GB 4789.4-2024National food safety standard - Food microbiological examination - Salmonella (English PDF)

2.1 Refrigerator: 2℃~8℃.

2.3 Homogenizer.

2.4 Oscillators.

2.5 Balance: with sensitivity of 0.1g.

2.6 Sterile Erlenmeyer flask: with capacity of 500mL, 250mL.

2.8 Sterile homogenization cups and sterile homogenization bags.

2.9 Sterile wide-mouth bottle: with capacity of 500mL.

2.10 Sterile pipette: 1mL (with scale of 0.01mL), 10mL (with scale of 0.1mL) or micropipette and tip.

2.11 Sterile petri dishes: with diameter of 60mm, 90mm.

2.15 pH meter or precision pH test paper.

2.16 Microbial biochemical identification system.

2.17 Biological safety cabinet.

3.1 Buffered peptone water (BPW): see A.1.

3.2 Sodium Tetrasulfonate Brilliant Green Bacteria Enrichment Solution (TTB): see A.2.

3.3 Magnesium chloride malachite green soy peptone (RVS) enrichment solution: see A.3.

3.5 HE agar: see A.5.

3.6 Xylose lysine deoxycholate (XLD) agar: see A.6.

3.7 Triple sugar iron (TSI) agar: see A.7.

3.8 Nutrient agar (NA): see A.8.

3.9 Semi-solid agar: see A.9.

3.12 Potassium cyanide (KCN) medium: see A.12.

3.13 Lysine decarboxylase test medium: see A.13.

3.14 Sugar fermentation medium: see A.14.

3.15 o-Nitrophenol β-D galactopyranoside (ONPG) medium: see A.15.

3.16 Sodium malonate medium: see A.16.

3.17 Salmonella chromogenic medium.

3.18 Salmonella diagnostic serum.

3.19 Biochemical identification kit.

5.1 Pre-enrichment

Aseptic operation: take 25g (mL) of sample; place it in a sterile homogenization cup containing 225mL of BPW, homogenize at 8000r/min~10000r/min for 1min~2min; or place it in a sterile homogenization bag containing 225mL of BPW, beat with a slap homogenizer for 1min~2min. For liquid samples, they can also be placed in a sterile Erlenmeyer flask or other suitable container containing 225mL of BPW and shaken to mix well. If adjustment of the pH value is required, use 1mol/L NaOH or HCl to adjust the pH to 6.8±0.2. Aseptically transfer the sample to a 500mL Erlenmeyer flask or other suitable container (if the homogenization cup itself has a non-porous cover or use a homogenization bag, the sample does not need to be transferred) and place it at 36℃±1℃ for 8h~18h.

For milk powder, aseptically weigh 25g of the sample and slowly pour it onto the surface of 225mL of BPW liquid in a wide-mouth bottle or homogenization bag. Do not adjust the pH and do not mix evenly. Let it stand at room temperature for 60min ± 5min before mixing. Place at 36℃±1℃ and incubate for 16h~18h.

If frozen samples need to be thawed, they shall be thawed in a water bath at 40°C ~ 45°C for no more than 15 min before sampling or slowly thawed in a refrigerator at 2°C ~ 8°C for no more than 18 h.

5.2 Selective enrichment

Gently shake the pre-enriched culture; transfer 0.1 mL into 10 mL RVS; mix and incubate at 42°C±1°C for 18h~24h. At the same time, transfer another 1 mL into 10 mL TTB and mix evenly. Samples with low background bacteria (such as deeply processed pre-packaged foods, etc.) are incubated at 36℃±1℃ for 18h~24h. Samples with high background bacteria (such as fresh poultry meat, etc.) are incubated at 42℃±1℃ for 18h~24h.

If necessary, the pre-enriched culture can be stored in a refrigerator at 2°C ~ 8°C for no more than 72 h before selective enrichment.

