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December 2012 1 Schedule 1 National Microbiological Database Programme
December 2012 3 Schedule 1 National Microbiological Database Programme
Prelims
Interpretation In this Schedule, unless the context otherwise requires,-
“alert” response means an immediate review of the process by an operator to identify and
document factors that may have compromised hygienic processing, and if appropriate, take
corrective and preventative action.
batch of samples means all the samples collected in one sampling period.
biosecurity (Campylobacter) means measures undertaken to prevent the introduction and
spread of Campylobacter into grower flocks.
birds means poultry broilers.
bobby calf means bovine calf that is at least 4 days old, generally unweaned and less than
45 kilograms dressed carcass weight.
bovine means beef, with 3 classes; bull, cow and prime.
broiler chicken means a male or female chicken kept primarily for meat production, but
does not include poussins.
bulk meat means bulk packed meat, not including IW cuts.
caprine means red meat species, goat.
carcass post slaughter and dressing means a red meat carcass after completion of
slaughter and dressing, after post-mortem inspection, prior to chiller entry at cold/warm
boning premises or just prior to quartering at hot-boning premises. For the purposes of NMD
post mortem inspection is considered to include removal of navel in bobby calves.
cervine means deer, with 2 classes defined for NMD; fallow and other (wapiti, red and
hybrid).
domestic premises means premises that supply the domestic market only.
dsBPW means double strength buffered peptone water.
equine means red meat species which includes horse, mules and donkeys.
extremes of temperature means, in relation to samples during collection and transport, less
than 0oC and greater than 25oC.
EU and US listed premises means premises that are listed for export to the EU and/or US
markets or premises supplying red meat to markets specifying EU and/or US.
farm means the location at which the poultry sheds are situated
HACCP means Hazard Analysis Critical Control Points.
initial item means is the first carcass, cut or bulk carton within the run to be sampled.
December 2012 4 Schedule 1 National Microbiological Database Programme
Prelims
initiation of analysis means
a. In relation to APC and E.coli analyses, from the time the sample is suspended and ready
for dilution; and
b. In relation to Salmonella analysis, the commencement of incubation of the BP
pre-enrichment.
ILCP means Inter-Laboratory Comparison Programme.
insulated container means a sample collection or transport container that is insulated.
IW cut means an individually wrapped cut.
MEGAREG means slang for US Pathogen Reduction HACCP Final Rule.
MIMM 2005 means Meat Industry Microbiological Methods, Edition Four, March 2005.
moving window means addition of 5 new samples to the bottom of the window to displace 5
samples from the top of the window.
NMT means National Microbiological Target.
non EU and non US premises means premises that do not export to EU and/or US, or any
markets specifying EU and/or US.
off-site laboratory means an independent consulting laboratory to which the operator of a
premises has appointed responsibility to enter results directly to NMD.
on-site laboratory means a laboratory facility located within the physical boundaries of a
premises.
ovine means species with 2 classes; sheep and lamb.
porcine means red meat species pig.
processing plant means poultry broiler slaughter house.
processing season means bovine, caprine, cervine, ovine, ostrich and emu, poultry, 1
October to 30 September or from the commencement of a short season such as bobby
calves, which may be before 1 October.
primal cuts means a primal cut from red meat carcass.
post chill carcass means carcass after chilling.
post slaughter and dressing carcass means a carcass after post mortem inspection of
carcasses. In the case of bobby calves after inspection, after the umbilicus/navel is removed
and prior to any further trimming.
PSW means a primary sampling window re Salmonella sampling programme.
RMP means a Risk Management Programme.
ssBPW means single strength buffered peptone water.
December 2012 5 Schedule 1 National Microbiological Database Programme
Prelims
shed means building purpose built for sustenance of a flock of poultry broilers.
SSW means secondary sampling window in relation to the Salmonella sampling programme.
sub-contracted Laboratory means where a premises has a LAS approved laboratory but
sub-contracts an off-site laboratory for specific analyses, the on-site laboratory may be sub-
contracted or seconded to train personnel to collect samples for the off-site laboratory.
TNTC means too numerous to count.
To means the time a poultry carcass takes to move from the point of selection on the chain to
the first drop point.
verifier means “official assurance verifier” which means a person accredited under section
103 of the Animal Products Act 1999 to undertake official assurance verification and includes
animal product officers employed by MPI.
VLT means very low throughput.
December 2012 6 Schedule 1 National Microbiological Database Programme
Administration and Participation
1. Administration and Participation
1.1 Species to which the National Microbiological Database Programme Applies
The species to which the NMD applies are bovine, ovine, bobby calf, caprine, cervine,
ostrich, emu, poultry and porcine. The NMD operates on the same principles for each
species with slight variations. A summary of the requirements for each species is listed
below in Table 1.
Note: If your premises is EU and/or US listed, whether or not you are exporting, you must
comply with the NMD programme for all species processed that are covered by the NMD
programme. If you are currently EU listed or US listed it is irrelevant whether the product is
going to an EU or US market that week, NMD sampling/analysis must be conducted each
processing week.
Domestic and export premises; even if not processing, a report to NMD is required to confirm
that there was no processing that week.
Table 1: Domestic and export application of NMD programme.
Species Domestic, non EU and non US Listed Premises
EU Listed Premises US Listed Premises
Bovine (excluding bobby calves)
Every processing week Every processing week Every processing week
Ovine Every processing week Every processing week Every processing week
Bobby Calves Every processing week Every processing week Every processing week
Caprine Every processing week Every processing week Every processing week
Cervine (farmed)
Every processing week Every processing week Currently no requirements for US. Domestic, non EU and non US or EU apply
Ostrich and emu Every processing week Every processing week Every processing week
Porcine Every processing week Exporting to the EU is prohibited unless prior notification is obtained from the Director General
Comply with the US Pathogen Reduction HACCP Final Rule. Must notify Director General of intentions
Poultry (broiler chickens)
Every processing week Exporting to the EU is prohibited unless prior notification is obtained from the Director General
Exporting to the US is prohibited
Ducks, geese and guineas
Not required Not required Exporting to the US is prohibited
Horses, mules, and other equines
Not required Not required Comply with the US Pathogen Reduction HACCP Final Rule. Must notify Director General of intentions.
December 2012 7 Schedule 1 National Microbiological Database Programme
Administration and Participation
Table 1: Domestic and export application of NMD programme (continued).
Species Domestic, non EU and non US Listed Premises
EU Listed Premises US Listed Premises
Wild game Not required Not required Wild games, excluding feral goats and pigs, can be exported to US. Not required
Game estate Not required Not required No negotiated entry requirements. Not required
1.2 Participation
All premises referred to in Table 1 must participate in the NMD programme as part of the
New Zealand standard. Once a premises is participating it is mandatory to comply with all
requirements of the NMD programme.
Premises may voluntarily participate in additional NMD programmes but if submitting results
to MPI must follow all requirements of the NMD.
The NMD sampling plan applies to all premises irrespective of throughput. Allowances are
made for premises classified as “VLT”; (see section 2.3 Table 2).
1.3 NMD Administration
On commencing participation in the NMD an operator must submit completed demographic
questionnaires to the NMD Administrator (these can be found on the MPI website).
If any of the following details change, the operator must inform the NMD Administrator within
7 working days of the change occurring.
a. name of premises;
b. name of plant manager and contact details: phone, cell phone and email;
c. address of premises, physical and postal;
d. name of NMD controller and contact details: phone, cell phone and email;
e. name of LAS approved laboratory conducting NMD analysis and coordinating the NMD
sampling programme;
f. name of the data submitter and whether this is a person employed by the premises or
the LAS approved laboratory conducting analysis;
g. species required to be sampled for NMD;
h. if species processed is VLT or standard throughput;
i. any process details as outlined on demographics questionnaire;
December 2012 8 Schedule 1 National Microbiological Database Programme
Administration and Participation
j. location and reference number for each farm and reference number for each shed for
which the operator processes poultry.
