500mL.
2.7 Aseptic pipette: 1mL (scale division of 0.01mL), 10mL (scale division of 0.1mL) or micropipettor and pipette tip.
2.8 Aseptic plate: 90mm in diameter.
2.9 Aseptic test tube: 16mm×l60mm.
2.10 Centrifugal tube: 30mm×100mm.
2.11 Aseptic injector: 1mL.
2.12 Listeria monocytogenes ATCC 19111 or CMCC 54004, or any other equivalent standard bacterial strain.
2.13 Listeria innocua ATCC 33090, or any other equivalent standard bacterial strain.
2.14 Listeria ivanovii ATCC 19119, or any other equivalent standard bacterial strain.
2.15 Listeria seeligeri ATCC 35967, or any other equivalent standard bacterial strain.
2.16 Staphylococcus aureus ATCC 25923 or other β-hemolysis-ring-generated staphylococcus aureus, or any other equivalent standard bacterial strain.
2.17 Rhodococcus equi ATCC 6939 or NCTC 1621, or any other equivalent standard bacterial strain.
2.18 Mouse: ICR weight 18g~22g.
2.19 Full-automatic microbe biochemical identification system.
3 Culture Medias and Reagents
3.1 Trypticase soy broth with 0.6% yeast extract (TSB-YE): see A.1.
3.2 Trypticase soy agar with 0.6% yeast extract (TSA-YE): see A.2.
3.3 Listeria enrichment broth (LB1, LB2): see A.3.
3.4 1% acriflavine HCl solution: see A.3.2.1, A.3.2.2.
3.5 1% naladixic acid solution: see A.3.2.1, A.3.2.2.
3.6 PALCAM agar: see A.4.
3.7 Gram stain: see A.5.
3.8 SIM motility medium: see A.6.
3.9 Buffered glucose peptone water [for methyl red (MR) and V-P test]: see A.7.
3.10 5%~8% sheep blood agar: see A.8.
3.11 Sugar fermentation tube: see A.9.
3.12 Hydrogen peroxide reagent: see A.10.
3.13 Listeria chromogenic medium.
3.14 Biochemical identification kit or full-automatic microbial identification system.
3.15 Buffered peptone water (BPW): see A.11.
Method I Qualitative Examination for Listeria Monocytogenes
4 Examination Procedures
See Figure 1 for the qualitative examination procedures for Listeria monocytogenes .
Figure 1 Qualitative Examination Procedures for Listeria Monocytogenes
5 Operation Steps
5.1 Enrichment
Take 25g (mL) sample by aseptic operation and put it into a homogenizing bag that contains 225mL LB1 enrichment broth, and homogenize for 1min~2min continuously with a slap type homogenizer; or put the sample into a homogenizing cup that contains 225mL LB1 enrichment broth, and homogenize for 1 min~2 min at a speed of 8000r/min~10000r/min. Cultivate the sample for 24h±2h at 30℃±1℃, transfer 0.1mL solution into 10mL LB2 enrichment broth to cultivate for 24h~2h at 30℃±1℃.
5.2 Isolation
Take LB2 secondary enrichment broth for streak inoculation on Listeria colorplate and PALCAM agar plate; cultivate for 24h~48h at 36℃±1℃ and observe the growing colonies on the plates. On PALCAM agar plate, the typical colony is small greyish green round colony with brownish black hydrolytic circle around while some have black dent; refer to the product instructions for determination of the colony characteristics on Listeria colorplate.
5.3 Primary screening
Respectively pick 3~5 typical or suspicious colonies from the selective agar plate, and inoculate them into the xylose fermentation tube and rhamnose fermentation tube to cultivate for 24h±2h at 36℃±1℃; meanwhile, streak on TSA-YE plate to cultivate for 18h~24h at 36℃±1℃; then select xylose-negative and rhamnose-positive pure culture to continue the identification.
5.4 Identification (or selecting biochemical identification kit or full-automatic microbial identification system, etc.)
5.4.1 Gram staining microscopic examination: Listeria is gram-positive brevibacterium with a size of (0.4μm~0.5μm)×(0.5μm~2.0μm); prepare the bacterial suspension with normal saline, observe under oil immersion lens or phase contrast microscope, and this bacterium has slight movement, like rotation or rolling over.
