Approved methods for microbiological testing of meat and meat products

Amended July 2022

The following is a list of Department of Agriculture, Fisheries and Forestry (DAFF) approved test methods for meat and meat products. From 31 January 2006 all testing of product relating to export certification, including carcass testing under National Carcase Microbiology Monitoring Program (formerly known as ESAM), must be by one of the methods listed with the modifications and options specified; no other modifications are permitted. Laboratory manuals and protocols must reflect the above and will be subject to audits to ensure compliance.

• Aerobic Plate Count/Total Viable Count (TVC)
• Escherichia coli O157:H7
• Shiga-toxin producing Escherichia coli  (STEC)
• Generic Escherichia coli
• Listeria monocytogenes
• Salmonella

Aerobic Plate Count/Total Viable Count (TVC)

• Australian Standard AS 5013.5-2016 Horizontal method for the enumeration of microorganisms – Colony count at 30°C by the pour plate technique
• AOAC 990.12 TVC Petrifilm™
• AOAC 2008.10 TEMPO TVC Method: Automated Enumeration of Total Viable Count in Food
• AOAC 010404 Compact Dry TC
• AOAC 091702 and MicroVal 2015LR52 MC-Media PAD AOAC 091702 is a validation study for incubation of MC-Media Pad at 35 ± 1°C for 24 -48 h and applies only to 50 g raw meats and other foods. MicroVal 2015LR52 is a validation study for 10 g samples, incubated at 30 ± 1°C for 72 h
• Other methods Any method that has been validated by an internationally recognised certification body using ISO 16140 (or equivalent ie AOAC guidelines) for the enumeration of total viable count in meat and meat products

Note where specific market access requirements exist for the methodology used to determine total viable count these requirements must be met.

Escherichia coli O157:H7

• ISO 16654:2001 Microbiology of food and animal feeding stuffs ¡V Horizontal method for the detection of Escherichia coli O157. Note - when analysing frozen or chilled samples, the temperature of broth and samples must be at 41.5 ± 1°C for a minimum of 6 h and subsequently for a further 12 to 18 hours.
• FSIS MLG 5 Detection, isolation, and identification of Escherichia coli O157:H7 from meat products. When analysing frozen or chilled samples, the temperature of broth and samples must be at 42 ± 1°C for a minimum of 15 hours. Note the method has been updated changing the initial enrichment to mTSB and the definition of E. coli O157:H7
• FDA BAM Chapter 4A(K) Diarrheagenic Escherichia coli - Enrichment and isolation of E. coli Serotype O157:H7 from Foods With the following modification; must use the IMS option and a sample size of 325 g for ground beef, analysed as five separate 65 g portions

Rapid methods

Where positive confirmation is required such confirmation must be by ISO 16654:2001, FDA BAM 4A(K) or FSIS MLG 5

Note all modifications/notes listed for each method must be followed

• FSIS MLG 5A
  FSIS procedure for the use of Escherichia coli O157:H7 screening tests
  
  (15-22h PCR based screening test using DuPont BAX MP)
  
  Note the method has been updated and only mTSB should be used for enrichment of samples and the definition of E. coli O157:H7 has been changed. Temperature of broth and samples must be at 42°C for a minimum of 15 hours.
   
• AOAC 031002 DuPont Qualicon BAX® System PCR Assay for Real-Time E. coli O157:H7
  This method is approved for 375 g composite samples in 1.5 L of BAX® System E. coli O157:H7 MP medium and incubation for 10-24 h at 42°C. Note temperature of broth and sample must be at 42°C for a minimum of 10 hours.
   
• AOAC 2005.04 Assurance GDS for Escherichia coli O157:H7 in Selected Foods
  This method is approved for using 375 g composite samples in 1.2 L mEHEC medium and incubation for 8-18 h at 42°C. Note temperature of broth and samples must be at 42°C for a minimum of 8 hours.
   
• AOAC 071001 MicroSEQ(R) Real-Time PCR System for Detection of E. coli O157:H7 in raw ground beef and beef trim
  This method is approved for 375 g composite samples in 1.5 L of BPW and incubation for 16 h at 42°C. Note temperature of broth and sample must be at 42°C for a minimum of 16 hours
   
• AOAC 2017.01 3M Molecular Detection Assay (MDA) 2 – E. coli O157 (including H7)
  Method: This method is approved for 375 g sample in 975 mL of ISO BPW and incubation for 10 – 18 h at 41 ± 1°C. Note – temperature of broth and sample must be at 41± 1°C for a minimum of 10 hours.
   