1.4 Health of Personnel
Personnel handling product for the NMD programme must comply with the specifications and
market access requirements for health of personnel, refer to Part 3 of the Animal Products
(Specifications for Products Intended for Human Consumption) Notice 2004.
1.5 Training of Samplers
All NMD sampling must be undertaken by persons trained and deemed competent for the
species being sampled. Persons who have attended an LAS approved sampling course
related to the species they will be sampling are collectively called “Certified Trainers”.
Certified trainers are qualified to select personnel who are technically competent and are
sufficiently familiar with the process associated with the species to be sampled, to be trained
as “Associate Trainers”. Both Certified and Associated Trainers may train others to sample.
Persons trained by Associate Trainers cannot train others. Samplers trained by Certified or
Associate Trainers who are not qualified to train others are referred to as “restricted
samplers”.
1.6 NMD analyses
All sample analysis must be performed in LAS approved laboratories. Modification or
substitution of methods is not permitted unless approved by LAS, and documented in LAS.
Each NMD analysis must be performed according to the method listed in section 4 of the this
Schedule. These analyses are based on those described in Meat Industry Microbiology
Methods Edition Four (MIMM 2005) or later edition, general procedures. NMD analyses
have been modified from those published in MIMM where appropriate, to reflect the specific
requirements of the NMD programme. Methods as written in NMD procedures for
application to the NMD programme take precedence over the MIMM 2005 or later edition.
As required under LAS all quality assurance functions and quality control procedures for
methods and media as per MIMM 2005 or later edition, Chapter 2 must be followed.
December 2012 9 Schedule 1 National Microbiological Database Programme
NMD Sampling Requirements - Red Meat and Poultry
2. NMD Sampling Requirements - Red Meat
and Poultry
2.1 Products sampled
Non EU and non US listed premises, including domestic premises must sample the
following:
• Bobby Calves and Caprine: all product types (fresh carcasses, primal cuts and bulk
meat) processed under their RMP
• Bovine (excluding bobby calves), Ovine, Ostrich and Emu: fresh carcasses
• Poultry broiler: fresh carcasses
• Cervine: fresh carcasses and primal cuts
• Porcine: fresh carcasses.
EU and US listed premises must sample the following:
• Bovine (excluding bobby calves), Bobby Calves and Caprine: all products types
(carcasses, primal cuts and bulk meat) processed under their RMP
• Ovine, Ostrich and Emu: fresh carcasses only
• Cervine: fresh carcasses and primal cuts
2.2 Number of samples per week
The number of samples is dependant on:
• whether the product is for the domestic market, non EU and non US markets or to
be exported to EU and/or US; and
• the species processed; and
• whether the species to be sampled is classified as standard throughput or VLT; and
• the type of analyses required.
(Refer also to section 2.3 Summary, Table 2: Number of samples required per week).
2.2.1 VLT provisions for domestic only and non EU and/or non US Iisted premises
These VLT provisions
• do not apply to ovine and porcine species; and
December 2012 10 Schedule 1 National Microbiological Database Programme
NMD Sampling Requirements - Red Meat and Poultry
• apply to premises that process product destined only for the local market and/or
markets other than EU and US or any markets specifying EU and US; and
• apply to bovine (excluding bobby calf) premises which process product destined only
for local markets and/or markets other than EU and US or any other markets
specifying EU and US, regardless of throughput.
Domestic only and non EU and/or non US listed red meat premises processing bovine as
above must sample:
• one fresh carcass each week of processing, starting from the first week of operation
and continuing for at least 16 processing weeks:
• where a VLT processing season is less than 16 weeks, the number of samples
collected per product per week must be increased to ensure that 16 samples are
collected within a season (for example an 8 week season would require collection of
2 samples per week for 8 weeks).
VLT premises for bobby calf and/or caprine are premises which process product destined
only for local markets and/or markets other than EU and US or any other markets
specifying EU and US and have a throughput of less than 400 bobby calf/caprine animals
in each processing week throughout the season.
Domestic only and non EU and/or non US listed premises processing bobby calf and/or
caprine qualifying as VLT premises must sample as follows:
• from the first week or part week of operation, including short seasonal operations or
where processing is intermittent, sampling of one of each product type (fresh
carcasses, primal cuts and bulk) must be conducted each processing week or part
week for at least 16 weeks or, if the number of processing weeks in the season is
less than 16 weeks, conducted each processing week or part week; or
• if at any time during the current season the weekly bobby calf and/or caprine
throughput exceeds 400 animals in a processing week, sampling of five samples of
each product type (fresh carcasses, primal cuts and bulk) each processing week is
required.
VLT premises for cervine species are defined as those that slaughter or cut/bone product
from 100 cervine animals or less per week (this is based on throughput over a whole
season). If at any time during the current season the weekly cervine throughput exceeds
100 cervine animals, cervine sampling must revert to standard throughput requirements.
Premises qualifying as VLT for cervine species must sample as follows:
• one of each of the following product types; fresh carcasses and primal cuts each
week of processing, starting at the first full week of operation within a season, and
continuing for at least 16 processing weeks:
December 2012 11 Schedule 1 National Microbiological Database Programme
NMD Sampling Requirements - Red Meat and Poultry
• where a VLT processing season is less that 16 weeks, the number of samples
collected per product per week must be increased to ensure that 16 sampled are
collected within a season (for example an 8 week season would require collecting on
2 samples per week for 8 weeks).
2.2.2 VLT for EU and US listed premises processing bobby calf, bovine, cervine and caprine
VLT premises are defined as those that slaughter or cut/bone product from 100
bovine/cervine animals or less, and/or 400 bobby calf/caprine animals or less per week (this
is based on throughput over a whole season).
If at any time during the season the weekly throughput exceeds the VLT limit, samples must
be collected at the standard rate for the remainder of the season.
Premises qualifying as VLT for a given bobby calf, bovine, caprine or cervine species or
product type of one of those species must sample as follows:
• at least one item of each product each week of processing, starting at the first full
week of operation within a season, and continuing for at least 16 processing weeks:
• where a VLT processing season is less than 16 weeks, the number of samples
collected per product per week shall be increased to ensure that 16 samples are
collected within a season (for example an 8 week season would require collection of
2 samples per week for 8 weeks).
2.2.3 Discontinuing VLT sampling
These VLT provisions do not apply to porcine and ovine. The following are the only
circumstances when VLT sampling may be discontinued:
• Bovine/bobby calf: These species have National Microbiological Targets (NMTs),
see sections 6.2.1 NMT (bovine species): Escherichia coli moving window (m) and
6.2.4 Bobby calf NMT 80th percentile m limits: Escherichia coli re limits specified for
E. coli. So, in addition to the above 16 week requirement, NMD sampling at the
required rate may only be discontinued after E. coli moving window of 15
consecutive samples that does not elicit an “m alert”, is completed:
• VLT premises processing cervine and caprine may discontinue weekly testing of a
product type after 16 samples of that product type have been collected.
VLT premises processing bobby calf, bovine, caprine and cervine may continue to sample as
per NMD requirements throughout the season if they wish. The NMD Administrator will
continue to accept results.
December 2012 12 Schedule 1 National Microbiological Database Programme
NMD Sampling Requirements - Red Meat and Poultry
2.2.4 Recommencing VLT sampling
The VLT sampling requirements above/in section 2 are to be recommenced each season
(which usually commences on 1st October) or whenever the process is changed such that
new process conditions could affect microbiological outcomes.
