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GB 4789.4-2024 PDF English

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GB 4789.4-2024: National food safety standard - Food Microbiological Testing - Salmonella Testing
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GB 4789.4: Evolution and historical versions

Standard IDContents [version]USDSTEP2[PDF] deliveryName of Chinese StandardStatus
GB 4789.4-2024English380 Add to Cart 0-9 seconds. Auto-delivery National food safety standard - Food Microbiological Testing - Salmonella Testing Valid
GB 4789.4-2016English155 Add to Cart 0-9 seconds. Auto-delivery National food safety standard - Food Microbiological Examination - Salmonella Test Obsolete
GB 4789.4-2010English839 Add to Cart 3 days National food safety standard -- Food microbiological examination: Salmonella Obsolete
GB/T 4789.4-2008English879 Add to Cart 6 days Microbiological examination of food hygiene -- Examination of Salmonella Obsolete
GB/T 4789.4-2003English519 Add to Cart 4 days Microbiological examination of food hygiene -- Examination of salmonella Obsolete
GB 4789.4-1994EnglishRFQ ASK 3 days Microbiological examination of food hygiene. Examination of Salmonella Obsolete
GB 4789.4-1984EnglishRFQ ASK 3 days Microbiological examination of food hygiene--Examination of salmonella Obsolete

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GB 4789.4-2024: National food safety standard - Food Microbiological Testing - Salmonella Testing


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GB NATIONAL STANDARD OF THE PEOPLE’S REPUBLIC OF CHINA National Food Safety Standard – Food Microbiological Examination – Examination of Salmonella Issued on: FEBRUARY 8, 2024 Implemented on: AUGUST 8, 2024 Issued by. National Health Commission of the People’s Republic of China; State Administration for Market Regulation.

Table of Contents

Foreword... 3 1 Scope... 4 2 Equipment and Materials... 4 3 Culture Media and Reagents... 5 4 Inspection Programs... 6 5 Operation Procedures... 8 6 Results and Reports... 16 Appendix A Culture Medium and Reagents... 17 Appendix B Common Salmonella Antigen Table... 28

1 Scope

This Standard specifies the test methods for Salmonella in food. This Standard applies to the detection of Salmonella in food.

2 Equipment and Materials

In addition to the routine sterilization and culture equipment of the microbiology laboratory, other equipment and materials are as follows.

3 Culture Media and Reagents

3.1 Buffered peptone water (BPW). see A.1. 3.2 Sodium Tetrasulfonate Brilliant Green Bacteria Enrichment Solution (TTB). see A.2. 3.3 Magnesium chloride malachite green soy peptone (RVS) enrichment solution. see A.3. 3.4 Bismuth sulfite (BS) agar. see A.4. 3.5 HE agar. see A.5. 3.6 Xylose lysine deoxycholate (XLD) agar. see A.6. 3.7 Triple sugar iron (TSI) agar. see A.7. 3.8 Nutrient agar (NA). see A.8. 3.9 Semi-solid agar. see A.9. 3.10 Peptone water, indigo matrix reagent. see A.10. 3.11 Urea agar (pH 7.2). see A.11. 3.12 Potassium cyanide (KCN) medium. see A.12. 3.13 Lysine decarboxylase test medium. see A.13. 3.14 Sugar fermentation medium. see A.14. 3.15 o-Nitrophenol β-D galactopyranoside (ONPG) medium. see A.15. 3.16 Sodium malonate medium. see A.16. 3.17 Salmonella chromogenic medium. 3.18 Salmonella diagnostic serum. 3.19 Biochemical identification kit.

4 Inspection Programs

The Salmonella inspection program is shown in Figure 1.

