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Depuration of bivalve shellfish

Although the Safe Food for Canadians Regulations (SFCR) came into force on January 15, 2019, certain requirements may apply in 2020 and 2021 based on food commodity, type of activity and business size. For more information, refer to the SFCR timelines.

On this page

Introduction

This document provides information for licensed operators that are conducting depuration as part of a decontamination plan under the Management of Contaminated Fisheries Regulations. More specifically, it describes preventive control measures associated with the process of depuration, and is intended to inform and support the development and implementation of a preventive control plan (PCP).

This document covers historical accepted and proven depuration parameters, as well as other example control measures. It is not intended as an exhaustive list of measures. Always ensure that the control(s) chosen are tailored to the uniqueness of your business and shown to be effective for your situation.

Definitions

Please refer to the Canadian Shellfish Sanitation Program manual for definitions.

Validation of the depuration process

Table 1A End Product Targets for Overall Depuration Establishment Performance Evaluation (Faecal coliforms/100 g)
Shellfish Species Geometric Mean Upper 10% Value Table Note 1
Soft clam
(Mya arenaria)
50 130
Hard clam (Mercenaria mercenaria, Protothaca staminea, Venerupis philippinarum, Nuttallia obscurata) 20 70
Blue Mussel
(Mytilus edulis)
20 70
Oyster (Crassostrea virginica, Crassostrea gigas) 20 70

Table Note

Table Note 1

Upper 10 percent level is where no more than 10 percent of the samples' results used in the evaluation may exceed the value established as the upper 10 percent level for each species.

Return to table note 1  referrer

Transportation

Pre-depuration storage

Pre-depuration storage in the marine environment

Pre-depuration dry storage

Post-depuration storage

Post-depuration storage in tanks

Post-depuration dry storage

Depuration water and facility operations

Deviations from these guidelines are possible if the depuration process validation studies show that the depuration process consistently yields bacteriologically acceptable product.

Depuration water

Depuration facility equipment

Shellfish separation

Handling

Mid depuration cycle cleaning

Post validation routine monitoring and verification

Table 1B End product targets for each lot (Faecal coliforms/100 g)
Number of Samples Shellfish Species Geometric Mean not to Exceed One Sample May Exceed No Sample to Exceed
1 Soft clam No value No value 170
1 Oyster, hard clams, mussel No value No value 100
2 Soft clam 125 No value 170
2 Oyster, hard clams, mussel 75 No value 100
3 Soft clam 110 No value 170
3 Oyster, hard clams, mussel 45 No value 100
5 Soft clam 50 100 170
5 Oyster, hard clams, mussel 20 45 100
10 Soft clam 50 130 170
10 Oyster, hard clams, mussel 20 70 100

Deviations

Laboratories

Independent Expert Assessment of On-site Laboratories

For facilities that engage an independent expert, consider the following.

Independent Expert Qualifications

CFIA will review the information submitted and will advise the facility whether the expert meets the criteria or not.

Assessment

Proficiency Testing

Appendix I

Conflict of Interest Questionnaire – Independent Expert

This questionnaire is intended for use by the licensed depuration facility.

A conflict of interest can be described as any situation in which personal assets, interests or activities affect in any way, or have the potential to appear to affect, the honest, impartial performance of duties or judgement.

  1. Is the independent expert performing the assessment an impartial professional, capable of giving a true and honest assessment? Yesspace No space
  2. Does the independent expert have any potential benefits or personal interests in the outcome of the assessment? Yes space No space
  3. Does the independent expert have any interest in the records or information obtained during the assessment such that a real, perceived or apparent conflict of interest with the assessment may exist or arise? Yes space No space
  4. Has the independent expert agreed to disclose to the facility all personal or professional activities that could place the assessor in a position of a real, perceived or apparent conflict of interest? Yes space No space

Signed:

Facility Representative
Date

Appendix II

Checklist for the Assessment of Laboratories in Depuration Facilities

This checklist is intended for use by the independent expert engaged to evaluate a laboratory conducting testing to monitor the processes in depuration facilities.