2.2.5 VLT for porcine premises
VLT porcine premises are defined as those that slaughter product from 10,000 pigs or fewer
per annum.
Porcine premises qualifying as VLT must sample at least one carcass each week or part
week of processing for analyses required.
2.2.6 VLT for poultry premises
VLT poultry premises are defined as those that slaughter product from one million
(1,000,000) birds or fewer per annum.
Poultry premises qualifying as VLT for carcasses must sample at least three carcasses each
week or part week of processing for analyses required.
December 2012 13 Schedule 1 National Microbiological Database Programme
NMD Sampling Requirements - Red Meat and Poultry
2.3 Summary Table
Table 2: Number of samples required per week
Species Product type
Technique Domestic and non EU and non US listed
EU and US listed standard throughput
EU and US listed VLT
Bovine Carcass Multiple Swab Technique
1 per week 5 per week 1 per week
Primal Cuts Multiple Swab Technique
Not required 5 per week 1 per week
Bulk Meat Product
Whole Tissue Composite Sampling
Not required 5 per week 1 per week
Post Chill Carcass
Multiple Swab Technique
Not required 5 per week for 6 weeks
1 per week for 6 weeks
Ovine Carcass Multiple Swab Technique
5 per week 5 per week Not applicable
Porcine Carcass Multiple Swab Technique
5 per week; VLT 1 per week
Bobby Calf and Caprine
Carcass Multiple Swab Technique
5 per week VLT: 1 per week for 16 weeks
5 per week 1 per week
Primal Cuts Multiple Swab Technique
5 per week VLT: 1 per week for 16 weeks
5 per week 1 per week
Bulk Meat product
Whole Tissue Composite Sampling
5 per week VLT: 1 per week for 16 weeks
5 per week 1 per week
Post Chill Carcass
Multiple Swab Technique
Not required 5 per week for 6 weeks
1 per week for 6 weeks
Cervine Carcass Multiple Swab Technique
3 per week VLT: 1 per week
3 per week 1 per week
Primal Cuts Multiple Swab Technique
2 per week VLT: 1 per week
2 per week 1 per week
Ostrich/Emu Carcass Multiple Swab Technique
1 per week 2 per week Not applicable
December 2012 14 Schedule 1 National Microbiological Database Programme
NMD Sampling Requirements - Red Meat and Poultry
Table 2 continued: Number of samples required per week
Species Product type
Technique Domestic and non EU and non US listed
EU and US listed standard throughput
EU and US listed VLT
Poultry Carcass Whole Carcass Rinse Method
3 per day VLT: 3 per week
Not applicable Not applicable
2.4 Post Chill Carcass Sampling Programme
Post chill carcass sampling is only required for bovine, bobby calf, and caprine species for
EU and US listed premises.
• Post chill carcass sampling must be commenced on starting the NMD programme
and then be undertaken annually at the start of the season.
• Post chill carcass sampling is required when the chiller operating conditions changes
significantly (calibration). The season is classified as beginning at 1st October or the
commencement of a short season such as bobby calves, which may be before 1st
October.
• Post chill carcass sampling provides a microbiological profile of chillers at normal
operating capacity for the particular species being tested.
• Post chill carcass sampling is required until 30 carcasses (at 5 carcasses per week
for 6 consecutive processing weeks) have been monitored. If sampling takes more
than 6 weeks it must be justified (for example, chiller capacity being lower than the
normal for the species, multiple species and concurrent Salmonella sampling
obligations exceeding laboratory capability for the sample collection and/or analysis).
• Post chill carcass sampling must be rotated on a weekly basis around the chillers, to
a maximum of 6 chillers (5 samples per chiller) during each annual period.
• Premises that qualify as VLT for bovine, bobby calf and/or caprine species are
required to have one post chill carcass sample per week for 6 weeks for each
season.
2.5 Salmonella Sampling Programme
There is a performance standard for Salmonella sampling for all products (excluding chilled
carcasses) from the following species:
• bovine
December 2012 15 Schedule 1 National Microbiological Database Programme
NMD Sampling Requirements - Red Meat and Poultry
• bobby calf
• caprine
• cervine
• ostrich/emu
• porcine (see section 2.5.2)
• poultry
(Note: Salmonella testing is not required on ovine species).
Salmonella sampling and performance standards are split into three groups:
Table 3: Salmonella groups
Group 1 – Salmonella Group 2 – Salmonella Group 3 – Salmonella
Bovine Bobby calf Caprine Cervine
Ostrich/Emu Porcine
Poultry
2.5.1 Group 1 (bovine, bobby calf, caprine, cervine) – seasonal Salmonella
Seasonal Salmonella sampling is required for each product type processed. The processing
season for bovine, caprine, and cervine is 1st October – 30th September. For bobby calves
the processing season is taken from the commencement of bobby calf processing during the
calendar year 1st January– 31st December.
An initial “primary sampling window” (PSW) of 16 consecutive weeks, within a processing
season must be conducted. Where a premises processing season is shorter than 16 weeks
the PSW carries over to the subsequent processing season.
A premises that has not detected Salmonella in a given product type of a given species in
the preceding PSW must test product for the initial six consecutive processing weeks of the
next season. (See Figure 1). This reduced sampling frequency is defined as a “secondary
sampling window” (SSW).
Where a premises doesn’t complete either a PSW or SSW in their processing season, this
then carries over to the new processing season. The carried over PSW or SSW does not
displace the new processing season requirement. The new seasonal Salmonella sampling
must commence on the completion of the carried over one. Examples:
• A bobby calf processing season, usually starting between March and August, might
commence with PSW15; indicating a carry over from the previous season. In the first
week of the new season the PSW of the previous season would be complete at
PSW16 (providing no Salmonella detection occurs). The processor is then required
to start a secondary sampling window (SSW1) in the following week.
December 2012 16 Schedule 1 National Microbiological Database Programme
NMD Sampling Requirements - Red Meat and Poultry
• Where a SSW reads SSW2, SSW3, SSW4, SSW5 or SSW6 at the beginning of a
new processing season this indicates an incomplete SSW from the previous season.
The full SSW of the previous season must be completed and the new season SSW1
must be started in the next processing week (providing no Salmonella detection
occurs).
A premises that detects Salmonella in a given product type of a given species during a PSW
or SSW must immediately begin another 16 week PSW for that species/product type. A
“sampling window” terminates on detection of Salmonella and week one of a new PSW
commences for that particular species/product type in the following processing week.
Figure 1: Salmonella sampling windows succession
Salmonella: detected (+) not detected (-)
• An operator who demonstrates to a verifier that Salmonella was not detected in
either a PSW or a SSW may, with approval of MPI VA, cease testing of that product
for the remainder of the processing season. Unless the PSW or SSW was carried
over from last season, where a SSW needs to be commenced for the current
processing season following completion of the carried over seasonal window.
Closures (seasonal or maintenance): An incomplete PSW or SSW carries over to the next
processing week following completion of maintenance.
Changes to process: A premises that is substantially modified in such a manner that it could
affect expected microbiological outcomes of the process must immediately begin a 16 week
PSW when processing recommences. Substantial modification includes:
• new premises under the same licence/registration number at the same or different
location; and
• major changes in processing methods (for example, change from traditional to
inverted dressing, installation of robotics, etc.) where changes in microbiological
performance are possible or probable.
December 2012 17 Schedule 1 National Microbiological Database Programme
NMD Sampling Requirements - Red Meat and Poultry
2.5.2 Group 2 (Ostrich/Emu and Porcine) - Salmonella
An operator must conduct ostrich/emu and porcine Salmonella sampling each processing
week.
