GB 4789.31-2013 English PDFUS$459.00 · In stock
Delivery: <= 4 days. True-PDF full-copy in English will be manually translated and delivered via email. GB 4789.31-2013: National Food Safety Standard Food Microbiological Examination -- Determination and testing of Enterobacteriaceae phage of Salmonella, Shigella and Diarrheagenic Escherichia coli Status: Valid GB 4789.31: Historical versions
Basic dataStandard ID: GB 4789.31-2013 (GB4789.31-2013)Description (Translated English): National Food Safety Standard Food Microbiological Examination -- Determination and testing of Enterobacteriaceae phage of Salmonella, Shigella and Diarrheagenic Escherichia coli Sector / Industry: National Standard Classification of Chinese Standard: C53 Classification of International Standard: 07.100.30 Word Count Estimation: 20,231 Older Standard (superseded by this standard): GB 4789.31-2003 Regulation (derived from): China Food & Drug Administration [2013] No. 234, November, 1, 2013 Issuing agency(ies): National Health and Family Planning Commission of the People's Republic of China Summary: This Standard, from June 1, 2014, substitutes GB/T 4789. 31-2003 Food Microbiological Examination Determination and testing of enterobacteriaceae phage of Salmonella, Shigella and Diarrheagenic Escherichia coli. GB 4789.31-2013: National Food Safety Standard Food Microbiological Examination -- Determination and testing of Enterobacteriaceae phage of Salmonella, Shigella and Diarrheagenic Escherichia coli---This is a DRAFT version for illustration, not a final translation. Full copy of true-PDF in English version (including equations, symbols, images, flow-chart, tables, and figures etc.) will be manually/carefully translated upon your order. National Food Safety Standard Food Microbiological Examination - Determination and testing of enterobacteriaceae phage of Salmonella, Shigella and Diarrheagenic Escherichia coli National Standards of People's Republic of China National Food Safety Standard Food Microbiology Inspection Salmonella, Shigella and diarrhea caused by Escherichia coli Enterobacteriaceae phage diagnostic tests Issued on. 2014-06-01 2013-11-29 implementation ForewordThis standard replaces GB/T 4789.31-2003 "Microbiological examination of food hygiene Salmonella, Shigella and diarrhea caused by Escherichia coli Enterobacteriaceae phage test methods. " This standard compared with GB/T 4789.31-2003, main changes are as follows. - Modify the procedure; - Added Appendix B. National Food Safety Standard Food Microbiology Inspection Salmonella, Shigella and diarrhea caused by E. coli Enterobacteriaceae phage diagnostic tests1 ScopeThis standard specifies the application Enterobacteriaceae phage diagnostic method for testing of Salmonella, Shigella and diarrhea caused by Escherichia coli. This standard applies to all types of foods and foodborne disease events samples Salmonella, Shigella and diarrhea caused by Escherichia coli test. This standard applies to the food industry practitioners intestinal Salmonella and Shigella carrier inspection.2 Equipment and MaterialsIn addition to conventional microbiological laboratory sterilization, training equipment, other equipment and materials as follows. a) incubator. 36 ℃ ± 1 ℃, 42 ℃ ± 1 ℃, 50 ℃ ± 1 ℃; b) anaerobic means. 41.5 ℃ ± 0.5 ℃; c) Microscope. 10 to 100 times; d) Level. bacteriophage test work surface should be adjusted to a horizontal position; e) 1 mL sterile disposable syringes (plastic material should be), the needle-free number before using measuring 5 L ~ 10 L per drop (per 100 mL dropwise to 200) are available; f) Micropipettes and tips (10 L and 5 L); g) and quantitative pipette tip (100 L and 50 L); h) sterile pipette. 10 mL, 5 mL, 1 mL; i) sterile capillary; j) sterile Petri dish. 90 mm; k) a sterile cotton swab; l) with lids sterilized small test tube. 8 mm × 50 mm; m) loading slide.3 media and reagents3.1 selenite cystine (SC) enrichment broth. See Appendix A for A.1. 3.2 four brilliant green, sodium sulfur (TTB) enrichment broth. See Appendix A A.2. 3.3 Shigella (BCT) enrichment broth. See Appendix A A.3. 3.4 nutrient broth (NB). See Appendix A in A.4. 