5 Operation Procedures

5.1 Pre-enrichment Aseptic operation. take 25g (mL) of sample; place it in a sterile homogenization cup containing 225mL of BPW, homogenize at 8000r/min~10000r/min for 1min~2min; or place it in a sterile homogenization bag containing 225mL of BPW, beat with a slap homogenizer for 1min~2min. For liquid samples, they can also be placed in a sterile Erlenmeyer flask or other suitable container containing 225mL of BPW and shaken to mix well. If adjustment of the pH value is required, use 1mol/L NaOH or HCl to adjust the pH to 6.8±0.2.Aseptically transfer the sample to a 500mL Erlenmeyer flask or other suitable container (if the homogenization cup itself has a non-porous cover or use a homogenization bag, the sample does not need to be transferred), and place it at 36℃±1℃ for 8h~18h. For milk powder, aseptically weigh 25g of the sample and slowly pour it onto the surface of 225mL of BPW liquid in a wide-mouth bottle or homogenization bag. Do not adjust the pH and do not mix evenly. Let it stand at room temperature for 60min ± 5min before mixing. Place at 36℃±1℃ and incubate for 16h~18h. If frozen samples need to be thawed, they shall be thawed in a water bath at 40°C ~ 45°C for no more than 15 min before sampling, or slowly thawed in a refrigerator at 2°C ~ 8°C for no more than 18 h. 5.2 Selective enrichment Gently shake the pre-enriched culture; transfer 0.1 mL into 10 mL RVS; mix and incubate at 42°C±1°C for 18h~24h. At the same time, transfer another 1 mL into 10 mL TTB and mix evenly. Samples with low background bacteria (such as deeply processed pre-packaged foods, etc.) are incubated at 36℃±1℃ for 18h~24h. Samples with high background bacteria (such as fresh poultry meat, etc.) are incubated at 42℃±1℃ for 18h~24h. If necessary, the pre-enriched culture can be stored in a refrigerator at 2°C ~ 8°C for no more than 72 h before selective enrichment. 5.3 Separation After shaking and mixing the selectively enriched cultures, use an inoculation loop with a diameter of 3 mm to take one loop of each selectively enriched culture and streak it onto a BS agar plate and an XLD agar plate (HE agar plate, Salmonella chromogenic medium plate or other suitable separation agar plate may also be used); respectively incubate them at 36℃±1℃ for 40h~48h (BS agar plate) or 18h~24h (XLD agar plate, HE agar plate, Salmonella chromogenic medium plate). Observe the colonies growing on each plate to see if they satisfy the colony characteristics in Table 1. If necessary, the selectively enriched culture can be stored in a refrigerator at 2°C ~ 8°C for no more than 72 h before separation. 5.4 Biochemical test 5.4.1 Select more than 4 typical or suspicious colonies for biochemical test. These colonies should come from different separation agars of different selective enrichment solutions. One of the typical or suspicious colonies may also be selected for testing first. If it is identified as non- Salmonella, then take the remaining colonies for identification. Inoculate typical or suspicious colonies into triple sugar iron agar, first draw lines on the slant; and then puncture the bottom layer. Simultaneously inoculate lysine decarboxylase test medium and nutrient agar (or other suitable non-selective solid medium) plates; and inoculate them at 36℃±1℃ for 18h~24h. The results and preliminary judgments of trisaccharide iron and lysine decarboxylase tests are shown in Table 2.Store the isolated agar plates with picked colonies at 2 ℃ ~ 8 ℃ for review when necessary. 5.4.2 If it is initially determined to be non-Salmonella, the results shall be reported directly. For suspicious Salmonella, pick its pure culture from the nutrient agar plate and inoculate it with peptone water (for indole test), urea agar (pH 7.2), potassium cyanide (KCN) medium; or inoculate trisaccharide iron agar and lysine decarboxylase test medium shall be incubated together with three biochemical test medium at the same time; incubate at 36℃±1℃ for 18h~24h, and judge the results according to Table 3. 