Chapter II. Growing Areas

PUBLIC HEALTH SERVICE

U.S. FOOD AND DRUG ADMINISTRATION
SHELLFISH PROGRAM IMPLEMENTATION BRANCH
SHELLFISH SAFETY TEAM
5100 PAINT BRANCH PARKWAY
COLLEGE PARK, MD 20740-3835
TEL. 301-436-2151/2147 FAX 301-436-2672

SHELLFISH LABORATORY EVALUATION CHECKLIST 

LABORATORY:

ADDRESS:

TELEPHONE: FAX: EMAIL:

DATE OF EVALUATION:
 

DATE OF REPORT:

LAST EVALUATION:

LABORATORY REPRESENTED BY:

TITLE:

LABORATORY EVALUATION OFFICER:

SHELLFISH SPECIALIST:

REGION:

OTHER OFFICIALS PRESENT:

TITLE:

Items which do not conform are noted by: 
C- Critical K - Key O - Other NA- Not Applicable Conformity is noted by a "√"
NSSP Form Lab-100 rev. 2001-11-27

Multiple Tube Fermentation Technique for Seawater (APHA)[PART II]

Multiple Tube Fermentation Technique for Seawater USING ma-1 [PART II]

Multiple Tube Fermentation Technique for Shellfish Meats (APHA)[PART III]

Standard Plate Count for Shellfish Meats [Part III]

Elevated Temperature Coliform Plate Method for Shellfish Meats [PART III ]

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a. Organization of the laboratory

b. Staff training requirements

c. Standard operating procedures

d. Internal quality control measures for equipment calibration, maintenance, repair and for performance checks.

e. Laboratory safety

f. Internal performance assessment

g. External performance assessment

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8

2. QA Plan Implemented

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11

3. Participates in a proficiency testing program annually.

Specify Program(s)________________________

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8, 11

1. Adequate for workload and storage.

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2. Clean, well lighted.

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3. Adequate temperature control.

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11

4. All work surfaces are nonporous, easily cleaned and disinfected.

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5. Microbiological quality and density of air is < 15 colonies/plate in a 15 minute exposure determined monthly and results recorded.

O

11

6. Pipette aid used, mouth pipetting not permitted.

O

9

1. To determine the pH of prepared media, the pH meter has a standard accuracy of 0.1 units.

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14

2. pH electrodes, consisting of pH half cell and reference half cell or equivalent combination electrode (free from Ag/AgCl or contains an ion exchange barrier preventing passage of Ag ions into the medium which may effect the accuracy of the pH reading).

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11

3. The effect of temperature on the pH is compensated for by an ATC probe or by manual adjustment.

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8

4. pH meter is calibrated daily or with each use and records are maintained.

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5. A minimum of two standard buffer solutions is used to calibrate the pH meter. The first must be near the electrode isopotential point (pH 7). The second near the expected sample pH (i.e. pH 4 or pH 10). (Standard buffer solutions are used once daily and discarded.

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8, 15

6. Electrode effectiveness is determined daily or with each use.

Method of determination_____________________________________.

K

9

7. Balance provides a sensitivity of at least 0.1 g at a load of 150 g.

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11,13

8. Balance checked monthly using NIST Class S or ASTM Class 1 or 2 weights or equivalent and records are maintained.

K

11

9. Refrigerator temperature(s) monitored at least once daily and recorded.

K

1

10. Refrigerator temperature maintained at 0° to 4° C.

C

9

11. The temperature of the incubator is maintained at 35 ± 0.5°C.

C

11

12. Thermometers used in the air incubator(s) are graduated at no greater than 0.5°C increments.

K

9

13. Working thermometer located on top and bottom shelves of use in the air incubator(s).

C

11

14. Temperature of the waterbath is maintained at 44.5 ± 0.2°C under any loading capacity.

C

9

15. The thermometers used in the waterbath are graduated in 0.1°C increments.

O

13

16. The waterbath has adequate capacity for workload.

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9

17. The level of water in the waterbath covers the level of liquid in the incubating tubes.

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18. Air incubator/waterbath temperatures are taken twice daily and recorded.

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13

19. Working thermometers are tagged with identification, date of calibration, calibrated temperature and correction factor.

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4

20. All working thermometers are appropriately immersed.

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11

21. A standards thermometer has been calibrated by NIST or one of equivalent accuracy at the points 0°, 35° and 44.5°C (45.5°C for ETCP). Calibration records maintained.

K

9

22. Standards thermometer is checked annually for accuracy by ice point determination. Results recorded and maintained.

Date of most recent determination________________________________.

K

13

23. Incubator and waterbath working thermometers are checked annually against the standards thermometer at the temperatures at which they are used. Records maintained.