Porcine Salmonella sampling is a 27 month programme which commenced week beginning
5 October 2009 and will cease on the week beginning 2 January 2012.
2.5.3 Group 3 (Poultry) – Salmonella
Poultry Salmonella sampling must be conducted by operators of;
• Standard throughput premises; each processing day
• Very Low Throughput premises; on one processing day of each processing week.
2.6 Poultry Campylobacter Sampling Programme
The Campylobacter sampling programme for poultry carcasses must be conducted by
operators of;
• Standard throughput premises; each processing day
• Very Low Throughput premises; on one processing day of each processing week.
2.7 Sampling Location within the Process
2.7.1 Red meat product selection
Refer to section 2.3 for products (carcasses post slaughter and dressing, post chill
carcasses, primal cuts and bulk meat) required to be sampled for each particular red meat
species NMD programme.
2.7.1.1 Carcasses post slaughter and dressing
NMD sampling for post slaughter and dressing aims is conducted to gain a representative
picture of microbiological contamination during the slaughter and dressing process.
Procedures that result in a significant transfer of microbes to the carcass are completed
before the post mortem inspection station. “Post slaughter and dressing” is defined, for the
purposes of NMD, as after post mortem inspection and after final dressing procedures
specified below.
The NMD sampling for post-slaughter and dressing is as follows:
• NMD sampling must commence as soon as is practical, but no later than 30 minutes
after post mortem inspection of carcasses.
• Carcasses detained by AsureQuality must not be sampled.
December 2012 18 Schedule 1 National Microbiological Database Programme
NMD Sampling Requirements - Red Meat and Poultry
• Carcasses must be sampled BEFORE any procedure that may impact on the NMD
carcass sampling sites. Processes following the post mortem inspection station may
redistribute contamination over a carcass, add some contamination by handling from
workers, or remove contamination due to particular process interventions. Such
procedures include carcass stacking (contact), gross hot trimming, carcass washing,
rail stimulation and worker contact during marshalling.
• In the case of bobby calves, samples are to be collected immediately after
inspection, after the navel is removed and prior to any further trimming.
• Positioning of carcass for sampling:
(i) Ovine, porcine, caprine and bobby calf carcasses can be moved to a dead rail for
sampling, or another suitable position that does not mask the microbiological
consequences of slaughter and dressing.
(ii) Bovine and cervine carcass sampling must be conducted on a moving rail (as
the carcass cannot be manually moved from the rail). This requires a greater level
of training to ensure sample collection is consistently from the correct sites, the
correct carcass side and with the correct sampling technique.
• The specified carcass sampling sites require wet/dry swab sampling. The sampling
sites will vary in location according to the species. The sites have been selected to
represent the points where microbes are most likely to be transferred to the carcass
during dressing procedures.
• Record the time of sampling, run, shift, species, class and process type; inverted,
traditional, gas de-pelted.
• Rotation of chains on a weekly basis in premises with multiple slaughter chains is
required.
2.7.1.2 Post chill carcasses
Post chill carcasses of cold and warm boning premises require wet/dry swab sampling. Refer
section 2.8.8. Post chill carcass sampling is not required for hot boning premises.
Cold and warm boning premises must sample the post chill carcass:
• no later than 24 hours after the onset of chilling; or
• when the chilling cycle is completed in less than 24 hours, at the completion of the
chilling cycle; and in either case
• in the chilling area itself prior to wrapping, transportation, freezing, boning or loadout.
Records must be taken of the:
• location of the chilled carcass at time of sampling; identifying the cooling floor,
chiller, side chiller, quarter chiller or other,
December 2012 19 Schedule 1 National Microbiological Database Programme
NMD Sampling Requirements - Red Meat and Poultry
• boning process (cold or warm), and
• number of hours chilled (timed from the onset of chilling).
2.7.1.3 Primal cuts and bulk meat product
Primal cuts and bulk meat product the microflora comprises organisms from two sources;
• those which remain attached to the carcass after the initial transfer during slaughter
and dressing; and
• those acquired by contact of the cut surfaces with other surfaces or materials (for
example condensate, aerosols) in the processing environment (cross
contamination).
Primal cuts and bulk meat manufactured at a premise, but from carcasses supplied by
another premises, shall be regarded as product of the boning premises.
Boning premises must record the premises of a carcass origin and enter that information in
the boning premises NMD report under the product description column of the NMD
database.
Only fresh product may be sampled for NMD (secondary processes and product that has
either been chilled in the carton, frozen or thawed from frozen is not required to be sampled
for NMD).
Primal cuts (cold, warm or hot boned, bone-in or bone-out).
Wet/dry swab samples must be taken from an outside (fell) surface of the cut, not an
exposed, freshly cut internal surface. The sampling site selected on the primal cut should
correspond to the equivalent carcass sampling site for the species concerned. Primal cut
samples are to be taken immediately prior to vacuum packaging, wrapping or bulk meat
packing into cartons.
• Bovine, bobby calf, caprine: hindquarter cuts or hindleg cuts
(refer Table 4).
• Venison: hindquarter cut (rump/topside) and forequarter cut (shank/shoulder). Removal of the silver skin (de-sinewing) of cuts is more commonly carried out in
venison further processing. Samplers must record whether the venison cuts
sampled are de-sinewed or intact.
December 2012 20 Schedule 1 National Microbiological Database Programme
NMD Sampling Requirements - Red Meat and Poultry
Table 4: Primal cut descriptors
Species Primal Cut Descriptor
Bobby Calf Hindleg Outside hindleg
Bovine ATS Bottom Round Eye of Round Flat Inside Outside Less Eye of Round Outside Round Outside Smalls SAT Silverside Topside
December 2012 24 Schedule 1 National Microbiological Database Programme
NMD Sampling Requirements - Red Meat and Poultry
Figure 9 (porcine) and Figure 10 (ostrich/emu). Figure 11 illustrates sampling from the
opposite sides of the carcass for APC/E. coli and Salmonella samples.
December 2012 25 Schedule 1 National Microbiological Database Programme
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2.8.1 Bovine
Three sites need to be swabbed on bovine. The three sites are as follows:
Rump: (situated on the hindleg, refer Figure 4)1: The 100cm2 site is centred on the fascia
overlying the semitendinosis muscle. The muscle is cut, packed and sold as the ‘eye round’.
The centre of the sampling site is halfway along the muscle on its superficial lateral margin.
Lateral to semimembranosis is the gluteobiceps muscle. This muscle is cut, packed, and
sold as the ‘outside flat’. The 100cm2 template may overlap both muscles.
Flank: The 100cm2 site is centred on the fascia overlying the cutaneous trunci muscle. The
centre of the site is 10cm lateral to the umbilicus on a line drawn along the ninth rib from the
spine and continued on to cut edge of the abdominal wall.
Brisket: The 100cm2 site is centred on the fascia overlying the cranial ventral part of the
cutaneous trunci muscle. The centre of the sampling area is 10cm off the central midline.
The margins of the sampling site may overlap onto the fascia overlying the caudal margin of
the pectoral profundis muscle. The lower edge of the sampling site is an imaginary line
drawn transversely along the thoracic wall at the level of the point of the elbow.
1The naming of the bovine hindleg sample site as rump is required under our equivalence agreement with the US.
December 2012 26 Schedule 1 National Microbiological Database Programme
NMD Sampling Requirements - Red Meat and Poultry
Figure 4: Sampling sites for bovine carcass, 100cm2
Rump site situated on the
hindleg
Flank site
Brisket site
Photographs courtesy of David Walkinshaw, Taylor Preston Limited
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NMD Sampling Requirements - Red Meat and Poultry
2.8.2 Ovine and Caprine
2.8.2.1 Ovine: traditional and inverted
A single ovine carcass site needs to be swabbed. The site is as follows:
Forequarter opening Y-cut: The Y-cut site is defined as the anterior aspect of the humero-
radial joint close to the brachial vein, which is clearly visible at the site but a little erratic in its
course.
