Food > Meat and Poultry Products > Manual of Procedures > Chapter 11
ANNEX(e) T
ANNEX T TESTING FOR Escherichia coli (E. coli) IN SLAUGHTER
ESTABLISHMENTS (ref. 9 CFR Part 310.25 and Part 381, Subpart K)
T.1 INTRODUCTION
The "Pathogen Reduction (PR) and HACCP Systems (HACCP); Final
Rule" requires that all slaughter establishments collect samples from the
type of livestock or poultry that it slaughters in largest
number and test these samples for generic Escherichia coli (E.
coli - Biotype I). The purpose of this requirement is to have slaughter
establishments use microbiological testing as a process control tool for
measuring the effectiveness of their dressing procedures.
In order to maintain USA export access, these requirements must be
implemented in an equivalent manner in Canadian establishments. Sampling for
E. coli shall be done in a manner consistent with the requirements set
out in this section.
T.1.1. Types Of Product To Be Sampled Under These
Requirements
Carcasses from the following species are covered by this requirement:
- Cattle (All types including steer, heifer, cow, bull, veal (hide-off and
hide-on));
- Swine
- Sheep
- Goats
- Horses, mules and other equines
- Chickens (All types including fryer, roaster, heavy broiler, fowl, rock
Cornish hen, etc.);
- Turkeys (All types)
- Ducks
- Geese
- Guineas
- Squabs
- Ratites
Affected establishments which slaughter any of the above species must take
samples for the species from the above list which it slaughters in the
greatest amount. Where an establishment only
slaughters species not found in the above list ( e.g. quails, bison, rabbits,
etc.) no sampling is required.
T.2 TECHNICAL REQUIREMENTS
T.2.1 Written Sampling Protocol And Sampling Procedures; Written
Records
The operator will develop and have a written protocol for
E. coli
sampling done at the establishment. The protocol shall be made available to the
Veterinarian-In-Charge (VIC) upon request. The written protocol shall contain
procedures covering all the establishments areas of responsibility. The
procedures shall be detailed to the extent necessary to enable verification.
This would include the following information:
- Sample selection: e.g., who carries out the sampling, when and where
sampling will be done, how random sampling of carcasses is achieved;
- Pre-sample preparation: e.g., check list of tasks to be performed prior to
sample collection, materials needed for sample collection, verification of the
suitability of materials before testing (ex: sponges checked to ensure they do
not have bactericidal properties);
- Sampling procedure to be used;
- Shipping procedures (if applicable): e.g., who packages, where is
packaging done, where are samples kept pending shipment, who ships samples;
where are samples shipped(laboratory)/how are samples shipped (shipping
agent);
- Tamper-proofing and protection of samples: e.g., how samples will be
handled/packaged and shipped to ensure sample integrity (temperature
maintenance and tamper proofing);
- Analytical method used: e.g., minimum sensitivity, official AOAC method
used (or method published by other scientific body;
- Compilation and analysis of results // record keeping activities: e.g.,
who receives test results, who reviews and compiles data, where will lab
reports/worksheets be kept, how long are records kept, how is CFIA
inspectors access to results provided;
- Process Verification Criteria: e.g., "m" and "M"
values, method used to set Process Verification Criteria when the sampling
method does not have pre-set "m" and "M" values;
- Corrective action procedures: e.g., for inconclusive results, for
non-compliance with procedures as found during the operators internal
verification activities, for
E. coli results exceeding action levels;
for
E. coli sampling program results indicating possible loss of
process control;
The VIC shall review the written program to verify that all the requirements
set out in this Annex are met. Reference may be made to an establishments
general sample collection procedures provided that the references are to the
procedure specific sections and that details specific to
E. coli
sampling procedures are provided. Procedures may also be written into the
establishment's HACCP plans.
T.2.2 Sampling Frequency:
Sampling frequency for
E. coli testing is determined by production
volume. With the exception of Very Low Production Volume establishments,
establishments shall collect samples at the rate set out in the following Table
or at a minimum of at least once a week.
Table T.2.2
E. coli Testing Frequencies
Cattle, sheep, goats, horses, mules and other equines |
1 test per 300 carcasses. |
Swine |
1 test per 1,000 carcasses. |
Chickens |
1 test per 22,000 carcasses |
Turkeys, ducks, geese and guineas, squabs and ratites |
1 test per 3,000 carcasses |
Notes:
- These testing frequencies do not apply to Very Low Volume Establishments.
See Section T.2.2.1
- A slaughter establishment with a HACCP System (recognized as per FSEP)
may substitute an alternate
E. coli sampling frequency in its HACCP
plan for the species, provided that the establishment has data to show that the
alternate frequency is suitable for verifying the effectiveness of the HACCP
plan (CCPs and controls). The alternate frequency and scientific
validation/justification would be assessed during the recognition process.
T.2.2.1 Sampling Rates for Very Low Volume
Establishments:
Some establishments may be classified as Very Low Volume Establishments. The
maximum yearly slaughter volumes for very low volume establishments are
described in the following table.
Table T.2.2.1
Maximum Yearly Livestock Slaughter Volumes for Very
Low
Volume Establishments
SLAUGHTER SPECIES |
CRITERIA (maximum yearly slaughter volume) |
Cattle, sheep, goats, horses, mules or other equines |
Not more than 6,000 head. |
Swine |
Not more than 20,000 head. |
Cattle, swine, sheep, goats, horses, mules or other equines |
Not more than 20,000 total, with not more than 6,000 cattle. |
Chicken |
Not more than 440,000 birds. |
Turkeys, ducks, geese, guineas and squabs |
Not more than 60,000 birds. |
Ratites |
Not more than 6,000 birds. |
Chicken, Turkeys, ducks, geese, guineas and squabs |
Not more than 440,000 birds total, with not more than 60,000 turkeys. |
Very Low Volume Establishments shall conduct one series of sampling annually
beginning the first full week that the establishment operates after June 11.
Sampling shall continue at the rate of one sample taken each week that the
establishment operates until:
- where the establishment uses a sampling procedure with pre-set Process
Verification Criteria (see section T.2.8):
- the establishment has taken a series of 13 tests that demonstrates that
their process in under control (meet m and M established criteria as per the
section T.2.8 );
- where the establishment uses a sampling procedure for which pre-set
Process Verification Criteria do not exist:
- June 1 of the following year until 13 samples have been collected which
ever comes first that demonstrates that the process is under control (see
section T.2.8 );
It will not be required to take any further samples during the year in Very
Low Volume Establishments unless:
- changes are made in establishment facilities, equipment, personnel or
procedures;
- the establishment or CFIA determines that the changes may affect the
adequacy of existing process control measures;
The VIC shall assess any changes made to determine if there is need for
further sampling. Records of the assessment shall be conserved (record under
E. coli inspection task). If it is determined that the change may
required further sampling, the VIC shall contact the Director, Programs Network
(DPN) to discuss the matter. If the DPN concurs that further sampling is
required, the establishment will be so advised in writing.
T.2.3 Pre-sampling Preparation Procedures:
Sample collection will be carried out by the individual designated in the
establishment's written sampling protocol. Sampling supplies, such as
sterile gloves, sterile sampling solutions, hand soap, sanitizing solution,
etc., as well as specific materials needed for sampling different carcass types
(i.e., specimen sponges in bags and template for sampling cattle or swine
carcasses), will need to be assembled prior to beginning sample collection.
When sponges are used, a verification of the sponges suitability must have
been done. Sponges must not possess antimicrobial properties which might reduce
bacterial counts. Supplier certification (e.g., letters of guarantee) shall be
obtained for each new lot or in-house verification of each new lot of sponges
shall be performed.
Such verification typically should consist of immersing a sponge for a
number of hours within a seeded bath of transport medium (ex.
Butterfields Phosphate Diluent) to simulate shipping conditions. The bath
containing indicator organisms in a known quantity is cultured at the end of
the period to verify if the concentration of bacteria has decreased
significantly, which would indicate that substances within the sponge are
killing bacteria.
For cattle, swine and equine carcass sampling, a template will be needed to
mark off the area to sample. The template can be made of metal or aluminum
foil, brown paper, flexible plastic, etc. Some disposable templates may come
sterilized and individually prepackaged. To make a reusable template for cattle
and swine, cut out a 10 centimetres
(cm) x 10 cm square from a sheet larger
than the area to be sampled (See Figure 1). For turkeys, ratites, sheep and goats, the
template shall be 5 cm x 10 cm.
