SN/VT 4684-2016

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This standard was drafted in accordance with the rules given in GB/T 1.1-
2009.

Please note that some content of this document may be patented. The issuing
authority of this document assumes no responsibility for identifying these
patents.
This standard is proposed and managed by the National Certification and
Accreditation Administration Commission.

The main drafting unit of this standard: Shandong Exit and Human
Environment Inspection and Quarantine Bureau of the People's Republic of
China. Perfect (China) Co., Ltd.

The main drafters of this standard: Zhao Ha, Cui Shuhua, Sun Jun, Hao Ying,
Tian Guoning. Wu Xinghai. Gao Yecheng, Jia Juntao, Duan Xiaohui. Li
Jianjun.

1 Scope

This standard specifies the detection method for Burkholderia cepacia in

imported and exported cosmetics.
This standard applies to the qualitative detection of Burkholderia cepacia
in imported and exported cosmetics.

2 Normative references

The following documents are essential for the application of this document.
For dated references, only the dated version applies to this article
pieces. For undated references, the latest edition (including all
amendments) applies to this document.

GB 7918.1 Microbiological Standard Test Methods for Cosmetics "General

GB 19489 General Requirements for Laboratory Biosafety

3 Equipment and materials

In addition to the routine sterilization and culture equipment in the

microbiology laboratory, other equipment and materials are as follows.
3.1 Human constant temperature incubator: 30C±11C.32C+1C.36CLtlTC.
3.2 Homogenizer.
3.3" Razor.
3.4 Optical microscope.
3.5 Balance: Sensitive amount 0.1 g.
3.6 Sterile pipettes: 1 mL, 10 mL (with 0.1 mL scale) or micropipettes and
tips.
3.7" sterile culture four: 90 mm in diameter (or other commercial
disposable sterile flats).
3.8 Inoculation loop: 3 mm diameter.
3.9 Homogenizing bags or bottles.
3.10 Fully automatic microbial biochemical identification system.

4" media and reagents

4.1 SCDLP enrichment solution containing polymyxin: see A.1.

4.2 BCSA enrichment solution containing gentamicin: see A.2.

4.3 BCSA agar containing polymyxin B. gentamicin: see A.3.

4.4 MacConkey agar (without crystal violet) containing polymyxin B.

gentamicin: see A.4.
4.5 Gram stain: see A.5.

4.6 Oxidase test reagent: see A.6.

4.7 Glucose oxidation/fermentation tube: see A.7.

4.8" Mangose Oxidation Medium: See A.8.

4.9 Simon's citrate medium: see A.9.

4.10 Aesculus medium: see A.10.

4.11 Biochemical identification kit.

5" inspection procedure

The Burkholderia cepacia inspection procedure is shown in Figure 1.

sample
10 g (mL) sample + 90 mL dilution

1 mL + 10 mL BCSA

BCSA agar <contains polymyxin B, gentamicin) <contains polymyxin B,

gentamicin)

Figure 1 Burkholderia cepacia inspection procedure

6 Operation steps

6.1 Enrichment culture

Prepare a 1:10 cosmetic dilution according to the method of GB 7918.1, take

10 mL of the dilution and add it to 90 mL of polymyxin B-containing
In SCDLP enrichment solution, culture at 36 cc ± 1 cc for 24 h, pipette 1
mL, and transfer it into 10 mL of BCSA enrichment solution containing
gentamicin.
Cultured at 36°C ± 1 CC for 18 h to 24 h.

6.2 Isolation and culture

Take a loop of BCSA enrichment solution with an inoculating loop, and

streak it on BCSA agar (containing polymyxin B, gentamicin) plate and
McConnell.
2
Kay agar (containing polymyxin B, gentamicin) plate, cultured at 32 people
C ± 1 for 48 h, and observed the colonies growing on each plate. Typical
bacteria
On the BCSA agar plate, it is a red round colony, the surface is smooth,
and the surrounding medium turns red; the typical colony on the MacConkey
agar plate is
Light yellow-brown round colonies with smooth surface and no discoloration
of the surrounding medium.

6.3 Identification
6.3.1 First time

More than 5 typical or suspicious colonies were picked from the selective
agar plates, and the same strain was inoculated into 2 glucose
oxidation/fat cells.
One tube was sealed with sterilized liquid paraffin, the other was not
sealed, and incubated at 36 nits and 1 cc for 24 h, both of which turned
yellow and became glucose.
Type, without adding Shixia tube becomes yellow but adding paraffin tube
does not change color is glucose oxidation type. At the same time, streak
purification on nutrient agar plates at 36 ℃
One person was cultured for 24 h. Select the glucose oxidizing type (add
stone nutrients to continue the identification.

6.3.2" Gram stain

Pick suspicious colony smears for Gram staining. under the microscope
Bacillus.

6.3.3 Oxidase test

Pick a single with a glass rod or disposable needle

Holder bacteria were positive for delayed oxidase (purple,
NOTE: Never use nickel/chromium materials in experiments.

6.3.4 Catalase test

Pick a single special 3% hydrogen peroxide solution with a glass rod or a

disposable inoculating needle (for temporary use)
Preparation) 2 mL, observe the bubble generation. Bubbles appear in the
onion.

6.3.5" Sucrose Oxidation

Pick suspicious colonies and inoculate them in sucrose oxidation culture. 7

colors are positive, blue is negative.
Burkholderia cepacia was positive.

6.3.6" Citrate Utilization

Pick suspicious colonies and inoculate them on Simon's citrate citrate

medium and turn blue as positive, light green
is negative. Burkholderia cepacia was positive.