5.4.2.1 Those that meet A1 in Table 3 are typical biochemical reactions of Salmonella, and the results shall be reported after serological identification. If one item among urea, potassium cyanide and lysine decarboxylase does not meet A1, judge the result according to Table 4.If two items among urea, potassium cyanide and lysine decarboxylase do not meet A1, judge it as non-Salmonella and report the results. 5.4.2.2 If the biochemical test results are consistent with A2 in Table 3, additional mannitol and sorbitol tests shall be performed. The mannitol and sorbitol test results of Salmonella (indole positive variant) are both positive, and the result report requires serological identification. 5.4.2.3 If the biochemical test results comply with A3 in Table 3, the ONPG test shall be performed additionally. The ONPG test result for Salmonella is negative and the lysine decarboxylase test result is positive; but the lysine decarboxylase test result for Salmonella NOTE. K. alkali production; A. acid production; +. positive; -. negative; +(-). most positive, a few negative; +/-. positive or negative. Trisaccharide iron 5.4.2.4 When necessary, perform biochemical identification of Salmonella species and subspecies according to Table 5. 5.4.3 If choosing a biochemical identification kit or microbial biochemical identification system, use pure cultures of typical or suspicious colonies on the separation plate, or pure cultures that are initially judged to be suspicious Salmonella according to Table 2; and use the instruction manual of the biochemical identification kit or microbial biochemical identification system for the identification. 5.5 Serological identification 5.5.1 Culture autoagglutination test Generally, pure cultures with an agar content of 1.2% ~ 1.5% are used for slide agglutination tests. First, perform an autoagglutination test. Add a drop of physiological saline to a clean glass slide. Mix an appropriate amount of the bacterial culture to be tested with it to form a homogeneous turbid suspension. Gently shake the glass slide for 30s ~ 60s. Under a black background, observe the reaction (use a magnifying glass if necessary). If visible bacterial agglutination appears, it is considered to have autoagglutination. Otherwise, there is no autoagglutination. Serological identification of cultures without autoagglutination is performed as follows. 5.5.2 Identification of polyvalent bacterial antigen (O) Mark out two areas of about 1cm × 2cm on the glass slide, pick out the bacterial culture to be tested; place about one ring of each area on the upper part of each area on the glass slide. And add a drop of polyvalent bacteria (O) serum to the lower part of one of the areas; add a drop of physiological saline to the lower part of another area as a control. Then use a sterile inoculation loop or needle to grind the bacterial cultures to be tested in the two areas into an emulsion with serum and physiological saline, respectively. Tilt, shake and mix the slide for 1 min; and observe against the dark background. Compared with the control, those with visible bacterial agglutination are considered positive reactions. When the O serum does not agglutinate, inoculate the strain on a medium with a higher agar content (such as 2% ~ 3%) and culture it before identification. If the agglutination reaction of the O serum is prevented due to the presence of Vi antigen, the bacterial culture to be tested shall be picked and make a concentrated bacterial solution in 1 mL of physiological saline; bathe it in boiling water for 20 ~ 30 min; and then cool it for identification. 5.5.3 Identification of polyvalent flagellar antigen (H) According to the operation of 5.5.2, replace the polyvalent bacterial (O) serum with the polyvalent flagellar (H) serum to identify the polyvalent flagellar antigen (H). When the H antigen is underdeveloped, inoculate the strain in the center of the semi-solid agar plate. When the colony spreads and grows, take bacteria from the edge for identification; or inoculate the strain into a small glass tube filled with semi-solid agar and culture it for 1 ~ 2 generations; the bacteria are taken from the remote site and then identified. 