• AOAC 022002 BACGene E. coli O157:H7 Workflow – This method is approved for 375 g composite samples in 750 mL (1:2) of mTSB and incubation for 10-24 h at 41.5 ± 1°C. Note temperature of broth and sample must be at 41 ± 1°C for a minimum of 10 hours.
   
• AOAC 2019.03 GENE-UP E. coli O157:H7 2 (ECO 2) – This method is approved for 375 g composite samples in 1,125 mL of buffer peptone water and incubation for 10-24 h at 42± 1°C. Note temperature of broth and sample must be at 42 ± 1°C for a minimum of 10 hours. The GENE-UP® E. coli O157:H7 2 kit (REF 423108) must be used in conjunction with the GENE-UP® Lysis kit (REF 414057).
  
  The following rapid methods are not to be used for the routine testing of export meat and meat products for E. coli O157. They are approved as backup methods for use when PCR methods are temporarily unavailable. They may be used for the testing of product under commercial arrangements when a methodology is not specified under that arrangement:
   
• AOAC 996.09 BioControl VIP (8-12 h and 18-28 h options)
  With the following modification: disregard plating steps for confirmation, confirmation must be by ISO 16654:2001, FSIS MLG 5.05 or FDA BAM using the IMS option. 8 h enrichment is carried out in mEHEC media Note 18-28h option for 375g samples incubated in 1L of mTSB+n media has been validated and is approved, 8-12h option for 375g in 1L of mEHEC media has been validated and approved. Temperature of broth and samples must be at 36°C for a minimum of 18 hours (for 18-28 h protocol) or at 42°C for a minimum of 8 hours (8-12 h protocol).
   
• AOAC 2000.13 Reveal (8-hours)
  With the following modification: disregard plating steps for confirmation, confirmation must be by ISO 16654:2001, FSIS MLG 5.04 or FDA BAM using the IMS option. Note longer incubation times (12-14h) are required for large samples (375g) diluted less than 1:10 in initial enrichment media ie in one litre. Temperature of broth and samples must be at 42°C for a minimum of 8 hours.
   
• AOAC 2000.14 Reveal (20-hours)
  With the following modification: disregard plating steps for confirmation, confirmation must be by ISO 16654:2001, FSIS MLG 5.04 or FDA BAM using the IMS option. Note samples can be enriched for 18-24h as specified in Journal of AOAC International 84(3), 2001: 737-751
   
• AOAC 070201 (Performance Tested Method) Rapid√ for Escherichia coli O157 Lateral Flow Assay
  With the following modifications, mTSB+n must be used for selective enrichment and enrichment can only be for 15 to 22h at 42±1ºC (as specified in MLG 5.05). The 8 hour enrichment option is not approved. Note ¡V temperature of broth and samples must be at 42 ± 1°C for a minimum of 15 hours.

Shiga-toxin producing Escherichia coli (STEC)

• FSIS MLG 5B Detection and isolation of non-O157 Shiga-toxin Producing Escherichia coli (STEC) from meat products