6.3.7 "Aesculin Utilization

The suspicious colonies were picked and inoculated into Aesculus medium,
and cultured for 24 h at 30 nits per person. If the medium turned black, it
was positive, and if it did not change color, it was negative.
Burkholderia cepacia was positive.
6.3.8 "Substitute Test

Biochemical identification kits or fully automatic microbial biochemical

identification systems can be selected, and according to the preliminary
judgment results in 6.3.
identification.

Gram-negative bacteria without budding

9 filter paper plates. Onion Burke

10 min.

7 Results report

Based on the above test results, it was reported that Burkholderia cepacia
was detected or not detected in 10 gCmL) samples.

8 Waste disposal

The waste in the testing process shall be disposed of in accordance with

the relevant requirements of GB 19489.

Appendix A
(normative appendix)
Media and Reagents
A.1 SCDLP enrichment solution containing polymyxin B
A.1.1 Composition
Casein and 17 g
Soy Protein Anal 3g
Sodium 58g
Dipotassium phosphate 2.5 g
Glucose 2.5 g
Lecithin 1 g
Tween 80 7g
Distilled water 1 000 mL
A.1.2 Production method

After mixing the above ingredients, heat to dissolve, adjust the pH to

7.2±0.1, divide into packs, sterilize by autoclaving at 121 Y for 20 min,
and set aside for later use.
Filter sterilization method to add polymyxin B to make the final
concentration of 250 000 UVL.

A.2 BCSA enrichment solution containing gentamicin

A.2.1 Composition

Coolin Anal
Yeast extract
Sodium chloride
lactose

Phenol red

Chang
purple crystal
distilled water
A.2.2 Production method

10g
1.5g

5g

10g

10g
0.08 g
0.002 g

1 000 mL

. Before use, use

After mixing the above ingredients, heat to dissolve, adjust the pH to 7.0
± 0.2, pack in aliquots, sterilize by autoclaving at 115 nits for 15 min,
and set aside. Before use, use
Gentamicin was added by filtration sterilization method to make the final
concentration of 10 mg/L.

A.3 BCSA agar containing polymyxin B. gentamicin

A.3.1 Composition
Coolin Anal

10g

A.5.2 "Gram iodine solution

A.5.2.1 Composition
Iodine 1.0g
Potassium iodide 2.0g
Distilled water 300 mL

A.5.2.2 Preparation method

Mix iodine and potassium iodide first, add a little distilled water and
shake well. After it is completely dissolved, add distilled water to 300
mL.
A.5.3 Sand Yellow Counterstain Solution
A.5.3.1 Composition

95% ethanol
distilled water

A.5.3.2 Preparation method

Dissolve sand yellow in ethanol, then distill
A.5.4 Dyeing method

The operation steps are as follows:

a) . The smear is fixed on the flame, and the crystals are added dropwise
b) Add gram iodine solution dropwise for 1 min
c) Add 95% ethanol dropwise to decolorize for about 15 s-
d) Add counterstain solution dropwise, counterstain for 1 min, water;

A.6 Oxidase test reagent

A.6.1 Composition
Tetramethyl-p-phenylenediamine hydrochloride 1 g
100 mL of distilled water
A.6.2 Preparation method

Dissolve tetramethyl-p-phenylenediamine hydrochloride in distilled water.

Freshly prepared reagents should be used, if placed in a refrigerator, they
should be prepared after preparation
Use within 7 days.

A.7 Glucose oxidation/fermentation tube

A.7.1 Composition
Protein Anal 1g
Beef Xiang 0.3 g

Sodium oxide 0.5g

Bromocresol Violet 0.004 g

Distilled water 100 mL

A.7.2 Manufacturing method

Heat and dissolve all components except the indicator, adjust the pH to
7.0±0.2 after cooling, add cresyl violet, dissolve fully, add 0.5 g
Glucose, mixed and dispensed into small test tubes, and autoclaved at 115 Y
for 15 min.

A.8 Sucrose oxidation medium

A.8.1 Composition
Protein cell 0.2 g
Sodium oxide 0.5 g
Dipotassium phosphate 0.03 g
0.5 flash thymol blue solution 0.5 mL
Distilled water 100 mL
A.8.2 Manufacturing method

Heat and dissolve all the components except the indicator, adjust the pH to
7.0 ± 0.2 after cooling, add thymol blue solution, 121 it is autoclaved.
Bacteria for 15 min, before use, add sucrose solution by filter
sterilization method to make the final concentration 1%, and aseptically
divide into small test tubes.

A.9 Simon's Citrate Medium

A.9.1 Composition
Sodium citrate 0.5g
Sodium oxide 0.5 g
Dipotassium hydrogen phosphate 0.1 g
Magnesium dihydrogen phosphate 0.1g
Magnesium sulfate (MgSO,.7H:O) 0.02 g
0.5 flash thymol blue solution 1.6 mL
Distilled water 100 mL

A.9.2 Manufacturing method

Heat and dissolve all the components except the indicator, adjust the pH to
7.0±0.2 after cooling, add the thymol blue solution, mix well and dispense
into small batches.
The test tube was sterilized by autoclaving at 121 min for 15 min.

A.10 Aesculus medium

A.10.1 Composition
Trypsin Anal 0.5 g
Beef Xiang 0.3 g
Aesculus 0.1 g

0.05g

Distilled water 100 mL

A.10.2 Preparation method

Heat and dissolve the above components, adjust the pH to 7.0±0.2 after
cooling, divide into small test tubes, and sterilize by autoclaving at 115
nits for 15 min.