Approved methods for microbiological testing of meat and meat products

Amended May 2012

The following is a list of approved test methods for meat and meat products. From 31st January 2006 all testing of product relating to export certification, including carcass testing under 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
• Escherichia coli non-O157 (top six STEC)
• Generic Escherichia coli
• Listeria monocytogenes
• Salmonella

Aerobic Plate Count/Total Viable Count (TVC)

• Australian Standard AS 5013.1-2004 Examination for specific organisms - Standard plate count
• AOAC 990.12 TVC Petrifilm™
• AOAC 2008.10 TEMPO TVC Method: Automated Enumeration of Total Viable Count in Food
• 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 – 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 5.05 Detection, isolation, and identification of Escherichia coli O157:H7 and O157:NM (nonmotile) 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+n 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 5.05
Note all rapid methods are approved for testing 375g trim samples using one litre of primary enrichment media, all modifications/notes listed for each method must be followed

• FSIS MLG 5A.02
  FSIS procedure for the use of Escherichia coli O157:H7 and O157:NM (nonmotile) screening tests
  (15-22h PCR based screening test using DuPont BAX MP)
  Note the method has been updated and only mTSB+n or BAX System Media can be used for enrichment of samples and the definition of E. coli O157:H7 has been changed. 8-24h option for 375g samples incubated in 1L of BAX systems media has been validated and is approved. Note - temperature of broth and samples must be at 42 ±1°C for a minimum of 15 hours (if use mTSB+n) or 8 hours (if use BAX MP medium).
• 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 ±1°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 996.10 BioControl Assurance (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 5.04 or FDA BAM using the IMS option. 8h 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 ±1°C for a minimum of 18 hours (for 18-24 h protocol) or at 42°C for a minimum of 8 hours (8 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 – temperature of broth and samples must be at 42 ± 1°C for a minimum of 15 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 031002 (Performance Tested Method) 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.

Non-O157 Escherichia coli (STEC)

• FSIS MLG 5B.01 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.01

• 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 – temperature of broth and samples must be 42 ± 1C for a minimum of 10 hours
• 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 – temperature of broth and samples must be at 39-42C for a minimum of 12 hours.
• 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 – temperature of broth and samples must be 42C for a minimum of 9 hours

Generic Escherichia coli

• Australian Standard AS 5013.15-2006 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 – 02/05 for testing of generic E. coli

Listeria monocytogenes

• Australian Standard AS 5013.24.1–2009 Food and animal feeding stuffs – Horizontal method for the detection and enumeration of Listeria monocytogenes. Detection method
• FSIS MLG 8.07 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 5013.24.1–2009 or FSIS MLG 8.07
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.04 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 (Jan 2003) Detection and Enumeration of Listeria monocytogenes in Foods
• AOAC 995.22 TECRA Listeria Visual Immunoassay
• AOAC 996.14 BioControl Assurance Listeria Immunoassay
• AOAC 997.03 BioControl Listeria Visual Immunoprepicipate (VIP) Assay
• AOAC 999.06 VIDAS LIS Assay for Listeria in Foods (AOAC 2004.06 - VIDAS LIS modification)
• AOAC 2002.09 TECRA Listeria Visual Immunoassay
• AOAC 2003.12 Automated BAX System for Detection of Listeria monocytogenes in Foods

Salmonella

• Australian Standard AS 5013.10-2009 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 4.04 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 5013.10-2009 or FSIS MLG 4.04

• FSIS MLG 4C.02 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 989.14 TECRA Visual Immunoassay
  With the following modification: pre-enrichment in buffered peptone water (BPW)
• AOAC 992.11 BioControl Assurance EIA
  With instructions specified for pre-enrichment in table 999.08 C (ie use of BPW + novobiocin)
• AOAC 996.08 VIDAS Salmonella (SLM) Assay
• AOAC 998.09 TECRA ULTIMA
  With the following modification: pre-enrichment in BPW
• AOAC 998.09 TECRA Visual Immunoassay (VIA)
  With the following modification: pre-enrichment in buffered peptone water (BPW).
  Only Option 1 is approved
• 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 2001.07 VIDAS Immuno Concentration Salmonella (ICS) and Selective Plate (HE BS SMID) Procedure
• AOAC 2001.08 VIDAS Immuno Concentration Salmonella (ICS) and Selective Plate (HE XLD BS) Method
• AOAC 2001.09 VIDAS Immuno Concentration Salmonella (ICS)
• AOAC 2003.09 BAX Automated System for Screening Salmonella in foods
• AOAC 2009.03 Assurance GDS™ Salmonella method for foods