Rapid methods

Where positive confirmation is required such confirmation must be by FSIS MLG 5B

• AOAC 071301 Assurance GDS® MPX Top 7 STEC for detection of top 7 pathogenic STEC in beef trim. Samples (375 g) are diluted in 1.5 L of pre-warmed (42°C) mEHEC medium. Incubation is carried out for 10 h at 42°C. Note ¡V temperature of broth and samples must be 42 ± 1°C for a minimum of 10 hours
• AOAC 091301 DuPont Qualicon BAX® System Real-Time PCR Assays for detection of selected STEC in beef trim. Samples (375 g) are diluted in 1.5 L pre-warmed (45-46°C) Bax® System MP enrichment broth. Samples are incubated at 39-42°C for 12-24 h. Note ¡V temperature of broth and samples must be at 39-42°C for a minimum of 12 hours.
• AOAC 0100701 IEH E. coli Test System for detection of non-O157 Shiga-toxin producing E. coli and E. coli O157 in raw ground beef. Samples (375 g) are diluted in 750 mL of pre-warmed IEH enrichment medium. Incubation at 42 ± 1°C for 9-48 hours. Note ¡V temperature of broth and samples must be 42°C for a minimum of 9 hours
• AOAC 061602  RapidFinderTM STEC Detection Workflow for detection of top 7 STEC serogroups in beef products. Samples (375 g) are diluted in 1.0 L of pre-warmed (48°C) Trypticase Soy Broth. Incubation at 42°C for 8 hours. Note ¡V temperature of broth and samples must be at 42°C for a minimum of 8 hours.
• AOAC 031401 GeneDisc(R) Plate  STEC Top 7 methods for detection of O157 and top 6 non-O157 Shiga toxin producing E. coli in raw ground beef and beef trim Samples (375 g) are diluted in 1.5 L of pre-warmed (41.5 ± 1„aC) BPW Broth. Incubation at 41.5 ± 1„aC for 10-20 hours. Note ¡V temperature of broth and samples must be at 41.5 ± 1„aC for a minimum of 10 hours
• AOAC 101502 Assurance GDS® MPX ID for Top 6 STEC
  Detection of Top 6 Shiga toxin-producing E. coli (O26, O45, O103, O111, O121 and O145) in beef trim as a secondary screening method following a positive result using the Assurance GDS® MPX Top 7 STEC assay (AOAC 071301). All screen positive samples must be confirmed by MLG 5B.
   
• AOAC 022003 BACGene STEC Top 7 Workflow – This method is approved for 375 g composite samples in 750 mL (1:2) of mTSB and incubation for 10-24 h at 41.5 ± 1°C. Note temperature of broth and sample must be at 41 ± 1°C for a minimum of 10 hours.
   
• AOAC 121806 GENE-UP EHEC Detection Method – This method is approved for 375 g composite samples in 1,125 mL of buffer peptone water and incubation for 10-24 h at 41.5 ± 1°C. Note temperature of broth and sample must be at 41 ± 1°C for a minimum of 10 hours. This method is approved for STEC confirmatory testings.
• AOAC 071902 - 3M Molecular Detection Assay (MDA) 2 – STEC Gene Screen (stx and eae) gene
• AOAC 081901 - NeoSeek STEC - This method is approved for STEC confirmatory testings.
• AOAC 012102 – SureTect E. coli O157:H7 and STEC Screening PCR Assay

Generic Escherichia coli

• Australian Standard AS 5013.15 General guidance for enumeration of presumptive Escherichia coli - Most probable number technique Note this is an update from the 2004 standard, the main change being that the temperature of incubation is now 44±1ºC
• AOAC 991.14 E. coli PetriFilm™ Butterfields or buffered peptone water or 0.1% Peptone Salt Solution must be used as diluent
• AOAC 998.08 E. coli PetriFilm™ Butterfields or buffered peptone water or 0.1% Peptone Salt Solution must be used as diluent
• AOAC 2005.03 SimPlate® Colour Indicator: Detection and Quantitation of Coliforms and E. coli in foods
• AOAC 2009.02 Tempo® EC AFNOR Bio 12/13 ¡V 02/05 for testing of generic E. Coli
• AOAC 110402 Compact Dry EC
• AOAC 070901 and MicroVal 2017LR71 MC-Media Pad EC . AOAC 070901 is a validation study that applies only to 50 g raw meats and other foods. MicroVal 2017LR71 is a validation study that applies to sample diluted 1:10.
• AOAC 2018.13 - 3M Petrifilm Rapid E. coli/Coliform Count Plate

Listeria monocytogenes

• Australian Standard AS 5013.24.1 Food and animal feeding stuffs ¡V Horizontal method for the detection and enumeration of Listeria monocytogenes. Detection method
• FSIS MLG 8 Isolation and identification of Listeria monocytogenes from red meat, poultry, egg, and environmental samples

Note alternative secondary enrichment has been included

Rapid methods

Where positive confirmation is required such confirmation must be by Australian Standard AS 5013.24.1 or FSIS MLG 8
Note the following bio-chemical test systems can be used for confirmation for all methods MICRO-ID® Listeria or API®-Listeria or VITE® 2 Compact, . β-lysin. CAMP factor discs (Remel #21-120, or equivalent) can be used instead of the traditional CAMP test procedure

