GB 4789.30-2025 PDF English
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National food safety standard - Food microbiological examination - Examination of Listeria monocytogenes
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| GB 4789.30-2016 | English | 115 |
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National food safety standard - Food microbiological examination - Examination of Listeria monocytogenes
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National food safety standard -- Food microbiological examination: Listeria monocytogenes
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| GB/T 4789.30-2008 | English | 679 |
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Microbiological examination of food hygiene -- Examination of listeria monocytogenes
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Microbiological examination of food hygiene -- Examination of listeria monocytogenes
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GB 4789.30-2025: National food safety standard - Food microbiological examination - Examination of Listeria monocytogenes
---This is an excerpt. Full copy of true-PDF in English version (including equations, symbols, images, flow-chart, tables, and figures etc.), auto-downloaded/delivered in 9 seconds, can be purchased online: https://www.ChineseStandard.net/PDF.aspx/GB4789.30-2025
GB
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
National food safety standard - Food microbiology test
Listeria monocytogenes test
Issued on. MARCH 16, 2025
Implemented on. SEPTEMBER 16, 2025
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 medium and reagents... 5
First method -- Qualitative test of Listeria monocytogenes... 6
4 Test procedure... 6
5 Operation steps... 7
6 Results and report... 10
Second method -- Plate count method for Listeria monocytogenes... 10
7 Test procedure... 10
8 Operation steps... 10
9 Results and report... 12
Third method -- MPN count method for Listeria monocytogenes... 14
10 Test procedure... 14
11 Operation steps... 15
12 Results and report... 15
Annex A Culture medium and reagents... 16
Annex B Table of most probable number (MPN) of Listeria monocytogenes... 27
Foreword
This Standard replaces GB 4789.30-2016 “National food safety standard - Food
microbiological test - Listeria monocytogenes test”.
Compared with GB 4789.30-2016, this Standard has the following major changes.
- MODIFY the scope of application;
- MODIFY the culture medium and reagents, and ADD the formula of agar Listeria
according to Ottaviani and Agosti;
- MODIFY the enrichment solution, selective culture medium, test procedure,
identification method, etc. of the first method -- Qualitative test of Listeria
monocytogenes;
- MODIFY the sample inoculation, colony count and confirmation, result count, and
result report of the second method -- Plate count method for Listeria
monocytogenes;
- MODIFY the sample inoculation of the third method -- MPN count method for
Listeria monocytogenes.
National food safety standards
Food microbiology test
Listeria monocytogenes test
1 Scope
This Standard specifies the test methods for Listeria monocytogenes in food.
The first method of this Standard applies to the qualitative test of Listeria
monocytogenes in food; the second method applies to the counting of Listeria
monocytogenes in food with a high content of Listeria monocytogenes; the third method
applies to the counting of Listeria monocytogenes in food with a low content of Listeria
monocytogenes.
2 Equipment and materials
In addition to routine sterilization and culture equipment of microbiology laboratories,
other equipment and materials are as follows.
2.1 Refrigerator. 2 ℃ ~ 8 ℃.
2.2 Constant temperature incubator. 30 ℃ ± 1 ℃, 36 ℃ ± 1 ℃, 25 ℃ ~ 30 ℃.
2.3 Homogenizer.
2.4 Microscope. 100× ~ 1000×.
2.5 Electronic balance. the sensitivity is 0.1 g, 0.1 mg.
2.6 Conical flask. 100 mL, 500 mL.
2.7 Sterile pipette. 1 mL (with 0.01 mL scale), 10 mL (with 0.1 mL scale) or pipette
(with a scale of 0.1 mL, 1 mL, 10 mL) and sterile pipette tip.
