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GB 4789.29-2020: National food safety standard - Food microbiological examination - Burkholderia gladioli
GB 4789.29-2020
GB
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
National food safety standard - Food microbiological
examination - Burkholderia gladioli
ISSUED ON: SEPTEMBER 11, 2020
IMPLEMENTED ON: MARCH 11, 2021
Issued by: National Health Commission of the PRC;
State Administration for Market Regulation.
Table of Contents
Foreword ... 3 
1 Scope ... 4 
2 Apparatus and materials ... 4 
3 Media and reagents ... 5 
4 Examination procedure ... 5 
5 Operation procedure ... 6 
6 Result report ... 10 
Appendix A Media and reagents ... 11 
National food safety standard - Food microbiological
examination - Burkholderia gladioli
1 Scope
This Standard specifies the examination method of Burkholderia gladioli
(Pseudomonas cocovenenans subsp. farinofermentans) in the food.
This Standard applies to the examination of Burkholderia gladioli
(Pseudomonas cocovenenans subsp. farinofermentans) in the food.
2 Apparatus and materials
2.1 Routine laboratory sterilization equipment.
2.2 Refrigerator: 2 °C~8 °C, -20 °C~-30 °C.
2.3 Constant-temperature incubator: 26 °C±1 °C, 36 °C±1 °C.
2.4 Constant-temperature water bath: 46 °C±1 °C.
2.5 Microscope: 10 times~100 times.
2.6 Homogenizer.
2.7 Centrifuge: 3000 r/min.
2.8 Electronic balance: Sensitivity is 0.1 g.
2.9 Nephelometer.
2.10 Conical flask: Capacity is 100 mL and 500 mL.
2.11 Sterile petri dish: Diameter is 90 mm and 150 mm.
2.12 Sterile transparent cellophane, filter paper.
2.13 Sterile gastric lavage device: 1 mL.
2.14 Microbial biochemical identification system.
2.15 Mice: 18 g~20 g. Each batch of test shall use the same strain of KM or ICR
saline is used as a control. For those agglutinating with multivalent serum, use
O-III, O-IV, O-V, O-VI, O-VII, O-VIII factor serum in order, to do test tube
agglutination test. According to the test results, determine the somatic antigen
type. Those self-coagulating in normal saline cannot be typed. For those which
meet the biochemical characteristics but cannot agglutinate with the above
serum, the strains need to be retained for further identification.
5.7 Toxicity test
5.7.1 Toxin-producing cultivation
Inoculate the strain, preliminarily identified as Burkholderia gladioli
(Pseudomonas cocovenenans subsp. farinofermentans), on a PDA plate;
CULTURE at 36 °C±1 °C for 24 h±2 h. USE a sterile inoculation loop to scrape
an appropriate amount of lawn; ADD it to a test tube of 3 mL of sterile normal
saline; prepare a 1 McFarland (MCF) bacterial suspension (approximately 108
CFU/mL). USE a sterile straw to suck up 0.5 mL; DROP it on a potato dextrose
semi-solid plate with a diameter of 150 mm spread with sterile cellophane; USE
a sterile L bar to spread evenly; incubate at 26 °C±1 °C for 5 d. REMOVE the
cellophane that carries bacteria; PLACE the semi-solid plate at 100 °C flowing
steam for 30 min of sterilization. After cooling at room temperature, place it in a
refrigerator at -20 °C~-30 °C overnight. THAW the frozen semi-solid plate at
room temperature; USE a sterile straw to suck out the freeze-thaw solution;
filter it through a filter paper into a sterile test tube or conical flask (this is the
crude toxin extract); STORE it at 4 °C in the dark.
At the same time, use a potato dextrose semi-solid plate with a diameter of 150
mm spread with sterile cellophane without inoculation of lawn as a negative
control. According to the same test method, prepare the negative control crude
extract and store at 4 °C in the dark.
5.7.2 Determination of virulence
TAKE the crude toxin extract, or the 5 ~ 10 times concentrated solution after
evaporation in a 100 °C water bath; gavage 3 mice, each with 0.5 mL; observe
for 7 d. If the strain produces bongkrekic acid, the mice will become ill within 20
min~24 h after gavage. The main symptoms are erected hair, flagging, and
restlessness, followed by staggering, numbness, paralysis, convulsions,
opisthotonos, rapid breathing, and death.