5.6 Serological typing (optional) 5.6.1 Identification of O-antigens A~F polyvalent O serum is used for slide agglutination test, and physiological saline is used as control. Those that self-coagulate in physiological saline are rough strains and cannot be typed. For those who are agglutinated by polyvalent O serum from A to F, the agglutination test shall be done with O4, O3, 10, O7, O8, O9, O2 and O11 factor serum in sequence. Based on the test results, determine the O group. For strains agglutinated by O3 and 10 serum, the agglutination test shall be performed with O10, O15, O34, and O19 single-factor serum to determine the E1 and E4 subgroups. According to the identification results of O single factor serum, each O antigen component is determined. If there is no O single-factor serum, use two O complex factor serum for identification. 5.6.2 Identification of H antigens For common bacterial types belonging to each O group from A to F, the H factor serum described in Table 6 is used to identify the H antigens in the Phases 1 and 2 in sequence. 5.6.2.1 Simple plate method Dry the surface moisture of the semi-solid agar plate; pick 1 loop of H factor serum of known phase; drop it on the surface of the semi-solid agar plate. Place the plate upright for a moment until the serum is absorbed; and inoculate the strain to be tested in the center of the place where the serum is dropped. After inverting the plate and placing it at 36°C ± 1°C for culture; pick out bacteria from the edge of the bacterial lawn that forms a spreading growth for identification. 5.6.2.2 Small glass tube method Melt 1 mL ~ 2 mL of semi-solid agar and cool it to about 48°C. Add 0.05 mL ~ 0.1 mL of H factor serum of known phase, mix well; and put it into a small glass tube of 3 mm × 50 mm with both ends open. After the agar solidifies, use an inoculation needle to pick out the bacteria to be tested and inoculate them into the agar at one end of the small glass tube. Place the small glass tube flat in a flat dish; place it for culture at 36 ℃ ± 1 ℃; and take moisturizing measures to prevent the water in the agar from evaporating and shrinking. Observe the results every day, and after the bacteria in the other phase are dissociated, pick the bacteria from the other end of the small glass tube for identification. The concentration of serum in the culture medium shall be at an appropriate ratio. If it is too high, bacteria cannot grow; and if it is too low, the motility of bacteria in the same phase cannot be inhibited. Generally, the adding amount is original serum of 1.(200~800). 5.6.2.3 Small casing method In a test tube containing about 10 mL of semi-solid agar culture medium, insert a 3 mm × 50 mm small glass tube with both ends open (a gap shall be left for the lower end opening, not flush). The upper end of the small glass tube shall be higher than the medium surface; autoclave at 121°C for 15 min and then make for later-use. Before use, heat and melt; and cool to about 48°C. Pick 1 loop of H factor serum of known phase; add it to the culture medium in the small glass tube; and stir slightly to mix. After the agar solidifies, inoculate the bacteria to be tested in the semi-solid surface layer of the small glass tube; culture it at 36°C ± 1°C; and observe the results every day. After the bacteria in the other phase is dissociated, take the bacteria from the semi-solid surface outside the small glass tube for identification, or transfer the collected bacteria to 1% agar slant and incubate at 36℃±1℃ before identification. 5.6.3 Identification of Vi antigen Identification is performed using Vi factor serum. The bacterial types known to have Vi antigen are. Salmonella typhi, Salmonella paratyphi C, and Salmonella Dublin. 5.6.4 Determination of serotype Based on the results of serological typing identification, determine the serotype according to Appendix B or the relevant Salmonella antigen table.

