GB 4789.30-2025National food safety standard - Food microbiological examination - Examination of Listeria monocytogenes (English PDF)

Foreword

This document was issued on 16 March 2025 by the State Administration for Market Regulation; Standardization Administration of the PRC and takes effect on 16 September 2025.

It is a GB standard without the /T suffix: compliance is mandatory in China.

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

GB 4789.30-2025 is the Chinese national food safety standard for detecting Listeria monocytogenes in food. Listeria is the pathogen that makes ready-to-eat food dangerous rather than merely spoiled: it grows at refrigeration temperature, which almost nothing else does, so a low count at production becomes a high one by the end of shelf life, and in pregnant women and the immunocompromised the case fatality is among the highest of any foodborne infection. The standard sets the equipment and materials, the culture media and reagents, and then the examination procedures, operating steps and reporting for the qualitative detection, the enumeration and the confirmation, in each case through selective enrichment, isolation on selective agar and biochemical and molecular confirmation. It replaces GB 4789.30-2016 and takes effect on 16 September 2025.

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 degrees C ~ 8 degrees C.

2.2 Constant temperature incubator. 30 degrees C +/- 1 degrees C, 36 degrees C +/- 1 degrees C, 25 degrees C ~ 30 degrees C.

2.3 Homogenizer.

2.4 Microscope. 100x ~ 1000x.

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 x 160 mm.

2.10 Centrifuge. 4000 r/min.

2.11 Sterile centrifuge tube. 30 mm x 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 beta-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 degrees C +/- 1 degrees C 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 degrees C +/- 1 degrees C 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 degrees C +/- 1 degrees C 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 degrees C +/- 1 degrees C 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 degrees C +/- 1 degrees C for 24 h +/- 2 h; at the same

time, streak on TSA-YE plates or sheep blood plates, and culture at 36 degrees C +/- 1 degrees C 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 um ~ 0.5 um) x (0.5 um ~ 2.0 um). 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 degrees C

+/- 1 degrees C 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 degrees C +/- 1 degrees C 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 degrees C +/- 1 degrees C 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 degrees C ~ 50 degrees C 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 degrees C +/- 1 degrees C 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 degrees C +/- 1 degrees C for

24 h +/- 2 h. Pipette 0.1 mL from each tube, inoculate to 10 mL of FB2 enrichment broth,

and culture at 30 degrees C +/- 1 degrees C 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 degrees C +/- 1 degrees C 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).

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