413 / 1195
Page
11
of
38
3M Molecular Detection Assay Salmonella –Test Portion Analysis
After incubation, samples were analyzed according to the 3M MDA
Salmonella
package insert. Prior to analysis,
lysis tubes were brought to room temperature (25 ± 2
o
C) by placing the tubes on a sanitized bench top for 18 ± 2
hours. A 20 µL aliquot of each sample was transferred to separate lysis tubes, and tubes were mixed by inverting
three times, ensuring that the resin in the bottom of the lysis tubes was dispersed throughout the sample. After
mixing, samples were placed in a dry bath incubator for 15 minutes at 100 ± 1
o
C. Following the heat lysis, samples
were transferred to a cooling block and were allowed to cool at 0°C-20°C for 10 minutes. After cooling, samples
were mixed by inverting the rack three times and then firmly tapping the rack onto a sanitized bench top three times
to settle the resin. Samples were allowed to sit undisturbed on a sanitized bench top for 5 minutes.
A 20 µL aliquot of each lysed sample and control was added to separate reagent tubes, one for each assay, and
samples were mixed by pipetting up and down 5 times. A matrix control tube was analyzed with the samples for
each matrix to verify that no interference with the assay was caused by the matrix. A 20 µL aliquot of a randomly
picked sample was added to the matrix control tube, mixed, and recapped. Using the 3M software, prompts were
followed to identify samples and controls. All samples were loaded into the Speed Loader Tray (SLT), placed into
the Molecular Detection System, and the 3M MDA
Salmonella
was
initiated and results were obtained within 75
minutes. Regardless of presumptive results, all test portions were confirmed following the appropriate reference
methods.
USDA/FSIS MLG 4.06 Isolation and Identification of Salmonella from Meat, Poultry, Pasteurized Egg and Catfish
Products
For the USDA/FSIS MLG 4.06 reference method, 25 ± 2.5 g test portions were enriched with 225 ± 22.5 mL of
buffered peptone water (BPW) and incubated at 35 ± 2°C for 22 ± 2 hours. For chicken carcass rinses, 30 ± 0.6 mL
of rinsate was combined with 30 ± 0.6 mL BPW and incubated at 35 ± 2°C for 22 ± 2 hours. Chicken carcass
sponges were enriched with 50 ± 0.6 mL of BPW and incubated at 35 ± 2°C for 22 ± 2 hours. After incubation, 0.1
± 0.02 mL of each sample was transferred to 10 mL of modified Rappaport-Vasilliadis broth (mRV) and 0.5 ± 0.05
mL into 10 mL of tetrathionate Hajna broth (TT Hajna). The broths were incubated in a water bath at 42 ± 0.5°C for
18-24 hours. Following incubation and for each sample, a loopful from each broth replicate was streaked to xylose-
lysine-tergitol™ 4 (XLT4) and brilliant green sulfa agar (BGSA). Both selective agars were incubated at 35 ± 2
o
C
for 18-24 hours. Presumptive positive
Salmonella
colonies from each selective agar were picked and transferred to
triple sugar iron agar (TSI) and lysine iron agar (LIA) slants and incubated at 35 ± 2°C for 24 ± 2 hours. Growth
from samples producing typical biochemical reactions in TSI was transferred to trypticase soy-tryptose broth
(TSTB) and incubated at 35 ± 2°C for 24 ± 2 hours. Growth from the TSTB was used to conduct the flagellar H
serological test. Growth from samples producing typical biochemical reactions in TSI and LIA, were streaked to
trypticase soy agar (TSA) slants and incubated at 35 ± 2
o
C for 18-24 hours. Growth from the TSA slant was used to
conduct the polyvalent O serological test for biochemical confirmation and final confirmations using VITEK
®
2 GN
following AOAC Official Method 2011.17.
FDA-BAM Chapter 5 Detection and Enumeration of Salmonella
Environmental sponges and 25 g samples were enriched with 225 mL of lactose broth and environmental swabs
were enriched with 10 mL of lactose broth. For sprout irrigation water, 375 mL test portions were enriched with
3375 mL of BPW with novobiocin. The enrichments were homogenized by stomaching or vortexing for 2 minutes
and placed at room temperature for 60 ± 5 minutes. If necessary, the pH of the enrichments was adjusted to 6.8 ±
0.2. No pH adjustment was needed for any matrix. Subsequently, the enrichments were incubated at 35 ± 2°C for
24 ± 2 hours. Following incubation and for each sample, 0.1 mL of primary enrichment was transferred into 10 mL
of Rappaport-Vassiliadis medium (RV) and 1.0 mL into 10 mL of tetrathionate (TT) broth. RV tubes were
incubated at 42 ± 0.2
o
C for 24 ± 2 hours. Aerobic plate counts for sprout irrigation water and dry dog food were <
10
4
, therefore, TT tubes were incubated at 35 ± 2
o
C for 24 ± 2 hours. Following incubation, a loopful from each
secondary enrichment was streaked to bismuth sulfite agar (BS), Hektoen enteric agar (HE), and xylose lysine
desoxycholate agar (XLD) and incubated at 35 ± 2
o
C for 24 ± 2 hours. If no visible colonies were present after 24
hours of incubation, BS plates were re-incubated for an additional 24 ± 2 hours at 35 ± 2
o
C. Up to 2 or more suspect
colonies from each selective agar were transferred to TSI and LIA and incubated at 35 ± 2°C for 24 ± 2 hours.
Growth from samples producing typical biochemical reactions in TSI was transferred to TSTB and incubated at 35
2013.09 /PTM Matrix Extension Report
March 2014
Expert Review Panel Use Only
AOAC Research Institute
Expert Review Panel Use Only