O

9

1. Utensils and containers are clean borosilicate glass, stainless steel or other noncorroding materials

K

9

2. Culture tubes are of a suitable size to accommodate the volume for nutritive ingredients and samples

K

9

3. Sample containers are made of glass or some other inert material (i.e. polypropylene).

O

9

4. Dilution bottles and tubes are made of borosilicate glass or plastic and closed with rubber stoppers, caps or screw caps with nontoxic liners.

K

9

5. Graduations are indelibly marked on dilution bottles and tubes or an acceptable alternative method is used to ensure appropriate volumes.

K

9

6. Pipettes used to inoculate the sample deliver accurate aliquots, have unbroken tips and are appropriately graduated. Pipettes larger than 10 ml are not used to deliver 1ml; nor, are pipits larger than 1ml used to deliver 0.1ml.

K

9

7. Reusable sample containers are capable of being properly washed and sterilized.

K

9

8. In washing reusable pipits, a succession of at least three fresh water rinses plus a final rinse of distilled/deionized water is used to thoroughly rinse off all the detergent.

C

9

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 current edition of Standard Methods for the Examination of Water and Wastewater. Records are kept.

Date of most recent testing________________________________

Average difference between Groups A and B_________________

Average difference between Groups B and D__________________

Detergent Brand____________________Lot #________________

K

11

10.Once during each day of washing several pieces of glassware (pipettes, sample bottles, etc.) from one batch are tested for residual acid or alkali w/aqueous 0.04% bromthymol blue. Records are maintained.

O

9

1. Autoclave(s) are of sufficient size to accommodate the workload.

O

8

2. Routine autoclave maintenance performed (e.g. pressure relief valves, exhaust trap, chamber drain) and records maintained.

O

8

3. Autoclave(s) and/or steam generators serviced annually or as needed by qualified technician and records maintained.

C

11

4. Autoclave(s) provides a sterilizing temperature of 121°C (tolerance 121 ± 2° C) as determined weekly using a calibrated working maximum registering thermometer or equivalent (thermocouples, platinum resistance thermometers).

K

11

5. An autoclave standards thermometer has been calibrated by the National Institute of

Standards and Technology (NIST) or its equivalent at 121° C.

K

16

6.    The autoclave standards thermometer is checked every five years for accuracy at either 121°C or at the steam point.

Date of most recent determination___________________________

K

1

7.    Working autoclave thermometers are checked against the autoclave standards thermometer at 121° C yearly.

Date of last check ______________ Method _____________________

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11

8.    Spore suspensions are used monthly to evaluate the effectiveness of the autoclave sterilization process. Results recorded.

O

11

9. Heat sensitive tape is used with each autoclave batch.

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11, 13

10.Autoclave sterilization records including length of sterilization, total heat exposure time and chamber temperature are maintained.

Type of record: Autoclave log, computer printout or chart recorder tracings (circle appropriate type or types)

K

11

11.For dry heat sterilized material, the hot-air sterilizing oven provides heating and sterilizing temperature in the range of 160° to 180° C.

K

9

12.A thermometer capable of determining temperatures accurately in the range of 160 to 180°C is used to monitor the operation of the hot-air sterilizing oven when in use.

K

13

13.Records of temperatures and exposure times are maintained for the operation of the hot-air sterilizing oven during use.

K

11

14.Spore strips are used quarterly to evaluate the effectiveness of the sterilization process in the hot-air oven. Records are maintained.

K

11

15.Resuable sample containers are sterilized for 60 minutes at 170° C in a hot-air oven or autoclaved for 15 minutes at 121°C.

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1

16.The sterility of reusable/disposable sample containers is determined for each batch/lot.

K

9

17.Reusable pipettes are stored and sterilized in aluminum or stainless steel canisters or equivalent alternative.

K

9

18. Reusable pipettes (in canisters) are sterilized in a hot-air oven at 170° C for 2 hours.

O

2

19. The sterility of reusable/disposable pipettes is determined with each batch/lot. Results are recorded and maintained.

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18

20. Hardwood applicators transfer sticks are properly sterilized.

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13

21. Spent broth cultures and agar plates are decontaminated by autoclaving for at least 30 minutes before conventional disposal.

C

11

6.       Make-up water is analyzed for residual chlorine monthly and is at a non-detectable level (≤ 0.1 ppm). Records are maintained.

Specify method of determination_______________________________.

K

11

7. Make-up water is free from trace (<0.05mg/L) dissolved metals, specifically Cd, Cr. Cu, Ni, Pb, and Zn as determined annually with total heavy metal content < or equal to 1.0mg/L and records are maintained.

K

11

8. Make-up water contains <1000 CFU/ml as determined monthly using the heterotrophic plate count method and records are maintained.

K

11

9. Media are sterilized according to the manufacturer's instructions.

K

9

10. Volume and concentration of media in the tube are suitable for the amount of sample inoculated.

C

11

11. Total time of exposure of sugar broths to autoclave temperatures does not exceed 45 minutes.

C

1

12. Media sterility and positive and negative controls are run with each lot of commercially prepared media or are run with each batch of media prepared from its components as a check of media productivity. Results recorded and records maintained.

