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GB 4789.11-2014National Food Safety Standard -- Microbiological Examination of Food Hygiene -- Examination of Streptococcus Hemolyticus (English PDF)

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Issued by

National Health and Family Planning Commission of the People's Republic of China

Level / Type

National · Mandatory

Issue date

December 1, 2014

Implementation date

May 1, 2015

Scope

GB 4789.11-2014 (National Food Safety Standard -- Microbiological Examination of Food Hygiene -- Examination of Streptococcus Hemolyticus) is available as an English-translated PDF.

GB 4789.11-2014 — This Standard specifies the inspection method for beta-type hemolytic streptococcus in foods. This Standard is applicable to inspection of beta-type hemolytic streptococcus in foods.

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Document preview — GB 4789.11-2014

National Standard of the People's Republic of China

Issued by: National Health and Family Planning Commission of the People's Republic of China

Foreword...3
1 Scope...4
2 Terms and Definitions...4
3 Equipment and Materials...4
4 Culture Media and Reagents...5
5 Inspection Procedure...5
6 Operation Procedure...6
7 Results and Reports...7
Appendix A Culture Media and Reagents...8

Foreword

This Standard replaces "Microbiological Examination of Food Hygiene - Examination

of Hemolytic Streptococcus" (GB/T 4789.11-2003).

Compared with GB/T 4789.11-2003, this Standard has the following main changes.

- The standard name was modified;

- The "Scope" was modified;

- The "Terms and Definitions" was added;

- The "Equipment and Materials" was modified;

- The "Culture Media and Reagents" was modified;

- The procedure of sample dilution with aseptic normal saline and the bacitracin

sensitivity test were deleted;

- The catalase test was added;

- Appendix A was added.

National Food Safety Standard –

Food Microbiological Examination –

Examination of beta-type Hemolytic Streptococcus

1 Scope

This Standard specifies the inspection method for beta-type hemolytic streptococcus in

foods.

This Standard is applicable to inspection of beta-type hemolytic streptococcus in foods.

2 Terms and Definitions

2.1 beta-type hemolysis

Totally transparent hemolytic rings are formed around the bacterial colony, and the

erythrocytes are fully dissolved.

2.2 beta-type hemolytic streptococcus

It can produce beta-type hemolytic streptococcus pyogenes (or A-group) and

streptococcus agalactiae (or B-group).

3 Equipment and Materials

In addition to the conventional sterilization and cultivation equipment in

microbiological laboratory, other equipment and materials are as follows.

4 Culture Media and Reagents

4.1 Modified tryptone soybean broth (mTSB). see A.1 of Appendix A.

4.2 Columbia CNA blood agar. see A.2 of Appendix A.

4.3 Columbia blood agar. see A.3 of Appendix A.

4.4 Gram stain solution. see A.4 of Appendix A.

4.8 3% hydrogen peroxide (H2O2) solution. see A.8 of Appendix A.

4.9 Biochemical identification kit or card.

5 Inspection Procedure

The inspection procedure of hemolytic streptococcus is detailed in Figure 1.

6 Operation Procedure

6.1 Sample Treatment and Bacteria Enrichment

Weigh 25 g (mL) of sample according to aseptic operation; put into a homogenizing

bag that contains 225 mL of mTSB;

6.2 Separation

The enrichment broth is subject to streak inoculation to the Columbia CNA blood agar

plate and anaerobic cultivation under 36°C±1°C for 18 h~24 h, then observe the

colony shape.

6.3 Identification

6.3.1 Separable pure cultivation

Select five suspicious colonies (if less than five, select all) to inoculate with Columbia

blood agar plate and TSB enrichment broth respectively, then cultivate under

36°C±1°C for 18 h~24 h.

6.3.2 Gram stain microscopy

Select suspicious colony for gram staining microscopic examination. The beta-type

hemolytic streptococcus is gram positive, spherical or oval, and generally shaped in

short chain.

6.3.4 Streptokinase test (optional item)

Suck 0.2 mL of potassium oxalate blood plasma into 0.8 mL of sterilized normal saline

and mix well; then add 0.5 mL of TSB culture solution of suspicious colony after

cultivation under 36°C±1°C for 18 h~24 h and 0.25 mL of 0.25% calcium chloride

solution; oscillate and shake well; place into the water bath of 36°C±1°C for 10 min;

6.3.5 Other inspections

Identify the suspicious colony with biochemical identification kit or biochemical

identification card.

7 Results and Reports

Upon the above test results, report the detected or un-detected hemolytic

streptococcus in every 25 g (mL) of samples.

Appendix A

Culture Media and Reagents

A.1 Modified tryptone soybean broth (mTSB) culture medium

A.1.1 Basal medium (Tryptone soybean broth [TSB])

A.1.2 Antibiotic solution

A.1.2.1 Polymyxin solution

Weigh 10 mg of polymyxin B into 10 mL of sterile purified water, shake to mix well,

filter and remove bacteria after fully dissolved.

A.1.2.2 Nalidixic acid sodium solution

Weigh 10 mg of nalidixic acid into 10 mL of sodium hydroxide solution (0.05mol/L),

shake to mix well, filter and remove bacteria after fully dissolved.

A.1.3 Complete medium

A.2.2 Preparation

Dissolve the compositions listed in A.2.1 into the distilled water, heat to dissolve,

calibrate the pH value to 7.3±0.2 and sterilize at 121°C for 12 min; after cooling to

around 50°C, add 50 mL of aseptic defibrinated sheep blood, shake well and pour to

the plate.

A.3 Columbia blood agar

A.3.1 Basal medium

A.3.1.1 Compositions

Animal tissue zymolyte 23.0 g

Starch 1.0 g

Sodium chloride 5.0 g

Agar 8.0 g~18.0 g

Distilled water 1 000.0mL

A.4 Gram stain solution

A.4.1 Basal medium of crystal violet staining solution

A.4.1.1 Compositions

Crystal violet 1.0 g

95% ethanol 20.0mL

1% ammonium oxalate solution 80.0 mL

A.4.1.2 Preparation

Fully dissolve the crystal violet into ethanol, and then mix with ammonium oxalate

solution.

A.4.2 Gram's iodine solution

A.4.2.1 Compositions

Iodine 1.0 g

Kalium iodide 2.0 g

Distilled water 300.0mL

A.4.3 Safranin solution

A.4.3.1 Compositions

Safranin 0.25 g

95% ethanol 10.0mL

Distilled water 90.0mL

A.4.3.2 Preparation

Dissolve safranin into the ethanol, and dilute with distilled water.

A.5 Tryptone soybean broth (TSB)

A.5.1 Compositions

Tryptone 17.0 g

Soybean peptone 3.0 g

Sodium chloride 5.0 g

Potassium dihydrogen phosphate (anhydrous) 2.5 g

Glucose 2.5 g

Distilled water 1000.0 mL

A.6 Potassium oxalate blood plasma

A.6.1 Compositions

Potassium oxalate 0.01 g

Human blood 5.0 mL

A.6.2 Preparation

Take 0.01 g of potassium oxalate into the small sterilizing test tube, add 5 mL of

human blood and mix them well, centrifuge the mixture and make it sediment, and

then absorb the supernatant, that is the potassium oxalate blood plasma.

A.7 0.25% calcium chloride (CaCl2) solution

A.7.1 Compositions

Calcium chloride (anhydrous) 22.2 g

Distilled water 1000.0 mL

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