1 Scope
This standard specifies the examination method for Salmonella in foods.
This standard is applicable to the examination of salmonellae in foods.
2 Apparatus and Materials
In addition to the apparatuses for conventional sterilization and cultivation in microbiological laboratory, other apparatuses and materials are as follows:
2.1 Refrigerator: 2℃ ~ 5℃.
2.2 Constant temperature incubator: 36℃ ± 1℃, 42℃ ± 1℃.
2.3 Homogenizer.
2.4 Oscillator.
2.5 Electronic balance: sensibility of 0.1g.
2.6 Sterile conical flask: capacity of 500mL and 250mL.
2.7 Sterile pipette: 1mL (scale division of 0.01mL), 10mL (scale division of 0.1mL) or micropipette and pipette tip.
2.8 Sterile culture dish: diameter of 60 mm and 90mm.
2.9 Sterile test tube: 3mm×50mm, 10mm×75mm.
2.10 pH meter or pH colorimetric tube or precise pH paper.
2.11 Full-automatic microbe biochemical identification system.
2.12 Sterile capillary.
3 Culture Media and Reagents
3.1 Buffered peptone water (BPW): see A.1.
3.2 Tetrathionate broth (TTB): see A.2.
3.3 Selenite cystine (SC) broth: see A.3.
3.4 Bismuth sulfite (BS) agar: see A.4.
3.5 HE agar: see A.5.
3.6 Xylose lysine deoxycholate (XLD) agar: see A.6.
3.7 Salmonella chromogenic medium.
3.8 Triple sugar iron (TSI) agar: see A.7.
3.9 Peptone water and indole reagent: see A.8.
3.10 Urea agar (pH 7.2): see A.9.
3.11 Potassium cyanide (KCN) medium: see A.10.
3.12 Lysine decarboxylase test medium: see A.11.
3.13 Sugar fermentation tube: see A.12.
3.14 O-Nitrophenyl-β-D-galactopyranoside (ONPG) medium: see A.13.
3.15 Semi-solid agar: see A.14.
3.16 Sodium malonate medium: see A.15.
3.17 Salmonella O, H and Vi diagnostic sera.
3.18 Biochemical identification kit.
4 Examination Procedure
See Figure 1 for the examination procedure for Salmonella.
Figure 1 Procedure for Salmonella Examination
5 Operation Procedure
5.1 Pre-enrichment
Aseptically weigh 25g (mL) sample into sterile homogenizing cup or other appropriate container with 225mL BPW and homogenize at 8000r/min ~ 10000r/min for 1min ~ 2min, or into sterile homogenizing bag with 225 mL BPW and slap with slap type homogenizer for 1min ~ 2min. If sample is in liquid state, obviate homogenization and shake well. Adjust pH, if necessary, to 6.8 ± 0.2 with sterile 1mol/mL NaOH or HCl. Aseptically transfer the sample into conical flask (500mL) or other appropriate container (if the homogenizing cup is provided with nonporous cap, the sample may not be transferred). if homogenizing bag is used, incubate directly at 36℃ ± 1℃ for 8h ~ 18h.
If product is frozen, thaw below 45℃ for at most 15min or thaw within 18h at 2℃ ~ 5℃.
5.2 Enrichment
Shake the incubated sample mixture gently; take 1mL of the mixture into 10mL TTB; and incubate for 18h~24h at 42℃ ± 1℃.
Meanwhile, take another 1mL of the mixture into 10mL SC; and incubate for 18h~24h at 36℃ ± 1℃.
5.3 Isolation
Streak 3mm loopful broth on BS agar plate and XLD agar plate (or HE agar plate or Salmonella chromogenic medium), incubate 40h ~ 48h (for BS agar plate) or 18h ~ 24h (for XLD agar plate, HE agar plate and Salmonella chromogenic medium plate) at 36℃ ± 1℃. Examine plates for growth and characteristics of colony, see Table 1.
Table 1 Colony Characteristics of Salmonella by Selective Agar Plate
Selective agar plate Salmonella
BS agar The colonies are black and have a metallic sheen, sepia or gray; and the culture medium around the colonies may appear in black or brown and that around some glaucous colonies of strains is unchanged.
HE agar Green-blue or blue, and most colonies present black centers or are almost completely black; some strains are yellow and produce black center, or are almost completely black.
XLD agar The colonies appear in pink with or without black centers. Some strains may present large lustrous black centers, or produce completely black colonies and some may produce yellow colonies with or without black centers.
Salmonella chromogenic medium Determine according to the instructions of chromogenic medium.
5.4 Biochemical tests
5.4.1 Pick more than 2 typical or suspicious colonies from selective agar plate to inoculate triple sugar iron agar by streaking slant and stabbing butt. Directly inoculate lysine decarboxylase test medium and nutrient agar plate with non-sterile inoculating needle. Incubate for 18h ~ 24h or, if necessary, for 48h, at 36℃ ± 1℃.The reaction results of salmonella in triple sugar iron agar and lysine decarboxylase test medium are listed in Table 2.
Contents
Foreword i
1 Scope
2 Apparatus and Materials
3 Culture Media and Reagents
4 Examination Procedure
5 Operation Procedure
6 Result and Report
Appendix A Culture Media and Reagents
Appendix B Common Salmonella Antigens
1 Scope
This standard specifies the examination method for Salmonella in foods.