O

9

13. Sterile phosphate buffered dilution water is used as the sample diluent.

K

11

14. pH is determined after sterilization to ensure that it is consistent with manufacturer's requirements and records are maintained.

O

9

1. Prepared culture media are stored in a cool, clean, dry space where excessive evaporation and the danger of contamination are minimized.

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2. Brilliant green bile 2% broth and A-1 media are stored in the dark.

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13

3. Stored media are labeled with expiration date or sterilization date.

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9

4. Storage of prepared culture media at room temperature does not exceed 7 days.

O

2

5. Storage under refrigeration of prepared media with loose fitting closures shall not exceed 1 month.

O

11

6. Storage under refrigeration of prepared media with screw-cap closures does not exceed 3 months.

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17

7. All prepared media stored under refrigeration are held at room temperature overnight prior to use. Culture tubes containing any type of precipitate or Durham tubes containing air bubbles are discarded.

C

11

1. Containers are of suitable size to contain at least 100 ml and to allow headspace for shaking. Seawater samples are collected in clean, sterile, water tight, properly labeled sample containers.

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1

2. Sample identified with collectors name, harvest area, time and date of collection.

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9

3. After collection, seawater samples shall be kept at a temperature between 0 and 10°C until examined.

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1

4. A temperature blank is used to determine the temperature of samples upon receipt at the laboratory. Results are recorded and maintained.

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9

5. Examination of the sample is initiated as soon as possible after collection. However, seawater samples are not tested if they are held beyond 30 hours of refrigeration.

C

9

1. Lactose broth or lauryl tryptose broth is used as the presumptive medium. (circle appropriate one)

C

9

2. Sample and dilutions of sample are mixed vigorously (25 times in a 12" arc in 7 seconds) before inoculation.

C

9

3. In a multiple dilution series not less than 3 tubes per dilution are used (5 tubes are recommended).

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6

4. In a single dilution series not less than 12 tubes are used (for depuration at least 5 tubes are used).

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6

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_________________________

K

9

6. Inoculated media are placed in an air incubator at 35 ± 0.5° C for up to 48 ± 3 hours.

K

2

7. Positive and negative control cultures accompany samples throughout the procedure. Records are maintained.

Positive Control _________________ Negative Control _________________

K

9

8. Inoculated media are read after 24 ± 2 hours and 48 ± 3 hours of incubation and transferred at both intervals if positive for gas.

C

9

1. Brilliant green bile 2% broth (BGB) is used as the confirmatory medium for total coliforms.

C

9

2. EC medium is used as the confirmatory medium for fecal coliforms.

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9, 11

3. Transfers made to BGB/EC by either sterile loop or sterile hardwood applicator stick from positive presumptives incubated for 24 and 48 hours (Circle the method of transfer).

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2

4. When the inoculation of both EC and BGB broths is performed using the same loop or transfer stick, the order of inoculation is EC first, followed by BGB.

C

9

5. BGB tubes are incubated at 35 ± 0.5°C.

K

9

6. BGB tubes are read after 48 ± 3 hours of incubation.

C

9

7. EC tubes are incubated in a circulating waterbath at 44.5 ± 0.2° C for 24 ± 2 hours.

C

9

8. The presence of any amount of gas or effervescence in the culture tube constitutes a positive test.

K

9

1. Results of multiple dilution tests are read from tables in Recommended Procedures, 4^th Edition.

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7

2. 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".

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7, 9

3. Results are reported as MPN/100 ml of sample.

C

5

1. Medium A-1 sterilized for 10 minutes at 121°C.

C

9

2. Sample and dilutions of sample are mixed vigorously (25 times in a 12" arc in 7 seconds) before inoculation.

C

9

3. In a multiple dilution series not less than 3 tubes per dilution are used (5 tubes are recommended).

C

6

4. In a single dilution series at least 12 tubes are used.

K

6

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_________________________

K

2

6. Positive and negative control cultures accompany samples throughout the procedure. Records are maintained.

Positive Control _________________ Negative Control _________________

C

2,5

7. Inoculated media are placed in an air incubator fat 35 ± 0.5°C for 3 ± 0.5 hours of resuscitation.

C

5

8. After 3 ± 0.5 hours resuscitation at 35°C, inoculated media are incubated at 44.5 ± 0.2° C in a circulating waterbath for the remainder of the 24 ± 2 hours.

C

5

9. The presence of any amount of gas or effervescence in the culture tube constitutes a positive test.

K

9

1. Results of multiple dilution tests are read from tables in Recommended Procedures, 4^th Edition.

K

7

2. 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".

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3. Results are reported as MPN/100 ml of sample.