3.5 nutrient agar (NA). A A.5 in the Appendix. 3.6 semi-solid nutrient agar (NSA). See Appendix A in A.6. 3.7 triple sugar iron agar (TSI). See Appendix A A.7. 3.8 dextrose agar ammonium. See Appendix A A.8. 3.9 diagnosis Enterobacteriaceae phage, 7 and 3. If necessary, the purchase of Escherichia coli phage typing (six kinds) set. Secure After BU open with a sterile capillary aspiration moved within sterilized small test tube, if the amount of each use small, can be divided into 2 or 3 installed in the test tube and stored at 5 ℃ refrigerator spare. 3.10 Salmonella factor serum, Shigella typing serum factor, causing diarrhea Escherichia coli typing serum factor. Category serum factor Depending on need.4 inspection procedures4.1 Salmonella test procedure Salmonella test procedure shown in Figure 1. Figure 1 salmonella testing procedures 4.2 Shigella inspection procedures Shigella inspection procedures shown in Figure 2. ONPG mannitol, sorbitol, Test sample O-1 cleavage Other not cleaved Non-described as left Various reactions result salmonella Serological test confirmed Non-Salmonella Salmonella Serological test confirmed salmonella Serological test confirmed Non-non-Salmonella Salmonella report Pre-enrichment, enrichment and separation Suspected colonies were picked TSI (slant, bottom gas, H2S) Peptone water (indole) Urea (pH7.2), KCN, lysine H2S, indole -, Urea -, KCN-, Lysine + Phage test H2S, indole, Urea -, KCN-, Lysine Various phage Not cleaved Non-described as left The various cracking Results H2S-, indole -, Urea -, KCN-, Lysine/- +, +, - -, - Figure 2 Shigella inspection procedures Sh cleavage and exhibits The various cracking mode Sh not cleaved Serological tests consistent with cleavage pattern And lysed 1 RTD Sh Serological tests and split mode does not match Or may not be cleaved 1 RTD Sh Biochemical grouping test Ammonium glucose test If necessary, a plus sodium acetate, sodium citrate Clinton, Mucus acid, esculin, salicin test Esculin \\ salicin test Shigella Clustering and classification results Glucose ammonium Or any other positive results Non-Shigella report Non-Shigella Non-Shigella Phage test Non-referred as left Species Reactivity Results Test sample Suspected colonies were picked TSI, glucose semisolid Enrichment and separation TSI underlying acid production ramp production base, H2S- No gas, no power 4.3 diarrheogenic Escherichia coli testing procedures Diarrhea caused by Escherichia coli testing procedures shown in Figure 3. Figure 3 diarrhea caused by Escherichia coli testing procedures Test sample And E or Sh, E-4 Phage not cracking Other phage lysis Strains isolated from different sources Its cleavage pattern have identity TSI, indole, pH7.2 Urea, KCN, Lysine, power TSI bottom +, H2S-, KCN-, urea - Non-described as left Various reactions result Serological tests ETEC Enrichment and separation And E or Sh, E-4 Phage lysis Various phage are not cleaved Oxidase -, G- bacilli EPEC Enterotoxigenic Escherichia coli Non-colon Coli Lysine -, indole, Power - (O124 power/-) Enterotoxin Non-described as left EIEC Various reactions result Non-colon Coli Large non-induced diarrhea Escherichia coli Intestinal invasive Escherichia coli Enteropathogenic Escherichia coli Lactose fermentation or unfermented 3 to 5 colonies Phage test STEC (O157) stx1, stx2, hly Shiga toxin Escherichia coli eae report5 steps5.1 pre-enrichment, enrichment and separation 5.1.1 Salmonella pre-enrichment, enrichment and separation carried out according to GB 4789.4. Salmonella bacteria by measuring sensitivity in Appendix B. in Intestinal colonization of Salmonella in the food industry practitioners test specimens collected should enrichment, should not be pre-enrichment. Samples must not be food poisoning before Enrichment, samples collected at the same time do not enrichment and enrichment step. Enrichment and separation 5.1.2 Shigella performed according to GB 4789.5. No laboratory anaerobic culture conditions can be used in Appendix A BCT Enrichment broth. Food poisoning patient stool samples at the same time do not enrichment and enrichment step. 5.1.3 diarrhea caused by E. coli bacteria and isolated