This standard supersedes "Microbiological Examination of Food Hygiene - Examination of Yersinia Enterocolitica" (GB/T 4789.8-2008).
Compared with GB/T 4789.8-2008, this standard has the following main changes:
- The standard name is revised as "National Food Safety Standard - Food Microbiological Examination: Yersinia Enterocolitica";
- Morphological description of typical colony is modified;
- Commercial names in biochemical identification are deleted;
- Method for serum identification is described.
National Food Safety Standard
Food Microbiological Examination:
Yersinia Enterocolitica
食品安全国家标准
食品微生物学检验
小肠结肠炎耶尔森氏菌检验
1 Scope
This standard specifies examination method of Yersinia enterocolitica in foods.
This standard is applicable to the examination of Yersinia enterocolitica in foods.
2 Apparatus and Materials
In addition to the conventional sterilization and cultivation equipment in microbiological laboratory, other apparatuses and materials are as follows:
2.1 Refrigerator: 0~4℃.
2.2 Constant temperature incubator: 26±1℃, 36±1℃.
2.3 Microscope: 10x~100x.
2.4 Homogenizer.
2.5 Balance: with sensibility of 0.1g.
2.6 Sterilized test tube: 16mm×160mm, 15mm×100mm.
2.7 Sterilized suction tube: 1mL (0.01mL scale), 10mL (0.1mL scale).
2.8 Conical flask: 200mL, 500mL.
2.9 Sterilized plate: 90mm in diameter.
2.10 Microbiological biochemical identification kit or system.
3 Media and Reagents
3.1 Modified phosphate buffer solution: see A.1.
3.2 CIN-1 medium (Cepulodin Irgasan Novobiocin Agar): see A.2.
3.3 Modified Y medium (Agar Y, Modified): see A.3.
3.4 Modified KIA iron medium: see A.4.
3.5 Sugar fermentation tube: see A.5.
3.6 Ornithine decarboxylase test medium: see A.6.
3.7 Semi-solid agar: see A.7.
3.8 Buffered glucose peptone water [for methyl red (MR) and V-P test]: see A.8.
3.9 Alkali treatment solution: see A.9.
3.10 Urea medium: see A.10.
3.11 Nutrient agar: see A.11.
3.12 Diagnostic serum of Yersinia enterocolitica.
4 Examination Procedures
Examination procedures of Yersinia enterocolitica are shown in Figure 1.
Figure 1 Examination Procedures of Yersinia Enterocolitica
5 Operation Steps
5.1 Enrichment
Take 25g (or 25mL) sample in sterile operation, put into aseptic homogenizing cup or homogenizing bag containing 225mL modified phosphate buffer solution enrichment broth, homogenize for 1min at 8000r/min or for 1min with slap type homogenizer. Liquid sample or powder sample shall be mixed uniformly by shaking. After homogenization, enrich for 48~72h at 26±1℃. Enrichment time may be determined according to the estimation on the sample contamination degree.
5.2 Alkali treatment
Except the milk and milk products, mix 0.5mL enrichment broth of other foods and 4.5mL alkali treatment solution sufficiently for 15s.
5.3 Separation
Inoculate the enrichment broth of milk and milk products or that of other foods subjected to alkali treatment in CIN-1 agar plate and modified Y agar plate, cultivate for 48±2h at 26±1℃. Typical colony is a dark red center, surrounded by colorless and transparent zone (red bull eye colony), at the size of 1~2mm, in CIN-1 and is colorless, transparent and non-mucoid colony in the modified Y agar plate.
5.4 Modified KIA iron test
Pick 3~5 suspicious colonies stated in 5.3 and inoculate them in modified KIA iron agar respectively; during inoculation, firstly streak on the inclined plane and then puncture at bottom layer, cultivate for 24h at 26±1℃ and conduct further biochemical identification for the anaerogenic culture with yellowed inclined plane and bottom.
5.5 Urease test and motility observation
Pick a full loop of suspicious culture obtained in 5.4 with inoculating loop, inoculate adequate amount of it in urea medium, shake for several seconds, cultivate for 2h~4h at 26±1℃. Inoculate positive colonies in urease test in two tubes of semi-solid medium and cultivate for 24h at 36±1℃. Streak and inoculate the suspicious bacterium culture with mobility at 26℃ and without mobility at 36℃ in nutrient agar plate for purification and culture, and conduct Gram stain microscopy and biochemical test with purified substance.
5.6 Gram stain microscopy
Conduct Gram staining for the purified suspicious bacterium. Yersinia enterocolitica is Gram-negative bacillus, sometimes it appears in elliptical or bacillar at a size of (0.8~3.0μm)× 0.8μm
5.7 Biochemical identification
5.7.1 Pick single colony from the nutrient agar plate described in 5.5, inoculate in biochemical reaction tube and perform the biochemical reaction at 26±1℃. Main biochemical characteristics of Yersinia enterocolitica and its difference from other similar bacteria are detailed in Table 1.
1 Scope
2 Apparatus and Materials
3 Media and Reagents
4 Examination Procedures
5 Operation Steps
6 Results and Report
Appendix A Media and Reagents
This standard supersedes "Microbiological Examination of Food Hygiene - Examination of Yersinia Enterocolitica" (GB/T 4789.8-2008).
