Methods of Test for Food Microorganisms- Test of Cronobacter 1. 2. 2.1. 100 15 15 CFU/ 2.2. 2.2.1. (Biological safety cabinet, BSC) (class II)( ) 2.2.2. 2.2.3. 2.2.4. (Blender) (Stomacher) 2.2.5. 2000 g 0.1 g 120 g 1 mg 2.2.6. 5±3 2.2.7. 1 ml 0.01 ml 5 ml 10 ml 0.1 ml 2.2.8. (Pipette aid) 2.2.9. 160 ml (polyethylene) (Teflon) 121 20 2.2.10. 9 cm 15 mm 2.2.11. 125 ml 250 ml 2 L 121 20 2.2.12. ph 2.2.13. ± 1 2.2.14. ±0.2 2.2.15. ( 3 mm) 2.2.16. 3 4 mm 45 55 mm 2.2.17. 10 100 mm 13 100 mm 13 120 mm 15 150 mm 16 150 mm 818
2.2.18. (Vortex mixer) 2.2.19. 1000 2.2.20. 2.2.21. 2.2.22. 2.2.23. 0.45 μm 2.2.24. (Durham fermentation tube) 9 22 mm 2.2.25. (sodium lauryl sulfate) No. 3 (bile salts No. 3) (neutral red) (crystal violet) (ferric ammonium citrate) (sodium desoxycholate) (sodium thiosulfate) (ammonium oxalate) O (safranin O) - (p-dimethyl aminobenzaldehyde) (N,N,N',N'-tetramethyl-ρ-phenylenediamine 2HCl) (NaH 2 PO 4 H 2 O) (Na 2 HPO 4 ) (K 2 HPO 4 ) (KH 2 PO 4 ) (glucose) (ox-gall) (urea) (o-nitrophenyl-β-d-galactopyranoside, ONPG) 5- -4- -3- -α-d- (5-bromo-4-chloro-3-indolyl-α-D-glucopyranoside) (Na 3 C 6 H 5 O 7 ) (brilliant green) (phenol red) (bromcresol purple) (NH 4 H 2 PO 4 ) (MgSO 4 ) (bromthymol blue) (creatine) (methyl red) α- (α-naphthol) 95% (95% ethanol) (absolute ethanol) (amyl alcohol) (isoamyl alcohol) (lactose) (sucrose) (dulcitol) (adonitol) (raffinose) (sorbitol) (D-arabitol) L- (L-lysine) L- (L-ornithine) L- (L-arginine) (mineral oil) (paraffin oil) (peptone) tryptose) (yeast extract) (agar) (tryptone) (soya peptone) (trypticase peptone) (phytone peptone) (beef extract) (polypeptone) (buffered peptone-water powder) 2.2.26. 819
2.2.26.1. 0.85% 8.5 g 1000 ml 121 15 2.2.26.2. (Kovacs reagent) - 5 g 75 ml 25 ml 4 2.2.26.3. (Methyl red indicator) 0.1 g 95% 300 ml 500 ml 2.2.26.4. (Voges-Proskauer test reagents, VP reagents) A α- 5 g 100 ml B 40 g 100 ml 2.2.26.5. 0.5% (0.5% potassium cyanide solution) 0.5 g 100 ml ( ) 2.2.26.6. 20 50 ml 1/2 121 30 2.2.26.7. (Gram stain solution) 2.2.26.7.1. (Hucker s) ( ) A 2 g 95% 20 ml B 0.8 g 80 ml A B 24 2.2.26.7.2. ( ) 2 g 1 g 5 10 1 ml 5 ml 10 ml 300 ml 2.2.26.7.3. ( ) O 2.5 g 95% 100 ml 10 ml 90 ml 820
2.2.26.8. (Oxidase reagent) 1 g 100 ml 1 2.2.26.9. 1M 6.9 g 45 ml 30% 3 ml ph 7.0 50 ml 4 2.2.26.10. (ONPG reagent) 80 mg 37 15 ml 1M 5 ml 0.0133M 4 37 2.2.27. 2.2.27.1. (Buffered peptone water, BPW) (peptone)..... 10 g...5 g (Na 2 HPO 4 )....3.5 g (KH 2 PO 4 )...1.5 g 1000 ml 121 15 ph 7.2±0.2 2.2.27.2. (Lauryl tryptose broth with 2% NaCl, 2% NaCl-LST) (tryptose) 20 g (lactose).... 5 g (KH 2 PO 4 ).. 2.75 g (K 2 HPO 4 )... 2.75 g.. 20 g (sodium lauryl sulfate)....0.1 g. 1000 ml 10 ml 121 15 ph 6.8±0.2 2.2.27.3. (Violet red bile glucose agar, VRBG) (yeast extract) 3 g (peptone)... 7 g...5 g 821
