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MLG) 4.06
Isolation and Identification of Salmonella from Red Meat, Poultry, Egg and Environmental Samples
1
and the Food and Drug Administration-Bacterialogical Analytical Manual(FDA-BAM) Chapter 5
Detection and
Enumeration of Salmonella
2
as indicated in Table A. Details of the enrichment modifications can be found in Table
B.
A background screen of each food matrix was conducted prior to inoculation and no natural contamination of the
target organisms was detected in the test matrices. Each matrix was inoculated with a different strain of
Salmonella
as specified in Table A. Each inoculum was prepared by transferring a pure isolated colony of the specified
organism from trypticase soy agar with 5% sheep’s blood (SBA) into brain heart infusion (BHI) broth and
incubating the inoculum at 35 ± 2
o
C for 24 ± 2 hours. Post incubation, the inoculum was diluted using BHI as the
diluent, added to a bulk lot of test material and homogenized. For chicken carcasses, the inoculum was added drop-
wise to the cavity of the birds. The inoculum for the dry dog food was suspended in 10% non-fat dry milk and
lyophilized prior to inoculation. For pasteurized American cheese and creamy peanut butter, the organism was heat
stressed by incubating the culture for 10 minutes at 50±1°C prior to inoculation. The heat stressed culture was plated
onto a selective agar, Xylose, Lycine Desoxycholate (XLD) and a non-selective agar, Tryptic Soy Agar (TSA) to
determine percent reduction. The degree of injury was estimated as
where
n
select
= number of colonies on selective agar and
n
non-select
= number of colonies on non-selective agar and
reported in Table C. For matrices using a non-stressed culture, the diluted culture was used to inoculate the matrices.
All perishable food matrices were held for 48-72 hours post inoculation at 2-8
o
C to allow for equilibration of the
organism within the matrix. The bulk samples of dry dog food and peanut butter were inoculated and held at room
temperature (24±2°C) for a minimum of 2 weeks.
For all food matrices the level of
Salmonella
was determined by Most Probable Number (MPN)
3
on the day of
analysis by analyzing 5 x 100 g, 20 x 25 g (reference method test portions) and 5 x 10 g inoculated test samples.
Each test portion was enriched with the appropriate reference method enrichment broth at a 1:4 dilution and
analyzed by the reference method procedure. The number of positives, from each of the 3 sample sets, were
included in the calculation of the MPN using the LCF MPN calculator.
( http://www.lcfltd.com/customer/LCFMPNCalculator.exe )Environmental surfaces and chicken carcass surfaces were inoculated at a level expected to yield fractional
recovery. For concrete and sealed, glazed, ceramic tile, replicate 4"x 4" surface areas were inoculated with 250 µL
of diluted culture and allowed to dry for 18-24 hours at room temperature (24 ±2
o
C). For stainless steel, replicate
1"x 1" surfaces areas were inoculated with 100 µL of diluted culture and allowed to dry for 18-24 hours at room
temperature (24 ±2
o
C). Additionally, stainless steel surface areas were inoculated with
Proteus vulgarius
ATCC
6380 at 10x the level of the target organism. For chicken carcass sponges, the back and thighs of each carcass were
inoculated with 500 µL of diluted culture. The carcasses were stored at 4±1C for 48-72 hours to allow the organism
to equilibrate on the carcass. The inoculation level for the environmental surfaces and chicken carcasses was
determined by plating the inoculum onto TSA in triplicate. For environmental surfaces, sampling sponges and swabs
were pre-moistened with Dey-Engley neutralizing (DE) broth. For chicken carcass sponges, sponges were pre-
moistened with neutralizing buffer and 50 sq. cm surface areas, from the backs and thighs, were sampled. The
environmental surfaces and chicken carcass sponges were sampled using both horizontal and vertical sweeping
motions. The sponges and swabs were held at room temperature (24 ±2
o
C) for at least 2 hours prior to analysis.
Safety Precautions
The 3M Molecular Detection Assay
Salmonella
is intended for use in a laboratory environment by
professionals trained in laboratory techniques. The user should read, understand and follow all safety
information in the instructions for the 3M Molecular Detection System and the 3M Molecular Detection Assay
Salmonella
and retain the safety instructions for future reference. Follow all instructions carefully. Failure to
do so may lead to inaccurate results.
The 3M™ Molecular Detection Instrument is intended for use with samples that have undergone heat
treatment during the assay lysis step, which is designed to destroy organisms present in the sample. Samples
2013.09 /PTM Matrix Extension Report
March 2014
Expert Review Panel Use Only
AOAC Research Institute
Expert Review Panel Use O ly