2.8.2.2 Caprine
Three carcass sites need to be swabbed for caprine. The three sites are as follows:
Outside hindleg: The outside hindleg site is defined as an area one third up on a vertical
line originating at the midpoint of a line between the ischial crest and stifle, and extending to
a line horizontal to the cut end of the hock.
Flap: The flap site is defined as an area ~50mm from the flap edge, midway between the
flap joint and the xiphoid cartilage.
Forequarter opening Y-cut: The Y-site is defined as the anterior aspect of the humero-
radial joint close to the brachial vein, which is clearly visible at the site but a little erratic in its
course.
December 2012 28 Schedule 1 National Microbiological Database Programme
NMD Sampling Requirements - Red Meat and Poultry
Figure 5: Ovine sampling site and caprine sampling sites, 5cm2 sampling area.
Ovine carcasses (both traditional and inverted dressing) require sampling of the opening Y
cut site only.
Caprine carcasses require sampling of all three sites shown below.
Photographs courtesy of Sandy Moorhead (AgResearch) and Monique Biss (MAF VA)
Outside leg
Opening Y-cut
Flap
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NMD Sampling Requirements - Red Meat and Poultry
2.8.3 Bobby calf
Three sites need to be swabbed on bobby calves. The three sites are: Fore rump: An area adjacent to the rectal canal, no greater than 50mm horizontally from the
edge of the rectal canal, vertically with midpoint on a horizontal line dissecting the forward
(lower) edge of the rectal canal.
Flank: The site is defined as an area ~50mm from the flank edge, midway between the flank
joint and the xiphoid cartilage.
Foreleg: An area on the outside surface
of the lateral head of the triceps brachii.
The side (left or right of the animal) of the carcass APC/E. coli swabs are collected from
must be recorded for bobby calf samples.
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Figure 6: Sampling sites for bobby calf carcasses, 25 cm2 sampling area
December 2012 31 Schedule 1 National Microbiological Database Programme
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2.8.4 Cervine
2.8.4.1 Cervine – traditional
Three sites need to be swabbed on traditional cervine. The three sites are as follows:
Posterior hindleg: The hindleg sample site is centred longitudinally and laterally on an
imaginary line drawn between the posterior aspect of the aitch bone and the archilles
tendon.
Sternum: The sternum site is immediately adjacent to the intersect of the abdominal cavity
opening and brisket, but outside the area of “standard brisket trim” (by <10mm at its closest
edge). Note: Tissue exposed by the standard trim must not be sampled.
Inside foreleg: The inside foreleg site is defined as the anterior aspect of the humero-radial
joint close to the brachial vein, which is clearly visible at the site but a little erratic in its
course.
2.8.4.2 Cervine – inverted
Three sites need to be swabbed on inverted cervine. The three sites are as follows:
Inside hindleg: The sample site is on the medial side and proximal end of the hindleg
immediately adjacent to the pelvic symphysis (midline).
Brisket: The brisket sample site is centred longitudinally on the brisket, but immediately
outside the area of “standard brisket trim” (by <10mm at its closest edge). Note: Tissue
exposed by the standard trim must not be sampled.
Inside foreleg: The inside foreleg site is defined as the anterior aspect of the humero-radial
joint close to the brachial vein, which is clearly visible at the site but a little erratic in its
course.
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NMD Sampling Requirements - Red Meat and Poultry
Figure 7: Sampling sites for cervine carcasses (traditional), 25cm2 sampling area
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Figure 8: Sampling sites for cervine carcasses (inverted), 25cm2 sampling area
December 2012 34 Schedule 1 National Microbiological Database Programme
NMD Sampling Requirements - Red Meat and Poultry
2.8.5 Porcine
Three sites need to be swabbed on porcine carcasses. The three sites are as follows:
Outside hindleg: Midway between the stifle and hip joint.
Lower flap: 50mm from the abdominal incision midway between the two lower nipples.
Outside shoulder: Above the forward top end of the shoulder blade.
Figure 9: Sampling sites for porcine carcasses, 25cm2 sampling area Photos courtesy Land Meats
Porcine NMD site 1: Outside Hindleg Porcine NMD site 2: lower flap between the
lower two nipples
Porcine NMD site 3: outside shoulder site
Whole carcass view of NMD sites
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NMD Sampling Requirements - Red Meat and Poultry
2.8.6 Ostrich and emu
LEG HUNG
A single ostrich and emu carcass site needs to be swabbed. The site is as follows:
Inside leg: Located longitudinally on the opening cut line and laterally where the edge of the
remaining flap contacts the leg. It is recommended that this site be alternated between left
right sides.
Figure 10: Sampling sites for leg hung ostrich and emu carcasses, 25cm2 sampling area
Photo courtesy of Venison Packers Fielding Limited
WING HUNG
No site identified at present as no NZ premises are using wing hung slaughter and dressing
practice.
Inside hindleg
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2.8.7 Salmonella sampling
Where Salmonella sampling is being undertaken the same sites on the opposite side of the
carcass from APC/E. coli samples are to be selected and sampled.
In the case of adult cattle the carcass is physically split. Substitute a side from another
carcass for sampling if the opposite side to that sampled for APC/E. coli is not available due
to condemnation.
Figure 11: Illustrates sampling from opposite sides of the carcass as required for APC/E.
coli and Salmonella samples.
2.8.8 Post chill carcass sampling
• Where post chill sampling is being undertaken, select the same sites on the opposite
side of the carcass from APC/E. coli sampling. For example, the carcasses post
slaughter and dressing can be tagged and re-sampled on the opposite side of the
carcass after chilling.
• Where Salmonella sampling of carcasses post slaughter and dressing coincides with
post chill sampling choose another carcass for post chill sampling that has
December 2012 37 Schedule 1 National Microbiological Database Programme
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undergone the same chilling conditions adjacent to the carcass originally selected
for carcass post slaughter and dressing sampling.
• Where matching is impractical (for example, pre-chill carcasses spread over several
chillers) post-chill carcasses may be selected randomly from a single chiller, rotating
weekly through all available chillers.
• NEVER sample a site that has been previously swabbed because the microbial load
will have been removed/modified during the first swab sampling procedure.
• Note: post-chill carcass sampling can be conducted on the same day as fresh
carcasses are sampled.
2.8.9 Poultry carcass sampling
The whole carcass is sampled for poultry.
2.9 Analysis Required
The following analyses are required:
Table 6: NMD analyses
Product Analyses required
Bovine, bobby calf, caprine, cervine, ostrich and emu, and porcine carcasses post slaughter and dressing
Aerobic plate count E. coli Salmonella
Ovine carcasses post slaughter and dressing
Aerobic plate count
Post chill carcasses Aerobic plate count E. coli
Primal cuts Aerobic plate count E. coli Salmonella
Bulk meat Aerobic plate count E. coli Salmonella
Poultry whole carcass Salmonella Campylobacter
December 2012 38 Schedule 1 National Microbiological Database Programme
NMD Sampling Requirements - Red Meat and Poultry
2.10 Summary
Table 7: Summary of NMD sampling requirements
Product to be sampled
Frequency Sites to be tested Analyses
Carcasses Post Slaughter and Dressing Bovine, bobby calf, caprine, cervine and porcine.
Weekly • 3 sites, one side of
carcass, alternating
between the leading
and trailing sides
• 3 sites, other side of
carcass
• APC/E. coli
• Salmonella (each
seasonal sampling
window, except porcine-
refer 2.5.2)
Carcasses post Slaughter and Dressing Ovine
Weekly • 1 site, one side of
carcass, alternating
between the leading
and trailing sides.