Compared with GB/T 4789.8-2008, this standard has the following main changes:
- The standard name is revised as "National Food Safety Standard - Food Microbiological Examination: Yersinia Enterocolitica";
- Morphological description of typical colony is modified;
- Commercial names in biochemical identification are deleted;
- Method for serum identification is described.
National Food Safety Standard
Food Microbiological Examination:
Yersinia Enterocolitica
食品安全国家标准
食品微生物学检验
小肠结肠炎耶尔森氏菌检验
1 Scope
This standard specifies examination method of Yersinia enterocolitica in foods.
This standard is applicable to the examination of Yersinia enterocolitica in foods.
2 Apparatus and Materials
In addition to the conventional sterilization and cultivation equipment in microbiological laboratory, other apparatuses and materials are as follows:
2.1 Refrigerator: 0~4℃.
2.2 Constant temperature incubator: 26±1℃, 36±1℃.
2.3 Microscope: 10x~100x.
2.4 Homogenizer.
2.5 Balance: with sensibility of 0.1g.
2.6 Sterilized test tube: 16mm×160mm, 15mm×100mm.
2.7 Sterilized suction tube: 1mL (0.01mL scale), 10mL (0.1mL scale).
2.8 Conical flask: 200mL, 500mL.
2.9 Sterilized plate: 90mm in diameter.
2.10 Microbiological biochemical identification kit or system.
3 Media and Reagents
3.1 Modified phosphate buffer solution: see A.1.
3.2 CIN-1 medium (Cepulodin Irgasan Novobiocin Agar): see A.2.
3.3 Modified Y medium (Agar Y, Modified): see A.3.
3.4 Modified KIA iron medium: see A.4.
3.5 Sugar fermentation tube: see A.5.
3.6 Ornithine decarboxylase test medium: see A.6.
3.7 Semi-solid agar: see A.7.
3.8 Buffered glucose peptone water [for methyl red (MR) and V-P test]: see A.8.
3.9 Alkali treatment solution: see A.9.
3.10 Urea medium: see A.10.
3.11 Nutrient agar: see A.11.
3.12 Diagnostic serum of Yersinia enterocolitica.
4 Examination Procedures
Examination procedures of Yersinia enterocolitica are shown in Figure 1.
Figure 1 Examination Procedures of Yersinia Enterocolitica
5 Operation Steps
5.1 Enrichment
Take 25g (or 25mL) sample in sterile operation, put into aseptic homogenizing cup or homogenizing bag containing 225mL modified phosphate buffer solution enrichment broth, homogenize for 1min at 8000r/min or for 1min with slap type homogenizer. Liquid sample or powder sample shall be mixed uniformly by shaking. After homogenization, enrich for 48~72h at 26±1℃. Enrichment time may be determined according to the estimation on the sample contamination degree.
5.2 Alkali treatment
Except the milk and milk products, mix 0.5mL enrichment broth of other foods and 4.5mL alkali treatment solution sufficiently for 15s.
5.3 Separation
Inoculate the enrichment broth of milk and milk products or that of other foods subjected to alkali treatment in CIN-1 agar plate and modified Y agar plate, cultivate for 48±2h at 26±1℃. Typical colony is a dark red center, surrounded by colorless and transparent zone (red bull eye colony), at the size of 1~2mm, in CIN-1 and is colorless, transparent and non-mucoid colony in the modified Y agar plate.
5.4 Modified KIA iron test
Pick 3~5 suspicious colonies stated in 5.3 and inoculate them in modified KIA iron agar respectively; during inoculation, firstly streak on the inclined plane and then puncture at bottom layer, cultivate for 24h at 26±1℃ and conduct further biochemical identification for the anaerogenic culture with yellowed inclined plane and bottom.
5.5 Urease test and motility observation
Pick a full loop of suspicious culture obtained in 5.4 with inoculating loop, inoculate adequate amount of it in urea medium, shake for several seconds, cultivate for 2h~4h at 26±1℃. Inoculate positive colonies in urease test in two tubes of semi-solid medium and cultivate for 24h at 36±1℃. Streak and inoculate the suspicious bacterium culture with mobility at 26℃ and without mobility at 36℃ in nutrient agar plate for purification and culture, and conduct Gram stain microscopy and biochemical test with purified substance.
5.6 Gram stain microscopy
Conduct Gram staining for the purified suspicious bacterium. Yersinia enterocolitica is Gram-negative bacillus, sometimes it appears in elliptical or bacillar at a size of (0.8~3.0μm)× 0.8μm
5.7 Biochemical identification
5.7.1 Pick single colony from the nutrient agar plate described in 5.5, inoculate in biochemical reaction tube and perform the biochemical reaction at 26±1℃. Main biochemical characteristics of Yersinia enterocolitica and its difference from other similar bacteria are detailed in Table 1.
Contents of GB 4789.8-2016
1 Scope
2 Apparatus and Materials
3 Media and Reagents
4 Examination Procedures
5 Operation Steps
6 Results and Report
Appendix A Media and Reagents
