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GB 4789.30-2016: National Food Safety Standard -- Food Microbiological Examination -- Examination of Listeria Monocytogenes
GB 4789.30-2016
GB
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
National food safety standard - Food microbiological
examination - Examination of Listeria monocytogenes
ISSUED ON. DECEMBER 23, 2016
IMPLEMENTED ON. JUNE 23, 2017
Issued by. National Health and Family Planning Commission;
China Food and Drug Administration.
Table of Contents
Foreword ... 3 
1 Scope ... 4 
2 Equipment and materials ... 4 
3 Media and reagents ... 5 
4 Testing procedures ... 6 
5 Operation procedures ... 6 
6 Test procedures ... 9 
7 Operation procedures ... 9 
8 Result count ... 11 
9 Results report ... 12 
10 Testing procedures ... 12 
11 Operation procedures... 13 
12 Results and reports ... 14 
Appendix A Medium and reagents ... 15 
Appendix B Listeria monocytogenes most probable number (MPN) search table
... 23 
National food safety standard - Food microbiological
examination - Examination of Listeria monocytogenes
1 Scope
This standard specifies the test method for Listeria monocytogenes in food.
The first method of this standard is applicable to the qualitative test of Listeria
monocytogenes in food; the second method is applicable to the counting of
Listeria monocytogenes in foods with higher Listeria monocytogenes; the third
method is applicable to the count of Listeria monocytogenes in foods with low
Listeria monocytogenes (< 100 CFU/g) and high bacterial content, especially
milk, water and food containing particulate matter which interferes with the
counting of colonies.
2 Equipment and materials
In addition to routine sterilization and culture equipment in microbiology
laboratories, other equipment and materials are as follows.
2.1 Refrigerator. 2 °C ~ 5 °C.
2.2 Constant temperature incubator. 30 °C ± 1 °C, 36 °C ± 1 °C.
2.3 Homogenizer.
2.4 Microscope. 10 x ~ 100 x.
2.5 Electronic balance. the sensitivity is 0.1 g.
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
micropipette and tip.
2.8 Sterile plate. 90 mm in diameter.
2.9 Sterile test tube. 16 mm × 160 mm.
2.10 Centrifuge tube. 30 mm × 100 mm.
2.11 Sterile syringe. 1 mL.
HOMOGENIZE it at 8000 r/min ~ 10000 r/min for 1 min ~ 2 min. CULTURE it at
30 °C ± 1 °C for 24 h ± 2 h, TAKE 0.1 mL, TRANSFER it to 10 mL of LB2
monocytogenes solution, CULTURE it at 30 °C ± 1 °C for 24 h ± 2 h.
5.2 Separation
TAKE the LB2 secondary monocytogenes solution, INOCULATE it onto the
Listeria chromogenic plate and the PALCAM agar plate in a line shape,
CULTURE it at 36 °C ± 1 °C for 24 h ~ 48 h, OBSERVE the colonies growing
on each plate. Typical colonies are small round gray-green colonies on
PALCAM agar plates with brown-black hydrolyzed circles around them, some
colonies have black depressions; colony characteristics on Listeria color-
developing plates are determined by reference to product specifications.
5.3 Initial screening
TAKE to five typical or suspect colonies from selective agar plates, respective
INOCULATE them into the xylose and rhamnose fermentation tubes, CULTURE
it at 36 °C ± 1 °C for 24 h ± 2 h, meanwhile MARK a line on the TSA-YE plate,
CULTURE it at 36 ° C ± 1 ° C for 18 h ~ 24 h, then SELECT the pure culture of
xylose-negative and rhamnose-positive for further identification.
5.4 Identification (or selection of biochemical identification kits or fully
automated microbial identification systems, etc.)
5.4.1 Staining microscopy. Listeria monocytogenes is a Gram-positive
Brevibacterium, the size is (0.4 μm ~ 0.5 μm) × (0.5 μm ~ 2.0 μm); the bacterial
suspension is made with physiological saline, which is observed in oil mirror or
under the phase contrast microscope, the bacteria shows slight rotation or
rolling-like motion.