2.8 Sterile culture dish. diameter of 90 mm.
2.9 Sterile test tube. 16 mm × 160 mm.
2.10 Centrifuge. 4000 r/min.
2.11 Sterile centrifuge tube. 30 mm × 100 mm.
2.12 Sterile syringe. 1 mL.
2.13 Oil immersion lens or phase contrast microscope.
2.14 Sterile coating stick.
2.15 Listeria monocytogenes ATCC 19111 or CMCC 54004 or other equivalent strains.
2.16 Listeria innocua ATCC 33090 or other equivalent strains.
2.17 Listeria ivanovii ATCC 19119 or other equivalent strains.
2.18 Listeria seeligeri ATCC 35967 or other equivalent strains.
2.19 Staphylococcus aureus ATCC 25923 or other equivalent strains, requiring the
production of β-hemolytic ring.
2.20 Rhodococcus equi ATCC 6939 or NCTC 1621 or other equivalent strains.
3 Culture medium and reagents
3.1 Tryptic soy broth with 0.6 % yeast extract powder (TSB-YE). see A.1 in Annex A.
3.2 Tryptic soy agar with 0.6 % yeast extract powder (TSA-YE). see A.2.
3.3 Fraser enrichment broth (FB1, FB2). see A.3.
3.4 Agar Listeria according to Ottaviani and Agosti. see A.4.
3.5 PALCAM medium. see A.5.
3.6 Gram staining solution. see A.6.
3.7 SIM motility medium. see A.7.
3.8 Buffered glucose peptone water [for methyl red (MR) and acetyl methyl alcohol
(VP) tests]. see A.8.
3.9 Sheep blood agar. see A.9.
3.10 Sterile phosphate buffer. see A.10.
3.11 Sterile normal saline. see A.11.
3.12 Sugar fermentation tube. see A.12.
4 Test procedure
The qualitative test procedure for Listeria monocytogenes is shown in Figure 1.
5 Operation steps
5.1 Enrichment
Take 25 g (mL) of sample by aseptic operation, place it in a sterile homogenizing cup
containing 225 mL of FB1 enrichment broth, and homogenize at 8000 r/min ~ 10000
r/min for 1 min ~ 2 min, or place it in a sterile homogenizing bag containing 225 mL of
FB1 enrichment broth, and beat it with a slapping homogenizer for 1 min ~ 2 min to
make a 1.10 sample homogenate. If the sample is in liquid form, it can also be mixed
by oscillation or stirring. Culture at 30 ℃ ± 1 ℃ for 24 h ± 2 h. Mix well, pipette 0.1
mL of FB1 enrichment broth, and inoculate in 10 mL of FB2 enrichment broth. Culture
at 30 ℃ ± 1 ℃ for 24 h ± 2 h.
5.2 Separation
Take the mixed FB2 enrichment broth and inoculate on agar Listeria according to
Ottaviani and Agosti (or other equivalent Listeria chromogenic medium) plates and
PALCAM medium plates respectively, culture at 36 ℃ ± 1 ℃ for 24 h ~ 48 h, and
observe the colonies grown on each plate. Typical colonies form round blue-green
colonies with a diameter of 1 mm ~ 3 mm on agar Listeria according to Ottaviani and
Agosti plates, surrounded by opaque halos. Typical colonies form round gray-green
colonies with a diameter of 1 mm ~ 3 mm on PALCAM medium plates, surrounded by
brown-black hydrolysis circles. After 48 h of culture, some colonies form black spots
and depressions in the center. The characteristics of colonies on other equivalent
Listeria chromogenic medium plates shall be determined according to the product
instructions.
NOTE 1.Some Listeria monocytogenes colonies on agar Listeria according to Ottaviani and Agosti
have weak or no halos around them. There are also some Listeria monocytogenes colonies on agar
Listeria according to Ottaviani and Agosti have halos that appear slowly, sometimes taking more
than 4 days to appear.
NOTE 2.The colony morphology of Listeria ivanovii on agar Listeria according to Ottaviani and
Agosti is similar to that of Listeria monocytogenes.
5.3 Pure culture
Pick 3 to 5 typical or suspicious colonies (select all if less than 3) from each plate (plate
that meet the requirements of 5.2), streak on TSA-YE plates or sheep blood plates, and
culture at 36 ℃ ± 1 ℃ for 18 h ~ 24 h. Listeria monocytogenes on TSA-YE plates or
sheep blood plates are grayish white, translucent, with neat edges, dew drop-shaped
colonies, and a diameter of 1 mm ~ 2 mm.