No. 3 (bile salts No. 3).. 1.5 g (lactose)...10 g (neutral red).0.03 g (crystal violet) 0.002 g (agar)... 15 g (glucose)..10 g...1000 ml 45 50 ph 7.4±0.2 20 ml 2 8 2.2.27.4. (Enterobacter sakazakii agar, ESA) (tryptone)... 15 g (soya peptone) 5.0 g. 5.0 g (ferric ammonium citrate)... 1.0 g (sodium desoxycholate).. 1.0 g (Na 2 S 2 O 3 5H 2 O)...1.0 g 5- -4- -3- -α-d- (5-bromo-4-chloro-3 -indolyl-α-d-glucopyranoside).... 0.1 g (agar)....15 g.. 1000 ml 121 15 ph 7.3±0.2 50 20 ml 2.2.27.5. (Enterobacteriaceae enrichment broth, EE broth) (peptone).. 10 g (glucose).....5 g (Na 2 HPO 4 )..... 8 g (KH 2 PO 4 )..... 2 g (ox-gall)..... 20 g (brilliant green)...0.015 g....1000 ml ph 7.2±0.2 90 ml 125 ml 822
2 8 2.2.27.6. (Trypticase soy agar, TSA) (trypticase peptone)..15 g (phytone peptone)...5.0 g. 5.0 g (agar)....15 g......1000 ml 121 15 ph 7.3±0.2 50 20 ml 5 ml 121 15 ph 7.3±0.2 4 5 cm 2 3 cm 2.2.27.7. (Urea broth) (urea).......20 g (yeast extract). 0.1 g (K 2 HPO 4 )... 9.1 g (Na 2 HPO 4 ).. 9.5 g (phenol red)..0.01 g..1000 ml 1.5 3 ml ph 6.8±0.2 2.2.27.8. (Motility test medium) (beef extract). 3 g (peptone).10 g...5 g (agar) 4 g.1000 ml 8 ml 121 15 ph 7.4±0.2 2.2.27.9. (Potassium cyanide broth) (polypeptone) 3 g...5 g (KH 2 PO 4 )...0.225 g (Na 2 HPO 4 )...5.64 g 823
.1000 ml 121 15 ph 7.6±0.2 0.5% 15 ml 1 1.5 ml ( ) 2.2.27.10. (Tryptone broth) (tryptone) 10 g....1000 ml 5 ml 121 15 ph 6.9±0.2 2.2.27.11. MR-VP (MR-VP broth) (buffered peptone-water powder)...7 g (glucose)...5 g (K 2 HPO 4 )..5 g.1000 ml 10 ml 118 121 15 ph 6.9±0.2 2.2.27.12. (Bromcresol purple broth) (peptone) 10 g (beef extract) 3 g.. 5 g (bromcresol purple) 0.04 g....1000 ml 2.5 ml 121 10 ph 7.0±0.2 50% (w/v) 0.278±0.002 ml 5% (w/v) 2.2.27.13. (Decarboxylase basal medium) (peptone)...5 g (yeast extract)...3 g (glucose)...1 g (bromcresol purple). 0.02 g.....1000 ml L- 5 g 824
121 10 ph 6.5±0.2 L- L- 2.2.27.14. (Simmons citrate agar)......5 g (Na 3 C 6 H 5 O 7 ) 2 g (K 2 HPO 4 ).....1 g (NH 4 H 2 PO 4 ).1 g (MgSO 4 ) 0.2 g (bromthymol blue)..0.08 g (agar)..15 g.1000 ml 5 ml 121 15 ph 6.8±0.2 4 5 cm 2 3 cm 2.3. 2.3.1. 100 g 10 g 1 g 45 900 ml 90 ml 9 ml 2 L 250 ml 125 ml ( ) ( ) 10 35 18 24 ( ) 2.3.2. 100 ml 10 ml 1 ml 900 ml 90 ml 9 ml ( ) 10 35 18 24 2.3.3. 2.3.3.1. 1 g 2% NaCl-LST 10 ml 44 18 24 2.3.3.2. ( ) 5 ml 10 ( 825