If a reusable template is used, it will need to be sanitized with an
approved sanitizing solution [e.g., hypochlorite (bleach) solution or alcohol]
for at least 2 to 3 minutes. However, the template needs to be totally
dry before placing it on the carcass. Aluminum foil or paper templates
can be used once and discarded. The foil for the template should be stored in a
manner to prevent contamination. Since the area enclosed by the template will
be sampled, take care not to touch this area with anything other than the
sampling sponge. Using dirty or contaminated material may lead to erroneous
results. If an autoclave is available, paper or aluminum foil templates can be
wrapped in autoclavable paper and sterilized.
Sterile sampling solutions, Butterfield's phosphate diluent (BPD), can
be stored at room temperature. However, at least on the day prior to sample
collection, check solutions for cloudiness. DO NOT use
solutions that are cloudy, turbid or contain particulate matter. Place the
number of containers of sampling solution (BPD) that will be needed for the
next day's sampling in the refrigerator. Ensure that shipping containers,
coolant packs and shipping documents are prepared as required.
Sterile Buffered Peptone Water (BPW) can be substituted for BPD. It must be
noted however that there may be a slight rise in bacteria counts following this
change (¼ log increase). If Process Verification Criteria have been
published by USDA for the sampling method used, marginal ("m") and
unacceptable ("M") limits are not allowed to be changed.
T.2.4 Procedure for the Random Selection of Carcasses:
Samples are to be taken randomly at the required frequency (See section
T.2.2, "Sampling Frequency"). For example:
- For cattle (1 test required/per every 300 head slaughtered): a plant which
slaughters 150 head of cattle an hour will select 1 sample at random for every
2 hour interval of production.
- For turkeys (1 test required/per every 3,000 turkeys slaughtered): a plant
which slaughters 1,000 turkeys an hour will select 1 sample at random for every
3 hour interval of production.
Cattle half-carcasses and swine, sheep, goat, equine and ratites carcasses should be
selected from those in the cooler 12 or more hours after
slaughter2. Poultry carcasses should be selected at random
after chilling3.
The operators written procedure must clearly explain how the random
selection of samples is achieved. Every carcass (for cattle, every
half-carcass, e.g. "leading" and "trailing" side) should
have an equal chance of being selected from all eligible
half-carcasses/carcasses. If multiple lines exist, randomly select the line for
sample collection for that interval. Repeat the random selection process for
the next sampling interval. Each line should have an equal chance of being
selected at each sampling interval.
Use of random numbers tables or similar random number generating systems
(ex. computer-generated, calculator generated, drawing cards, etc.) is
required. To ensure that all carcasses have an equal chance of being selected,
the procedure must ensure:
- that the TIME when sample is to be selected is
determined in a random manner (alternatively, random selection of the carcass
sequence number achieves this - see Note 2);
- that, where there is more than one possible site for selecting the
sample, the SITE is determined in a random manner. For
example:
- Selection of beef, hog, sheep, goat and equine carcasses when selection is
done within coolers (12 or more hours after slaughter) or on transfer chains,
grading chains or other rails: the selection of the cooler and site within the
cooler where the carcass is to be selected for the sampling period (i.e.
days production) is determined in a random manner; AND the selection of
the half-carcass or side of carcass to sample is randomly determined;
- Selection of poultry carcass when the selection is done after chilling, at
the end of the drip line or last readily accessible point prior to packaging
(e.g., carcasses which are subjected to immersion freezing) - see note 3:
that the selection of the chiller/drip line/packaging line where the sample is
to be selected for the sampling period (i.e. days production) is
determined in a random manner.
- where the sample selection procedure involves selecting a
CARCASS/HALF-CARCASS at a randomly determined sampling time/sampling
site, the following procedure is used to eliminate bias and ensure
random selection.
After having identified the carcass or half-carcass selected by the random
procedure from the predetermined point, count back five (5)
carcasses/half-carcasses and select the next carcass/half-carcass for sampling.
Each carcass/half-carcass must have an equal chance of being selected. The
reason for counting back five carcasses/half-carcasses is to avoid any possible
bias during selection.
Additionally, for poultry carcasses
only: because only WHOLE (untrimmed) carcasses are to be
sampled, if the fifth carcass is not a WHOLE (untrimmed) carcass, count back an
additional five carcasses for sample selection. Repeat if necessary.
If more than one shift is operating at the plant, sample(s) can be taken on
any shift, provided the number of samples corresponds to the total days
production and that TIME, SITE and CARCASS requirements have been met.
T.2.5 Procedures to Ensure Aseptic Techniques/Sampling:
Micro-organisms from the environment, hands, clothing, sample containers,
sampling devices, etc., may lead to erroneous analytical results. Stringent
requirements for microbiological analysis are necessary: use of aseptic
sampling techniques and clean, sanitized equipment and supplies are of utmost
importance.
There should be an area designated for preparing sampling supplies, etc. A
stainless steel, wheeled cart or table would be useful during sampling. A small
tote or caddy could be moved to the location of sampling and could be used for
carrying supplies, supporting sample bags when adding sterile solutions to
sample bags, etc.
Sterile gloves should be used for collecting samples. The only items which
may contact the external surface of the glove are the exposed sample being
collected and/or the sterile sample utensil (specimen sponge). Keep in mind
that the outside surfaces of the sample container are not sterile. Do not
handle the inside surface of the sterile sample containers. Do not touch
anything else. The following procedure for putting on sterile gloves can be
followed when collecting samples:
(a) | Peel open the package of sterile gloves from the top without
contaminating (touching, breathing on, contacting, etc.) the exterior of the
gloves. |
(b) | Remove the first glove by holding it from the wrist-side opening inner
surface. Avoid any contact with the outer surface of the glove. Insert the
washed and sanitized hand into the glove, taking care not to puncture the
glove. |
(c) | Remove the second glove by holding it by the cuff (outer surface). Avoid
contaminating the outside of the first glove when pulling on the second glove.
Take care not to touch the face, skin, clothes and other non-sterile
surfaces. |
(d) | If at any time you are concerned that a glove may be contaminated,
discard it and begin again with Step (a) above. |
T.2.5.1 Preparation for Sample Collection:
Prior to collecting samples, review appropriate sampling steps, random
selection procedures, and other information that will aid in sample
collection.
On the day prior to sample collection, after checking for cloudiness/
turbidity, place the number of BPD containers that will be needed for the next
day's sampling in the refrigerator/cooler. If samples are to be shipped to
an off-site facility, pre-chill shipping container and refrigerator packs.
On the day of sampling, gather all sample collection bags, sterile gloves,
sanitizer, hand soap, sterile solutions for sampling, and specific materials
listed under the Materials section (i) of the sample
collection section for the type of carcass to be sampled. Ensure that all
sampling supplies are on hand and readily available before beginning sample
collection.
Label the sample bags before starting the sampling procedure. Use permanent
ink. If you are using paper labels, it is important that the label be applied
to the bag at normal room temperature; it will not stick if applied in the
cooler.
Outer clothing (frocks, gloves, head gear, etc.) worn in other areas of the
plant should be removed before entering the sampling area or preparing to
collect samples. Replace outer clothing removed earlier with clean garments
(i.e., laboratory coat) that have not been directly exposed to areas of the
plant outside of the sampling area.
Sanitize the sample work area surfaces by wiping with a clean disposable
paper towel dipped in a freshly prepared 500 ppm (parts per million) sodium
hypochlorite solution (0.05% sodium hypochlorite) or other approved sanitizer
which provides an equivalent available chlorine concentration. The sample work
area surfaces must be free of standing liquid before sample supplies and/or
product containers are placed on them.
Before sampling, thoroughly wash and scrub hands to the mid-forearm. Use
antibacterial hand soap. If available, this should include a sanitizer at 50
ppm equivalence available chlorine. Dry the hands using disposable paper
towels.
T.2.5.2 Specific Sample Collection Procedures:
The establishments sample collection procedures must be clearly
described in writing. The following procedures are those referred to in the
USDA Pathogen Reduction and HACCP Systems Final Rule. Establishments
which do sampling of
E. coli on beef and hogs must use either the
excision or carcass sponging technique described in this section.
Establishments which do sampling of
E. coli on
ratites, sheep, goats and equine
must use the carcass sponging technique described in this section.
Establishments testing for
E. coli in chickens, ducks and guineas
shall use the carcass wash technique described in this section. Establishments
testing for
E. coli in turkeys and geese can either use the carcass
wash technique or the carcass sponging technique described in this section.
Other sample collection procedures (ex. Excision of samples instead of
swabbing) may be acceptable provided they have been assessed by CFIA and found
satisfactory. Establishments wishing to use alternative sampling methods should
forward their proposal to the FAOD.
