SN/T 4684-2016 in English

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This standard is developed in accordance with the rules given in GB/T 1.1-2009.

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Determination of Burkholderia cepacia in cosmetics for import and export

1 Scope

This standard specifies the determination of Burkholderia cepacia in cosmetics for import and export.

It is applicable to the qualitative determination of Burkholderia cepacia in cosmetics for import and export.

2 Normative references

The following referenced documents are indispensable for the application of this document. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies.

GB 7918.1 Standard methods of microbiological examination for cosmetics - General rules
GB 19489 Laboratories - General requirements for biosafety

3 Equipment and materials

In addition to the conventional sterilization and culture equipment in microbiological laboratory, other equipment and materials are as follows.

3.1 Thermostatic incubator: 30℃±1℃, 32℃±1℃ and 36℃±1℃.

3.2 Homogenizer.

3.3 Oscillator.

3.4 Optical microscope.

3.5 Balance: with sensibility of 0.1g.

3.6 Aseptic pipette: 1mL, 10mL (0.1mL-scale) or micropipettor and sucker.

3.7 Aseptic culture dish: 90mm in diameter (or other commercial disposable aseptic culture dish).

3.8 Inoculation ring: 3mm in diameter.

3.9 Homogeneous bags or bottles.

3.10 Full-automatic microbe biochemical identification system.

4 Culture media and reagents

4.1 SCDLP enrichment broth containing polymyxin B: see A.1.

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

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

4.4 McConaughey agar containing polymyxin B and gentamicin (excluding crystal violet): see A.4.

4.5 Gram staining solution: see A.5.

4.6 Oxidase test reagent: see A.6.

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

4.8 Sucrose oxidation medium: see A.8.

4.9 Simmons citrate medium: see A.9.

4.10 Esculin medium: see A.10.

4.11 Biochemical identification kit.

5 Determination procedures

See Figure 1 for the determination procedures of Burkholderia cepacia.



Sample to be tested
10g(mL) sample + 90mL diluent


10mL + 90mL SCDLP enrichment broth (containing polymyxin B)


1mL + 10mL BCSA enrichment broth (containing gentamicin)





BCSA agar (containing polymyxin B and gentamicin) MacConkey agar (containing polymyxin B and gentamicin)




Select more than 5 suspicious colonies


Carry out biochemical test, or select biochemical identification kit or full-automatic microbe biochemical identification system


Report

Figure 1 Determination procedures of Burkholderia cepacia

6 Operation steps

6.1 Enrichment culture

Prepare 1:10 cosmetic diluent according to GB 7918.1 and add 10mL of diluent to 90mL of SCDLP enrichment broth containing polymyxin B, then culture at 36℃±1℃ for 24h, transfer 1mL of the obtained solution to 10mL of BCSA enrichment broth containing gentamicin, and culture at 36℃±1℃ for 18h to 24h.

6.2 Isolated culture

Take a loop of BCSA enrichment broth with an inoculating loop respectively, subject it to streak inoculation on BCSA agar (containing polymyxin B and gentamicin) plate and MacConkey agar (containing polymyxin B and gentamicin) plate, and culture at 32℃±1℃ for 48h to observe the colonies growing on each plate. Typical colonies are red round colonies on BCSA agar plate and are smooth-faced, with the surrounding culture medium turns red; they are light yellow-brown round colonies on McConnell agar plate and are smooth-faced, with the color of surrounding culture medium unchanged.

6.3 Identification

6.3.1 Primary screening

Pick more than 5 typical or suspicious colonies respectively from the selective agar plate. Inoculate the same strain into two glucose oxidation/fermentation tubes, seal one tube with sterilized liquid paraffin and do not seal the other tube. Then culture at 36℃±1℃ for 24h. If two tubes turn to yellow, it is glucose fermentation type; if the color of tube sealed with paraffin is not changed but the other tube turns to yellow, it is glucose oxidation type. At the same time, streak and purify on nutrient agar plate and culture at 36℃±1℃ for 24h. Select the pure culture of glucose oxidation type (the color of tube sealed with paraffin is not changed and the other tube turns to yellow) for further identification.

Foreword i
1 Scope
2 Normative references
3 Equipment and materials
4 Culture media and reagents
5 Determination procedures
6 Operation steps
7 Result report
8 Disposal of waste
Annex A (Normative) Culture media and reagents
GB/T
QC/T