This standard is applicable to the examination of salmonellae in foods.
2 Apparatus and Materials
In addition to the apparatuses for conventional sterilization and cultivation in microbiological laboratory, other apparatuses and materials are as follows:
2.1 Refrigerator: 2℃ ~ 5℃.
2.2 Constant temperature incubator: 36℃ ± 1℃, 42℃ ± 1℃.
2.3 Homogenizer.
2.4 Oscillator.
2.5 Electronic balance: sensibility of 0.1g.
2.6 Sterile conical flask: capacity of 500mL and 250mL.
2.7 Sterile pipette: 1mL (scale division of 0.01mL), 10mL (scale division of 0.1mL) or micropipette and pipette tip.
2.8 Sterile culture dish: diameter of 60 mm and 90mm.
2.9 Sterile test tube: 3mm×50mm, 10mm×75mm.
2.10 pH meter or pH colorimetric tube or precise pH paper.
2.11 Full-automatic microbe biochemical identification system.
2.12 Sterile capillary.
3 Culture Media and Reagents
3.1 Buffered peptone water (BPW): see A.1.
3.2 Tetrathionate broth (TTB): see A.2.
3.3 Selenite cystine (SC) broth: see A.3.
3.4 Bismuth sulfite (BS) agar: see A.4.
3.5 HE agar: see A.5.
3.6 Xylose lysine deoxycholate (XLD) agar: see A.6.
3.7 Salmonella chromogenic medium.
3.8 Triple sugar iron (TSI) agar: see A.7.
3.9 Peptone water and indole reagent: see A.8.
3.10 Urea agar (pH 7.2): see A.9.
3.11 Potassium cyanide (KCN) medium: see A.10.
3.12 Lysine decarboxylase test medium: see A.11.
3.13 Sugar fermentation tube: see A.12.
3.14 O-Nitrophenyl-β-D-galactopyranoside (ONPG) medium: see A.13.
3.15 Semi-solid agar: see A.14.
3.16 Sodium malonate medium: see A.15.
3.17 Salmonella O, H and Vi diagnostic sera.
3.18 Biochemical identification kit.
4 Examination Procedure
See Figure 1 for the examination procedure for Salmonella.
Figure 1 Procedure for Salmonella Examination
5 Operation Procedure
5.1 Pre-enrichment
Aseptically weigh 25g (mL) sample into sterile homogenizing cup or other appropriate container with 225mL BPW and homogenize at 8000r/min ~ 10000r/min for 1min ~ 2min, or into sterile homogenizing bag with 225 mL BPW and slap with slap type homogenizer for 1min ~ 2min. If sample is in liquid state, obviate homogenization and shake well. Adjust pH, if necessary, to 6.8 ± 0.2 with sterile 1mol/mL NaOH or HCl. Aseptically transfer the sample into conical flask (500mL) or other appropriate container (if the homogenizing cup is provided with nonporous cap, the sample may not be transferred). if homogenizing bag is used, incubate directly at 36℃ ± 1℃ for 8h ~ 18h.
If product is frozen, thaw below 45℃ for at most 15min or thaw within 18h at 2℃ ~ 5℃.
5.2 Enrichment
Shake the incubated sample mixture gently; take 1mL of the mixture into 10mL TTB; and incubate for 18h~24h at 42℃ ± 1℃.
Meanwhile, take another 1mL of the mixture into 10mL SC; and incubate for 18h~24h at 36℃ ± 1℃.
5.3 Isolation
Streak 3mm loopful broth on BS agar plate and XLD agar plate (or HE agar plate or Salmonella chromogenic medium), incubate 40h ~ 48h (for BS agar plate) or 18h ~ 24h (for XLD agar plate, HE agar plate and Salmonella chromogenic medium plate) at 36℃ ± 1℃. Examine plates for growth and characteristics of colony, see Table 1.
Table 1 Colony Characteristics of Salmonella by Selective Agar Plate
Selective agar plate Salmonella
BS agar The colonies are black and have a metallic sheen, sepia or gray; and the culture medium around the colonies may appear in black or brown and that around some glaucous colonies of strains is unchanged.
HE agar Green-blue or blue, and most colonies present black centers or are almost completely black; some strains are yellow and produce black center, or are almost completely black.
XLD agar The colonies appear in pink with or without black centers. Some strains may present large lustrous black centers, or produce completely black colonies and some may produce yellow colonies with or without black centers.
Salmonella chromogenic medium Determine according to the instructions of chromogenic medium.
5.4 Biochemical tests
5.4.1 Pick more than 2 typical or suspicious colonies from selective agar plate to inoculate triple sugar iron agar by streaking slant and stabbing butt. Directly inoculate lysine decarboxylase test medium and nutrient agar plate with non-sterile inoculating needle. Incubate for 18h ~ 24h or, if necessary, for 48h, at 36℃ ± 1℃.The reaction results of salmonella in triple sugar iron agar and lysine decarboxylase test medium are listed in Table 2.
Contents of GB 4789.4-2016
Contents
Foreword i
1 Scope
2 Apparatus and Materials
3 Culture Media and Reagents
4 Examination Procedure
5 Operation Procedure
6 Result and Report
Appendix A Culture Media and Reagents
Appendix B Common Salmonella Antigens