Name of Independent Assessor:

Address:

Phone:
Fax:

E-mail:

Depuration facility laboratory:

Address:

Phone:
Fax:

E-mail:

Date of Laboratory Evaluation:

Laboratory Represented by (list names)

Title (list titles or function)

Acceptable Methods - Check all that are in use in the laboratory
9222 B Standard Total Coliform Membrane Filter Procedure, Standard Methods for the Examination of Water and Wastewater, 20th ed, American Public Health Association.
Multiple Tube Fermentation Technique for Seawater (APHA Recommended Procedures for the Examination of Sea Water and Shellfish, Part III, A)
Multiple Tube Fermentation Technique for Seawater using MA-1 (AOAC Official Method 978.23 Fecal Coliforms in Shellfish-Growing Waters)
Multiple Tube Fermentation Technique for Shellfish Meats (APHA Recommended Procedures for the Examination of Sea Water and Shellfish, Part III, B)
Standard Plate Counts for Shellfish Meats (APHA Recommended Procedures for the Examination of Sea Water and Shellfish, Part III, B)
Elevated Temperature Coliform Plate Method for Shellfish Meats
MFHPB-19, Enumeration of Coliforms, Faecal coliforms and of E. coli in foods using the MPN method (Health Canada's Health Protection Branch Method, Compendium of Analytical Methods, Volume 2)

Complete the checklist by indicating Yes (Y), No (N) or Not Applicable (NA) in the appropriate column after observing each listed requirement or the records for the requirement. Provide a description of any observed non-conformances with as much detail as possible (include equipment ID numbers, media lots numbers, sample IDs, dates etc.). If necessary use a separate page to record the information and attach it to the checklist.

1- Written plan incorporated into the facility's preventive control plan including
CSSP Requirement Conforms
(Y, N or NA)
Description of the non-conformance, corrective action required and/or Comments
1.1 organization of the laboratory
1.2 staff training requirements/records
1.3 standard operating procedures/ methods
1.4 internal quality control procedures for equipment calibration, maintenance, repair, performance checks
1.5 laboratory safety Table Note 2
1.6 Included in facility's internal audit or assessment plan. All laboratory activities covered. Records of any findings and corrective actions maintained.
1.7 Participation in approved PT program annually. Where participation in proficiency testing is not possible, the laboratory is monitoring the validity of the test by quantifying a certified reference material and comparing the obtained result to the certified value.

Table Note

Table Note 2

Assessing compliance with specific safety requirements or standards is outside the scope of this assessment.