Sampling/testing methods used for
E. coli testing must achieve a
sensitivity of a least 5 cfu/cm2.
For methods where USDA has published Process Verification Criteria (see
Section T.2.8), the establishment must adhere to the method and use the pre-set
Process Verification Criteria. Where USDA Process Verification Criteria have
not been published, the establishment should adhere to the procedure and must
develop establishment specific Process Verification Criteria (see Section
T.2.8.2).
T.2.5.2.1 Carcass Sample Collection Procedures - Cattle (including
veal), sheep, goats, horses, mules and other equines.
T.2.5.2.1.1 Surface sponging method
(i) Materials
- Sterile specimen sponge in sterile wire twist top-type bag or
equivalent
- 25 ml sterile Butterfield's phosphate diluent (BPD)
- Sterile plastic self-sealing-type or stomacher bag
- Template for 100 cm2 sampling
area ( except for sheep and goats where a template of 50 cm2 is required)
- Sterile gloves
- Wheeled ladder, sampling platform, or step ladder
- Sanitizing solution
- Small tote or caddy for carrying supplies
(ii) Collection
Read sections T.2.3 "Pre-Sampling Preparation" and T.2.5.1
"Preparation for Sample Collection" before beginning the sampling
procedure. Use the random selection procedures for selecting the half-carcass
(ref. section T.2.4).
A sampling sponge (which usually comes dehydrated and prepackaged in a
sterile bag) will be used to sample all three sites on the carcass (flank,
brisket, and rump--see Figure T.2). It is important to swab the areas in the
order of least to most contamination in order to avoid spreading any
contamination. Therefore, swab the areas in the sequence indicated in this
sampling protocol.
Nondestructive surface sampling will be conducted as follows:
- Ensure that all bags have been pre-labeled and all supplies are on hand,
including the sampling template. (An assistant may be helpful during the
sampling process.)
- IF a reusable template is used, immerse the sampling template in an
approved sanitizing solution for at least 1-2 minutes. Just prior to swabbing
the first sample site on the carcass (step 13), retrieve the sampling template
from the sanitizing solution. Shake excess solution from the utensil, then
protect the portion of the template that will contact the carcass from
contamination.
- Locate the flank, brisket, and rump sampling sites using illustrations
and directions in Figure T.2 (cattle carcass sampling locations).
- Position the wheeled ladder, sampling platform, or step ladder near the
carcass so the rump sample area (Figure T.2) is within easy reach from the
ladder.
- While holding the sponge bag at the top corner by the wire closure, tear
off the clear, perforated strip at the top of the bag.
- Remove the cap from sterile BPD bottle, being careful not to touch the
bottle opening.
- Carefully pour about half the contents of the sterile BPD bottle
(approximately 10 ml) into the sponge bag to moisten the sponge.
- Close the top of the bag by pressing the wire closures together. Use hand
pressure from the outside of the bag and carefully massage the sponge until it
is FULLY HYDRATED (moistened).
- With the bag still closed, carefully push the moistened sponge to the
upper portion of the bag orienting one narrow end of the sponge up toward the
opening of the bag. Do NOT open the bag or touch the sponge with your fingers.
While holding the bag, gently squeeze any excess fluid from the sponge using
hand pressure from the outside. The whole sponge should still be in the
bag.
- Open the bag containing the sponge, being careful not to touch the inner
surface of the bag with your fingers. The wire closure at the top of the bag
should keep the bag open. Set bag aside.
- Put on a pair of sterile gloves.
- Carefully remove the moistened sponge from the bag with the thumb and
fingers (index and middle) of your sampling hand.
- With the other hand, retrieve the template by the outer edge, taking
care not to contaminate the inner edges of the sampling area of the
template.
- Locate the flank sampling area (Figure T.2). Place the template over
this location.
- Hold the template in place with one gloved hand (Remember, only the
sponge should touch the sampling area. Take care not to contaminate this area
with your hands)
- With the other hand, wipe the sponge over the enclosed sampling area(10
cm x 10 cm)
for a total of approximately 10 times in the vertical and 10 times in the
horizontal directions. The pressure for swabbing would be as if you were
removing dried blood from the carcass. However, the pressure should not be too
hard as to crumble or destroy the sponge. (Note: The template may need to be
"rolled" from side to side during swabbing since the surface of the carcass is
not flat. This ensures that the 100 cm2 area is enclosed
while swabbing.)
- Repeat steps 14-16 for the brisket area, using the SAME side or surface
of the sponge used to swab the flank area.
- After swabbing the brisket area, transfer the template to the same hand
holding the sponge. Do not contaminate the sponge or inner edges of the
sampling area of the template.
- Climb the ladder or platform, holding onto the handrail with the hand
used to hold the template. Once at a convenient and safe height for sampling
the rump, transfer template back to "climbing" hand (hand used to
hold onto the rail while climbing the ladder), taking care not to contaminate
the inner edges of the template.
- Repeat steps 14-16 for the rump area, using the "clean"
surface or side (the side that was NOT previously used to swab the
flank/brisket areas) of the sponge.
- After swabbing the rump area, carefully place the sponge back in the
sponge sample bag, taking care not to touch the sponge to the outside of the
sample bag.
- While holding the handrail, climb down from the ladder.
- Add the additional BPD (about 15 ml) to the sample bag to bring the
total volume to approximately 25 ml.
- Expel excess air from the bag containing the sponge and fold down the
top edge of the bag 3 or 4 times to close. Secure the bag by folding the
attached wire tie back against the bag. Place closed sponge bag into second bag
and close the second bag securely.
- (a) If samples are to be analyzed at an ON-SITE LABORATORY, begin sample
preparation (T.2.7 "ANALYTICAL METHODS")
(b) If samples are to be analyzed at an OUTSIDE (OFF-SITE) LABORATORY,
follow procedure in section T.2.6 "Sample Shipment".
- Results must be analyzed using establishment specific statistical
process control - the moving window and pre-set ("m") and
("M") criteria CANNOT BE USED (see section T.2.8.2.).
T. 2.5.2.1.2 Excision method - Cattle (including veal)
This procedure is available upon request from the FAOD. When this method is
used, the Process Verification Criteria found in section T.2.8 must be
utilized.
T.2.5.2.2 Carcass sample collection procedure - Swine
T.2.5.2.2.1 Carcass sponging method - Swine
(i) Materials
- Sterile specimen sponge in sterile wire twist top type-bag or
equivalent
- 25 ml sterile Butterfield's phosphate diluent (BPD)
- Sterile plastic self-sealing-type bag or stomacher-type bag
- Template for a 100 cm2 sampling area
- Sterile gloves
- Wheeled ladder, sampling platform, or step ladder
- Sanitizing solution
- Small tote or caddy for carrying supplies
(ii) Collection
Read sections T.2.3 "Pre-Sampling Preparation" and T.2.5.1
"Preparation for Sample Collection" before beginning the sampling
procedure. Use the random selection procedures for selecting the half-carcass
(ref. section T.2.4).
A sampling sponge (which usually comes dehydrated and prepackaged in a
sterile bag) will be used to sample all three sites on the swine carcass
(belly, ham, and jowl-see Figure T.3). It is important to swab the areas in the
order of least to most contamination in order to avoid spreading any
contamination. Therefore, swab the areas in the sequence indicated in this
sampling protocol. Nondestructive surface sampling will be conducted as
follows:
- Ensure that all supplies are on hand. (An assistant may be helpful during
the sampling process.)
- If a reusable template is used, immerse the sampling template in a
sanitizing solution for at least 1-2 minutes. Just prior to swabbing the first
sample site on the swine carcass (step 12), retrieve the sampling template from
the sanitizing solution. Shake excess solution from the utensil, then protect
the portion of the template that will contact the carcass from
contamination.
- Locate the belly, ham, and jowl sampling sites using illustrations and
directions in Figure T.3 (swine carcass sampling locations).
- Position the wheeled ladder, sampling platform, or step ladder near the
carcass so the ham sample area (Figure T.3) is within easy reach from the
ladder.
- Hold the sponge bag at the top corner by the wire closure, then tear off
the clear perforated strip at the top of the bag. Open the bag.
- Remove the cap from sterile BPD bottle, being careful not to touch the
bottle opening. Do not contaminate the lid.
- Carefully pour about half of the contents of the sterile BPD bottle (10
ml) into the sponge bag to moisten the sponge. Put the lid back on the BPD
bottle.
- Close the top of the bag by pressing the wire closures together. Use hand
pressure from the outside of the bag and carefully massage the sponge until it
is FULLY HYDRATED (moistened).
