行政院公報第 021 卷第 003 期衛生勞動篇 Methods of Test for Food Microorganisms- Test of Cronobacter CFU/ (Biological safe

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1 Methods of Test for Food Microorganisms- Test of Cronobacter CFU/ (Biological safety cabinet, BSC) (class II)( ) (Blender) (Stomacher) g 0.1 g 120 g 1 mg ± ml 0.01 ml 5 ml 10 ml 0.1 ml (Pipette aid) ml (polyethylene) (Teflon) cm 15 mm ml 250 ml 2 L ph ± ± ( 3 mm) mm mm mm mm mm mm mm 818

2 (Vortex mixer) μm (Durham fermentation tube) 9 22 mm (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) 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)

3 % 8.5 g 1000 ml (Kovacs reagent) - 5 g 75 ml 25 ml (Methyl red indicator) 0.1 g 95% 300 ml 500 ml (Voges-Proskauer test reagents, VP reagents) A α- 5 g 100 ml B 40 g 100 ml % (0.5% potassium cyanide solution) 0.5 g 100 ml ( ) ml 1/ (Gram stain solution) (Hucker s) ( ) A 2 g 95% 20 ml B 0.8 g 80 ml A B ( ) 2 g 1 g ml 5 ml 10 ml 300 ml ( ) O 2.5 g 95% 100 ml 10 ml 90 ml 820

4 (Oxidase reagent) 1 g 100 ml M 6.9 g 45 ml 30% 3 ml ph ml (ONPG reagent) 80 mg ml 1M 5 ml M (Buffered peptone water, BPW) (peptone) g...5 g (Na 2 HPO 4 ) g (KH 2 PO 4 ) g 1000 ml ph 7.2± (Lauryl tryptose broth with 2% NaCl, 2% NaCl-LST) (tryptose) 20 g (lactose) g (KH 2 PO 4 ) g (K 2 HPO 4 ) g.. 20 g (sodium lauryl sulfate) g ml 10 ml ph 6.8± (Violet red bile glucose agar, VRBG) (yeast extract) 3 g (peptone)... 7 g...5 g 821

5 No. 3 (bile salts No. 3) g (lactose)...10 g (neutral red).0.03 g (crystal violet) g (agar) g (glucose)..10 g ml ph 7.4± ml (Enterobacter sakazakii agar, ESA) (tryptone) g (soya peptone) 5.0 g. 5.0 g (ferric ammonium citrate) g (sodium desoxycholate) g (Na 2 S 2 O 3 5H 2 O) g α-d- (5-bromo-4-chloro-3 -indolyl-α-d-glucopyranoside) g (agar) g ml ph 7.3± ml (Enterobacteriaceae enrichment broth, EE broth) (peptone).. 10 g (glucose) g (Na 2 HPO 4 ) g (KH 2 PO 4 ) g (ox-gall) g (brilliant green) g ml ph 7.2± ml 125 ml 822

6 (Trypticase soy agar, TSA) (trypticase peptone)..15 g (phytone peptone) g. 5.0 g (agar) g ml ph 7.3± ml 5 ml ph 7.3± cm 2 3 cm (Urea broth) (urea) g (yeast extract). 0.1 g (K 2 HPO 4 ) g (Na 2 HPO 4 ) g (phenol red) g ml ml ph 6.8± (Motility test medium) (beef extract). 3 g (peptone).10 g...5 g (agar) 4 g.1000 ml 8 ml ph 7.4± (Potassium cyanide broth) (polypeptone) 3 g...5 g (KH 2 PO 4 ) g (Na 2 HPO 4 ) g 823

7 .1000 ml ph 7.6± % 15 ml ml ( ) (Tryptone broth) (tryptone) 10 g ml 5 ml ph 6.9± MR-VP (MR-VP broth) (buffered peptone-water powder)...7 g (glucose)...5 g (K 2 HPO 4 )..5 g.1000 ml 10 ml ph 6.9± (Bromcresol purple broth) (peptone) 10 g (beef extract) 3 g.. 5 g (bromcresol purple) 0.04 g ml 2.5 ml ph 7.0±0.2 50% (w/v) 0.278±0.002 ml 5% (w/v) (Decarboxylase basal medium) (peptone)...5 g (yeast extract)...3 g (glucose)...1 g (bromcresol purple) g ml L- 5 g 824

8 ph 6.5±0.2 L- L (Simmons citrate agar) g (Na 3 C 6 H 5 O 7 ) 2 g (K 2 HPO 4 ) g (NH 4 H 2 PO 4 ).1 g (MgSO 4 ) 0.2 g (bromthymol blue) g (agar)..15 g.1000 ml 5 ml ph 6.8± cm 2 3 cm g 10 g 1 g ml 90 ml 9 ml 2 L 250 ml 125 ml ( ) ( ) ( ) ml 10 ml 1 ml 900 ml 90 ml 9 ml ( ) g 2% NaCl-LST 10 ml ( ) 5 ml 10 ( 825