Return to table note 2  referrer

2- Work area
CSSP Requirement Conforms
(Y, N or NA)
Description of the non-conformance, corrective action required and/or Comments
2.1 Adequate for workload and storage.
2.2 Clean, well-lighted.
2.3 Adequate temperature control.
2.4 Work surfaces are non-porous, easily cleaned and disinfected.
2.5 air quality < 15 colonies/plate in 15 min
3- Equipment
CSSP Requirement Conforms
(Y, N or NA)
Description of the non-conformance, corrective action required and/or Comments
3.1 pH meter has a standard accuracy of 0.1 pH unit
3.2 pH electrode consisting of pH half-cell and reference half-cell or equivalent (free from Ag/AgClor with an ion exchange barrier preventing the passage of Ag ions into the medium which may affect the accuracy of the pH reading)
3.3 automatic temperature compensation (ATC) probe or manual temperature adjustment
3.4 pH meter calibrated daily or as-used and records maintained
3.5 pH meter calibration requirements- minimum 2 standard buffers, pH 7 and one near the pH of the expected sample or material being measured (pH4, pH 10) Stock standard buffer protected from contamination.
3.6 Electrode effectiveness determined
3.7 Balance provides a sensitivity of at least 0.1 g at a load of 150 g
3.8 Balance verified monthly using NIST Class S or ASTM Class 1 or 2 weights
3.9 Refrigerator temperatures monitored at least daily and recorded.
3.10 Refrigerator temperature maintained between 2°C to 4°C
3.11 Incubator is 35°C +/- 0.5°C.
3.12 Thermometers are graduated at no greater than 0.5 degree increments.
3.13 Sufficient number of working thermometers located throughout incubators.
3.14 Waterbath is at 44.5°C+/- 0.2.
3.15 Thermometers in waterbaths are graduated in 0.1 degree increments
3.16 Waterbath has adequate capacity
3.17 Level in waterbath covers level of liquid in incubating tubes
3.18 Air incubator/waterbath temperatures are taken daily and recorded
3.19 Working thermometers tagged with identification, date of calibration, calibrated temperature and correction factor
3.20 All working thermometers are appropriately immersed
3.21 A standard thermometer has been calibrated by NIST or one of equivalent accuracy at 0, 35, and 44.5 degrees (45.5 for Elevated Temperature Coliform Plate (ETCP)); calibration records maintained
3.22 Standard thermometer is checked annually for accuracy by ice point determination; results recorded and maintained
3.23 Incubator and waterbath working thermometers are checked annually against the standard thermometer at the temperatures at which they're used; records maintained
4- Labware and Glassware Washing
CSSP Requirement Conforms
(Y, N or NA)
Description of the non-conformance, corrective action required and/or Comments
4.1 Utensils and containers are clean borosilicate glass, stainless steel or other non-corroding material
4.2 Culture tubes are of a suitable size to accommodate the volume of ingredients and samples
4.3 Sample containers are made of glass or some inert material (polypropylene)
4.4 Dilution bottles and tubes are made of borosilicate glass or plastic and closed with rubber stoppers, caps or screw caps
4.5 Graduations are indelibly marked on dilution bottles or an acceptable alternative is used to ensure appropriate volumes
4.6 Pipettes are used to inoculate the sample delivering accurate aliquots, have unbroken tips, are appropriately graduated. Pipettes to deliver 1 mL are not larger than 10 mL. Pipettes to deliver 0.1 mL are not larger than 1 mL.
4.7 Reusable sample containers are capable of being properly washed and sterilized
4.8 In washing reusable pipettes, a succession of at least 3 fresh water rinses plus a final rinse of distilled/deionized water
4.9 In washing reusable sample containers, glassware and plasticware, the effectiveness of the rinsing procedure is established annually or when detergent (brand or lot) is changed by the Inhibitory Residue Test as described in the Standard methods for the Examination of Water and Wastewater. Records are kept.
4.10 Once during each day of washing several pieces of glassware from one batch are tested for residual acid or alkali using a 0.4% aqueous solution of bromothymol blue
5- Sterilization and Decontamination
CSSP Requirement Conforms
(Y, N or NA)
Description of the non-conformance, corrective action required and/or Comments
5.1 Autoclave(s) are of sufficient size to
accommodate workload
5.2 Routine autoclave maintenance is carried out and records maintained.
5.3 Autoclave(s) and/or steam generators serviced annually or as needed, and records maintained
5.4 Autoclave(s) provides a sterilizing temperature as determined weekly using a calibrated working maximum registering thermometer or equivalent
5.5 An autoclave standard thermometer has been calibrated by NIST or its equivalent at 121°C