5.4.2 Motility test: pick purely-cultivated single suspicious colony and penetrate it into semi-solid or SIM motility medium to cultivate for 48h at 25℃~30℃; Listeria has motility and it can grow in umbrella shape; if the umbrella-shaped growth is not apparent, cultivate for another 5d and then observe the result.
5.4.3 Biochemical identification: pick purely-cultivated single suspicious colony to conduct the catalase test; the catalase-positive colony shall be subjected to further sugar fermentation test and MR-VP test. The major biochemical characteristics of Listeria monocytogenes are detailed in Table 1.
5.4.4 Hemolytic test: divide the bottom surface of fresh sheep blood agar plate into 20~25 grids, pick purely-cultivated single suspicious colony and inoculate one colony in each grid of the blood plate by stabbing, and also inoculate the positive contrast bacteria (Listeria monocytogenes, Listeria ivanovii and Listeria seeligeri) and negative contrast bacteria (Listeria innocua) by stabbing. Approach to the bottom as much as possible when stabbing but not touch the bottom, meanwhile, avoid the agar cracking, cultivate for 24h~48h at 36℃±1℃ and observe at bright place. Listeria monocytogenes shall present narrow, clear and bright hemolytic circle; Listeria seeligeri shall present weak transparent hemolytic circle around the stabbing point; Listeria innocua shall not have hemolytic circle and Listeria ivanovii shall present wide β-hemolysis area with clear boundary. If the result is inapparent, place the plate in 4℃ refrigerator for another 24h~48h.
Note: streak inoculation is also applicable.
5.4.5 cAMP test (optional): inoculate staphylococcus aureus and Rhodococcus equi on sheep blood agar plate by parallel streaking, and pick purely-cultivated single suspicious colony to inoculate between the parallel lines by perpendicular streaking, with the perpendicular line keeping 1mm~2mm away from the parallel line. Meanwhile, inoculate Listeria monocytogenes, Listeria innocua, Listeria ivanovii and Listeria seeligeri to cultivate them for 24h~48h at 36℃±1℃. Listeria monocytogenes shall present approx. 2mm β-hemolysis enhancement area near the staphylococcus aureus; Listeria seeligeri shall also present weak hemolysis enhancement area; Listeria ivanovii shall present approx. 5mm~10mm "arrow-like" β-hemolysis enhancement area near the Rhodococcus equi; Listeria innocua shall not present hemolysis. If the result is inapparent, place the plate in 4℃ refrigerator for another 24h~48h.
Note: 5%~8% Listeria monocytogenes show enhanced hemolysis on the end of Rhodococcus equi.
Table 1 Difference in Biochemical Characteristics
between Listeria Monocytogenes and Other Listeria Bacteria
Bacteria Hemolytic reaction Glucose Maltose MR-VP Mannitol Rhamnose Xylose Esculin
L.monocytogenes + + + +/+ - + - +
L.grayi - + + +/+ + - - +
L.seeligeri + + + +/+ - - + +
L.welshimeri - + + +/+ - V + +
L.ivanovii + + + +/+ - - + +
L.innocua - + + +/+ - V - +
Note: + means positive; - means negative; V means uncertain reaction.
5.5 Mouse virulence test (optional)
Inoculate the pure culture with the aforesaid characteristics in TSB-YE to cultivate for 24h at 36℃±1℃, then centrifuge for 5min at 4000r/min; abandon the supernatant and prepare the remaining solution to 1010CFU/mL bacterium suspension with stroke-physiological saline solution; inject 0.5mL of the bacterium suspension into the abdominal cavity of 3~5 mice respectively, and observe the death of mice. The mice inoculated with pathogenic strain die in 2d~5d. Listeria monocytogenes pathogenic strain and aseptic normal saline control are set for the test. Listeria monocytogenes and Listeria ivanovii are pathogenic to mice.
5.6 Result and report
The detection of Listeria monocytogenes in 25g (mL) sample shall be reported by combining the above biochemical tests and hemolytic test results.
Contents of GB 4789.30-2016
Contents
Foreword i
1 Scope
2 Apparatus and Materials
3 Culture Medias and Reagents
4 Examination Procedures
5 Operation Steps
6 Examination Procedures
7 Operation Steps
8 Result Enumeration
9 Result Report
10 Examination Procedures
11 Operation Steps
12 Result and Report
Appendix A Media and Reagent
Appendix B Most Probable Number (MPN) of Listeria Monocytogenes