according to GB 4789.6 and GB 4789.36 conduct. Food poisoning were done simultaneously and enrichment No enrichment step. 5.2 Phage test 5.2.1 Prepare medium Nutrient agar plate (agar containing 1% to 1.5%, in order to prevent the spread of the growth of Proteus, may be added in an amount of 0.02% sodium lauryl sulfate), After heating melted nutrient agar was added to each dish 9 cm to about 20 mL ~ 25 mL, on a level table wait until solidified. Flip the plate, At 36 ℃ 1 ℃ incubator half open pan upside down about 1 h, or half-open within 50 ℃ 1 ℃ incubator pan upside down for about 30 min, the medium to dry Surface moisture. 5.2.2 Test preparation broth When picked up two more typical on selective agar plates were from 5.2.2.1 or suspicious colonies Salmonella test, picked lactose negative yield H2S or H2S-producing colonies, and colonies of lactose-positive production of H2S. Picked lactose-positive or lactose-negative test when Escherichia coli bacteria drop. Picked typical or suspicious colonies were inoculated test when Shigella TSI, semi-solid and nutrient agar slant of each one, and placing 36 ℃ 1 ℃ Cultured 20 h ~ 24 h after selected triple sugar iron underlying acid production ramp production base, did not produce H2S, no gas unpowered colonies. The following two methods When selected for the preparation of test bacteria. 5.2.2.2 Method One. to be tested colonies were inoculated into nutrient broth tube, cultured 14 h ~ 24 h at 36 ℃ 1 ℃. Picked this broth culture 1 full-ring (about 5 L), diluted in containing within 1 mL ~ 2 mL of nutrient broth tube, so that became the 1.200 to 1. 400 dilution of broth, about bacteria 1 × 106 CFU/mL. 5.2.2.3 Method Two. inoculation needle on the identification plate suspected colonies were picked, diluted in containing within 1mL ~ 2 mL nutrient broth tubes, bacteria About 1 × 106 CFU/mL. 5.2.3 bacilli smear test 5.2.3.1 spots smear method. nutrient agar surface starting from the center is divided into three equal portions or bisecting each aliquot applied for a bacterial culture. Each picked a full ring test bacteria, smear a diameter of about 1 cm a plaque. Per plant culture smear plaque 7, the outer ring 5, the inner ring 2 A. 5.2.3.2 swab smear method. with a sterile cotton swab dipped slightly squeeze test bacteria and excess fluid in the above-third of the agar surface area Apply within. Maintaining appropriate distance between the three applicator region, to be dried bacteria. 5.2.4 solution of phage With a basis weight of 1 mL disposable syringe (without the needle number, 1 mL of about 100 to 200 drops, the equivalent of 5 L ~ 10 L per drop), Phage dropped on each plaque. Or with a basis weight of 10 L or 5 L micropipette dropped a drop on each phage plaque, Each phage solution of one kind should replace a syringe or a suction head, but each pair or each injector tip can drop finished with various strains of bacteria One kind of bacteriophage. Phage were dropping OI, C, Sh, E, CE, E-4 and Ent. Should be dropped on the phage agar plates The level of the table, should be suspended droplets dripping, not contaminated needles or dripper. After seven kinds of phages are the addition was complete, wait a few minutes and so little phage Dried body fluids. Flip the plate, cultured in 36 ℃ 5 h ~ 6 h, and 14 h ~ 24 h each observation time result. If only a few of several strains of bacteria for the phage test, available 3 mm diameter inoculation ring picked phages, each the equivalent of five full ring L, successively added to the plaque, minimize time at room temperature. 