• FSIS MLG 8A FSIS procedure for the use of Listeria monocytogenes BAX screening test Note the method has been updated to include testing of liquid egg products
• FDA BAM Cha 10 Detection and Enumeration of Listeria monocytogenes in Foods
• AOAC 2003.12 Automated BAX System for Detection of Listeria monocytogenes in Foods
• AOAC 999.06 VIDAS LIS Assay for Listeria in Foods (AOAC 2004.06 - VIDAS LIS modification)
• AOAC 996.14 BioControl Assurance Listeria Immunoassay
• AOAC 997.03 BioControl Listeria Visual Immunoprepicipate (VIP) Assay
• AOAC 070401 Foodproof Listeria mon9ocytogenes Detection Kit, 5¡¦Nuclease and Hybridization Probes
• AOAC 031204 Pall GeneDisc(R) method for the detection of Listeria monocytogenes in food and environmental samples
• AOAC 011022 MicroSEQ(R) Real-Time PCR System for Detection of Listeria monocytogenes in food
• NF BIO 12/33 – 05/12 VIDAS UP Listeria method (VIDAS LPT)
• Solus Listeria ELISA – NF SOL 37/02 – 06/13
• AOAC 071304 Thermo Scientific SureTect Listeria species PCR Assay (AFNOR UNI 03/09 - 11/13)
• AOAC 121402 DuPontTM BAX® System Real-Time PCR Assay for Listeria monocytogenes
• AOAC 2016.08 3MTM Molecular Detection Assay (MDA) 2 – Listeria monocytogenes Method
• AOAC 061703 BACGene Listeria monocytogenes

 Salmonella

• Australian Standard AS 5013.10 Microbiology of food and animal feeding stuffs - Horizontal method for the detection of Salmonella spp The following options are required: Second agar choice must be capable of detecting H2S negative Salmonella (internationally validated Salmonella media e.g. BGA, BGS, Rambach, ChromAgar)
• FSIS MLG 4 Isolation and identification of Salmonella from meat, poultry and egg products

Rapid methods

When positive confirmation is required such confirmation must be by Australian Standard AS 5013.10 or FSIS MLG 4

• FSIS MLG 4C FSIS procedure for the use of the BAX system PCR assay for screening Salmonella in raw meat, carcass sponge samples, whole bird rinses, ready-to-eat meat and poultry products and pasteurised egg products Note procedure to follow when a PCR indeterminate or signal-error occurs has been updated
• AOAC 2009.03 Assurance GDS™ Salmonella method for foods
• AOAC 992.11 BioControl Assurance EIA With instructions specified for pre-enrichment in table 999.08 C (ie use of BPW + novobiocin)
• AOAC 999.08 BioControl Assurance Gold pre-enrichment with BPW + novobiocin as per instructions
• AOAC 999.09 BioControl VIP With instructions specified for pre-enrichment in table 999.08 C (ie use of BPW + novobiocin)
• AOAC 996.08 VIDAS Salmonella (SLM) Assay
• AFNOR BIO 12/16 ¡V 09/05 VIDAS EASY Salmonella method
• AOAC 071101 VIDAS UP Salmonella method (VIDAS SPT)
• AOAC 2001.09 VIDAS Immuno Concentration Salmonella (ICS)
• AOAC 2009.03 Assurance GDS™ Salmonella method for foods and Assurance GDS Salmonella Tq method
• AOAC 100701 IEH PCR assay for detection of Salmonella in carcass and environmental sponges or swabs
• AOAC 120301 foodproof Salmonella Detection Kit, 5¡¦Nuclease and Hybridization Probes
• AOAC 100201 DuPont Qualicon BAX (R) System Salmonella2 PcR Assay
• AOAC 2013.02 DuPont Qualicon BAX(R) System real-time PCR assay for Salmonella
• AOAC 031001 MicroSEQ(R) Real-Time PCR System for Detection of Salmonella in food
• AOAC 050602 Assurance GDS for Salmonella
• AOAC 2014.01 3M Petrifilm Salmonella Express System
• AOAC 011404 Veriflow Salmonella species (SS)
• NF SOL 37/01 – 06/13 Solus Salmonella ELISA
• AOAC 051303 Thermo Scientific SureTect Salmonella spp PCR Assay (AFNOR UNI 03/07 – 11/13)
• AOAC 2016.01 3MTM Molecular Detection Assay (MDA) 2 – Salmonella Method​​
• AOAC 121501 BACGene Salmonella spp.