5.6 Working autoclave thermometers are checked against the autoclave standards thermometer at 121°C yearly
5.7 Spore suspensions used monthly, and results recorded
5.8 Heat sensitive tape used with each autoclave batch
5.9 Autoclave sterilization records including length of sterilization, time and pressure
5.10 For dry heat sterilized materials, the hot air sterilizing oven provides heating and sterilizing temperature in the range 160° to 180°C
5.11 A thermometer capable of determining temperatures accurately to monitor operation of hot-air sterilizing oven
5.12 Records of temperatures and exposure times are maintained for hot-air oven
5.13 Spore strips used quarterly to evaluate effectiveness of sterilization process in hot air oven
5.14 Reusable sample containers are sterilized for 60 minutes at 170°C in hot air oven or 15 minutes at 121°C in autoclave
5.15 Sterility of reusable sample containers is determined for each batch/lot
5.16 Reusable pipettes are stored and sterilized in aluminum or stainless steel canisters; an equivalent method is acceptable.
5.17 Reusable pipettes (in canisters) are sterilized in a hot-air oven at 170°C for 2 h
5.18 Sterility of reusable pipettes is determined with each batch/lot. Results recorded.
5.19 Hardwood applicator transfer sticks are properly sterilized.
5.20 Spent broth cultures and agar plates are decontaminated by autoclaving for at least 30 min before conventional disposal
6- Media Preparation
CSSP Requirement Conforms
(Y, N or NA)
Description of the non-conformance, corrective action required and/or Comments
6.1 Commercially available media used except for Medium A-1 and modified MacConkey
6.2 Dehydrated media and components are properly stored in a cool, clean, dry place.
6.3 Dehydrated media are labelled with date of receipt and date opened.
6.4 Make-up water is distilled or deionized and exceeds 0.5 megohm resistance or <2  micro-siemens (µS)conductivity at 25°C to be tested and recorded monthly for resistance or conductivity
6.5 Make-up water is analyzed for residual chlorine monthly and is at a non-detectable level (<0.1 mg/L) Records are maintained.
6.6 Make-up water is free from trace dissolved metals as determined annually.
6.7 Make-up water contains <1000 cfu/ml Heterotrophic Plate Count (HPC) as determined monthly
6.8 Media sterilized according to manufacturer's instructions
6.9 Volume and concentration of media in tubes are suitable for the amount of sample inoculated
6.10 Total time of exposure of sugar broths to autoclave temperatures does not exceed 45 min
6.11 Media sterility, and positive and negative controls are run with each lot of commercially prepared media or run with each batch of media prepared from its components; results recorded
6.12 Sterile phosphate buffered dilution water or 0.5% peptone water is used as a sample diluent
6.13 pH of media is determined after sterilization
6.14 Stored media is labelled with expiration date or sterilization date.
6.15 All prepared culture media is stored in accordance with manufacturer's recommendations.
6.16 All culture media is used within the expiration date recommended by the manufacturer.
6.17 Culture tubes containing precipitate or Durham tubes containing air bubbles are discarded.
7- Control Cultures
CSSP Requirement Conforms
(Y, N or NA)
Description of the non-conformance, corrective action required and/or Comments
7.1 All required control organisms available and properly stored and documented
7.2 Organisms obtained from a suitable source such as ATCC or source able to provide documented characterization
Identify Source:
7.3 Procedure for storing and maintaining control cultures technically valid so suitable controls are always available
7.4 All working cultures labeled with name and date of subculture
8- Collection and Transportation of Samples
CSSP Requirement Conforms
(Y, N or NA)
Description of the non-conformance, corrective action required and/or Comments
8.1 Containers are of a suitable size to contain at least 100 mL and allow head space for shaking. Seawater samples are collected in clean, sterile, water tight, properly labelled sample containers.
8.2 Sample identified with collectors name, harvest area, time and date of collection if appropriate
8.3 After collection, seawater samples shall be immediately placed in a cooler which is maintained between 0-10°C degrees
8.4 A temperature blank is used to determine the temperature of samples upon receipt at the lab, and results recorded.
8.5 Examination of the sample is initiated as soon as possible after collection, preferably within 8 hours. However, seawater samples are not to be tested if they are held beyond 30 hours of collection, regardless of refrigeration.
9- 9222 B Standard Total Coliform Membrane Filter Procedure
(Standard Methods for the Examination of Water and Wasterwater, 20th ed, Americal Public Health Association.)
CSSP Requirement Conforms