- With the bag still closed, carefully push the moistened sponge to the
upper portion of the bag orienting one narrow end of the sponge up toward the
opening of the bag. Do NOT open the bag or touch the sponge with your fingers.
While holding the bag, gently squeeze any excess fluid from the sponge using
hand pressure from outside. The whole sponge should still be inside the
bag.
- Open the bag containing the sponge, being careful not to touch the inner
surface of the bag with your fingers. The wire closure at the top of the bag
should keep the bag open.
- Put on a pair of sterile gloves.
- Carefully remove the moistened sponge from the bag with the thumb and
fingers (index and middle) of your sampling hand.
- With the other hand, retrieve the template by the outer edge, taking
care not to contaminate the inner edges of the sampling area of the
template.
- Locate the belly sampling area (Figure T.3). Place the template over
this location.
- Hold the template in place with one gloved hand. Remember, only the
sponge should touch the sampling area. Take care not to contaminate this area
with your hands.
- With the other hand, wipe the sponge over the enclosed sampling area (10
cm x 10 cm) for a total of approximately 10 times in the vertical and 10 times
in the horizontal directions. The pressure for swabbing would be as if you were
removing dried blood from the carcass. However, the pressure should not be too
hard as to crumble or destroy the sponge.
Note: The template may need to be "rolled" from side to side
during swabbing since the surface of the carcass is not flat. This ensures that
the 100 cm2 area is enclosed while swabbing.
- After swabbing the belly area, transfer the template to the same hand
that is holding the sponge. Do not contaminate the sponge or the inner edges of
the sampling area of the template.
- Climb the ladder or platform, holding onto the handrail with the hand
used to hold the sampling template in place. Once at a convenient and safe
height for sampling the ham, transfer template back to the "climbing"
hand (hand used to hold onto the rail while climbing the ladder), taking care
not to contaminate the sponge or the inner edges of the template.
- Repeat steps 14-16 for the ham sampling area, using the SAME surface of
the sponge used to swab the belly area.
- After swabbing the ham area, carefully place the template back to the
same hand that is holding the sponge. Do not contaminate the sponge or the
inner edges of the sampling area of the template.
- While holding the handrail, climb down from the ladder.
- Transfer the template back to the "climbing" hand (hand used
to hold onto the rail while descending the ladder), taking care not to
contaminate the sponge or the inner edges of the template.
- Repeat steps 14-16 for the jowl area, using the "clean"
surface or side (the side that was not previously used to swab the belly/ham
areas).
- After swabbing the jowl area, carefully place the sponge back into the
sponge bag. Do not touch the surface of the sponge to the outside of the sponge
bag.
- Add the additional BPD (about 15 ml) to the bag to bring the total
volume to approximately 25 ml.
- Press wire closures of the sponge bag together, expel excess air, then
fold down the top edge of the bag 3 or 4 times. Secure the bag by folding the
attached wire tie back against the bag. Place the closed sponge bag into the
second bag and close the second bag securely.
- (a) If samples are to be analyzed at an ON-SITE LABORATORY, begin sample
preparation (T.2.7 "ANALYTICAL METHODS")
(b) If samples are to be analyzed at an OUTSIDE (OFF-SITE) LABORATORY,
follow procedure in section T.2.6 "Sample Shipment".
- Results must be analyzed using establishment specific statistical
process control - the moving window and pre-set ("m") and
("M") criteria CANNOT BE USED (see section T.2.8.2.).
T.2.5.2.2.2 Carcass Excision Method - Swine
This procedure is available upon request from the Food of Animal Origin
Division. When this method is used, the Process Verification Criteria found in
section T.2.8 must be utilized.
T.2.5.2.3 Chicken, Ducks, Guineas and Squabs Carcass Rinse Sampling
Procedure
Note: There is no approved excision or carcass swabbing
method for chicken carcasses.
i) Materials
- 2 Sterile 3500 millilitre
(ml) stomacher-type or self-sealing type bags
or equivalent. (The bag must be sterile and should be large enough to hold the
carcass while rinsing.)
- 400 ml sterile, Butterfield's phosphate diluent (BPD).
- Plastic tie wraps or equivalent (if needed to secure the bag).
- Sterile gloves.
- Optional--(See alternate sampling--step 10)--Sterile leak-proof
container.
(ii) Collection
Read sections T.2.3 "Pre-Sampling Preparation" and T.2.5.1
"Preparation for Sample Collection" before beginning the sampling
procedure. Use the random selection procedures for selecting the carcass (ref.
section T.2.4). Handle the selected carcass in the following manner:
- Ensure all sampling supplies are present and have been properly labeled.
An assistant may be helpful during sampling.
- Open a large stomacher-type bag without touching the sterile interior of
the bag. (Rubbing the top edges of the bag between the thumb and forefinger
will cause the opening to gap for easy opening.)
- Put on sterile gloves.
- With one hand, push up through the bottom of the sampling bag to form a
"glove" over one hand with which to grab the bird, while using your
other hand to pull the bag back over the hand that will grab the bird. This
should be done aseptically without touching the exposed interior of the
bag.
- Using the hand with the bag reversed over it, pick up the bird by the
legs (hocks) through the stomacher bag. (The bag functions as a 'glove'
for grabbing the bird's legs.) Take care not to contaminate the exposed
interior surface of the bag. Allow any excess fluid to drain before reversing
the bag back over the bird. (Alternately, have an assistant hold open the bag.
Using your gloved hand, pick up the bird by the legs, allow any fluid to drain,
and place the bird in the sampling bag.)
- Rest the bottom of the bag on a flat surface. While still holding the top
of the bag slightly open, add the sterile BPD (400 ml) to the bag containing
the carcass, pouring the solution over the carcass. (Alternately, with the aid
of an assistant holding the bag open, add the sterile BPD (400 ml) to the bag
containing the carcass, pouring the solution over the carcass.)
- Expel most of the air from the bag, then close the top of the bag. While
securely holding the bag, rinse the bird inside and out using a rocking motion
for 30 shakes (approximately one minute). This is done by holding the bird
through the bottom of the bag with one hand and the closed top of the bag with
the other hand. Hold the bird securely and rock it in an arcing motion,
alternating the weight of the bird from one hand to the other (motion like
drawing an invisible rainbow or arch), assuring that all surfaces (interior and
exterior of the carcass) are rinsed.
- Rest the bag with the bird on a flat surface and, while still supporting
the bird, open the bag.
- With a gloved hand, remove the carcass from the bag. Since the carcass
was rinsed with a sterile solution, it can be returned to the chill tank. Be
sure not to touch the interior of the bag with your gloved hand.
- Secure the top of the bag so that the rinse fluid will not spill out or
become contaminated. (Alternately, at least 30 millilitres of
rinse fluid can
be poured into a sterile leak-proof container to be sent to the lab for
analysis.)
- Place the sample bag (or leak-proof container) into another bag and
secure the opening of the outer bag.
- (a) If samples are to be analyzed at an ON-SITE LABORATORY, begin sample
preparation for the selected method of analysis.
(b) If samples are to be analyzed at an OUTSIDE (OFF-SITE) LABORATORY,
follow the procedure in section T.2.6 "Sample Shipment".
- Results must be analyzed using the moving window and pre-set
("m") and ("M") (see section T.2.8.2.).
T.2.5.2.4 Turkey, geese and ratites carcass sample collection
procedures:
T.2.5.2.4.1 Carcass Rinse Sampling Procedure - Turkey and
Geese
(i) Materials
- 2 Sterile 3500 ml stomacher-type or self-sealing type-bags or equivalent.
(The bag must be sterile and should be large enough to hold the carcass while
rinsing, the bags USDA used for their sampling program measure approximately
18" x 24". Large turkeys should be placed in a plain, clear
polypropylene autoclave bag , about 24" x 30" to 36").
- 600 ml sterile, Butterfield's phosphate diluent (BPD)
- Plastic tie wraps or thick rubber bands or equivalent, if needed to
secure sample bag
- Sterile gloves
- Optional--sterile, leak-proof container (see step 12 Alternate
procedure)
(ii) Collection
Read sections T.2.3 "Pre-Sampling Preparation" and T.2.5.1
"Preparation for Sample Collection" before beginning the sampling
procedure. Use the random selection procedures for selecting the half-carcass
(ref. section T.2.4). Handle the selected carcass in the following manner:
- Ensure that all supplies are on hand and readily available. An assistant
will be needed to hold the bag for collecting the bird.