5.4 Biochemical test

5.4.1 Select more than 4 typical or suspicious colonies for biochemical test. These colonies should come from different separation agars of different selective enrichment solutions. One of the typical or suspicious colonies may also be selected for testing first. If it is identified as non- Salmonella, then take the remaining colonies for identification. Inoculate typical or suspicious colonies into triple sugar iron agar, first draw lines on the slant; and then puncture the bottom layer. Simultaneously inoculate lysine decarboxylase test medium and nutrient agar (or other suitable nonselective solid medium) plates; […]

5.5 Serological identification

5.6 Serological typing (optional)

A.1 Buffered peptone water (BPW)

A.1.1 Ingredients

A.1.2 Preparation method

Add each ingredient to distilled water (or other experimental water that meets the requirements, the same below); mix well; heat to dissolve; adjust pH if necessary; and autoclave at 121°C for 15 min. The pH of the sterilized culture medium at 25°C is 7.2±0.2.

A.2 Tetrasulfonate brilliant green bacteria enrichment solution (TTB)

A.2.1 Basic solution

Add each ingredient to distilled water; stir well and heat to dissolve. Boil, no need to autoclave. The pH of the boiled medium at 25°C is 7.6±0.2.

A.2.3 Brilliant green solution

After dissolving brilliant green in distilled water; store it in a cool and dark place for no less than 1 day.

A.2.4 Preparation method

On the day of use, add brilliant green solution to the cooled basic solution aseptically and shake well; add iodine solution; shake well again; […]

A.3.1 Ingredients

A.3.2 Preparation method

Add each ingredient to distilled water; mix well; heat to dissolve; adjust pH if necessary; quantitatively distribute into test tubes; and autoclave at 115°C for 15 min. The pH of the sterilized medium at 25°C is 5.2 ± 0.2.

A.4 Bismuth sulfite (BS) agar

A.4.1 Ingredients

A.4.2 Preparation method

Add each ingredient to distilled water; mix well; heat to dissolve; and adjust pH if necessary. Boil it; do not overheat; do not autoclave. Cool to 48℃±2℃ and pour into the plate. The pH of the boiled culture medium at 25℃ is 7.5±0.2.

NOTE: This culture medium shall be prepared and poured into plates one day before use;

stored in a dark place at room temperature; and used the next day. Storing the prepared culture medium for more than 48 h shall reduce its selectivity.

A.5 HE agar

A.5.1 Ingredients

A.5.2 Preparation method

Add each ingredient to distilled water; mix well; heat to dissolve; and adjust pH if necessary. Boil it; do not overheat; do not autoclave. Cool to 48℃±2℃ and pour into the plate. The pH of the boiled culture medium at 25℃ is 7.5±0.2.

A.6 Xylose lysine deoxycholate (XLD) agar

A.6.1 Ingredients

A.6.2 Preparation method

Add each ingredient to distilled water; mix well; heat to dissolve; and adjust pH if necessary. Boil it; […]

A.7 Triple sugar iron (TSI) agar

A.7.1 Ingredients

A.7.2 Preparation method

Add each ingredient to distilled water; mix well; heat to dissolve; and adjust pH if necessary. Dispense quantitatively into test tubes and autoclave at 115°C for 15 min. After sterilization, it is made into a slope; and the depth of the bottom layer is no less than 2.5cm. The pH of the sterilized culture medium at 25°C is 7.4 ± 0.2.

A.8 Nutrient agar (NA)

A.8.1 Ingredients

A.8.2 Preparation method

Add each ingredient to distilled water; mix well; heat to dissolve; adjust pH if necessary, and autoclave at 121°C for 15 min. The pH of the sterilized culture medium at 25°C is 7.3±0.2.

A.9 Semi-solid agar

A.9.1 Ingredients

A.10 Peptone water, indole reagent

A.10.1 Peptone water

A.10.1.1 Ingredients

A.11 Urea agar (pH 7.2)

A.11.1 Ingredients

A.12 Potassium cyanide (KCN) culture medium

A.12.1 Ingredients

A.13 Lysine decarboxylase test medium

A.13.2 Preparation method

Add ingredients other than lysine to distilled water; mix well and heat to dissolve. Add Llysine or DL-lysine. […]

A.14 Sugar fermentation medium

A.15 O-Nitrophenol β-D galactopyranoside (ONPG) medium

A.15.1 Ingredients

A.16 Sodium malonate medium

A.16.1 Ingredients