GB 4789.4-2024 PDF in English
GB 4789.4-2024 (GB4789.4-2024) PDF English
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National Food Safety Standards--Food Microbiological Testing--Salmonella Testing
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Standards related to: GB 4789.4-2024
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GB 4789.4-2024: PDF in English GB 4789.4-2024
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
National Food Safety Standard – Food
Microbiological Examination – Examination of Salmonella
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.
2.1 Refrigerator: 2℃~8℃.
2.2 Constant temperature incubator: 36℃±1℃, constant temperature device: 42℃±1℃,
48℃±2℃.
2.3 Homogenizer.
2.4 Oscillators.
2.5 Balance: with sensitivity of 0.1g.
2.6 Sterile Erlenmeyer flask: with capacity of 500mL, 250mL.
2.7 Sterile graduated cylinder: with capacity of 50mL.
2.8 Sterile homogenization cups and sterile homogenization bags.
2.9 Sterile wide-mouth bottle: with capacity of 500mL.
2.10 Sterile pipette: 1mL (with scale of 0.01mL), 10mL (with scale of 0.1mL) or micropipette
and tip.
2.11 Sterile petri dishes: with diameter of 60mm, 90mm.
2.12 Sterile test tube: 10mm×75mm, 15mm×150mm, 18mm×180mm or other suitable
specifications.
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
NOTE: There may be differences in the composition, identification procedures and result judgment of
Salmonella diagnostic serum from different manufacturers. When using commercial Salmonella
diagnostic sera for serological identification, the product instructions shall be followed.
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.
Those who are not agglutinated by polyvalent O serum from A to F are first identified with 9
polyvalent O sera. If one of the sera is agglutinated, the O group sera included in this serum are
used to identify one by one to determine the O group. The O group sera included in each
polyvalent O serum are as follows:
O polyvalent 1: A, B, C, D, E, F groups (including groups 6 and 14)
O polyvalent 2: 13, 16, 17, 18, 21 groups
O polyvalent 3: 28, 30, 35, 38, 39 groups
O polyvalent 4: 40, 41, 42, 43 groups
O polyvalent 5: 44, 45, 47, 48 groups
O polyvalent 6: 50, 51, 52, 53 groups
O polyvalent 7: 55, 56, 57, 58 groups
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.
Appendix A
Culture Medium and Reagents
A.1 Buffered peptone water (BPW)
A.1.1 Ingredients
Peptone: 10.0g
Sodium chloride: 5.0g
Disodium hydrogen phosphate (containing 12 crystal waters): 9.0g
Potassium dihydrogen phosphate: 1.5g
Distilled water: 1000mL
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
Peptone: 9.0g
Beef dipping powder: 4.5g
Sodium chloride: 2.7g
Calcium carbonate: 40.5g
Sodium thiosulfate (containing 5 crystal waters): 50.0g
Ox bile salt: 5.0g
Distilled water: 1000mL
Add each ingredient to distilled water; stir well and heat to dissolve. Boil, no need to autoclave.
The pH of the boiled medium at 25°C is 7.6±0.2.
A.2.2 Iodine solution
Potassium iodide: 25.0g
Iodine: 20.0g
Distilled water: 100mL
Dissolve potassium iodide in a small amount of distilled water; add iodine; and shake until all
iodine is dissolved. Add distilled water to 100mL; transfer to a brown bottle; seal the bottle
tightly and store in refrigerator.
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
Soy peptone: 4.5g
Sodium chloride: 7.2g
Potassium dihydrogen phosphate: 1.26g
Dipotassium hydrogen phosphate: 0.18g
Magnesium chloride (containing 6 crystal waters): 28.6g
Malachite green: 0.036g
Distilled water: 1000mL
Phenol red: 0.08g
Agar: 15.0g
Distilled water: 1000mL
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
Peptone: 20.0g
Beef dipping powder: 5.0g
Lactose: 10.0g
Sucrose: 10.0g
Glucose: 1.0g
Phenol red: 0.025g
Sodium chloride: 5.0g
Ferrous ammonium sulfate (containing 6 crystal waters): 0.2g
Sodium thiosulfate: 0.2g
Agar: 12.0g
Distilled water: 1000mL
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.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
Peptone: 1.0g
Sodium chloride: 5.0g
Glucose: 1.0g
Potassium dihydrogen phosphate: 2.0g
Phenol red: 0.012g
Agar: 20.0g
Distilled water: 900mL
20% urea solution: 100mL
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.