- Have an assistant open the large sterile stomacher-type bag (designated
for rinsing the carcass) and be ready to receive the turkey carcass. (Rubbing
the top edges of the bag between the thumb and index finger will cause the
opening to gap open). Alternately: If no assistant is available, place the
closed large sampling bag into a bucket or pail (e.g., use the bag to
"line" a bucket like a trash-can liner), then open the bag. The
bucket will be used as a holder or stand to support the bag. Do not contaminate
the inner surfaces of the sampling bag.
- Put on sterile gloves.
- Remove the selected turkey from the drip line by grasping it by the legs
and allowing any fluid to drain from the cavity.
- Place the turkey carcass, vent side up, into a sterile sampling bag. Only
the carcass should come in contact with the inside of the bag.
- Manipulate the loose neck skin on the carcass through the bag and
position it over the neck bone area to act as a cushion and prevent puncturing
of the bag. The assistant will need to support the carcass with one hand on the
bottom of the bag.
- While still supporting the bottom of the bag, have the assistant open the
bag with the other hand. Alternately, rest the bottom of the bag on a
pre-sanitized surface (i.e., a table), and while still supporting the carcass
in the bag, open the bag with the other hand.
- Add the sterile BPD (600 ml) to the bag containing the carcass, pouring
the diluent over the carcass.
- Take the bag from the assistant and expel excess air from the bag and
close the top. While securely holding the bag, rinse the bird inside and out
using a rocking motion for 30 shakes (approximately one minute). This is done
by holding the carcass through the bag with one hand and the closed top of the
bag with the other hand. Holding the bird securely with both hands, rock in an
arcing motion alternating the weight of the bird from one hand to the other
(motion like drawing an invisible rainbow or arch), assuring that all surfaces
(interior and exterior of the carcass) are rinsed.
- Hand the bag back to the assistant.
- With a gloved hand, remove the carcass from the bag letting excess fluid
drain back into the bag. Since the carcass was rinsed with a sterile solution,
it can be returned to the chill tank. Be sure not to touch the interior of the
bag with your gloved hand.
- Expel excess air, taking care not to expel any rinse fluid. Secure the
top of the bag so that the rinse fluid will not spill out or become
contaminated. (Alternately, at least 30 millilitres of
rinse fluid can be
poured into a sterile, leak-proof container and sent to the lab for
analysis.)
- Place the sample bag (or container) into another bag and secure the
opening of the outer bag.
- (a) If samples are to be analyzed at an ON-SITE LABORATORY, begin sample
preparation (T.2.7 "ANALYTICAL METHODS")
(b) If samples are to be analyzed at an OUTSIDE (OFF-SITE) LABORATORY,
follow procedure in section T.2.6 "Sample Shipment".
- Results must be analyzed using establishment specific statistical
process control - the moving window and pre-set ("m") and
("M") criteria CANNOT BE USED (see section T.2.8.2.).
T.2.5.2.4.2 Carcass Sponging Sampling Procedure - Turkeys,
Geese and Ratites
(i) Materials
- 1. Sterile specimen sponge in sterile wire twist top type-bag or
equivalent
- 2. 25 ml sterile Butterfield's phosphate diluent (BPD)
- 3. Sterile plastic self-sealing-type bag or stomacher-type bag
- 4. 5 x 10 cm sampling area template
- 5. Sterile gloves
- 6. Sanitizing solution
- 7. Small tote or caddy for carrying supplies
(ii) Collection
Read sections T.2.3 "Pre-Sampling Preparation" and T.2.5.1
"Preparation for Sample Collection" before beginning the sampling
procedure. Use the random selection procedures for selecting the carcass (ref.
section T.2.4).
A sampling sponge (which usually comes dehydrated and prepackaged in a
sterile bag) will be used to sample two sites on the turkey carcass (thigh and
back - see figure T.4.) It is important to swab the areas in the order of least
to most contamination in order to avoid spreading any contamination (i.e. back
then thigh). Therefore, swab the areas in the sequence indicated in this
sampling protocol. Nondestructive surface sampling will be conducted as
follows:
- Ensure that all supplies are on hand. (An assistant may be helpful during
the sampling process.)
- If a reusable template is used, immerse the sampling template in a
sanitizing solution for at least 1-2 minutes. Just prior to swabbing the first
sample site on the carcass (step 12), retrieve the sampling template from the
sanitizing solution. Shake all excess solution from the utensil, then protect
the portion of the template that will contact the carcass from
contamination.
- Place the carcass to be swabbed on a work surface which has been cleaned,
disinfected and dried. To prevent the carcass from sliding, place paper towels
or other suitable material between the carcass and the work surface. Place the
carcass on its breast to allow access to both sampling sites. It is ok if the
carcass leans to one side, provided the sampling sites are not
contaminated.
- Locate the back and thigh sampling sites using illustrations and
directions in Figure T.4 (turkey carcass sampling locations).
- Hold the sponge bag at the top corner by the wire closure, then tear off
the clear perforated strip at the top of the bag. Open the bag.
- Remove the cap from sterile BPD bottle, being careful not to touch the
bottle opening. Do not contaminate the lid.
- Carefully pour about half of the contents of the sterile BPD bottle (10
ml) into the sponge bag to moisten the sponge. Put the lid back on the BPD
bottle.
- Close the top of the bag by pressing the wire closures together. Use hand
pressure from the outside of the bag and carefully massage the sponge until it
is FULLY HYDRATED (moistened).
- With the bag still closed, carefully push the moistened sponge to the
upper portion of the bag orienting one narrow end of the sponge up toward the
opening of the bag. Do NOT open the bag or touch the sponge with your fingers.
While holding the bag, gently squeeze any excess fluid from the sponge using
hand pressure from outside. The whole sponge should still be inside the
bag.
- Open the bag containing the sponge, being careful not to touch the inner
surface of the bag with your fingers. The wire closure at the top of the bag
should keep the bag open.
- Put on a pair of sterile gloves.
- Carefully remove the moistened sponge from the bag with the thumb and
fingers (index and middle) of your sampling hand.
- With the other hand, retrieve the template by the outer edge, taking
care not to contaminate the inner edges of the sampling area of the
template.
- Locate the back swabbing area (Figure T.4). Place the template over this
location. The template should straddle the backbone with the long axis of the
template parallel to the spine.
- Hold the template in place with one gloved hand. Remember, only the
sponge should touch the sampling area. Take care not to contaminate this area
with your hands.
- With the other hand, wipe the sponge over the enclosed sampling area (5
cm x 10 cm) for a total of approximately 10 times in the vertical and 10 times
in the horizontal directions. The pressure for swabbing would be as if you were
removing dried blood from the carcass. However, the pressure should not be too
hard as to crumble or destroy the sponge.
Note: The template may need to be "rolled" from side to side during swabbing
since the surface of the carcass is not flat. This ensures that the 50 cm2 area is enclosed while swabbing.
- After swabbing the back area, transfer the template to the same hand
that is holding the sponge. Do not contaminate the sponge or the inner edges of
the sampling area of the template.
- Repeat steps 14-16 for the thigh sampling area, using the surface of the
sponge WHICH WAS NOT USED to swab the back area. The template needs to be
locates in the following manner: the long axis of the template is placed
parallel to the thigh bone and the top of the template is just below the hip
joint.
- After swabbing the thigh area, carefully place the sponge back into the
sponge bag. Do not touch the surface of the sponge to the outside of the sponge
bag.
- Add the additional BPD (about 15 ml) to the bag to bring the total
volume to approximately 25 ml.
- Press wire closures of the sponge bag together, expel excess air, then
fold down the top edge of the bag 3 or 4 times. Secure the bag by folding the
attached wire tie back against the bag. Place the closed sponge bag into the
second bag and close the second bag securely.
- (a) If samples are to be analyzed at an ON-SITE LABORATORY, begin sample
preparation (T.2.7 "ANALYTICAL METHODS")
(b) If samples are to be analyzed at an OUTSIDE (OFF-SITE) LABORATORY,
follow procedure in section T.2.6 "Sample Shipment".
- Results must be analyzed using establishment specific statistical
process control - the moving window and pre-set ("m") and
("M") criteria CANNOT BE USED (see section T.2.8.2.).
T.2.6 Sample Shipment Procedures:
To obtain the most accurate results, samples should be analyzed as soon as
possible after collection. However, if samples must be sent off-site, they need
to be maintained at refrigeration temperatures until transport and shipped
refrigerated to the laboratory performing the analysis. The sample must be
analyzed no later than the day after collection. The following
section gives information on shipping containers and transporting samples to
off-site facilities.