9 15 ) 50 1 ml 2% NaCl-LST 10 ml % NaCl-LST 10 ml ( ) ml 90 ml 160 ml VRBG ESA ( ) VRBG (bile acids) ESA VRBG ESA TSA ml 9 ml ( ) 0.1 ml VRBG ESA VRBG ESA TSA (Mixed culture) VRBG ESA VRBG ESA TSA (Oxidase test)

10 (Yellow pigment production test) TSA 5 TSA (Urease test) 35 24± (Motility test) ± (Gram stain) TSA 35 24±2 (1) (2) 1 5 (3) 1 (4) 95% (5) 30 (6) (7) TSA (ONPG test) 0.85% 0.2 ml

11 (KCN test) ± (Indole test) 35 48± ml (VP test) MR-VP 35 48±2 1 ml A 0.6 ml B 0.2 ml (Methyl red test) MR-VP 35 48±2 5 ml (Citrate utilization test) 35 96± (Arginine dihydrolase test) 1 2 cm (Lysine decarboxylase test) 1 2 cm

12 (Ornithine decarboxylase test) 1 2 cm (Fermentation test) 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

13 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) % ( ) 75 89% V 25 74% ( ) 10 24% 0 9% 830

14 ( ) ( ) 0.1, 0.01, (g ml) ( ) ( ) (MPN/g MPN/mL) ( ) [ ( ) g ml] 95% ( ) [ ( ) g ml] 95% MPN/ ml (g) MPN/ ml (g) < VRBG ESA ( ) MPN 1100 (1) ( ) 0.1, 0.01, (g ml) 1100 (MPN/g MPN/mL) (2) ( ) 100, 10, 1 (g ml) (MPN/g MPN/mL)

15 real-time PCR DNA (real-time polymerase chain reaction, real-time PCR) 2.1. DNA real-time PCR real-time PCR Real-time PCR ( 1) Applied Biosystems 7500 Real-Time PCR System (Biological safety cabinet, BSC) (class II) ( ) (Micro refrigerated centrifuge) g nm 280 nm (-20 C) (Vortex mixer) (ph meter) g 0.1 g 100 g 1 mg DNA DNA 832

16 ( 2) Real-time PCR ( 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 carboxy-fluorescein (FAM) 3 Black Hole Quencher-1 (BHQ1) TaqMan Fast Reagents Starter Kit ( Applied Biosystems 7500 Real-Time PCR System) real-time PCR DNA DNA ( 3) (Micropipette) 2μL 10 μl 20 μl 100μL 200 μl 1000 μl (Pipette tip) 10 μl 20 μl 200 μl 1000 μl μl 600 μl 1.5 ml 2 ml Real-time PCR 100 μl Real-time PCR 96 Applied Biosystems 7500 Real-Time PCR System 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 μl 5 μm R μl 833

17 5 μm μl TaqMan Fast Reagents Starter Kit μl DNA μl μl μl 4 Real-time PCR 2.6. DNA DNA C ml 1.5 ml g ml g 3 1 ml 10 DNA DNA DNA DNA DNA 1.5 ml DNA DNA 1 ml 1.5 ml g ml DNA DNA DNA DNA 260 nm 280 nm (O.D.) 260 nm 50 ng/μl DNA DNA O.D. 260 /O.D ( 5)

18 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 C 20 sec C 15 sec C 40 sec Real-time PCR DNA real-time PCR real-time PCR 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

19 100 g 10 g 1 g 900 ml 90 ml 9 ml( ) ( 1) ml 90 ml VRBG ESA TSA real-time PCR ( 2) real-time PCR 836

1.適用範圍：本方法適用於禽畜肉、蛋及蛋製品等中單核球增多性李斯特菌之檢驗，惟乳品除外。

1.適用範圍：本方法適用於禽畜肉、蛋及蛋製品等中單核球增多性李斯特菌之檢驗，惟乳品除外。 286 ISSN 0255-6162 GPN 2006900031 286 93 10 20 161-2 0226531318 http://www.nlfd.gov.tw SCI Expanded 藥物食品化粧品檢驗分析 SCI Expanded 1 286 ( ) Lester A. Mitscher (Kansas Univ., USA) ( ) Haruki Yamada (Kitasato

行政院公報第 021 卷第 003 期 衛生勞動篇 Methods of Test for Food Microorganisms- Test of Cronobacter CFU/ (Biological safe

行政院公報第 021 卷第 003 期衛生勞動篇 Methods of Test for Food Microorganisms- Test of Cronobacter CFU/ (Biological safe