(Y, N or NA)
Description of the non-conformance, corrective action required and/or Comments
9.1 Dehydrated Lawrence Experimental Station (LES) Endo agar or M-Endo medium is used. Manufacturer's directions for rehydration are followed.
9.2 Sample size of 100 mL is analyzed.
9.3 Sterile filtration units at the beginning of each filtration series are used. Filtration units are sterilized in accordance with the manufacturer's instructions.
9.4 Sterile forceps are used to place sterile membrane filter over receptacle.
9.5 Sample is filter under partial vacuum. Interior surface of the funnel is rinsed with sterile dilution water prior to processing samples.
9.6 For agar based medium, the membrane filter is placed directly on agar, dish inverted, and incubated for 22-24 hours at 35 +/- 0.5°C.
9.7 For liquid medium, a pad is placed in the dish and saturated with at least 2 mL of M-Endo and decanted. The filter is placed on the pad, dish inverted and incubated for 22-24 hours at 35 +/- 0.5°C.
9.8 Positive and negative controls accompany samples throughout the procedure. Records are maintained.
Positive Control:
Negative Control:
9.9 For counting, a low power binocular wide field dissecting microscope or other suitable optical device with cool white fluorescent light source is used to provide optimal viewing of sheen.
9.10 Coliform verification: Monthly verification of at least 10 sheen colonies and a representative number of atypical colonies using the lactose fermentation test or alternative coliform verification tests (using test reactions for cytochrome oxidase and B-galactosidase) is conducted.
9.11 Results are reported as total coliforms per 100 mL. Where no coliform colonies are observed, report as < 1 coliform/100 mL. For verified counts, the initial count is adjusted based on the positive verification percentage. Results are reported as verified coliform count/100 mL.
10- Multiple Tube Fermentation Technique for Seawater, Presumptive Test
(APHA Recommended Procedures for the Examination of Sea Water and Shellfish, Part III, A)
CSSP Requirement Conforms
(Y, N or NA)
Description of the non-conformance, corrective action required and/or Comments
10.1 Lactose broth or lauryl tryptose broth used as the presumptive media (circle the appropriate one)
10.2 Sample and dilutions mixed vigorously (25 times in a 30 cm arc in 7 sec) before inoculation
10.3 In a multiple dilution series 5 tubes per dilution are used
10.4 For depuration, a single dilution series between 5 and 12 tubes may be used
10.5 In a single dilution series, the volumes examined are adequate to meet the needs of routine monitoring. Sample volume inoculated:
Range of MPN:
Strength of media used:
10.6 Inoculated tubes incubated at 35°C ± 0.5°C for up to 48 ± 3 hours
10.7 Positive and negative controls accompany samples throughout the procedure. Records are maintained.
Positive Control:
Negative Control:
10.8 Inoculated media are read after 24 ± 2 hours and at 48 ± 3 hours and transferred at both intervals if positive for gas.
11- Multiple Tube Fermentation Technique for Seawater, Confirmed Test
(APHA Recommended Procedures for the Examination of Sea Water and Shellfish, Part III, A)
CSSP Requirement Conforms
(Y, N or NA)
Description of the non-conformance, corrective action required and/or Comments
11.1 Brilliant green bile 2% broth (BGB) is used as the confirmatory medium for total coliforms.
11.2 EC medium is used as the confirmatory medium for fecal coliforms.
11.3 Transfers are made to BGB/EC either by sterile loop or sterile hardwood applicator stick from presumptive positives incubated for 24 and 48 hours. (Circle the method of transfer)
11.4 When inoculation is performed using the same loop or applicator stick the order of inoculation is EC first followed by BGB.
11.5 BGB tubes are incubated at 35 ± 0.5°C.
11.6 BGB tubes are read after 48 ± 3 hours.
11.7 EC tubes are incubated in a circulating waterbath at 44.5 ± 0.2°C for 24 ± 2 hours.
11.8 The presence of any amount of gas or effervescence in a culture tube constitutes a positive test
11- Computation of results
CSSP Requirement Conforms
(Y, N or NA)
Description of the non-conformance, corrective action required and/or Comments
11.9 Results of multiple dilution tests are read from tables in Recommended Procedures 4th Edition
11.10 Results from single dilution series are calculated from Hoskins equation or interpolated from figure 1 Public Health report 1621 entitled "Most Probable Numbers for Evaluation of Coli aerogenes Tests by Fermentation tube Method"
11.11 Results are reported as MPN/100 mL of sample
12- Multiple Tube Fermentation Technique for Seawater using MA-1
(AOAC Official Method 978.23 Fecal Coliforms in Shellfish-Growing Waters)
CSSP Requirement Conforms
(Y, N or NA)
Description of the non-conformance, corrective action required and/or Comments
12.1