T.2.6.1 Shipping Containers and Coolant Packs:
It is important that samples fit easily into the shipping containers so that
the sample bags do not break. Correct use of refrigerant gel-ice packs and
proper packing of the shipping container are necessary to ensure that samples
arrive at the laboratory at an acceptable temperature. Some bacteria may be
damaged by temperatures that are too cold, while temperatures that are too warm
can allow bacteria to reproduce. Maintaining samples at improper temperatures
may cause inaccurate sample results. Frozen samples or samples which
are too warm (>10°C) are not considered valid and must not be
analyzed.
The sample should be kept refrigerated, NOT FROZEN, in the
shipping container prior to pickup by the courier service. The shipping
container, itself, should not be used as a refrigerator. However, multiple
samples (if needed) for one day may be stored in an open shipping container
kept in the cooler or refrigerator.
The following packaging procedure can be used:
- Prechill shipping container by placing the open shipping container in the
refrigerator at least the day before sampling.
- Place the appropriately-labeled, double-bagged sample(s) in the
prechilled shipping container in an upright position to prevent spillage.
Newspaper may be used for cushioning the sample and holding it in the upright
position. If more than one sample is collected during the day, take steps to
ensure that samples are maintained at refrigeration temperature. Refrigeration
temperatures help limit multiplication of any microorganisms present which
ensures the most accurate results.
- Place a corrugated cardboard pad on top of samples. This corrugated
cardboard pad prevents direct contact of frozen gel packs with the samples.
Next place the frozen gel pack(s) on top of the corrugated pad. Use sufficient
frozen coolant to keep the sample refrigerated during shipment to the
designated laboratory. Insert foam plug and press it down to minimize shipper
head space.
- Ship samples (via overnight delivery or courier) to the assigned
laboratory.
When shipping samples, the method used to protect against sample tampering
shall be described in the written sampling protocol.
T.2.7 Analytical Methods:
The laboratory shall not analyze samples which are frozen or too warm (e.g.,
> 10°C). Samples must be analyzed using one of the
E. coli (Biotype
I) quantitation methods found in the Official Methods of
Analysis of the Association of Official Analytical Chemists (AOAC),
International, 16th edition4, or by any method which is validated by a
scientific body in collaborative trials against the three tube Most Probable
Number (MPN) method and agreeing with the 95% upper and lower confidence limits
of the appropriate MPN index.
T.2.7.1 Suggested Quantitation Schemes:
If a generic 1 ml plating technique is used for
E. coli
quantitation for cattle or swine carcass sponging sample analysis, the plate
count needs to be divided by 12 to equal the count per cm2 of
carcass surface area, according to the following calculations:
Total surface swabbed: | 3 x 100 cm2
= 300 cm2 |
Total quantity (volume) of fluid: | 10 + 15 ml
= 25 ml |
Conversion factor calculation: |
if 25 ml for 300 cm2
then 1 ml for x cm2
and x = 300 ÷ 25 = 12
|
Where a generic 1 ml plating technique is used for
E. coli
quantitation for turkey carcasses, the plate count needs to be divided by 4 to
obtain the count per cm2 (i.e.: 100 ÷ 25 = 4 ).
Similar conversions are not necessary for quantitation of
E. coli/ml of poultry rinse fluid samples.
To cover the marginal and unacceptable range for
E. coli levels
(described in section T.2.8.1), the undiluted sample extract, as well as 1:10,
1:100, 1:1,000 and 1:10,000 dilutions should be plated, preferably in
duplicate. Higher or lower dilutions may need to be plated based on the
specific product.
If a hydrophobic grid membrane filtration method were used, the only
difference is that filtrations shall be performed on 1 ml of the undiluted
sample extract, as well as of 1:10, 1:100, 1:1,000 and 1:10,000 dilutions.
Additional dilutions of the original extract may need to be used if a three
tube Most Probable Number (MPN) protocol is used. The three highest dilutions
that were positive for
E. coli are to be used to calculate the MPN.
For cattle and swine samples collected by swabbing technique, MPN values from
the appropriate MPN Table
represent the count per ml of original extract and therefore needs to be divided
by 12 to obtain the count per cm2 of
carcass surface area (divide by 4 for turkey samples collected by
swabbing.)
Laboratories shall report the exact
E. coli count. Where values are
quantified below 1 cfu/cm2, the exact count should be reported (ex.
0,01 cfu/cm2) - zero (0) shall not be reported as a test result.
Methods used must have a minimum sensitivity of at least 5 cfu/cm2
carcass surface area for cattle and swine.
T.2.8 Analysis of Results and Statistical Process
Controls
The goal of generic E. coli (biotype
I) testing is to assess the
effectiveness of a process through microbiological data and statistical process
control. The objective is to meet the specific Process Verification Criteria
(PVC) developed for the specified combination of process sampled and sample
collection method used at the establishment.
The USDA published PVC values for specific combinations of processes sampled
and sample collection methods. These are found in table T.2.8. If the
establishment is using one of the listed combinations, they must use the PVC
provided in the table. If they are not using one of the listed combinations,
they MUST develop their own specific PVC criteria using Statistical Process
Control (SPC) techniques and cannot use the PVC criteria developed for a
different combination.
Note:
- Establishments testing chickens are obliged to use the carcass rinse
sampling method and the published PVC.
- Beef and hog slaughter establishments are not required to sample carcasses
by excision and the corresponding PVC. The sponging methods may be used.
- There are no PVC published for sampling by sponging sampling method and
turkeys, ducks, geese and guineas carcasses rinse sampling methods.
Establishments using these methods will have to develop their own specific PVC
criteria.
Table T.2.8
Process Verification Criteria for the assessment of
generic E. coli (biotype I) results
Combination process sampled x sampling method
|
Process Verification Criteria (PVC) to use
|
Process sampled
|
Sampling method
|
Size of moving window ("n")
|
Max. # of marginal results ("c")
|
Acceptable results
|
Marginal limit ("m")
|
Unacceptable limit ("M")
|
Cattle, sheep, goats and equine slaughter |
excision |
13
|
3
|
Negative1 |
Positive1 |
cfu/cm2 |
sponge |
No published PVC - the operator must develop their own PVC
using Statistical Process Control (SPC) techniques.. |
Swine slaughter |
excision |
13
|
3
|
10 cfu/cm1 or less |
10 cfu/cm1 |
cfu/cm2 |
sponge |
No published PVC - the operator must develop their own PVC
using SPC techniques. |
Chicken, ducks, guineas and squabs slaughter |
carcass2 rinse |
13
|
3
|
100 cfu/ml or less |
100 cfu/ml |
1,000 cfu/ml |
sponge |
Not applicable - the operator must use the carcass rinse
method. |
Turkey and geese slaughter |
carcass rinse |
No published PVC - the operator must develop their own PVC using SPC techniques. |
sponge |
No published PVC - the operator must develop their own PVC using SPC techniques. |
Ratite slaughter |
carcass rinse |
Not applicable - the operator must use the sponge method. |
sponge |
No published PVC
- the operator must develop their own PVC
using SPC techniques. |
Notes 1: The test used for analyzing samples must have a minimum sensitivity
of 5 cfu/cm2.
2: PVC applicable only to chicken
|
Statistical Process Control techniques available:
The use of statistical process control (SPC) techniques is based on the
following principles :
- every product is manufactured through a process
- every process is subject to variation
- every process can be understood, described and/or measured in mathematical
or statistical terms
- the ongoing performance of the process can be assessed if regular
measurements are made and improvements to the process can be made until an
optimum level is reached.
According toSPC principles, a process is said to be "in control"
when it is stable and performs in an acceptable manner i.e. test results range
close to average and are within predicted limits. SPC techniques allow the
detection of process variations which would not have been identified otherwise.
Through early detection of unexpected variations, deficiencies can be readily
detected and corrected.
The development of SPC programs involve an initial collection of data to
determine baseline parameters characterizing the process of interest (also
called baseline data; baseline level; process control level). These parameters
describe what the typical or "normal" process looks like. Using a
statistical method, the baseline data is used to develop Process Verification
Criteria (PVC). Many different statistical approaches can be used for SPC (for
example, the moving window approach, Shewhart Charts, trends analysis, standard
deviations, time plots, CUSUM, control charts, etc.) Once the SPC approach has
been selected and the PVC developed, regular monitoring is performed and
results checked against the PVC to verify if the process is operating within
"normal" limits. If not, this indicates that the process is not
"in control" and it is time to find and correct the possible causes
for the abnormal results.