5.4 Preliminary identification
Pick an individual colony on TSA-YE plates or sheep blood plates, inoculate in xylose
and rhamnose fermentation tubes, and culture at 36 ℃ ± 1 ℃ for 24 h ± 2 h; at the same
time, streak on TSA-YE plates or sheep blood plates, and culture at 36 ℃ ± 1 ℃ for 18
h ~ 24 h to obtain pure culture for the next step of identification. Then select the pure
culture that is xylose negative and rhamnose positive to continue identification.
5.5 Identification
NOTE. It can first pick one strain from each plate (plate that meets the requirements of 5.2) for
identification test. If it is identified as Listeria monocytogenes, the result of “detected” can be
directly reported according to the provisions of “6 Results and reports”; the result of “not detected”
can be reported only after 3 to 5 typical or suspicious colonies (select all if less than 3) selected
according to the requirements of 5.3 are identified as non- Listeria monocytogenes.
5.5.1 Microscopic examination. Pick an individual colony of pure culture for 18 h ~ 24
h for Gram staining microscopic examination. Listeria is a Gram-positive
brevibacterium with a size of (0.4 μm ~ 0.5 μm) × (0.5 μm ~ 2.0 μm). Use normal saline
to make a bacterial suspension, observe under an oil immersion lens or phase contrast
microscope, the bacteria show slight rotation or tumbling motion.
5.5.4 Hemolysis test. Divide the bottom of a fresh sheep blood agar plate into 20 ~ 25
small grids, pick an individual colony of pure culture for 18 h ~ 24 h and inoculate on
the blood plate, inoculate one colony per grid, and inoculate positive control bacteria
(Listeria monocytogenes, Listeria ivanovii and Listeria seeligeri) and negative control
bacteria (Listeria innocua). When puncturing, try to get as close to the bottom as
possible, but do not touch the bottom surface, and avoid agar rupture. Culture at 36 ℃
± 1 ℃ for 24 h ~ 48 h, and observe in a bright place.
5.5.5 Cooperative hemolysis test CAMP (optional). Streak Staphylococcus aureus and
Rhodococcus equi in parallel lines on sheep blood agar plates. Pick an individual colony
of pure culture for 18 h ~ 24 h and streak vertically between the parallel lines, the two
ends of the vertical line shall not touch the parallel lines and the distance is 1 mm ~ 2
mm. At the same time, inoculate Listeria monocytogenes, Listeria innocua, Listeria
ivanovii and Listeria seeligeri and culture at 36 ℃ ± 1 ℃ for 24 h ~ 48 h.
5.6 Mouse toxicity test (optional item)
Inoculate the pure culture that meets the above characteristics into TSB-YE, culture at
36 ℃ ± 1 ℃ for 24 h, centrifuge at 4000 r/min for 5 min, discard the supernatant, and
use sterile normal saline to prepare a bacterial suspension with a concentration of 1010
CFU/mL. Take this bacterial suspension and inject it intraperitoneally into 3 ~ 5 mice,
0.5 mL per mouse, and observe the death of the mice at the same time. If the mice die
within 2 d ~ 5 d, it is a pathogenic strain. In the test, set up a pathogenic strain of Listeria
monocytogenes and a sterile normal saline control group. Listeria monocytogenes and
Listeria ivanovii are pathogenic to mice.
6 Results and report
Combining the identification results of 5.4 and 5.5, report whether Listeria
monocytogenes is detected or not detected in 25 g (mL) of sample. If Listeria
monocytogenes is not detected in 25 g (mL) of sample, it can also be reported as 0/25 g (mL).
7 Test procedure
The plate count procedure for Listeria monocytogenes is shown in Figure 2.