TAKE the negative control crude extract, or the 5 ~ 10 times concentrated
solution after evaporation in a 100 °C water bath; gavage 3 mice, each with 0.5
mL; observe for 7 d. The mice shall survive healthy.
5.7.3 Determination of bongkrekic acid
Appendix A
Media and reagents
A.1 GVC enrichment solution
A.1.1 Potato dextrose water (PD water)
A.1.1.1 Composition
Potato (peeled): 300 g
Dextrose: 20 g
Distilled water: 1000 mL
pH: 7.0±0.2
A.1.1.2 Preparation method
WEIGH 300 g of peeled potatoes and chop them into pieces; ADD 1000 mL of
distilled water; BOIL for 10 min~20 min. USE gauze to filter and add distilled
water to 1000 mL. ADD dextrose, heat to melt, dispense; autoclave at 121 °C
for 20 min.
A.1.2 Gentian violet aqueous solution
A.1.2.1 Composition
Gentian violet: 0.1 g
Distilled water: 100 mL
A.1.2.2 Preparation method
TAKE 0.1 g of gentian violet; USE a small amount of distilled water to dissolve;
ADD distilled water, to dilute to 100 mL; STORE it in a brown bottle. Filter and
sterilize before use.
A.1.3 Chloramphenicol solution
A.1.3.1 Composition
Chloramphenicol: 20.0 mg
Distilled water: 10 mL
Polymyxin sulfate B: 50000 U
Lincomycin: 30000 U
A.3.4 Preparation method
After heating, melting and cooling the PCFA basal medium to 50 °C, add
optional additives; MIX well and pour it into a sterile petri dish for later use.
A.4 Potato dextrose semi-solid agar
A.4.1 Composition
Potato (peeled): 300 g
Dextrose: 20 g
Agar: 5 g
Distilled water: 1000 mL
pH: 7.0±0.2
A.4.2 Preparation method
WEIGH 300 g of peeled potatoes and chop them into pieces; ADD 1000 mL of
distilled water; BOIL for 10 min~20 min. USE gauze to filter and add distilled
water to 1000 mL. ADD dextrose and agar, heat to melt, dispense; autoclave at
121 °C for 20 min.
A.5 Yolk agar
A.5.1 Composition of the basal medium
Meat infusion: 1000 mL
Peptone: 15 g
Sodium chloride: 5 g
Agar: 25 g~30 g
pH: 7.0±0.2
A.5.2 50% yolk saline suspension
A.5.3 Preparation of yolk agar
Prepare basal medium and pack 100 mL per bottle. Autoclave at 121 °C for 15
A.6.4 Staining method
A.6.4.1 FIX the smear on the flame; ADD dropwise crystal violet staining
solution, to stain for 1 min; and use water to wash.
A.6.4.2 ADD dropwise Gram iodine solution for 1 min and use water to wash.
A.6.4.3 ADD 95% ethanol dropwise to decolorize, about 15 s to 30 s, until the
staining solution is washed away. Do not over decolorize. USE water to wash.
A.6.4.4 ADD the counterstaining solution dropwise; counterstain for 1 min. USE
water to wash, wait to dry; inspect under a microscope.
A.7 Oxidase reagent
A.7.1 Composition
N, N, N', N'-tetramethyl-p-phenylenediamine hydrochloride: 1.0 g
Distilled water: 100.0 mL
A.7.2 Preparation method
Prepare a small amount freshly; STORE in the refrigerator away from light; and
use within 7 d.
A.7.3 Test method
TAKE a single characteristic colony; SPREAD it on filter paper moistened with
oxidase reagent. If the filter paper does not turn purplish red, purple or dark
blue within 10 s, the oxidase test is negative. Otherwise, the oxidase test is
positive.
Note: Do not use nickel/chromium materials in the test.
A.8 Hugh-Leifson medium (for O/F test)
A.8.1 Composition
Peptone: 2 g
Sodium chloride: 5 g
Dipotassium hydrogen phosphate: 0.3 g
Agar: 4 g
Dextrose: 10 g
A.9.3.1 K...