This section only discusses the basic principles of the moving window
approach which the USDA used to develop published PVC for assessing generic
E. coli biotype
I in slaughter processes. The reader should refer to
one of the following specialized references for more information on other SPC
methods:
- Ishikawa, K. 1986 - Guide to Quality control. Kraus International
Publications, White Plains, NY
- Kane, V.E. 1989 - Defect Prevention. ASQ Quality Press, Milwaulkee WI
- Kume, H. 1985 - Statistical Mehtods for Quality Improvement. UNIPUB/Kraus
International publications, White Plains, NY
- Surak, J.G. 1999 - SPC for the food processing Industry. Clemson University,
Clemson, SC
- Surak, J.G. 1999 - Integrating HACCP and SPC. Clemson University, Clemson,
SC.
Note: Many of these documents were developed for industrial applications
other than food and examples used may not lend themselves to food
production.
T.2.8.1.2 The moving window approach used by the USDA
The USDA used a statistical process control technique referred to as the
"moving window approach" when they developed the PVC listed in table
T.2.8. The following paragraphs describe in laymans terms how the
"moving window approach" used by the USDA was designed and how it can
be used to assess
E. coli testing results.
Establishment of "M", "m", "n" and
"c"
- Data is collected in order to obtain a baseline for the process.
- The data is ranked from the lowest result to the highest and converted into
percentiles i.e. divided into 100 classes with equal size (containing an equal
number of units/individuals), for example,
the 1st percentile = 1st
class of results
the 50th percentile = 50th class of results (within
that class, the median is the value for which 50% of the results are smaller or
larger)
the 100th percentile = 100th class of results
- The size of the moving window ("n" value") is established and
the control limits are defined. The moving window approach used by the USDA
sets the size of the moving window at "n"= 13 and uses two control
limits, an Absolute Control Limit ("M" value) and a Lower Control
Limit ("m" value).
- The Absolute control limit is the maximum value beyond which any single
result means that the process is no longer "in control" and
corrective action is to take place. The moving window approach used by the USDA
sets the "M" value at the "M"=98th percentile of
results.
- The Lower control limit ("m" value) is an intermediate value
somewhere between the average and "M". Test results which are between
"m" and "M" are normal and the process is still "in
control" when this happens. However, if the number of these test results
exceeds a pre-determined number ("c" value), this trend indicates
that the process is no longer "in control". Different combinations of
"m" and "c" values may be used when evaluating a process.
The selection of the combination to use is based on statistical factors (for
ex., confidence level sought, size of population sampled, prevalence of defect,
etc.) and consultation with a statistician usually takes place when the
sampling plan is designed. The moving window approach used by the USDA sets
these values at "c"= 3 and "m"= 80th percentile
of results.
Use of the moving window after "m" and "M"
values have been identified:
- The operator conducts testing as per requirements.
- Results are recorded as they become available either in a log book or on a
control diagram.
- The results are analyzed on an ongoing basis, i.e. each time a new result is
received and recorded.
- When analyzing the results, only the results which appear in the window
(i.e., last "n" number of results, in this case the last 13 results)
are used to assess the process.
- If the "M" value is exceeded at any time within the window (last
13 results), the process is said to be not "in control". Immediate
corrective actions are required as soon as the "M" value is
exceeded.
- If the "m" value is exceeded more than 3 times ("c"
value) within the window (last 13 results), the process is said to be not
"in control". Immediate corrective actions are required as soon as
the "m" value is exceeded more than 3 times..
- Each time a new result is received, the window moves forward by 1 result and
the results are analyzed again on the basis of the 13 most recent results. This
is why the method is called the "moving window approach".
- Periodically, as the process improves, the 80th and
98th percentile can be recalculated and new "m" and
"M" values used.
Example:
The following example corresponds to records of results for a chicken
slaughter plant required to do two tests per day (using a carcass wash
technique). As per table T.2.8 the "M" and "m" values for
E. coli (biotype
I) are set to 1000 and 100 cfu/ml respectively, c to
3 times and n to 13 results.
Test #
|
Date
|
Time collected
|
Test result (cfu/ml)
|
Is the result above the "unacceptable" limit?
> ("M")
|
Is the result above the "marginal" limit?
> ("m")
|
# of results exceeding the "marginal" limit in last
13 tests
|
Is the process "under control"?
(# of results > "m") # 3
AND no result >"M"
|
1
|
10-07(Mon) |
08:50
|
200
|
No
|
Yes
|
1
|
Yes
|
2
|
............ |
14:00
|
50
|
No
|
No
|
1
|
Yes
|
3
|
10-08 (Tue) |
07:10
|
500
|
No
|
Yes
|
2
|
Yes
|
4
|
............ |
13:00
|
5
|
No
|
No
|
2
|
Yes
|
5
|
10-09(Wed) |
10:00
|
80
|
No
|
No
|
2
|
Yes
|
6
|
............ |
12:20
|
50
|
No
|
No
|
2
|
Yes
|
7
|
10-10 (Thu) |
09:20
|
800
|
No
|
Yes
|
3
|
Yes
|
8
|
............ |
13:30
|
50
|
No
|
No
|
3
|
Yes
|
9
|
10-11 (Fri) |
10:50
|
80
|
No
|
No
|
3
|
Yes
|
10
|
............ |
14:50
|
50
|
No
|
No
|
3
|
Yes
|
11
|
10-14(Mon) |
08:40
|
500
|
No
|
Yes
|
4
|
No
|
12
|
............ |
12:00
|
80
|
No
|
No
|
4
|
No
|
13
|
10-15 (Tue) |
09:30
|
80
|
No
|
No
|
4
|
No
|
14
|
............ |
15:20
|
50
|
No
|
No
|
3
|
Yes
|
15
|
10-16(Wed) |
07:30
|
80
|
No
|
No
|
3
|
Yes
|
16
|
............ |
11:40
|
5
|
No
|
No
|
2
|
Yes
|
17
|
10-17 (Thu) |
10:20
|
80
|
No
|
No
|
2
|
Yes
|
18
|
........... |
14.45
|
80
|
No
|
No
|
2
|
Yes
|
19
|
10-18 (Fri) |
08:30
|
50
|
No
|
No
|
2
|
Yes
|
20
|
........... |
16:00
|
80
|
No
|
No
|
2
|
Yes
|
21
|
10-19 (Sat) |
09:10
|
50
|
No
|
No
|
1
|
Yes
|
22
|
.......... |
13:00
|
1500
|
Yes
|
Yes
|
2
|
No
|
23
|
10-21(MMonon) |
10:30
|
80
|
No
|
No
|
2
|
Yes
|
The following observations can be made upon studying this example:
- As of 10-14 at 08:40, there are four results in the last 11 which exceed
the "marginal" limit ("m"); this exceeds the maximum of
tests above "m" permitted ("c" = 3) within the window
("n" = 13). The process can no longer be said to be "in
control".
- The limit of 3 results above "m" in 13 consecutive samples
stays exceeded for the next two tests. The process is still considered not to
be "in control", but since no new result above the
"marginal" or "unacceptable" limits have occurred, the
conclusion should not be considered as evidence of a new problem. The log or
documentation of corrective action taken following the initial loss of control
will indicate if action was taken to address the identified deviation.
- On 10-15 at 15:20 the number of results above the "marginal"
limit (>"m") in the last 13 tests goes down to 3 because the
original result above "m" (10-07 at 08:50) is no longer in the window
after it moves ahead by one. The process can again be said to be "in
control".
- The result for 10-19 at 13:00 exceeds the "unacceptable" limit
(result > "M"). Even if the number of results above the marginal
limit ("m") is less than the maximum allowed
("c" = 3), the process is still considered to no longer be
"in control". Corrective action must be undertaken and logged by the
operator because of this unacceptable result.
- The result for 10-21 at 10:30 is below the "marginal" limit
(<"m"). The total of marginal results is 2. The process is said to
be "in control".
Figure T.5 shows the same results as the above example but the results are
displayed in chart form. The numbers along the horizontal axis of the graph
(x-axis), refers to the test number in the chart above. The information for
each test result, such as the time and date the sample was collected could also
be recorded on the chart.
T.2.8.2 Establishments with either a process and/or E. coli
sampling method which does not correspond to published
USDA Process
Verification Criteria listed in table T.2.8
An establishment which does generic E. coli (biotype I) testing for
a process and which is and/or uses a sampling method for which there is as yet
no USDA published Process Verification Criteria (PVC) (see table T.2.8), the
establishment cannot use the published PVC and must develop
establishment-specific PVC using SPC techniques (see Section
T.2.8).
