Attachment_3_Final.docx

Attachment 3

FDA-SOL-1116253

Microbiological Analysis Methods

Microbiological methods for each analysis shall be approved by the FDA. Sample sizes may vary depending on the sample type tested.

Approved methods include but are not limited to:

· Salmonella analysis shall be performed using a modified version of the FDA- Bacteriological Analytical Manual (BAM) (http://www.fda.gov/Food/FoodScienceResearch/LaboratoryMethods/ucm070149.htm) or any appropriate Salmonella method found in the Official Methods of Analysis (OMA: Official Methods of Analysis of AOAC INTERNATIONAL, 19th Ed., 2012. AOAC INTERNATIONAL, Gaithersburg, MD), or another appropriate method agreed upon by the CO or COR. The modifications include:

· Pre-enrichment in modified buffered peptone water, using 1:10 dilution of sample to buffer for all samples.

· All RV enrichments incubated at 42C and all TT enrichments incubated at 43C

· Plating onto XLT-4, HE plus 5ug/ml novobiocin and BS agars

· Presumptive Salmonella colonies maybe confirmed by automated methods such as the Vitek or Vitek 2.

· E. coli STEC (O157 and non-0157) analysis shall be performed as by Strawn et. al. (Strawn, L. K., et al., 2013. Landscape and Meteorological Factors Affecting Prevalence of Three Food-Borne Pathogens in Fruit and Vegetable Farms. Appl. Environ. Microbiol. 79:588-600.), any appropriate E. coli STEC (O157 and non-O157) method found in the Official Methods of Analysis (OMA: Official Methods of Analysis of AOAC INTERNATIONAL, 19th Ed., 2012. AOAC INTERNATIONAL, Gaithersburg, MD), or another appropriate method agreed upon by the CO or COR.

· Strawn et al method:

· For O157 STEC’s: Samples in Whirl-Pak bags were diluted 1:10 with pTSB and incubated for 2 h at room temperature (23 ± 2°C) to aid in the recovery of injured cells (32). Enrichments were transferred to 42 ± 2°C and incubated for 24 h. Enrichments were subjected to immunomagnetic separation (IMS) to concentrate E. coli O157:H7 cells as previously described (33). Washed IMS beads (50 μl) were plated onto two selective and differential media: modified sorbitol-MacConkey agar (mSMAC; Becton, Dickinson, Franklin Lakes, NJ) supplemented with 20 mg/liter of novobiocin and 2.5 mg/liter of potassium tellurite (Sigma-Aldrich, St. Louis, MO) and CHROMagar O157 agar (CHROMagar, Paris, France). CHROMagar O157 and mSMAC plates were incubated at 37 ± 2°C for 24 and 48 h, respectively. Up to 10 presumptive E. coli O157:H7 colonies were sub-streaked onto BHI and incubated at 37 ± 2°C for 24 h.

· Presumptive colonies may be confirmed by PCR or real-time PCR using a commercially available kit as approved by the COR.

· For Non-O157 STECs: To isolate non-O157 STEC, a 1-ml aliquot of the nonselective enrichment was transferred to 9 ml of EC broth (Oxoid) and incubated at 37°C with shaking for 24 h. A 50-μl aliquot of E. coli broth was plated onto washed sheep's blood agar (Hemostat, Dixon, CA) with 10 mM CaCl2 and 0.5 μg/ml mitomycin C (WBMA; Fisher Scientific, Hampton, NH) and incubated at 35 ± 2°C for 24 h. Up to 20 colonies that demonstrated enterohemolysis were sub-streaked to sorbitol MacConkey agar (SMAC) plates and incubated at 37 ± 2°C for 24 h. Up to 10 colonies that rapidly fermented sorbitol were sub-streaked to BHI and incubated at 37 ± 2°C for 24 h.

· Presumptive non-O157 STEC colonies may be confirmed by testing for the presence of the shiga-toxin genes, stx1 and stx2, by PCR or real-time PCR by a commercially available kit or other method approved by the COR.