5.4.2 Dynamic test. PICK a single suspicious colony to puncture semi-solid or
SIM dynamic medium in pure culture, CULTURE it at 25 °C ~ 30 °C for 48 h.
Listeria is motivated and has an umbrella shape growth above semi-solid or
SIM medium. If the umbrella growth is not obvious, it can continue to culture it
for 5 d, then observe the results.
5.4.3 Biochemical identification. PICK the single suspicious colony in pure
culture to perform catalase test. Colonies with positive catalase reaction is
continued to be subject to sugar fermentation test and MR-VP test. The main
biochemical characteristics of Listeria monocytogenes are as shown in Table 1.
5.4.4 Hemolysis test. DIVIDE the bottom surface of fresh sheep blood agar
plate into 20 ~ 25 small cells, PICK a single suspicious colony of pure culture,
INOCULATE it onto the blood plate by puncture, one colony per cell, and
INOCULATE the positive control bacteria by puncture (Listeria monocytogenes,
Listeria ivanovii and Listeria seeligeri) and negative control bacteria (Listeria
without additives, HOMOGENIZE it continuously for 1 min ~ 2 min on a tapping
homogenizer, or HOMOGENIZE it for 1 min ~ 2 min at 8000 r/min ~ 10000 r/min.
SHAKE and MIX the liquid sample uniformly, to make it into 1.10 sample
homogenate.
7.1.2 USE a 1 mL sterile pipette or micropipette to pipette 1 mL of 1.10 sample
homogenate, along the tube wall, INJECT it slowly into a sterile test tube which
contains 9 mL of buffered peptone or LB broth without additives (note avoiding
the pipette or tip end from touching the dilution solution surface), SHAKE the
test tube or USE another 1 mL sterile pipette to repeatedly mix it uniformly, to
prepare it into 1.100 sample homogenate.
7.1.3 PREPARE 10 times serial dilution sample homogenate in accordance with
the procedure of 7.1.2. For each incremental dilution, replace one 1 mL sterile
pipette or tip.
7.2 Inoculation of samples
In accordance with the estimation of the contamination status of the sample,
SELECT 2 ~ 3 sample homogenate of appropriate continuous dilution (the liquid
sample may include the original solution), respectively TAKE 1 mL of each
dilution of the sample homogenate, at the inoculation amount of 0.3 mL, 0.3 mL,
0.4 mL, respectively ADD it onto three Listeria chromogenic plates, USE the
sterile L bar to cover it over the entire plate, PAY attention to avoiding touching
the plate edge. Before use, if there is water drops on the surface of the agar
plate, it can be dried in an incubator at 25 °C ~ 50 °C until the water drops on
the surface of the plate disappear.
7.3 Culture
7.3.1 Under normal circumstances, after coating, ALLOW the plate to stand for
10 min. If the sample solution is not easily absorbed, it may place the plate in
an incubator at 36 °C ± 1 °C for 1 h; after the sample homogenate is absorbed,
INVERT the plate, PLACE it into the incubator upside down, to culture it at 36 °C
± 1 °C for 24 h ~ 48 h.
7.4 Typical colony count and confirmation
7.4.1 Colony characteristics of Listeria monocytogenes on Listeria chromogenic
plates are subject to product description.
7.4.2 SELECT a plate which has typical Listeria monocytogenes colonies, and
the total number of colonies in the same dilution of 3 plates between 15 CFU
and 150 CFU, COUNT the number of typical colonies. In case.
a) The plate colony count of only one dilution is between 15 CFU ~ 150 CFU
and there is typical colony, it shall count the typical colonies on the plate
Appendix A
Medium and reagents
A.1 Tryptic soy broth (TSB-YE) containing 0.6% yeast extract
A.1.1 Ingredients
Tryptone 17.0 g
Polyad tryptone 3.0 g
Yeast extract...