There is no requirement to use the moving window approach that was used by
USDA to develop the published PVC. The moving window approach as described in
section T.2.8.1 may be used, provided that the establishment use its own
historical data to develop a baseline and establish PVC. Percentile values can
be easily determined through computer spreadsheets, use of calculators with
statistical functions and even manually. The establishment must make available
to the CFIA upon request the raw data used to calculate their
"m"=80th percentile and "M"=98th percentile values. Because
sponging methods are less sensitive than excision, the establishments
"m" and "M" values should not be higher than those
published for the same process with the excision method.
In many cases it may not be appropriate for the establishment to use the
moving window approach with m=80th percentile M=98th percentile. An example of
this is when an establishment samples hog carcasses using a swabbing collection
method and obtains results consisting mostly of values equal or close to zero.
If the establishment used the same moving window approach as USDA, their
"m" value could be near the detection level for the test and the
"M" value very low (e.g., 10 cfu/cm2).
Unless there are published "m" and "M" values for the
combined process x sampling technique, the operator can choose to use a
different SPC(for ex., trend analysis approach, 3 standard deviations, etc.).
In such cases, the operator needs to make available to the CFIA information
regarding how they developed their PVC. This information should include details
information regarding the SPC method which was used (with appropriate
references where possible), the baseline data used to define the PVC, etc.
Provided the approach used is reasonable, the PVC developed will be
acceptable.
T.2.9 Corrective Actions when the Process is not "in
control"
It is expected that the establishment will occasionally find that their
process is not "in control". This situation does not correspond to an
inspection finding of deficiency or a failure to meet generic E. coli (biotype I) testing requirements. It does however mean that something in
the establishments process has caused a variance of test results from
normal. The establishment must look for the cause of the variance and implement
any required corrective measures. These actions (operators investigation
and measures taken) must be documented in writing. If the measures are
effective, subsequent test results should indicate that the process is again
"in control"
Situations where test results indicate repeatedly that the process is not
"in control" are not usual. This situation indicates that the
operator is not successfully identifying and correcting the cause(s) of the
variance and is not successfully bringing the process back "in
control". However, because PVC are not performance standards, this
situation in itself does not warrant the same type of CFIA enforcement response
as would repeated failure to meet the Salmonella Performance
Standards.
The CFIA I/VIC conducts an investigation at the establishment to verify that
the generic E. coli (biotype I) testing is being done according to
requirements and that test results are being assessed properly. They also
review the corrective measures taken by the operator each time that the process
was found to be not "in control". The I/VIC tries to determine what
the establishment is doing when the PVC values are exceeded and if this is
acceptable. For example:
- If the establishment is consistently exceeding the PVC values but does
nothing, this is unacceptable.
- If the establishment is taking action, but the action is always the same
and the problem keeps repeating itself, this is unacceptable.
- If the establishment is making a deliberate effort to find the cause
behind the higher than normal results, for ex., is trying different things, but
these measures havent worked, this is conditionally acceptable, i.e., the
establishment must continue to work towards a solution which corrects the
situation in a definitive manner.
Part 5 of the Basic Compliance Checklist should be completed by the I/VIC at
this time. If there is a "No" answer to any question on the
checklist, the I/VICis to contact the Program Network Director (PND). In cases
where testing requirements are not being met, the procedure outlined in section
Q.3 applies.
In cases where the testing requirements are being met but there is a chronic
inability to meet PVC, the procedure outlined in section Q.3. DOES NOT apply,
but the PND may consider other measures (for ex., Salmonella testing
by the operator, HACCP review of the process by CFIA.)
T.2.10 Record Keeping:
Results of each test result must by recorded in terms of colony forming
units per square centimetre
(cfu/cm2) for sponging or excision
methods, and in terms of colony forming units per ml rinse fluid (cfu/ml) for
whole bird carcass rinse methods. Results must be reported to the minimum
sensitivity of the test used. Zero shall not be reported as a test result. A
process control table or chart can be used to record the results and facilitate
evaluation.
Results should be recorded as they are collected and need to be logged in
the order of sample collection. Records should also include information useful
for determining appropriate corrective actions when problems occur. The
information needed for each sample must include date and time of sample
collection, and, if more than one slaughter line exists, the slaughter line
from which the sample was collected.
E. coli testing records are to be maintained at the establishment
for twelve months and must be made available to Inspection
staff on request.
T.2.11 CFIA VERIFICATION ACTIVITIES
CFIA inspectors will verify that establishments meet E. coli
testing requirements through The BCC and Multi-Commodity Activity Program
(MCAP) verification.
If the VIC discovers that the establishment is failing to perform required
testing and/or to record test results, the provisions outlined in Annex Q,
section Q.3 apply.
In principle the FSIS requirements for
E. coli testing are that the operator
must develop testing procedures, conduct routine testing of carcasses in
slaughter operations and record results as provided in this annex. The operator
is also responsible to develop and implement an action plan at the satisfaction
of the veterinarian-in-charge (VIC) upon receiving results exceeding the
established process verification criteria.
If the test results indicate a possible loss of control over the process,
the VIC verifies that the establishment has taken or is taking the required
actions (see T.2.9). The VIC also ensures that dressing procedures remain in
compliance with program requirements and, if required, take further action as
appropriate (ex: increased verification at boning room carcass re-inspection
station or shipping re-inspection station).
The Process Verification Criteria are not standards, enforcement activities
can not be initiated because of failure to meet Process Verification Criteria
only. Whenever the VIC finds that there is repeated failure to meet the Process
Verification Criteria and that the actions taken by the operator are not
effective, they shall contact the Program Network Director to advise the
situation and discuss further action. At this point one option would be to
conduct an in-dept inspection of the establishment.
T.2.12 Figures And Illustrations
Figure T.1 SWABBING TEMPLATE FOR CATTLE AND SWINE - EXAMPLE
Figure T.2 SAMPLING LOCATIONS FOR SWABBING- CATTLE, SHEEP, GOATS AND EQUINE
Figure T.3 SAMPLING LOCATIONS FOR SWABBING - SWINE
Figure T.4 SAMPLING LOCATIONS FOR SWABBING - TURKEYS AND RATITES
Figure T.5 PROCESS CONTROL CHART - EXAMPLE
* These drawings and descriptions of sampling sites are available
from CFIA inspectors.
1 Where an establishment’s normal operating week is less than 5
days/week, testing shall begin following the first week that the establishment
operates during all of it’s scheduled operating days. June 1 was set as the
starting date for sampling because the highest number of food borne diseases
occur during the 3 month period from June to September.
2 The selection of carcasses (or half-carcasses) can be carried
out before the carcass enters the cooler. The selected carcass can be tagged and
placed in the coolers with other carcasses and directed to the sample taking
site after 12 hours of chilling has been completed (where conventional chilling
methods are used). Carcasses should not be placed in a special area at the
beginning of the chilling process as this may misrepresent actual carcass
chilling conditions.
3 Where an establishment has been authorized to slaughter, dress
or chill types of livestock or poultry by using non-traditional methods (ex:
hot boning of swine and poultry, chilling of split turkey carcasses) and is not
able to collect samples in the cooler after 12 hours or is unable to sample a
whole bird carcass at the end of the drip line, sample collection may be done
after the final wash. For poultry, where it is otherwise impractical or unsafe
to select the carcass after the chiller, the carcass may be selected at the end
of the evisceration line; (ref. Federal Register,
Vol. 42, No. 220, Nov. 14, 1997)
4 AOAC
has approved the following methods for generic
E. coli
quantitation in foods:
- 3-tube MPN method - AOAC
17.2.01 - 17.2.02;
- Modified 3-tube MPN
method - AOAC
17.3.07 - Substrate Supporting Disc Method (ColiComplete®).
- Modified 3-tube MPN
method - AOAC
17.4.01 - Fluorogenic Assay for Glucuronidase, Lauryl sulfate tryptose broth
with added 4-methylumbelliferyl-β-D-glucuronide (MUG) is used in a 3-tube MPN
method.
- Plating Method - AOAC
17.3.04 - Dry Rehydratable Film (Petrifilm
E. coli Count
Plate) Method (Note: Use in lieu of plating ONLY - Not for use as a carcass
direct contact medium: see AOAC
method for details.)
- Filtration /Plating Method - AOAC
17.3.09 - Hydrophobic Grid Membrane Filter/MUG (ISO-GRID) Method
Annex C |
Annex D |
Annex D-1 |
Annex E |
Annex J (PDF) |
Annex K (PDF) |
Annex L |
Annex M |
Annex Q |
Annex R |
Annex S |
Annex T |
Annex U |
Annex W |
Annex W-1 |
Annex W-1 |
Annex X |
Annex Y |
Annex Z |
Annex Z-1 |
Annex Z-2 |
|