PART III - SHELLFISH SAMPLES

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1. A representative sample of shellstock is collected.

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2. Shellstock is collected in clean, waterproof, puncture resistant containers.

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9

3. Shellstock labeled with collector's name, type of shellstock, the source, the harvest area, time, date and place (if market sample) of collection.

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9

4. Shellstock samples are maintained in dry storage between 0 and 10°C until examined.

C

1

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.

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2,11

1. Shucking knives, scrub brushes and blender jars are (autoclave) sterilized for 15 minutes prior to use.

O

2

2. Blades of shucking knives are not corroded.

O

9

3. Prior to scrubbing and rinsing debris off shellstock, the hands of the analyst are thoroughly washed with soap and water.

O

2

4. The faucet used to provide the potable water for rinsing the shellstock does not contain an aerator.

K

9

5. Shellstock are scrubbed with a stiff, sterile brush and rinsed under water of drinking water quality.

O

9

6. Shellstock are allowed to drain in a clean container or on clean towels prior to opening.

K

9

7. Prior to opening, the hands (or gloved hands) of the analyst are thoroughly washed with soap and water and rinsed in 70% alcohol.

K

9

8. Shellstock are not shucked directly through the hinge.

C

9

9. Contents of shellstock (liquor and meat) are shucked into a sterile, tared blender jar or other sterile container.

K

9

10. At least 200 grams of shellfish meat is used for analysis.

K

2, 19

11. The sample is weighed to the nearest 0.1gram and an equal amount by weight of (tempered for ETCP) diluent is added.

O

9

12. Sterile phosphate buffered dilution water is used as the sample diluent.

K

3

13. Sterile phosphate buffered saline is used as a sample diluent for the ETCP procedure.

C

9

14. Samples are blended at high speed for 60 to 120 seconds.

K

9

15. For other shellstock, APHA Recommended Procedures are followed for the examination of freshly shucked and frozen shellfish meats.

C

9

1. Appropriate strength lactose or lauryl tryptose broth is used as presumptive media in the analysis. (circle appropriate choice)

K

9

2. Immediately (within 2 minutes) after blending, the ground sample is diluted and inoculated into tubes of presumptive media.

C

9

3. No fewer than 5 tubes per dilution are used in a multiple dilution MPN series.

C

9

4. Allowing for the initial 1:1 dilution of the sample, appropriate portions are inoculated (i.e., 2 ml of original 1:1 dilution for the 1 g portion) and diluted for subsequent inoculation (i.e., 22 ml of l:1 diluted sample to 88 ml of diluent or the equivalent for 0.1 g portion.

K

6

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_________________________

C

2

6.       Positive and negative control cultures accompany samples throughout the procedure. Records are maintained.

Positive Control _________________ Negative Control _________________

K

9

7. Inoculated media are incubated at 35 ± 0.5°C.

K

10

8. Presumptive tubes are read at 24 ± 2 hours of incubation and transferred if positive.

C

9

1. EC medium is used as the confirmatory medium.

K

9, 11

2. Transfers are made to EC medium by either sterile loop or hardwood sterile applicator sticks from positive presumptives incubated for 24 hours (circle the method of transfer).

C

9

3. EC tubes are incubated in a circulating waterbath at 44.5 ± 0.2°C for 24 ± 2 hours.

K

9

4. EC tubes are read for gas production after 24 ± 2 hours of incubation.

C

9

5. The presence of any amount of gas or effervescence in the Durham tube constitutes a positive test.

K

9

1. Results of multiple dilution tests are read from tables in Recommended Procedures, 4^th Edition and multiplied by the appropriate dilution factor.

K

7

2. 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".

K

9

3. Results are reported as MPN/100 grams of sample.

O

20

1. A standard plate count analysis is performed in conjunction with the analysis for fecal coliform organisms.

K

9

2. In the standard plant count procedure at least four plates, duplicates of two dilutions are used to provide 30 to 300 colonies per plate.

K

2

3. Fifteen to 20 ml of tempered sterile plate count agar is used.

K

9

4. Agar tempering bath maintains the agar at 44 to 46°C.

O

9

5. Temperature control of the plate count agar is used in the tempering bath.

K

9

6. Not more than 1 ml nor less than 0.l ml of sample or sample dilution is plated.

C

9

7. Samples or sample dilutions to be plated are mixed vigorously (25 times in a 12" arc in 7 seconds) before plating.

K

11

8. Control plates are used to check the sterility of the air, agar and the diluent.

K

9, 21

9. Solidified plates are incubated at 35 ± 0.5°C for 48 ± 3 hours inverted and stacked no more than four high.

K

9

10. Quebec Colony Counter or its equivalent is used to provide the necessary magnification and visibility for counting plates.

K

1

11. A hand tally or its equivalent is used for accuracy in counting.

K

9

1. Colony counts determined in accordance with Part III, A, Sections 4.31 through 4.33 Recommended Procedures, 4^th Edition.