15 ) 50 1 ml 2% NaCl-LST 10 ml 44 18 24 2% NaCl-LST 10 ml ( ) 44 18 24 2.4. 2.4.1. 2.3. 10 ml 90 ml 160 ml 35 18 24 2.4.2. 2.4.2.1. 2.4.1. VRBG ESA ( ) 35 18 24 VRBG (bile acids) ESA VRBG ESA TSA 35 18 24 2.4.2.2. 2.4.1. 1 ml 9 ml 10-4 10-6 ( ) 0.1 ml VRBG ESA 35 18 24 VRBG ESA TSA 35 18 24 2.4.3. (Mixed culture) VRBG ESA VRBG ESA 35 18 24 TSA 35 18 24 2.5. 2.5.1. 2.5.1.1. (Oxidase test) 10 15 826
2.5.1.2. (Yellow pigment production test) TSA 5 TSA 25 48 72 2.5.1.3. (Urease test) 35 24±2 2.5.1.4. (Motility test) 0.5 35 24 48±2 2.5.1.5. (Gram stain) TSA 35 24±2 (1) (2) 1 5 (3) 1 (4) 95% (5) 30 (6) (7) TSA 2.5.2. 2.5.2.1. (ONPG test) 0.85% 0.2 ml 35 6 24 827
2.5.2.2. (KCN test) 35 24 48±2 2.5.2.3. (Indole test) 35 48±2 0.2 0.3 ml 10 2.5.2.4. (VP test) MR-VP 35 48±2 1 ml A 0.6 ml B 0.2 ml 2 4 2.5.2.5. (Methyl red test) 2.5.2.4. MR-VP 35 48±2 5 ml 5 6 2.5.2.6. (Citrate utilization test) 35 96±2 2.5.2.7. (Arginine dihydrolase test) 1 2 cm 35 4 24 2.5.2.8. (Lysine decarboxylase test) 1 2 cm 35 4 24 828
2.5.2.9. (Ornithine decarboxylase test) 1 2 cm 35 4 24 2.5.2.10. (Fermentation test) 35 2 5 24 2.6. Cronobacter E. cloacae E. aerogenes E. agglomerans E. gergoviae (1) Cronobacter E. cloacae E. aerogenes E. agglomerans E. gergoviae (lysine decarboxylase) (arginine dihydrolase) (ornithine decarboxylase) (citrate utilization) [ ] (urease) (indole) d d (KCN) d (yellow pigment production) d (MR) d ND ND (VP) d ND ND (1) 1 2 [ ] (89% ) 1 4 d ( 11 80% ) 7 ND 829
Cronobacter E. cloacae E. aerogenes E. agglomerans E. gergoviae (1) Cronobacter E. cloacae E. aerogenes E. agglomerans E. gergoviae (sucrose) ( ) (dulcitol) ( ) ( ) ( ) (adonitol) ( ) (raffinose) V (sorbitol) V (D-arabitol) ( ) (1) 90 100% ( ) 75 89% V 25 74% ( ) 10 24% 0 9% 830
2.7. 2.6. ( ) ( ) 0.1, 0.01, 0.001 (g ml) ( ) ( ) (MPN/g MPN/mL) ( ) [ ( ) g ml] 95% ( ) [ ( ) g ml] 95% MPN/ ml (g) MPN/ ml (g) 0.1 0.01 0.001 0.1 0.01 0.001 0 0 0 < 3.0 -- 9.5 2 2 0 21 4.5 42 0 0 1 3.0 0.15 9.6 2 2 1 28 8.7 94 0 1 0 3.0 0.15 11 2 2 2 35 8.7 94 0 1 1 6.1 1.2 18 2 3 0 29 8.7 94 0 2 0 6.2 1.2 18 2 3 1 36 8.7 94 0 3 0 9.4 3.6 38 3 0 0 23 4.6 94 1 0 0 3.6 0.17 18 3 0 1 38 8.7 110 1 0 1 7.2 1.3 18 3 0 2 64 17 180 1 0 2 11 3.6 38 3 1 0 43 9 180 1 1 0 7.4 1.3 20 3 1 1 75 17 200 1 1 1 11 3.6 38 3 1 2 120 37 420 1 2 0 11 3.6 42 3 1 3 160 40 420 1 2 1 15 4.5 42 3 2 0 93 18 420 1 3 0 16 4.5 42 3 2 1 150 37 420 2 0 0 9.2 1.4 38 3 2 2 210 40 430 2 0 1 14 3.6 42 3 2 3 290 90 1000 2 0 2 20 4.5 42 3 3 0 240 42 1000 2 1 0 15 3.7 42 3 3 1 460 90 2000 2 1 1 20 4.5 42 3 3 2 1100 180 4100 2 1 2 27 8.7 94 3 3 3 1100 420 -- VRBG ESA ( ) 3-3-2 MPN 1100 (1) ( ) 0.1, 0.01, 0.001 (g ml) 1100 (MPN/g MPN/mL) (2) ( ) 100, 10, 1 (g ml) 1100 1000 1.1 (MPN/g MPN/mL) 2.8. 831