• APC only.
• Salmonella is not
required
Carcasses Post Slaughter and Dressing Ostrich and emu
Weekly • 1 site, one side of
carcass, alternating
between the leading
and trailing sides
• 1 site, other side of
carcass
• APC/ E. coli
• Salmonella
Carcasses Post Chill Only required for bovine, bobby calf and caprine species Not required for hot boning premises
Weekly, for a 6 consecutive week annual sampling window, at the start of the season
• 3 sites, one side of
carcass, alternating
between the leading
and trailing sides
• APC/ E. coli
• Salmonella is not
required
December 2012 39 Schedule 1 National Microbiological Database Programme
NMD Sampling Requirements - Red Meat and Poultry
Table 7: Summary of NMD sampling requirements (continued)
Product to be sampled
Frequency Sites to be tested Analyses
Primal Cuts
Bovine, bobby calf,
caprine, and cervine
Weekly • 1 site on required
number of separate
cuts (same site as
on carcass)
• APC/ E. coli
• Diluent remaining after
APC/E.coli test is
composited for
Salmonella
• Salmonella (each
seasonal sampling
window)
Bulk Meat
Bovine, bobby calf,
and caprine.
Weekly • 10g from 5 locations
within each carton
composited, from
which a 25g
analysis sample is
taken
• APC/ E. coli
• Diluent remaining after
APC/E. coli tests is
composited for
Salmonella
• Salmonella (each
seasonal sampling
window)
Poultry Whole Carcass Rinse
Every
processing
day
One
processing
day a week for
VLT
• Whole carcass is
rinsed with ssBPW
• Campylobacter for each
carcass.
• Additionally - Salmonella
for one carcass.
December 2012 40 Schedule 1 National Microbiological Database Programme
NMD Sampling Requirements - Red Meat and Poultry
2.11 Weekly Sampling Plan for Red Meat – Random Rotational Sampling
To ensure that a complete evaluation of the process is made, samples must be selected
randomly such that every animal has an equal chance of selection.
The sampling plan for red meat must include a randomly selected time each week to sample
all products types (carcasses, cuts and bulk as per premises scope and as required for a
particular species) for each species.
When there are multiple red meat species processed at a premises, a single selected time
period to sample all species can be used (same day, shift and run for all species) if you have
enough samplers and laboratory personnel to process the samples. Alternatively, a different
day of the week or time in the day may be randomly selected for the other species from the
remaining days or part days.
Red meat VLT premises must base their sampling plan on the farmed animal/cervine plan as
above and randomly select a day, shift and run for each week or part week of processing.
2.11.1 Sample selection
First priority: to sample the required carcasses, cuts and bulk for each species each week.
MPI can provide a random sample selection macro available to assist (contact the NMD
Administrator).
The order of selection; day, class, shift, run, time can be varied. The selection can be made
in any order as long as all the parameters required by NMD random rotational sampling
programme are met.
2.11.1.1 Day: random/rotational selection of day of week
Ascertain the days in the week processing for each particular species is taking place.
All processing or part processing days (including statutory holidays and weekends) must be
eligible for selection.
Randomly select an order of days from days available by randomly selecting one and
rotation through the remainder (in any order).
Random sampling only, without rotation through all available days is not permitted. All
available processing days must be sampled from.
For example if there are 5 days in the week available for processing over 5 weeks all days
will be selected for sampling.
December 2012 41 Schedule 1 National Microbiological Database Programme
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Table 8: Random rotational of weekdays
Week number
1 2 3 4 5
Day Wednesday Monday Friday Tuesday Thursday
Ovine species: Random/rotational sampling of sampling days is required for ovine species.
However, where reasonable practical constraints exist, a specified day may be excluded
from sampling without the scientific proof required under section 2.11.3 and on agreement
with MPI VA. Where not all days in a processing week are available for sampling,
random/rotational sampling must occur through the remaining days.
2.11.1.2 Class: random/rotational sampling of class
Where there is more than one class random rotational sampling of classes is required. This
is not dependent on throughput.
The proportion of livestock classes processed at premises is irrelevant under the NMD
programme and must not be used to deliberately bias the selection. Throughput only affects
the number of samples collected if the premises is classified as Very Low Throughput.
The class selected should be used for all product types (carcasses, cut, bulk) of that species
that week.
Ovine – 1 class, lamb, is required to be sampled except on processing weeks where only
sheep is being processed, in which case sheep must be sampled.
Ostrich and emu – 2 classes if both ostrich and emu are being processed.
Cervine – 2 classes; fallow and ‘other’ - wapiti, red, hybrid and sika grouped as one class.
Similar to tossing a coin for heads or tails randomly select the first class of fallow or ‘other’ to
be sampled every 2 weeks.
Bovine – 3 classes; bull, cow, prime. Randomly select one of the 3 classes for week 1, then
randomly selected week 2 from the remaining two classes. The class remaining will be the
one for week 3.
Porcine – 4 classes: suckers, porkers, baconers, choppers. Randomly select one of the 4
classes for week 1, randomly select week 2 from the remaining three classes, and week 3
from the remaining two classes and the class remaining will be the one for week 4. Variation
to this may be required to ensure choppers are selected for one week of each month if
choppers are being processed that month.
December 2012 42 Schedule 1 National Microbiological Database Programme
sample itself, primal cut and bulk original peptone APC/E. coli suspensions, and the final BPW
suspensions.
All Salmonella samples must be in an insulated container maintained between 0˚C and 5˚C (not
frozen), and not exceeding 10ºC in transit, and/or stored in a refrigerator operating between 0˚C
and 5ºC until commencement of analysis.
Following receipt of samples by the laboratory initiation of Salmonella analysis is defined as the
commencement of incubation of the BPW pre-enrichment broth. This may mean cooling of these
original suspensions during APC/E. coli analysis and use of pre-chilled dsBPW to <5ºC to make
up the final Salmonella sample suspension.
December 2012 65 Schedule 1 National Microbiological Database Programme
Sampling
3.5.5 Poultry samples
Figure 15: Poultry sampling timetable
Select whole carcass in process room day shift or
night shift. Record time of collection of
first carcass.*
Initiate analysis within 24 hours after first carcass
selected.
Rinse carcass immediately in process room.
Transport carcass in an insulated container to the laboratory within 15 – 30 minutes of
sampling.
Rinse carcass immediately.
Commence analysis immediately or store at <5ºC.
Transport sample to on-site or off-site laboratory.
Insulated, arriving at or below 10ºC and stored at
<5ºC.
The maximum period may be extended to 30 hours, not routinely, with reasons recorded
*First carcass in each discrete consignment of samples dispatched to the laboratory for that processing day.
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Sampling
3.5.6 Preparation of poultry samples for transportation
See also 3.4.3
The process for the preparation of poultry samples for transportation is as follows:
1. Tightly seal the sample bag, transportation vial, or bag containing the carcass to be
sampled; and
2. All samples must be immediately placed into an insulated container with frozen coolant
ensuring the sample bags or transport vials are not in direct contact with the frozen
coolant.
3.5.7 Transport and temperature requirements for poultry whole carcass samples
The carcass rinse sample or the carcass itself must be transported to the laboratory in an
insulated container with ice packs, but not in direct contact with ice packs, and not frozen.
1. Whole carcasses not rinsed immediately in the process room, but transported to the
laboratory for rinsing must be transported in insulated containers with sufficient frozen
coolant to cool the sample. The temperature of the carcass on arrival at the laboratory
does not need to be measured.