Medium A-1 is prepared according to the following recipe (preformulated medium is unacceptable):

  • Lactose - 5.0 g
  • Tryptone - 20.0 g
  • NaCl - 5.0 g
  • Salicin - 0.5 g
  • Triton X-100 - 1.0 mL
  • Distilled/deionized Water - 1.0 L

Suspend the above ingredients in 1.0 L of distilled or deionized water. Mix thoroughly then add 1 mL of Triton X-100 and continue mixing until dissolved completely. Double strength media is prepared using the above amounts dissolved in 500 mL of water. Dispense 10 mL aliquots into tubes containing inverted Durham tubes. Autoclave at 121°C for 10 minutes. The pH of the medium should be 6.9 after sterilization.

12.2 Prepared A-1 media is stored in the dark at room temperature and used within 7 days.
12.3 Sample and dilutions mixed vigorously (25 times in a 30 cm arc in 7 sec) before inoculation
12.4 In a multiple dilution series 5 tubes per dilution are used
12.5 For depuration, a single dilution series between 5 and 12 tubes may be used
12.6 In a single dilution series, the volumes examined are adequate to meet the needs of routine monitoring. Sample volume inoculated:
Range of MPN:
Strength of media used:
12.7 Positive and negative controls accompany samples throughout the procedure. Records are maintained.
Positive Control:
Negative Control:
12.8 Inoculated media are incubated at 35°C ± 0.5°C for 3 ± 0.5 hours of resuscitation.
12.9 After 3 ± 0.5 hours resuscitation at 35°C, inoculated media are incubated at 44.5 ± 0.2°C in a circulating water bath for the remainder of the 24 ± 2 hours.
12.10 The presence of any amount of growth, gas or effervescence in a culture tube constitutes a positive test
12- Computation of results
CSSP Requirement Conforms
(Y, N or NA)
Description of the non-conformance, corrective action required and/or Comments
12.10 Results of multiple dilution tests are read from tables in Recommended Procedures 4th Edition
12.11 Results from single dilution series are calculated from Hoskins equation or interpolated from figure 1 Public Health report 1621 entitled "Most Probable Numbers for Evaluation of Coli aerogenes Tests by Fermentation tube Method"
12.13 Results are reported as MPN/100 mL of sample