O

19

2. Colony counts reported as APC/g of sample.

K

9

1. Sample homogenate is cultured within 2 minutes of blending.

K

3

2. Double strength Modified MacConkey Agar is used.

C

3

3. Hydrated double strength Modified MacConkey Agar is heated to boiling, removed from the heat, and boiled again. This agar is never autoclaved.

K

2, 3

4. Twice boiled, double strength Modified MacConkey Agar and sterile phosphate buffered saline are maintained in a tempering bath at 45 to 50°C until used. Prepared Modified MacConkey Agar is used on the day it is made.

C

2, 3

5. The equivalent of 6 grams of the homogenate is placed into a sterile container and the contents brought up to 60 ml with tempered, sterile phosphate buffered saline.

K

3

6. Sixty (60) ml of tempered, twice boiled double strength Modified MacConkey Agar is added.

K

2, 3, 22

7. The container is gently swirled or rotated to mix the contents, which are then, distributed uniformly over 6 to 8 petri plates.

C

1

8. Media and diluent sterility are determined with each use. Results are recorded and records maintained.

C

1

9. To determine media productivity, positive and negative control cultures are pour plated in an appropriate concentration to accompany samples throughout the procedure.

Positive control_______________ Negative control _______________

C

3, 13

10. Plates are incubated inverted within 3 hours of plating in air at 45.5 ± 0.5°C for 18 to 30 hours. Plates are stacked not more than four high.

C

3

11. Incubator temperature is maintained at 45.5 ± 0.5°C.

K

11

1. Quebec Colony counter or its equivalent is used to provide the necessary magnification and visibility.

O

1

2. A hand tally or its equivalent is used to aid in counting.

C

3, 6

3. All brick red colonies greater than 0.5mm in diameter are totaled over all the plates and multiplied by a factor of 16.7 to report results as CFU/100 grams of sample.

1.

American Public Health Association. 1984. Compendium of Methods for the Microbiological Examination of Foods, 2^nd Edition. APHA, Washington, D.C.

2.

Good Laboratory Practice.

3.

"Interim Guides for the Depuration of the Northern Quahog, Mercenaria mercenaria. 1968. Northeast Marine Health Sciences Laboratory, North Kingstown, RI.

4.

U.S. Department of Commerce. 1976. NBS Monograph 150. U.S. Department of Commerce, Washington, D.C.

5.

Association of Official Analytical Chemists (AOAC). 2000. Official Methods of Analyses of the Association of Official Analytical Chemists. 17^th Edition, Chapter 17.305, page 22. AOAC, Arlington, VA.

6.

Wilt, D.S. (ed.). 1974. Proceedings of the 8^th National Shellfish Sanitation Workshop. U.S. Food and Drug Administration, Washington, D.C.

7.

U.S. Public Health Service (PHS). 1947. Public Health Report, Reprint #1621. PHS, Washington, D.C.

8.

Association of Official Analytical Chemists (AOAC). 1991. Quality Assurance Principles for Analytical Laboratories. AOAC, Arlington, VA.

9.

American Public Health Association (APHA). 1970. Recommended Procedures for the Examination of Sea Water and Shellfish, 4^th Edition. APHA, Washington, D.C.

10.

Interstate Shellfish Sanitation Conference (ISSC). 1986. Shellfish Sanitation Interpretation #SS-39. ISSC, Columbia, S.C.

11.

American Public Health Association (APHA). 1992. Standard Methods for the Examination of Water and Wastewater,18^th Edition. APHA/AWWA/WEF, Washington, D.C.

12.

Title 21, Code of Federal Regulations, Part 58, Good Laboratory Practice for Nonclinical Laboratory Study. U.S. Government Printing, Washington, D.C.

13.

American Public Health Association (APHA). 1992. Standard Methods for the Examination of Dairy Products, 16^th Edition. APHA, Washington, D.C.

14.

Fisher, J. 1985. Measurement of pH. American Laboratory 16:54-60.

15.

Consult pH electrode product literature.

16.

Association of Official Analytical Chemists (AOAC). 1999. AOAC Methods Validation and Technical Programs - Criteria for Laboratories Performing Food Testing. AOAC, Arlington, VA.

17.

U.S. Environmental Protection Agency (EPA). 1975. Handbook for Evaluating Water Bacteriological Laboratories. EPA-670/9-75-006. U.S. EPA, Cincinnati, OH

18.

Adams, W.N. 1974. NETSU. Personal communication to Dr. Wallace Andrews, FDA.

19.

U.S. Food and Drug Administration (FDA). 1995. Bacteriological Analytical Manual. U.S. FDA, 8^th Edition, AOAC, Arlington,VA.

20.

U.S. Food and Drug Administration (FDA) and Interstate Shellfish Sanitation Conference (ISSC). 1997. NSSP Guide to the Control of Molluscan Shellfish. FDA/ISSC, Washington, D.C. and Columbia, S.C.