· Listeria analysis shall be performed as by Strawn et. al. (Strawn, L. K., et al., 2013. Landscape and Meteorological Factors Affecting Prevalence of Three Food-Borne Pathogens in Fruit and Vegetable Farms. Appl. Environ. Microbiol. 79:588-600.), or any appropriate L. monocytogenes method found in the Official Methods of Analysis (OMA: Official Methods of Analysis of AOAC INTERNATIONAL, 19th Ed., 2012. AOAC INTERNATIONAL, Gaithersburg, MD), or another method agreed upon by the CO or COR.

· Strawn et al method:

· Samples diluted 1:10 with buffered Listeria enrichment broth (BLEB; Becton, Dickinson, Franklin Lakes, NJ). These enrichments were incubated at 30 ± 2°C for 4 h. At 4 h, Listeria selective enrichment supplement (Oxoid, Cambridge, United Kingdom) was added. After 24-h and 48-h incubations at 30 ± 2°C, 50 μl of each enrichment was streaked onto modified Oxford agar (MOX; Becton, Dickinson, Franklin Lakes, NJ) and L. monocytogenes plating medium (LMPM; Biosynth International, Itasca, IL). MOX and LMPM plates were incubated for 48 h at 30 and 35 ± 2°C, respectively. Up to 10 L. monocytogenes presumptive colonies were sub-streaked to brain heart infusion (BHI) agar (Becton, Dickinson, Franklin Lakes, NJ). BHI agar plates were incubated for 37 ± 2°C for 24 h.

· Presumptive colonies should be confirmed by PCR as outlined in FDA-BAM(http://www.fda.gov/Food/FoodScienceResearch/LaboratoryMethods/ucm279532.htm)or another method as approved by the COR.

· Vibrio analysis shall be performed as in the FDA-BAM (http://www.fda.gov/Food/ScienceResearch/LaboratoryMethods/BacteriologicalAnalyticalManualBAM/ucm070830.htm), or any appropriate Vibrio method found in the Official Methods of Analysis (OMA: Official Methods of Analysis of AOAC INTERNATIONAL, 19th Ed., 2012. AOAC INTERNATIONAL, Gaithersburg, MD), or another appropriate method agreed upon by the CO or COR. The modifications include:

· Scaling of pre-enrichment broth to accommodate for smaller sample size than listed in the current method.

· Norovirus and Hepatitis A analysis shall be performed as below or any appropriate Norovirus/Hepatitis A/enteric virus method found in the Official Methods of Analysis (OMA: Official Methods of Analysis of AOAC INTERNATIONAL, 19th Ed., 2012. AOAC INTERNATIONAL, Gaithersburg, MD), or another method agreed upon by the CO or COR.

· For vegetation, produce and soil samples:

· For concentrated water samples:

Concentration and Extraction of Viruses in Sewage

Place 40mL of sewage in ultra-centrifuge tubes Centrifuge at 37K rpm, 4C, 1Hour (Tubes should be within 0.05) Discard Supernatant Re-suspend Pellet in 4mL 0.05M Glycine Buffer Incubate on ice 30min (shake occasionally) Add 4mL 2x PBS Shake to Mix Centrifuge at 4800rpm, 4C, 20Min Save supernatant to clean tube Add 1x PBS to ¾ full Balance tubes with 0.05g of one another Centrifuge at 37K rpm, 4C, 1Hour Discard Supernatant Re-suspend Pellet in 800uL 1x PBS Split into 4-2mL centrifuge tubes To one tube add 400uL 6M GITC and vortex to dissolve pellet Sample should incubate for up to 30 minutes, RT Add equal volume 70% EtOH & invert twice to ppt RNA (50% EtOH for samples with more debris) Transfer 700uL to mini spin column (RNeasy Qiagen Kit) Centrifuge 8 to 10,000g, 1min Discard eluent & add remaining sample to column, repeat (Preheat your TE buffer to 70C) Add 700uL RWI wash buffer to column Incubate at RT 5-10min Centrifuge 8 to 10,000g, 1min & discard eluent Add 500uL RPE Incubate at RT, 5-10min Centrifuge 8 to 10,000g, 1min & discard eluent Repeat Change collection tubes Add 35uL preheated Primer TE buffer to column Centrifuge 8 to 10,000g, 1min. (do NOT discard eluent) Add 25uL preheated Primer TE to column Additionally, add the eluent back to column Centrifuge 8 to 10,000g, 1min Save Eluent

For RT-PCR detection of enteric viruses:

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