Shellfish Samples

13- Collection and Transportation of Samples
CSSP Requirement Conforms
(Y, N or NA)
Description of the non-conformance, corrective action required and/or Comments
13.1 A representative sample of shellstock is collected (min 10-12 live animals)
13.2 Shellstock is collected in clean, water- proof, puncture resistant containers
13.3 Shellstock is labeled with collector's name, type of shellstock, the source, the harvest area, date and place (if market sample) of collection
13.4 Shellstock samples are maintained in dry storage between 0 -10°C degrees until examined
13.5 Examination of the sample is initiated as soon as possible after collection. However, shellfish samples are not examined if the time interval between collection and examination exceeds 24 hours
14- Preparation of Shellstock for Examination
CSSP Requirement Conforms
(Y, N or NA)
Description of the non-conformance, corrective action required and/or Comments
14.1 Shucking knives, scrub brushes and blender jars are sterilized (autoclaved) for 15 min prior to use.
14.2 Blades of shucking knives are not corroded
14.3 Prior to scrubbing and rinsing debris off shellstock, the analyst's hands are washed with soap and water
14.4 The faucet used to provide the potable water for rinsing shellstock does not contain an aerator
14.5 Shellstock are scrubbed with a stiff, sterile brush and rinsed under water of drinking water quality.
14.6 Prior to opening the molluscs, the hands or gloves are rinsed with 70% alcohol
14.7 Shellstock are not shucked directly through the hinge
14.8 Contents of the shellstock (liquor and meat) are shucked into a sterile tared blender jar or other sterile container.
14.9 At least 100 g of shellfish meat is used for analysis (a minimum of 10-12 live animals)
14.10 The sample is weighed to the nearest gram and an equal amount by weight of diluent (tempered for ETCP) is added (a 1 in 2 dilution)
14.11 Sterile phosphate buffered dilution water or 0.5% peptone water is used as the diluent.
14.12 Sterile phosphate buffered saline is used as diluent for ETCP procedure
14.13 Samples are blended at high speed for 60 to 120 seconds
14.14 For other than shellstock APHA Recommended Procedures are followed for examination of freshly shucked or frozen shellfish meats.
15- Multiple Tube Fermentation Technique for Shellfish Meats, Presumptive Test
(APHA Recommended Procedures for the Examination of Sea Water and Shellfish, Part III, B)
CSSP Requirement Conforms
(Y, N or NA)
Description of the non-conformance, corrective action required and/or Comments
15.1 Appropriate strength lactose or lauryl tryptose broth is used as presumptive media in the analysis (circle the appropriate broth)
15.2 Immediately after blending (within 2 minutes) the ground sample is diluted and inoculated into tubes of presumptive media.
15.3 Must use a 5 tube MPN
15.4 From the initial 1 in 2 dilution a 1 in 10 dilution is prepared (20 g of 1 in 2 added to 80 g of diluent). From the 1 in 10 a 1 in 100 dilution is prepared (10 g of 1 in 10 added to 90 g diluent). A 5 tube dilution series is inoculated using 10 mL and 1mL from the 1 in 10 dilution and 1 mL from the 1 in 100 dilution
15.5 In a single dilution series the volumes examined are adequate to meet the needs of routine monitoring
Sample volume inoculated:
Range of MPN:
Strength of media used:
15.6 Positive and negative control cultures accompany samples throughout the procedure. Records maintained.
Positive control:
Negative control:
15.7 Inoculated media are incubated 35°C ± 0.5°C
15.8 Presumptive tubes are read at 24 ± 2 hours and transferred if positive.
16a- Multiple Tube Fermentation Technique for Shellfish Meats, Confirmed Test
(APHA Recommended Procedures for the Examination of Sea Water and Shellfish, Part III, B)
CSSP Requirement Conforms
(Y, N or NA)
Description of the non-conformance, corrective action required and/or Comments
16.1 EC medium is used as the confirmatory medium
16.2 Transfers are made to EC medium by either sterile loop or sterile hardwood applicator sticks. (circle the method of transfer)
16.3 EC tubes are incubated in a circulating water bath at 44.5°C ± 0.2°C for 24 h ± 2 hours.
16.4 EC tubes are read for gas production after 24 h ± 2 hours incubation.
16.5 Presence of any amount of gas or effervescence in Durham tubes constitutes a positive test.
16b- Computation of results
CSSP Requirement Conforms
(Y, N or NA)
Description of the non-conformance, corrective action required and/or Comments
16.6 Results of multiple dilution tests are read from tables in Recommended Procedures 4th Ed and multiplied by the appropriate dilution factor
16.7 Results from single dilution series are calculated from Hoskins equation or interpolated from figure 1-Public Health Report 1621 entitled "Most Probable Numbers for Evaluation of Coli aerogenes Tests by Fermentation tube method"
16.8 Results are reported as MPN/100 g of sample
17a- Standard Plate Counts for Shellfish Meats
(APHA Recommended Procedures for the Examination of Sea Water and Shellfish, Part III, B)
CSSP Requirement Conforms
(Y, N or NA)
Description of the non-conformance, corrective action required and/or Comments
17.1 In the standard plate count procedure at least four plates, duplicates of two dilutions are used to provide 30-300 colonies per plate
17.2 15 to 20 mL of tempered sterile plate count agar (PCA) is used
17.3 Agar tempering bath maintains the agar at 44°C to 46°C
17.4 Not more than 1mL nor less than 0.1 mL of the sample or dilution is plated
17.5 Samples or sample dilutions to be plated are mixed vigorously (shaken 25 times in a 30 cm arc in 7 sec) before plating
17.6 Control plates are used to check the sterility of the air, agar and the diluent
17.7 Solidified plates are incubated at 35°C ± 0.5°C for 48 ± 3 hours inverted and stacked no more than 4 high
17.8 Quebec Colony Counter or equivalent to provide necessary magnification and visibility for counting plates.
17.9 A hand tally or equivalent is used for accuracy in counting
17b- Computation of Results
CSSP Requirement Conforms
(Y, N or NA)
Description of the non-conformance, corrective action required and/or Comments
17.10 Colony counts are determined in accordance with Part III, A, Sections 4.31 through 4.33 Recommended Procedures 4th Edition
17.11 Colony Counts reported as ACC/g of sample
18a- Elevated Temperature Coliform Plate Method for Shellfish Meats
CSSP Requirement Conforms
(Y, N or NA)
Description of the non-conformance, corrective action required and/or Comments
18.1 Sample homogenate is cultured within 2 minutes of blending
18.2