21.

U.S. Environmental Protection Agency. 1978. Microbiological Methods for Monitoring the Environment, Water and Wastes. EPA/600/8/78/017. EPA, Washington, D.C.

22.

Furfari, Santo. March 21, 1972. Personal Communication to Dan Hunt, FDA.

SHELLFISH LABORATORY EVALUATION CHECKLIST
 

SUMMARY OF NONCONFORMITIES

LABORATORY STATUS

LABORATORY

DATE

LABORATORY REPRESENTATIVE:

MICROBIOLOGICAL COMPONENT: (Part I-III)

A. Results

Total # of Critical (C) Nonconformities in Parts I-III

Total # of Key (K) Nonconformities in Parts I-III

Total # of Critical, Key and Other (O)

Nonconformities in Parts I-III

____________________

____________________

____________________

B. Criteria for Determining Laboratory Status of the Microbiological Component:

1. Does Not Conform Status: The Microbiological component of this laboratory is not in conformity with NSSP requirements if:

A. The total # of Critical nonconformities is > 4 or

B. The total # of Key nonconformities is > 13 or

C. The total # of Critical, Key and Other is > 18

2. Provisionally Conforms Status: The microbiological component of this

laboratory is determined to be provisionally conforming to NSSP requirements if

the number of critical nonconformities is > 1 but < 3

C. Laboratory Status (circle appropriate)

Does Not Conform        Provisionally Conforms        Conforms

Acknowledgment by Laboratory Director/Supervisor:

All corrective Action will be implemented and verifying substantiating documentation received by the Laboratory Evaluation Officer on or before ____________________
 

Laboratory Signature:_________________________ Date:___________________

LEO Signature:______________________________ Date:___________________

PUBLIC HEALTH SERVICE

U.S. FOOD AND DRUG ADMINISTRATION

SHELLFISH PROGRAM IMPLEMENTATION BRANCH

SHELLFISH SAFETY TEAM

5100 PAINT BRANCH PARKWAY

COLLEGE PARK, MD 20740-3835

TEL. 301-436-2151/2147 FAX 301-436-2672

SHELLFISH LABORATORY EVALUATION CHECKLIST

LABORATORY:

ADDRESS:

TELEPHONE: FAX: EMAIL:

DATE OF EVALUATION:

DATE OF REPORT:

LAST EVALUATION:

LABORATORY REPRESENTED BY:

TITLE:

LABORATORY EVALUATION OFFICER:

SHELLFISH SPECIALIST:

REGION:

OTHER OFFICIALS PRESENT:

TITLE:

Items which do not conform are noted by:
 

C- Critical K - Key O - Other NA - Not Applicable Conformity is noted by a "√"

K

1. Written Plan adequately covers all the following: (check √ those that apply)

a.____ Organization of the laboratory.

b.____ Staff training requirements.

c.____ Standard operating procedures.

d.____ Internal quality control measures for equipment, calibration, maintenance, repair and performance.

e.____ Laboratory safety.

f.____ Quality assessment.

g.____ Proper animal care.

C

2. QA plan implemented.

O

1. Adequate for workload and storage.

O

2. Clean and well lighted.

O

3. Adequate temperature control.

O

4. All work surfaces are nonporous and easily cleaned.

C

5. A separate, quiet area with adequate temperature control for mice acclimation and injection is maintained.

O

1. The pH meter has a standard accuracy of 0.1 unit.

K

2. pH paper in the appropriate range (i.e. 1-4) is used with minimum accuracy of 0.5 pH units.

K

3. pH electrodes consist of pH half cell and reference half cell or equivalent combination electrode (free from Ag/AgCl or contains an ion exchange barrier to prevent passage of Ag ions into the medium that may result in inaccurate pH readings).

K

4. pH meter is calibrated daily or with each use. Records maintained.

K

5. Effect of temperature has been compensated for by an ATC probe or by manual adjustment.

K

6. A minimum of two standard buffer solutions (2 & 7) is used to calibrate the pH meter. Standard buffer solutions are used once and discarded.

K

7. Electrode efficiency is determined daily or with each use following either slope or millivolt procedure.

K

8. The balance provides a sensitivity of at least 0.1g at a load of 150 grams.

K

9. The balance calibration is checked monthly using NIST Class S or ASTM Class 1or 2 weights or equivalent. Records maintained.

K

10. Refrigerator temperature is maintained between 0 and 4˚C.

O

13. Freezer temperature is monitored at least once daily. Record maintained.

O

14. All glassware is clean.

O

15. Once during each day of washing, several pieces of glassware from each batch washed are tested for residual detergent with aqueous 0.04% bromthymol blue solution. Records are maintained.