A.11.3 Test method
Pick the culture medium and inoculate it on the urea agar slope; culture it at 36 ℃ ± 1 ℃ for
24 h; and observe the results. If the urea agar slope turns rose red, it is urease positive.
NOTE: Agar-free urea medium can also be used.
A.12 Potassium cyanide (KCN) culture medium
A.12.1 Ingredients
Peptone: 10.0g
Sodium chloride: 5.0g
Potassium dihydrogen phosphate: 0.225g
Disodium hydrogen phosphate: 5.64g
Distilled water: 1000mL
0.5% potassium cyanide: 20.0mL
A.12.2 Preparation method
Add ingredients other than potassium cyanide to distilled water; mix well; heat to dissolve; and
autoclave at 121°C for 15 min. After sufficient cooling, add 2.0 mL of newly prepared 0.5%
potassium cyanide solution (final concentration: 1:10000) to every 100 mL of culture medium;
distribute it into sterile test tubes; and immediately plug it tightly with a sterile tube stopper. At
the same time, the culture medium without adding potassium cyanide is used as the control
culture medium and is distributed for later use.
A.12.3 Test method
Use physiological saline to prepare a pure culture of the bacteria to be tested into a bacterial
suspension with a turbidity of 0.5 McFarland. Add 2 to 3 drops of the bacterial suspension to
the potassium cyanide (KCN) culture medium. Mix evenly and then add a layer of sterile liquid,
and seal with paraffin. In addition, add 2 to 3 drops of bacterial suspension to the control culture
medium. Incubate at 36℃±1℃ for 24h~48h and observe the results. If there is bacterial growth
in potassium cyanide (KCN) culture medium, it is positive (not inhibited), and if there is no
bacterial growth after 48 h of culture, it is negative (inhibited).
NOTE: Potassium cyanide is a highly toxic drug, so gloves shall be worn during operation to avoid
contamination. The main reason for test failure is false positive reactions caused by loose sealing.
A.13 Lysine decarboxylase test medium
A.13.1 Ingredients
A.14.3 Test method
Pick a small amount of culture medium and inoculate it into the sugar fermentation tube; culture
it at 36℃±1℃ for 24h~48h; and observe the results. If the culture medium turns yellow, it is
considered positive. For those who are suspected of delayed fermentation of lactose after 48 h
of culture, the culture can be continued for 3 ~ 5 days before observing the results.
A.15 O-Nitrophenol β-D galactopyranoside (ONPG) medium
A.15.1 Ingredients
o-Nitrophenol β-D galactopyranoside (ONPG): 60.0mg
0.01mol/L sodium phosphate buffer (pH 7.5): 10.0mL
1% peptone water (pH 7.5): 30.0mL
A.15.2 Preparation method
Dissolve ONPG in the buffer; add peptone water; filter and sterilize; then dispense into sterile
small test tubes and plug the tubes tightly.
A.15.3 Test method
Pick the culture medium and inoculate it into ONPG medium; culture it at 36℃±1℃ for 3 h
and observe the results. If the medium turns yellow, it is β-galactosidase positive. If the culture
medium does not change color, continue culturing for 24 h. If the culture medium turns yellow,
it is β-galactosidase positive, otherwise it is negative.
A.16 Sodium malonate medium
A.16.1 Ingredients
Yeast extract powder: 1.0g
Ammonium sulfate: 2.0g
Dipotassium hydrogen phosphate: 0.6g
Potassium dihydrogen phosphate: 0.4g
Sodium chloride: 2.0g
Sodium malonate: 3.0g
Bromothymol blue: 0.025g
Distilled water: 1000mL
......
Source: Above contents are excerpted from the PDF -- translated/reviewed by: www.chinesestandard.net / Wayne Zheng et al.
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