2. Whole carcass for rinsing in the laboratory must be preferably delivered to the laboratory
within 15 minutes (no later than 30 minutes) of selection of the whole carcass from the
processing chain and immediately rinsed.
3. Rinse samples transported to the laboratory must be transported in insulated containers
with frozen coolant. The temperature of rinse samples within the insulated container
used for transport should be maintained between 0˚C and 5ºC (not frozen), and must not exceed 10ºC. The temperature of the diluent, not just the airspace in the container
must be maintained in this temperature range.
4. All samples are to be delivered to the laboratory as soon as possible after collection.
5. The time of collection of the whole carcass sample must be recorded.
6. The temperature of the rinse sample on arrival at the laboratory must be recorded.
7. Samples that are frozen shall be rejected by the laboratory and replacement samples
collected from the same days processing if possible.
8. Premises must be able to verify that analysis can be initiated within 24h from time of
collection for the first sample. This may be extended to 30h under exceptional
circumstances, not routinely, with reasons recorded.
9. Laboratories, including off-site laboratories, are responsible for training on-plant
personnel seconded to collect samples. Off-site laboratories are responsible for
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Sampling
ensuring that transport companies, e.g. on-site personnel, couriers are aware of the
transport and storage temperature and time requirements.
10. Off-site and on-site laboratories are responsible to ensure night shifts are informed of
storage temperatures, transport to laboratory and time requirements.
December 2012 68 Schedule 1 National Microbiological Database Programme
Laboratory Analysis
4. Laboratory Analysis
4.1 Dilutions Required
Table 23: Dilutions required
Analyses Red Meat Poultry
APC30 zero to 10-4 (dilution series of 5)
E. coli zero to 10-3 (dilution series of 4) 100 to 10-2 (dilution series of 3)
Campylobacter 100 and 10-1 plus higher dilutions if required.
Note: zero is the undiluted sample.
4.1.1 High end dilutions
All dilutions must be plated in duplicate until the premises data indicates at what dilution a
colony count of greater than the maximum allowable is never, or rarely exceeded.
The maximum allowable counts are:
300 CFU for spread plates;
150 CFU for spread plate half plates;
250 CFU for PetrifilmTM APC;
150 CFU for PetrifilmTM E. coli.;
150 CFU for mCCDA direct plate Campylobacter
When a valid count exceeds (>300, >250 or >150 on the highest dilution) the dilution series
shall be immediately extended until such time as the premises demonstrates that the higher
counts are not a consistent trend.
It is very important to have a valid actual count of higher results for statistical purposes. A
TNTC count can be any value greater than the valid count value of 300, 250 or 150 on the
highest dilution tested and is thus more variable and unknown than a not detected count. A
TNTC result can range from as low as a 2 log10 value up to any higher value (infinite). It
could be 5 log10 or 10 log10. If a dilution series is not great enough this will lead to TNTC
results and such high counts cannot be included in the database, which, in turn, leads to a
poor representation of the true range of results.
When insufficient dilutions are prepared and counts are above the maximum allowable count
on the lowest dilution the laboratory must report to NMD a too numerous to count result
which will be classed as a technical failure.
A TNTC must be regarded as “detection” and/or “higher than expected count” when
assessing compliance with premises performance criteria under HACCP and RMPs.
December 2012 69 Schedule 1 National Microbiological Database Programme
Laboratory Analysis
Note that the area sampled will have an effect on the number of dilutions required. More
bacteria are expected to be recovered from a larger sample area therefore more dilutions will
be required.
As regulatory limits will be applied to poultry broiler carcass Campylobacter enumeration
results laboratories need to ensure that a suitable series of dilutions is plated to minimise the
chance of TNTC results. If a TNTC result occurs it will be recorded as exceeding the CPT
limit. Refer sections 4.10 and 6.9.
When a TNTC count occurs the test does not need to be repeated.
Upon receipt of a TNTC result the laboratory must extend the dilution series, in anticipation
of higher counts, for at least 3 subsequent poultry processing days (standard throughput) or
3 subsequent processing weeks (VLT) following the TNTC result notification.
4.1.2 Low end dilutions
All cases – Undiluted (zero dilution) plates or PetrifilmTM plates that contain less than the
lowest counting range of 30, 25, or 15 are to be counted and reported.
Table 24: Summary of acceptable counting ranges
Dilutions Spreadplate APC
Spreadplate APC ½ plate
mCCDA direct plate
PetrifilmTM APC PetrifilmTM
E. coli
100 0-300 0-150 0-250 0-150
10-1, 10-2,…..10-n 30-300 15-150 25-250 15-150
4.2 Duplicate Plating/Analytical Precision
Plating must be performed in duplicate, i.e. two agar plate, both sides of a ½ plate or two
PetrifilmTM inoculated from each dilution until laboratory precision data (for all analysts
performing testing) indicates otherwise. Duplicate plating (APC30 and E. coli) must be
undertaken to give statistical confidence to the results obtained. Use the Analytical Precision
calculation (for a single operator) in chapter 5 Appendix 1.7 of MIMM to determine a Z score
which is then compared to a limit of 1.96.
Record all duplicate plate counts that fall within the acceptable counting ranges as specified
in section 4.1.
If a laboratory demonstrates that it meets the Analytical Precision criteria outlined in MIMM
2005 or later edition, it may use singlet plating for all dilutions other than the undiluted
(zero) dilution sample.
For ILCP analyses related to NMD APC and E. coli tests use the NMD procedures described
in this publication. Although successful precision analysis may have eliminated the need for
duplicates on all dilutions for routine NMD analyses, duplicates are required for all dilutions
for the corresponding ILCP tests.
December 2012 70 Schedule 1 National Microbiological Database Programme
Laboratory Analysis
4.3 Preparation of Dilutions
4.3.1 Introduction
When conducting a quantative bacterial analysis, sample suspensions must be diluted to an
appropriate level that will result in 30 – 300 colony forming units (CFU) on the plate, 25 – 250
CFU if using APC PetrifilmTM or 15 – 150 CFU if using half plates, E. coli PetrifilmTM or
mCCDA direct plate.
Dilutions for viable bacterial counts are aseptically prepared in ten fold steps.
All dilutions are performed using 0.1% peptone + 0.85% NaCI diluent. Reference: ISO
17604:2003.
The dilutions volume used must be sufficient to inoculate duplicate plate count agar plates,
duplicate PetrifilmTM, enough volume for the composite Salmonella pre-enrichment if
required and leaving enough volume in case of error.
Two dilution volumes are recommended:
a. 9ml in 25ml universal bottles or vials, with 1ml transfer volume.
b. 4.5ml in microcentrifuge or microdilution tubes, with 0.5ml transfer volume.
The use of pre-poured dilution volume is allowed only if a laboratory can provide evidence
that they do not lose volume during autoclaving (as defined in MIRINZ Bulletin 35 or MIMM
2005 or later edition, Section 2.5.6), and that they have an appropriate QA programme in
place for ongoing verification.
Always use a fresh pipette or pipette tip for each transfer of diluted sample.
Ensure that dilutions are as homogenous as possible at all stages by:
• Carefully following the instructions for the use of micropipettors;
• Mixing each dilution thoroughly prior to the next dilution step to ensure that the
bacteria are randomly distributed;
4.3.2 Definition of zero dilutions for calculation purposes
The swab suspension (swabs immersed in the initial volume of either 10ml or 15ml) is an
undiluted sample referred to as the “zero dilution”. The swab suspension is entered into the
calculations as the 100 dilution.
The whole tissue suspension is dilution but in order to maintain consistency with plate
labelling, it is entered into the calculation as the 100 dilution (undiluted) and accounted for in
the final numeric multiplier.
4.4 Preparation of Swab Samples for APC and E. coli Analysis
Add to each vial with swabs.