Double strength Modified MacConkey Agar (MMA) is prepared according to the following recipe:

Peptone - 34.0 g
Polypeptone - 6.0 g
Lactose - 20.0 g
Bile Salts No. 3 - 1.5 g
Agar - 27.0 g
Neutral Red - 0.06 g
Crystal Violet - 0.02 g
Distilled/deionized Water - 1.0 L
Suspend the above ingredients in 1.0 L of distilled/deionized water. Mix thoroughly. Heat with frequent agitation until boiling. Remove from heat and boil again (do not autoclave). Temper in water bath at 45 - 50°C for up to six hours.

18.3 Hydrated double strength Modified MacConkey Agar is heated to boiling, removed from the heat, and boiled again. This agar is never autoclaved.
18.4 The twice boiled, double strength Modified MacConkey Agar and sterile phosphate buffered saline are maintained in a tempering bath at 45-50°C until used. Prepared MMA is used the day it is made.
18.5 The equivalent of 6 grams of the homogenate is placed into a sterile container and the volume is brought up to 60 mL with tempered sterile phosphate buffered saline.
18.6 60 mL of the tempered MMA is added
18.7 Container is gently swirled or rotated to mix the contents which are then distributed evenly over 6 to 8 petri plates.
18.8 Media and diluent sterility are determined with each use. Results are recorded and records are maintained.
18.9 To determine media productivity, negative and positive control cultures are pour plated in an appropriate concentration to accompany samples throughout the procedure.
Positive control:
Negative control:
18.10 Plates are incubated, inverted stacked no more than 4 high within 3 hours of plating at 45.5°C ± 0.5°C for 18 to 30 h.
18b- Expression of Results
CSSP Requirement Conforms
(Y, N or NA)
Description of the non-conformance, corrective action required and/or Comments
18.11 Quebec Colony Counter or equivalent is used to provide the necessary magnification and visibility
18.12 A hand tally or its equivalent is used to aid in counting
18.13 All brick red colonies greater the 0.5 mm in diameter are totalled over all plates and multiplied by a factor of 16.7 to report results as CFU/100 grams of sample.
19a- MFHPB-19 Enumeration of Coliforms, Faecal Coliforms and of E. coli in Foods using the MPN Method
CSSP Requirement Conforms
(Y, N or NA)
Description of the non-conformance, corrective action required and/or Comments
19.1 For all shellfish, 0.5 % peptone water is used for all dilutions
19.2 Appropriate strength lauryl tryptose broth is used as presumptive media in the analysis
19.3 For shellfish, immediately after blending (within 2 minutes) the ground sample is diluted and tubes are inoculated.
19.4 Must use a 5 tube MPN
19.5 From the initial 1 in 2 dilution a 1 in 10 dilution is prepared (20 g of 1 in 2 added to 80 g of diluent). From the 1 in 10 a 1 in 100 dilution is prepared (10 g of 1 in 10 added to 90 g diluent). A 5 tube dilution series is inoculated using 10 mL and 1mL from the 1 in 10 dilution and 1 mL from the 1 in 100 dilution.
19.6 In a single dilution series the volumes examined are adequate to meet the needs of routine monitoring
Sample volume inoculated:
Range of MPN:
Strength of media used:
19.7 Inoculated media are incubated 35°C ± 0.5°C
19.8 Presumptive tubes are read at 24 ± 2 hours and transferred if positive.
19b- Confirmation steps for Coliforms
CSSP Requirement Conforms
(Y, N or NA)
Description of the non-conformance, corrective action required and/or Comments
19.9 One loopful from each positive Lauryl Sulphate Tryptose (LST) broth tube is transferred to a tube of Brilliant Green Lactose Bile (BGLB) broth.
19.10 BGLB broth tubes are incubated at 35°C for 24 +/- 2 h, examined for gas formation and re-incubated for an additional 24 +/- 2 h.
19.11 Formation of gas during 48 +/- 4 h incubation constitutes a positive confirmed test.
19.12 MPN of confirmed coliforms per 100 g of shellfish is calculated following the instructions in Appendix D of the Health Canada Compendium of Analytical Methods.
19c- Confirmation steps for faecal coliforms
CSSP Requirement Conforms
(Y, N or NA)
Description of the non-conformance, corrective action required and/or Comments
19.13 One loopful of each positive LST tube is transferred to EC broth.
19.14 EC tubes are incubated in a water bath at 44.5°C for 24 +/- 2 h.
19.15 MPN of confirmed faecal coliforms per 100 g of shellfish is calculated following the instructions in Appendix D of the Health Canada Compendium of Analytical Methods.

References:

  1. American Public Health Association (APHA). 1970. Recommended Procedures for the Examination of Sea Water and Shellfish, 4th Edition. APHA, Washington, D.C.
  2. American Public Health Association 1984. Compendium of Methods for the Microbiological Examination of Foods, 2nd Edition. APHA, Washington, D.C.
  3. "Interim Guides for the Depuration of the Northern Quahog, Mercenaria mercenaria." 1968. Northeast Marine Health Sciences Laboratory, North Kingstown, RI.
  4. Association of Official Analytical Chemists (AOAC). 2000. Official Methods of Analyses of the Association of Official Analytical Chemists. 17th Edition, Chapter 17.305, page 22. AOAC, Arlington, VA.
  5. U.S. Public Health Service (PHS). 1947. Public Health Report, Reprint #1621. PHS, Washington, D.C.
  6. American Public Health Association (APHA). 1992. Standard Methods for the Examination of Water and Wastewater, 18th Edition. APHA/AWWA/Water Environment Federation (WEF), Washington, D.C.
  7. Health Canada's Health Protection Branch, MFHPB-19, Enumeration of Coliforms, Faecal coliforms and of E. coli in foods using the MPN method, Compendium of Analytical Methods, Volume 2.

Appendix III

Summary Report Template for the Assessment of On-Site Laboratory

This report template is intended for use by the independent expert.

Laboratory name and location:
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Date of assessment:
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Name of assessor:
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Brief description of the visit -no more than one or two paragraphs

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List of non-conformances with outcome/ resolution, date of acceptance, and basis for acceptance (state the evidence or information provided).

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Final statement or attestation:

I have assessed this laboratory against the requirements of the checklist in Appendix L: Guidelines for On-Site laboratories at Registered Bivalve Shellfish Establishments. All non-conforming items that were identified and listed in this summary have been corrected.

Signature and date

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