5.2.5 determination of test results If necessary (for example, can not be measured to complete serotype), can absorb a little of the remaining peptone water cultures for indole test, colon Egypt Hippocratic generally indole positive bacteria, Salmonella indole negative. Bacteriophage test results are shown in Table 1. Table 1 bacteriophage test results No. Phage lysis mode Judgement result OI C Sh E CE E-4 Ent 1 CL - - - - - - Salmonella 2 CL - - - - CL - Salmonella (indole -), Escherichia coli (indole) 3 - CL - - (-) (-) - Frau Dietrich Citrobacter group a 4 - - CL vvv - Shigella b, Escherichia coli 5 CL - CL vvv - Escherichia coli 6 v - - CL vv - Escherichia coli 7 - - - - CL v - Frau Dietrich Citrobacter group a, Escherichia coli 8 - - - - - CL - Escherichia coli 9 - (-) v - - - CL Enterobacter cloacae 0 - - - - - - - not cleaved phage cultures were identified by biochemical tests NOTE. CL indicates fusion cleavage; - for not cracking; (-) indicates a small amount of strain cracking; v represents cleavage or no cleavage. Frau Dietrich containing a Citrobacter, Citrobacter Young, cloth Lei Keshi Citrobacter, Citrobacter and Gillen's Workman's Citrobacter. b Table 3 retrieves and do serological typing test. 5.2.6 rapid test for salmonella phage simplify diagnostics Using the following three kinds of phages. Salmonella phage OI; E polyvalent phage, containing E and Sh; C polyvalent phage, containing C, CE and Ent. Medium and bacteria were ready to test with the former method. Smear test bacteria. spot smearing agar surface of the plate is divided into four equal portions or 6 equal portions, each decile spreadable a bacterial culture, Apply 3 per plant culture plaque, the outer ring 2, the inner one. Swab smear method can be applied to the test strip bacteria on agar plates, Each plate can be applied to about five bacteria band, set a few minutes, until the plaque dry. It was added dropwise the above-described three kinds of phages. Spots smear method, and can be dropped in the OI phage plaque on the inner ring. Strip smear method, OI can be dropped phage bacteria left the band. After phage droplets dry, flip the plates and incubated at 36 ℃ 5 h ~ 6 h, and 14 h ~ 24 h each observation time result. Table 2 judgment result. Table 2 phage test simplify diagnostics OI E C multivalent polyvalent determination result CL - - Salmonella v CL v Escherichia coli - - CL Frau Dietrich Citrobacter group a - - - Not phage lysate cultures NOTE. CL indicates fusion cleavage; - for not cracking; v represents cleavage or no cleavage. In addition to a note in Table 1 of the outer, and contains few Enterobacter cloacae and Escherichia coli. 5.3 does not fragment phage cultures were supplemented biochemical tests Salmonella minority cultures are not OI phage lysate, Escherichia coli minority culture is not appropriate phage lysis, not all Culture was inoculated phage lysis triple sugar iron agar, according to GB 4789.4 do five biochemical, serological typing after identification of the determination result. 5.4 serotyping identification and other complementary tests 5.4.1 Identification of Salmonella Typing Carried out according to GB 4789.4. If it is determined salmonella, should be the outcome of a complete serotyping identified. 5.4.2 Escherichia coli cause diarrhea identification 5.4.2.1 according to GB 4789.6 and GB 4789.36 conduct. After isolates should also be the same number of phage lysis, with colorectal Coli phage typing test, these strains should have the same cleavage pattern, the simultaneous determination of the case 1 RTD phage lysis. 5.4.2.2 enterotoxigenic E. coli confirmed, should enterotoxin tests. 5.4.2.3 invasive Escherichia coli, a typical biochemical characteristics. lysine decarboxylase test was negative, no power, gas or no gas (O124 serotypes may also think that they have power, no gas), indole test positive. Guinea pig cornea can make further tests, the results should be positive Resistance, plasmid electrophoresis proved to be 120 ~ 140 Mdal large plasmids, PCR tests proved ipaC or ipaH genes. 5.4.2.4 Shiga toxin-producing Escherichia coli O157. H7, the typical biochemical characteristics. lactose, sucrose, acid production, acid production and glucose more Number of gas, negative hydrogen sulfide, indole-positive, sorbitol slow fermentation. PCR tests proved Shiga toxin gene stx1, stx2 and Hemolysin gene hly. 1RTD of E-2 phage lysis test can be E-2 bacteriophage lysis (lysis include 1RTD degree from CL A few plaques). For Shiga toxin and hemolysin other serotypes of Escherichia coli, in accordance with the procedures 5.4.2.3 Kam set. Typical biochemical characteristics 5.4.2.5 intestinal pathogenic Escherichia coli having the Escherichia coli, eae gene (shielded adhesion gene) PCR test was positive. Shiga toxin-producing Escherichia coli eae test may also be positive. EAF (adhesion factor plasmid gene) or bfp (bacteria PCR test hair bundling gene) can be further confirmed. 