December 2012 71 Schedule 1 National Microbiological Database Programme
Laboratory Analysis
Table 25: Swab suspension volumes required
Bovine, bobby calf, cervine, ostrich/emu, porcine
Ovine and caprine
15ml sterile 0.1% peptone diluent to each vial
10ml sterile 0.1% peptone diluent to each vial
Make certain there are 4 – 9 sterile glass or plastic beads in each vial.
Close the vial and shake vigorously to loosen the cotton bud of the swab for 2 minutes
(ensure consistency). Shaking can be performed by hand or by using a combination of initial
shaking by hand and a vortex mixer in 3 x 10 second bursts. Note that shaking by hand is
usually sufficient and enables several vials to be processed at once.
The cotton buds should be well broken up by this treatment. The cotton swabs must be left
in the diluent until all the dilution and plate inoculation procedures are completed. Do not
remove the swabs from the vials at any stage.
4.5 Preparation of Red Meat Separate Carcass Site Samples Swabbed for Salmonella
Analysis
Note: Salmonella testing to support official assurances for beef exported to Sweden and
Finland or to any other country with the same requirements, eg Iceland, must use the
required sampling and method specified for routine official test 2.4.2.
Table 26: Preparing Salmonella sample suspensions
Species Bovine 5 carcasses
Bobby calf, Porcine 5 carcasses
Cervine 3 carcasses
Ostrich/Emu 2 carcasses
Caprine 5 carcasses
Total number of swabs
6 swabs x 3 sites x 5 carcasses =
90 swabs
4 swabs x 3 sites x 5 carcasses =
60 swabs
4 swabs x 3 sites x 3 carcasses =
36 swabs
4 swabs x 1 site x 2 carcasses = 8
swabs
2 swabs x 3 sites x 5 carcasses =
30 swabs
Volume of ssBPW
250 ml 150 ml 150 ml 30 ml 75 ml
VLT (Very low throughput) premises with only one carcass sample (no composite)
Species Bovine VLT Bobby calf, Porcine VLT
Cervine VLT Ostrich/Emu VLT
Caprine VLT
Total number of swabs
6 swabs x 3 sites x 1 carcass =
18 swabs
4 swabs x 3 sites x 1 carcass =
12 swabs
4 swabs x 3 sites x 1 carcass =
12 swabs
4 swabs x 1 site x 1 carcass =
4 swabs
2 swabs x 3 sites x 1 carcass =
6 swabs
Volume of ssBPW
50 ml 30 ml 30 ml 15 ml 15 ml
Add an appropriate number of glass beads to the suspension, close the bottle and shake
vigorously for 2 minutes to remove the bacteria from the swabs into the diluent.
December 2012 72 Schedule 1 National Microbiological Database Programme
Laboratory Analysis
4.6 Preparation of Bulk Meat (Whole Tissue) Samples for APC, E. coli
Note: Salmonella testing to support official assurances for beef exported to Sweden and
Finland or to any other country with the same requirements, for example, Iceland, must use
the required sampling and method specified for routine official test 2.4.2
• Place the whole tissue samples (5 X ~10g) onto a sterile chopping board and finely
dice (size reduce) with a sterile knife or other suitable aseptic means.
• Re-sterilise or use separate chopping boards and knives etc for each sample.
• Aseptically weigh 25g of diced tissue directly into a stomacher bag.
• To each bag add 225ml of sterile diluent (0.1% peptone + 0.85% NaCl).
• Stomach for 2 minutes.
4.7 Aerobic Plate Count, APC30
Applicable to red meat NMD programmes.
Not applicable to poultry NMD programmes.
The following APC methods are approved for the red meat NMD programmes:
1. Spread Plate method (Routine Official test 2.1.2)
2. PetrifilmTM Aerobic Plate Count method (Routine official test 2.1.3)
3. Spiral Plater method (Routine official test 2.1.4)
4.7.1 General considerations for plating
The following are general considerations for plating:
1. Pre-poured plate count agar plates must be dried before use. Plates may be dried
using one of the following methods:
i. incubation for 24h at 30-37ºC inverted with lids in place;
ii. inverting with lids ajar in a laminar flow cabinet for 20-30 minutes;
iii. inverting with lids ajar in a still air incubator at 30-37ºC for 1 ½ to 2h;
iv. inverting with lids ajar in a forced air incubator at 30-37ºC for 30 minutes.
2. The swab samples in diluent with beads must be shaken for 2 minutes, bulk meat
samples 25g/225ml peptone diluent must be stomached for 2 minutes before
commencing analysis.
3. The same pipette/tip may be used when working up a dilution series from the most dilute
level to the most concentrated level.
4. The required incubation temperature for the APC is 30ºC +/- 1ºC (ISO 17604:2003).
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5. Initiation of analysis for APC is defined as the time when the sample is suspended and
ready for serial dilution.
6. The incubation time is 48 hours. Where plates cannot be counted at 48h plates may be:
I. incubated for up to but not longer than 72h
II. removed from the incubator and stored for no longer than 48h in a laboratory
refrigerator set at not greater than 5ºC.
The extra storage time of the plates must be recorded on the result sheet.
7. Count only plates with between 30 to 300 colonies, 25 to 250 colonies for PetrifilmTM
APC, 15 to 150 colonies for ½ plates and PetrifilmTM E. coli plates (ISO 17604:2003).
See table 22: Acceptable counting ranges.
8. If colonies are indeterminate at 48h estimate a count, re-incubate for a further 18-24h
and recount. The total incubation time must be recorded on the result sheet.
9. Express the result as the number of colony forming units (CFU)/cm2 or /g of sample.
When there are nil counts on both of the duplicate zero dilution plates, express the result
as “not detected”.
4.7.2 APC30 by spread plate method
(Reference MIMM 2005, Chapter 6 or later edition). Carry out all quality control procedures
for the media and methods using Pseudomonas aeruginosa NZRM 981 or Lactobacillus
viridescens NZRM 3313 as the positive control.
4.7.2.1 Method
The following is the APC30 by spread plate method:
1. Dispense 0.1ml (100µl) volumes of the appropriate dilutions (100 non-diluted, 10-1, 10-2,
10-3, 10-4) onto duplicate, dried plate count agar plates, or a single ½ plate.
2. Start with the highest (most dilute) level of dilution. Mix the dilutions thoroughly before
dispensing. The same pipette/tip may be used for all inoculations.
3. Spread the inoculum over the surface of the agar as evenly and as quickly as possible
using a sterile spreader (bent glass rods or disposable plastic spreaders). In order to
prevent cross-contamination, sterilise the spreader between plates both within and
between dilutions.
4. Allow the inoculum to soak into the agar surface. This should occur within 15 minutes. If
liquid does not soak in (i.e. the plates were not dried sufficiently), the viable bacteria
present in the inoculum may start to replicate and spread, possibly resulting in an
inaccurate count.
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5. Invert the plates before incubation so that the agar is in the upper half of the petri dish.
Inversion prevents condensation dropping onto the surface of the agar reducing
contamination and preventing the spread of motile micro-organisms. Stack if necessary,
and incubate at 30±1ºC for not less than 48h.
6. Count all colonies. Record all counts on the undiluted plates and only those between
30-300 on full plates or 15-150 on half plates.
7. Express the results as the number of colony forming units CFU/cm2 or /g of sample.
8. If colonies are not detected on both of the zero dilution plates express the results as “not
detected”.
9. Where counts are greater than 300 on full plates or 150 on half plates on the highest
dilution plate or half plate record the result as to numerous to count (TNTC). The TNTC
must be regarded as a “detection” and /or “higher than expected count” when assessing
compliance with premises performance criteria under HACCP and RMPs.