real-time PCR 1. 2. DNA (real-time polymerase chain reaction, real-time PCR) 2.1. DNA real-time PCR real-time PCR Real-time PCR ( 1) 2.2. 2.2.1. Applied Biosystems 7500 Real-Time PCR System 2.2.2. 2.2.3. (Biological safety cabinet, BSC) (class II) ( ) 2.2.4. 2.2.5. (Micro refrigerated centrifuge) 20000 g 4 2.2.6. 2.2.7. 260 nm 280 nm 2.2.8. (-20 C) 2.2.9. (Vortex mixer) 2.2.10. (ph meter) 2.2.11. 2000 g 0.1 g 100 g 1 mg 1 2.3. 2.3.1. DNA DNA 832
( 2) 2.3.2. Real-time PCR 2.3.2.1. 2.3.2.1.1. ( MMS operon) F 5 - GGG ATA TTG TCC CCT GAA ACA G -3 R 5 - CGA GAA TAA GCC GCG CAT T - 3 P 5 -(FAM)- AGA GTA GTA GTT GTA GAG GCC GTG CTT CCG AAA G -(BHQ1)-3 PCR 78 bp 2-20 5 6-carboxy-fluorescein (FAM) 3 Black Hole Quencher-1 (BHQ1) 2.3.2.2. TaqMan Fast Reagents Starter Kit ( Applied Biosystems 7500 Real-Time PCR System) real-time PCR DNA 2.3.3. DNA ( 3) 2.4. 2.4.1. (Micropipette) 2μL 10 μl 20 μl 100μL 200 μl 1000 μl 2.4.2. (Pipette tip) 10 μl 20 μl 200 μl 1000 μl 2.4.3. 200 μl 600 μl 1.5 ml 2 ml 2.4.4. Real-time PCR 100 μl 2.4.5. Real-time PCR 96 Applied Biosystems 7500 Real-Time PCR System 2.4.6. 50 ml 100 ml 250 ml 500 ml 1000 ml 2000 ml 3 DNase ( 4) 2.5. Real-time PCR Applied Biosystems 7500 Real-Time PCR System 5 μm F... 2.0 μl 5 μm R... 2.0 μl 833
5 μm... 1.5 μl TaqMan Fast Reagents Starter Kit...12.5 μl DNA... 5.0 μl... 2.0 μl...25.0 μl 4 Real-time PCR 2.6. DNA 2.6.1. DNA 2.3.1. 35 C 18 24 1 ml 1.5 ml 15000 g 3 2.6.1.1. 1 ml 15000 g 3 1 ml 10 DNA -20 2.6.1.2. DNA DNA DNA DNA 1.5 ml DNA -20 2.6.2. DNA 1 ml 1.5 ml 10 15000 g 3 1.5 ml DNA -20 2.6.1.2. DNA 2.6.3. DNA DNA 260 nm 280 nm (O.D.) 260 nm 50 ng/μl DNA DNA O.D. 260 /O.D. 280 1.7 2.0 ( 5) 2.7. 834
2.7.1. Real-time PCR DNA 1.5 ml 2.5. real-time PCR TaqMan Fast Reagents Starter Kit 20 μl real-time PCR DNA 5 μl real-time PCR 200 g real-time PCR 2.7.1.1. 1. 95 C 20 sec 2. 95 C 15 sec 3. 52 C 40 sec 2 3 40 2.7.2. Real-time PCR DNA real-time PCR real-time PCR 2.7.3. DNA real-time PCR DNA real-time PCR real-time PCR 5 Real-time PCR Applied Biosystems 7500 Real-Time PCR System real-time PCR 835
100 g 10 g 1 g 900 ml 90 ml 9 ml( ) ( 1) 35 18 24 10 ml 90 ml 35 18 24 VRBG ESA 35 18 24 2 TSA 35 18 24 real-time PCR ( 2) 1 45 2 real-time PCR 836