5.4.3 Shigella type identification 5.4.3.1 picked cultures triple sugar iron agar, by phage lysis mode, use the appropriate Shigella typing serum factor, do slide Agglutination test. Serotype identification results are shown in Table 3. Results Table 3 phage test and Shigella serotyping identified Phage lysis mode Results Shigella serotyping identified OI C Sh E CE E-4 Ent 1 - - CL CL CL CL - Freund 1-5, (Powell 11) 2 - - CL CL CL - - Freund 1,4, dysentery 2, Powell 5,7,11,16,17, (within the Song's I) 3 - - CL CL - - - within the Song's I, dysentery 2, 4 flexneri, boydii 5,16 4 - - CL CL - CL - Song's inner II, Freund 3 5 - - CL - - - - Freund's 6, Powell 1-4, even in 6 to 18 (except 16 outside), dysentery 3-12 6 - - CL - CL - - boydii 9,15 7 - - CL - - CL - dysentery 1, (Song's inner II) 8 CL - CL - - - - boydii 13a NOTE. CL indicates fusion cleavage; - for not cracking. a Powell's 13-type DNA homology determination does not comply with Shigella. 5.4.3.2 such as by phage lysis model results for the Shigella Shigella type 1 to 5, the first with Freund's polyvalent serum agglutination test. As presented coagulation Set, and then separately with each type checking group and serum factors, to determine the relevant serotypes and subtypes (see GB 4789.5). 5.4.3.3 such as by phage lysis model results for Freund type 6, or all types of dysentery Pauls 3-12 type, first with Freund's 6 Serum agglutination test Experience. Flexneri type 6 in serum agglutination who, with Martin's polyvalent serum or dysentery 3-12 polyvalent serum agglutination test, agglutination if presented again Respectively, with each type factor serology. 5.4.3.4 such as by phage lysis mode results in the Song's phase II within the bacteria without being Song's serum agglutination, you can press the phage lysis mold Style and rough determination of colony characteristics. 5.4.3.5 Press phage lysis model results do not aggregated by serological tests, or, having found that it is a sub-type of a serum agglutination factor with the bite Cell lysis pattern does not match the person or, being consistent with the phage lysate mode but not the Sh 1 RTD phage lysis, shall make glucose Ammonium Test with the Escherichia coli phase identification. Ammonium glucose test positive for the E. coli. Necessary to do the following biochemical tests. vinegar Salt, using experimental Klinefelter citrate and mucous acids, lysine and ornithine decarboxylase test, esculin decomposition test. In addition to Song within coli Martin's 13 type of ornithine decarboxylase positive, and within a few Song coli strain available mucus acid, the Shigella cultures were negative result. Any positive test results described above are suggestive of Escherichia coli (according to GB 4789.5). 5.4.3.6 The detection of Shigella culture should be consistent with the biochemical characteristics of the population. Shigella flexneri type 6 in line with the characteristics Martin Shigella bacteria. Indole-positive serotypes of Shigella dysentery type 2, type 7 & 8, Powell 5,7,9,11,13,15,16 and 17 type. Freund Shigella 1-5 type indole reaction is weak. Indole-negative serotypes of Shigella dysentery type 1, type 3-6, 9-12 type, Freund 6 type, Pauls 1 to 4,6,8,10,12,14 and 18, in the Song coli. 5.4.3.7 Shigella boydii type 13 is the only strain of Salmonella OI can be cleaved, this strain is only a high-titer bacteriophage lysis Sh. 5.5 Results Report 5.5.1 Salmonella test results report 5.5.1.1 OI phage lysis, other phage lysates were not, after Salmonella serotypes report. 5.5.1.2 variety of phages not cleaved, biochemical test results for salmonella, Salmonella serotyping by the report, can not be reported as undetermined type. Results 5.5.2 Shigella inspection report 5.5.2.1 Sh phage lysis, showing a variety of cleavage patterns, serological tests and cleavage pattern coincidence, could report Shigella serotypes. 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