8 Operation steps
8.1 Sample dilution
8.2 Sample inoculation
8.2.1 Based on the estimation of the sample contamination status, select 2 to 3 sample
homogenates with appropriate serial dilutions (liquid samples may include stock
solution), pipette 0.1 mL of sample homogenate for each dilution, and inoculate on 1
agar Listeria according to Ottaviani and Agosti (or other equivalent Listeria
chromogenic medium) plate. Use a sterile coating stick to coat the entire plate, being
careful not to touch the edge of the plate. Before use, if there are water droplets on the
surface of the agar plate, place it in an incubator at 25 ℃ ~ 50 ℃ to dry until the water
droplets on the surface of the plate disappear.
8.2.2 For food samples with low content of Listeria monocytogenes, pipette 1 mL of
the sample homogenate with lowest dilution and coat it on 3 agar Listeria according to
Ottaviani and Agosti (or other equivalent Listeria chromogenic medium) plates with
inoculation volumes of 0.3 mL, 0.3 mL, and 0.4 mL respectively. The coating method
is the same as 8.2.1.
8.3 Culture
8.3.1 Under normal circumstances, after coating, place the plate upright on a horizontal
table for 10 min ~ 20 min, then turn the plate over and place it in an incubator to culture
at 36 ℃ ± 1 ℃ for 24 h ~ 48 h.
8.4 Count and confirmation of typical colony
8.4.1 Select plates with typical or suspicious Listeria monocytogenes colonies. If.
9 Results and report
9.1 Count method
9.1.1 Formula (1)
9.2 Result report
9.2.1 When the number of colonies is less than 100 CFU, it shall be rounded off
according to the principle of “rounding off” and reported as an integer.
9.2.2 When the number of colonies is greater than or equal to 100 CFU, the third digit
shall be rounded off according to the principle of “rounding off”, and the first two digits
shall be taken, and the digits behind shall be replaced by 0.It can also be expressed in
the form of 10 exponentials, and two significant digits shall be retained after rounding
off according to the principle of “rounding off”.
9.2.3 Report the number of colonies of Listeria monocytogenes per g (mL) of sample,
expressed as CFU/g (mL). If the T value is 0, the method using the 0.1 mL inoculum
volume shall be reported as less than 10 multiplied by the lowest dilution factor. The
method using the 0.3 mL, 0.3 mL, 0.4 mL inoculum volumes shall be reported as less
than 1 multiplied by the lowest dilution factor.
10 Test procedure
The test procedure for MPN count method for Listeria monocytogenes is shown in
11 Operation steps
11.1 Sample dilution
Phosphate buffer is used as sample diluent. The sample dilution method is the same as 8.1.
11.2 Inoculation and culture
11.2.1 Based on the estimation of sample contamination status, select 3 sample
homogenates (liquid samples may include stock solution) with appropriate serial
dilutions and inoculate them in 10 mL of FB1 enrichment broth. Inoculate 3 tubes for
each dilution, and inoculate 1 mL in each tube. If the inoculation volume is 10 mL,
inoculate it in 10 mL of double-mix FB1 enrichment broth. Culture at 30 ℃ ± 1 ℃ for
24 h ± 2 h. Pipette 0.1 mL from each tube, inoculate to 10 mL of FB2 enrichment broth,
and culture at 30 ℃ ± 1 ℃ for 24 h ± 2 h.
11.2.2 Use inoculation loops to transfer 1 loop from each tube of FB2 enrichment broth,
inoculate on agar Listeria according to Ottaviani and Agosti (or other equivalent
Listeria chromogenic medium) plates, and culture at 36 ℃ ± 1 ℃ for 24 h ~ 48 h.
11.3 Confirmation test
Pick 3 ~ 5 typical or suspicious colonies (select all if less than 3) from each plate (plate
that meets the requirements of 5.2) and identify them according to 5.3, 5.4, and 5.5.
12 Results and report
Based on the number of test tubes confirmed to be positive for Listeria monocytogenes,
lookup the MPN retrieval table (see Annex B) and report the most probable number of
Listeria monocytogenes per gram (ml) of sample, expressed as MPN/g (mL).
......
Source: Above contents are excerpted from the full-copy PDF -- translated/reviewed by: www.ChineseStandard.net / Wayne Zheng et al.
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