6 Results and Reports

Based on the results of the above biochemical tests and serological identification, it is reported that Salmonella is detected or not detected in the 25g (mL) of sample.

Appendix A

Culture Medium and Reagents A.1 Buffered peptone water (BPW) A.1.1 Ingredients A.1.2 Preparation method Add each ingredient to distilled water (or other experimental water that meets the requirements, the same below); mix well; heat to dissolve; adjust pH if necessary; and autoclave at 121°C for 15 min. The pH of the sterilized culture medium at 25°C is 7.2±0.2. A.2 Tetrasulfonate brilliant green bacteria enrichment solution (TTB) A.2.1 Basic solution A.2.2 Iodine solution A.2.3 Brilliant green solution Brilliant green. 0.5g Distilled water. 100mL After dissolving brilliant green in distilled water; store it in a cool and dark place for no less than 1 day. A.2.4 Preparation method Basic solution. 1000mL Brilliant green solution. 2.0mL Iodine solution. 20.0mL On the day of use, add brilliant green solution to the cooled basic solution aseptically and shake well; add iodine solution; shake well again; and dispense into sterile test tubes. The culture medium added with brilliant green and iodine solution is used on the same day and cannot be heated again. A.3 Magnesium chloride malachite green soy peptone (RVS) enrichment solution A.3.1 Ingredients A.3.2 Preparation method Add each ingredient to distilled water; mix well; heat to dissolve; adjust pH if necessary; quantitatively distribute into test tubes; and autoclave at 115°C for 15 min. The pH of the sterilized medium at 25°C is 5.2 ± 0.2. A.4 Bismuth sulfite (BS) agar A.4.1 Ingredients Peptone. 10.0g Beef dipping powder. 5.0g Glucose. 5.0g Ferrous sulfate. 0.3g Disodium hydrogen phosphate. 4.0g Brilliant green. 0.025g Bismuth ammonium citrate. 2.0g Sodium sulfite. 6.0g Agar. 18.0g Distilled water. 1000mL A.4.2 Preparation method Add each ingredient to distilled water; mix well; heat to dissolve; and adjust pH if necessary. Boil it; do not overheat; do not autoclave. Cool to 48℃±2℃ and pour into the plate. The pH of the boiled culture medium at 25℃ is 7.5±0.2. NOTE. This culture medium shall be prepared and poured into plates one day before use; stored in a dark place at room temperature; and used the next day. Storing the prepared culture medium for more than 48 h shall reduce its selectivity. A.5 HE agar A.5.1 Ingredients A.5.2 Preparation method Add each ingredient to distilled water; mix well; heat to dissolve; and adjust pH if necessary. Boil it; do not overheat; do not autoclave. Cool to 48℃±2℃ and pour into the plate. The pH of the boiled culture medium at 25℃ is 7.5±0.2. A.6 Xylose lysine deoxycholate (XLD) agar A.6.1 Ingredients A.6.2 Preparation method Add each ingredient to distilled water; mix well; heat to dissolve; and adjust pH if necessary. Boil it; do not overheat; do not autoclave. Cool to 48℃±2℃ and pour into the plate. The pH of the boiled culture medium at 25℃ is 7.4±0.2. A.7 Triple sugar iron (TSI) agar A.7.1 Ingredients A.7.2 Preparation method Add each ingredient to distilled water; mix well; heat to dissolve; and adjust pH if necessary. Dispense quantitatively into test tubes and autoclave at 115°C for 15 min. After sterilization, it is made into a slope; and the depth of the bottom layer is no less than 2.5cm. The pH of the sterilized culture medium at 25°C is 7.4 ± 0.2. A.8 Nutrient agar (NA) A.8.1 Ingredients A.8.2 Preparation method Add each ingredient to distilled water; mix well; heat to dissolve; adjust pH if necessary, and autoclave at 121°C for 15 min. The pH of the sterilized culture medium at 25°C is 7.3±0.2. A.9 Semi-solid agar A.9.1 Ingredients A.9.2 Preparation method Add each ingredient to distilled water; mix well; heat to dissolve; adjust pH if necessary; and autoclave at 121°C for 15 min. Cool to 48°C ± 2°C and pour into plates or into small glass tubes. The pH of the sterilized culture medium at 25°C is 7.4 ± 0.2. A.10 Peptone water, indole reagent A.10.1 Peptone water A.10.1.1 Ingredients A.10.1.2 Preparation method Add each ingredient to distilled water; mix well; heat to dissolve; and adjust pH if necessary. Divide into small test tubes and autoclave at 121°C for 15 min. The pH of the sterilized culture medium at 25°C is 7.4 ± 0.2. A.10.2 Indole reagent A.10.2.1 Kovacs reagent. Dissolve 5.0g of p-dimethylaminobenzaldehyde in 75mL of amyl alcohol; and then slowly add 25mL of concentrated hydrochloric acid. A.10.2.2 Ou-Bo reagent. Dissolve 1.0g of p-dimethylaminobenzaldehyde in 95mL of 95% ethanol; and then slowly add 20mL of concentrated hydrochloric acid. A.10.3 Test method Pick a small amount of culture medium and inoculate it in peptone water; and inoculate it at 36℃±1℃ for 24h~48h. Add about 0.5mL of Kovacs reagent and shake the test tube gently. If the reagent layer turns dark red, it is positive. Or take about 0.5mL of Ou-Bo reagent; flow it down along the wall of the tube; and cover the surface of the culture medium solution. If the contact point of the liquid surface turns rose red, it is positive. A.11 Urea agar (pH 7.2) A.11.1 Ingredients A.11.2 Preparation method Except for urea, add other ingredients to 900mL distilled water; mix well; heat to dissolve; and adjust pH if necessary. Autoclave at 121°C for 15 min. Cool to 48℃±2℃; add 100mL of filtered and sterilized 20% urea solution; and distribute it into sterile test tubes to make a slope. The pH of the sterilized culture medium at 25°C is 7.2±0.2. ......
Source: Above contents are excerpted from the full-copy PDF -- translated/reviewed by: www.ChineseStandard.net / Wayne Zheng et al.


      

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