170 / 1195
10
(c)
Cluster tubes and racks. –For lysis.
1
(d)
Capping/de-capping tools.–
For removing and sealing cluster tube caps and PCR tube caps
2
without jarring the contents.
3
(e)
Thermal block(s) with inserts.–
For maintaining lysis tubes at 37±2°C, 95±3°C, and 4±3°C.
4
(f)
Pipettes.–
For transferring reagents; two adjustable mechanical pipettes covering 20-200
µ
L
5
and 5-50
µ
L; one repeating pipette; and one multi-channel pipette covering 8 channels and
6
5-50
µ
L. Pipettes should be calibrated to deliver required volumes within 10%.
7
(g)
Pipette tips with barriers.–
0.5-250
µ
L, 0.5-100
µ
L extended barrier; 5 mL repeater pipette
8
tips.
9
(h)
BAX® System PCR tubes.–
12 strips of 8 tubes containing one PCR tablet per tube.
10
(i)
Flat optical caps.
11
(j)
PCR tube holders.–
For transferring a rack of tubes from the cooling block to the
12
cycler/detector.
13
(k)
Incubators.–
For maintaining enrichment media at 35
°±
1
°
C and 39-42
°
C.
14
(l)
Stomacher®.–
Seward model 400 or equivalent.
15
(m)
Stomacher® type bags, or equivalent.
16
(n)
Blender.–
High speed (16,000 – 18,000 rpm) with sterile receptacle.
17
18
C.
Media and Reagents
19
20
(a)
BAX® System lysis buffer.–
Two bottles of 12 mL each.
21
(b)
BAX® System protease.–
One vial of 400 µL.
22
(c)
Lysis reagent.–
Pipette 150 µL of protease into on 12 mL bottle of lysis buffer. Reagent will
23
remain stable for up to 2 weeks if stored at 2 - 8
°
C.
24
(d)
BAX® System MP media.–
DuPont Qualicon catalog D12404925 (bulk powder), D12745725
25
(StatMedia).
26
(e)
Lactose broth.–
Dissolve 3 g of beef extract, 5 g of peptone and 5 g of lactose into 1 L of
27
distilled water. Dispense into containers and autoclave 15 minutes at 121
°
C. Final pH is
28
6.9
±
0.2.
29
(f)
Modified Tryptic Soy Broth containing glucose and 2 mg/L novobiocin (TSB + n)
, Difco
30
catalog number 211825 or equivalent, BD, Franklin Lakes, NJ, 07417, USA
31
(g)
Buffered Peptone Water (BPW).–
Oxoid CM 0509 or equivalent, Oxoid, Hampshire, United
32
Kingdom, RG24 8PW.
33
(h)
Universal preenrichment broth.–
Add 5 g tryptone, 5 g proteose peptone, 15 g potassium
34
phosphate, 7 g sodium phosphate, 5 g sodium chloride, 0.5 g dextrose, 0.25 g magnesium
35
sulfate, 0.1 g ferric ammonium citrate and 0.2 g sodium pyruvate to 1 L distilled water. Heat
36
ingredients with gentle agitation to dissolve, dispense and autoclave at 121°C for 15 min. The
37
final pH should be 6.3 ± 0.2. Difco catalog number 223510 or equivalent, BD, Franklin Lakes, NJ,
38
07417, USA.
39
(i)
Brain Heart Infusion (BHI) broth.–
Oxoid CM 1032 or equivalent.
40
Collaborative Study Approved Protocol
Expert Review Panel Use Only