C

1. Opened PSP reference stand solution (100 µg/ml) is not stored.

K

2. PSP working standard solution (1 µg/ml) and all dilutions are prepared with dilute HCl, pH 3 water, using 'Class A' volumetric glassware (flasks and pipettes) or prepared gravimetrically.

K

3. Refrigerated storage of PSP working standard solution (1µg/ml) does not exceed 6 months and is checked gravimetrically for evaporation loss.

K

4. PSP working dilutions are discarded after use.

K

5. Make up water is distilled or deionized (circle one) and exceeds 0.5 megohm resistance or is less than 2 µ Siemens/cm conductivity at 25°C to be tested and recorded monthly for resistance or conductivity (circle the appropriate).

O

6. Make up water is analyzed for residual chlorine monthly and is at a nondetectable level (≤ 0.1 ppm). Records maintained.

K

7. Make up water is free from trace (< 0.5 mg/l) dissolved metals specifically Cd, Cr, Cu, Ni, Pb, and Zn as determined annually with total heavy metal content ≤1.0 mg/l. Records maintained.

O

8. Makeup water contains < 1000 CFU/ml as determined monthly using the heterotrophic plate count method. Records maintained

O

1. Shellstock are collected in clean, waterproof, puncture resistant containers.

K

2. Samples are appropriately labeled with the collector's name, harvest area and time and date of collection.

K

3. Immediately after collection, shellstock samples are placed in dry storage for transport (e.g. cooler) which is maintained between 0 and 10°C. Upon receipt at the lab, samples are placed under refrigeration

K

4. The time from collection to completion of the bioassay should not exceed 24 hours.
However, if there are significant transportation delays, then shellstock samples are processed immediately as follows (circle the appropriate choice):

a. Washed, shucked, drained, frozen until extracted;

b. Washed, shucked, drained, homogenized and frozen;

c. Washed, shucked, drained, extracted, the supernatant decanted and refrigerated (best choice); or

d. The laboratory has an appropriate contingency plan in place to handle samples which can't be analyzed within 24 hours due to transportation issues.

K

5. Frozen shucked product or homogenates are allowed to thaw completely and all liquid is included as part of the sample before being processed further.

PART II - EXAMINATION OF SHELLFISH FOR PSP TOXIN

C

1. At least 12 animals are used per sample or the laboratory has an appropriate contingency plan for dealing with non-typical species of shellfish.

O

2. The outside of the shell is thoroughly cleaned with fresh water.

O

3. Shellstock are opened by cutting adductor muscles.

O

4. The inside of the shell is rinsed with fresh water to remove sand or other foreign material.

O

5. Shellfish meats are removed from the shell by separating adductor muscles and tissue connecting at the hinge.

K

6. Damage to the body of the mollusk is minimized in the process of opening.

O

7. Shucked shellfish are drained on a #10 mesh sieve (or equivalent) without layering for 5 minutes.

K

8. Pieces of shell and drainage are discarded.

C

9. Drained meats or thawed homogenates are blended at high speed until homogenous (60 - 120 seconds).

K

1. 100 grams of homogenized sample is weighed into a beaker.

K

2. An equal amount of 0.1 N/0.18 N HCl is added to the homogenate and thoroughly mixed (circle the appropriate normality).

C

3. pH is checked and, if necessary adjusted to between pH 2.0 and 4.0.

C

4. Adjustment of pH is made by the dropwise addition of either the acid (5 N HCl) or base (0.1N NaOH) while constantly stirring the mixture.

C

5. The homogenate/acid mixture is promptly brought to a boil, 100 ± 1°C, then gently boiled for 5 minutes.

O

6. The homogenate/acid mixture is boiled under adequate ventilation (i.e. fume hood).

O

7. The extract is cooled to room temperature.

C

8. The pH of the extract is determined and adjusted, if necessary to between pH 2 and 4, preferably to pH 3 with the stirred dropwise addition of 5 N HCl to lower the pH or 0.1N NaOH to raise the pH.

K

9. The extract volume (or mass) is adjusted to 200 mls (or grams) with dilute HCl, pH 3 water.

K

10. The extract is returned to the beaker, stirred to homogeneity and allowed to settle to remove particulates; or, if necessary, an aliquot of the stirred supernatant is centrifuged at 3,000 RPM for 5 minutes before injection.

K

11. If mice cannot be injected immediately then the supernatant should be removed from the centrifuge tubes and refrigerated for up to 24 hours.

K

12. Refrigerated extracts are allowed to reach ambient temperature before being bioassayed.

O

1. A 26-gaugue hypodermic needle is used for injection.

K

2. Healthy mice in the weight range of 17 -23 grams (19 - 21 grams preferable) from a stock colony are used for routine assays. Mice are not reused for bioassay.

Stock strain used _______________ Source of mice_______________

C

3. Mice are allowed to acclimate for at least 24 hours prior to injection. In some cases up to 48 hours may be required.

C

4. A conversion factor (CF) has been determined as ___________. Month and year when current CF determined___________________.

