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GB 4789.4-2024: National Food Safety Standards--Food Microbiological Testing--Salmonella Testing
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...