C

5. CF value is checked weekly if assays are done on several days during the week, or, once each day that assays are performed if they are performed less than once per week.

Date of most recent CF check ______________

CF verified/CF not verified (Circle appropriate choice)

C

6. If the CF is not verified, 5 additional mice are injected with the dilution used in the CF check to complete a group of 10 mice. Ten additional mice are also injected with this dilution to produce a second group of 10 mice. The CF is calculated for each group of 10 mice and averaged to give the CF to be used in sample toxicity calculations for the day's or week's work only. All subsequent work must make use of the original laboratory CF value unless this value continues to fail to be verified by routine CF checks.

C

7. If the CF fails to be verified, the cause is investigated and the situation corrected. If the cause cannot be determined with reasonable certainty and fails > 3 times per year, the bioassay is restandardized.

O

8. Mice are weighed to the nearest 0.5 gram.

C

9. Mice are injected intrapertioneally with 1 ml of the acid extract.

K

10. For the CF check, at least 5 mice are used.

C

11. At least 3 mice are used per sample in routine assays.

C

12. Elapsed time is accurately determined and recorded.

K

13. If death occurs, the time of death to the nearest second is noted by the last gasping breath.

C

15. If median death time( 2 out of 3 mice injected die) is < 5 minutes, a dilution is made with dilute HCl, pH 3 water, to obtain a median death time in the range of 5 to 7 minutes.

C

1. The death time of each mouse is converted to mouse units (MU) using Sommer's Table (Table 6 Recommended Procedures, 4^th edition). The death time of mice surviving beyond 60 minutes is considered to be < 0.875 MU.

K

2. A weight correction in MU is made for each mouse injected using Table 7 in Recommended Procedures, 4^th edition.

C

3. The death time of each mouse in MU is multiplied by a weight correction in MU to give the corrected mouse unit (CMU) for each mouse.

C

4. The median value of the array of corrected mouse units (CMU) is determined to give the median corrected mouse unit (MCMU).

C

5. The concentration of toxin is determined by the formula, MCMU x CF X Dilution Factor X 200.

C

6. Any value greater than 80µg/l00 grams of meat is actionable.

1. Adams, W.N. and S.A. Furfari. 1984. Evaluation of laboratory performance of the AOAC method for PSP toxin in shellfish. J. Assoc. Off. Anal. Chem. Vol 67, 6:1147-1148.

2. American Public Health Association. 1970. Recommended Procedures for the Examination of Sea Water and Shellfish, 4^th Edition. APHA, Washington, D.C.

3. American Public Health Association. 1992. Standard Method for the Examination of Dairy Products, 16^th Edition. APHA, Washington, D.C.

4. Association of Official Analytical Chemists International. 1990. Methods of Analysis, 15^th Edition. AOAC, Arlington, VA.

5. APHA/WEF/AWWA. 1992. Standard Methods for the Examination of Water and
Wastewater, 18^th Edition. APHA, Washington, D.C.

6. Title 21, Code of Federal Regulations, Part 58, Good Laboratory Practice for Nonclinical Laboratory Study. U.S. Government Printing, Washington, D.C.

7. National Research Council. 1996. Guide for the Care and Use of Laboratory Animals. National Academy Press, Washington, D.C.

8. Personal communication with USFDA Washington Seafood Laboratory Branch,
Office of Seafood, CFSAN, 1998-1999.

LABORATORY:

DATE OF EVALUATION:

SHELLFISH LABORATORY EVALUATION CHECKLIST

SUMMARY OF NONCONFORMITIES

LABORATORY STATUS

LABORATORY

DATE

LABORATORY REPRESENTATIVE:

PARALYTIC SHELLFISH POISON COMPONENT: PARTS I and II

A. Results

Total # of Critical (C) Nonconformities

Total # of Key (K) Nonconformities

Total # of Critical, Key and Other (O) nonconformities

____________________

____________________

____________________

B.      Criteria for Determining Laboratory Status of the PSP Component

1. Does Not Conform Status The PSP component of this laboratory is not in conformity with NSSP requirements if:

A. The total # of Critical nonconformities is > 3 or

B. The total # of Key nonconformities is > 6 or

C. The total # of Critical, Key and Other is > 10

2.      Provisionally Conforms Status: The PSP component of this laboratory is determined to be provisionally conforming to NSSP requirements if the number of critical nonconformities is > 1 but < 3

C. Laboratory Status (circle appropriate)

Does Not Conform - Provisionally Conforms - Conforms

Acknowledgment by Laboratory Director/Supervisor:

All corrective Action will be implemented and verifying substantiating documentation received by the Laboratory Evaluation Officer on or before ____________________

Laboratory Signature:_________________________ Date:___________________

LEO Signature:______________________________ Date:___________________