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U.S. Department of Health and Human Services

Food

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M-I-05-3c 2400a STANDARD PLATE COUNT, COLIFORM, AND SIMPLIFIED COUNT METHODS

June 2, 2005
 
SAMPLES
  1. __________ Laboratory sample requirements (see CP item 33 & 34)
    STANDARD PLATE AND COLIFORM
    METHODS DILUTING SAMPLES
  2. __________ Work Area
    1. __________ Level plating bench not in direct sunlight
    2. __________ Sanitized immediately before start of plating
  3. __________ Selecting Dilutions
    1. __________ Standard Plate Count
      1. __________ Plate two decimal dilutions per sample
      2. __________ Select dilutions to yield one plate with 25–250 colonies
        1. __________ Raw milk is normally diluted to 1:100 and 1:1000
        2. __________ Finished products are normally diluted to 1:10 and 1:100
        3. __________ The above are general guidelines and may have to be adjusted on a case by case basis (dilutions below 1:10 not required)
    2. __________ Coliform Counts
      1. __________ For milk samples, 1 mL direct and/or decimal dilutions
      2. __________ For all other products, distribute 10 mL of a 1:10 dilution among three plates, generally high fat and viscous products
  4. __________ Identifying Plates
    1. __________ Label each plate with sample identification and dilution
    2. __________ Arrange plates in order before preparation of dilutions
  5. __________ Sample Agitation
    1. __________ When appropriate, wipe top of unopened containers with sterile, ethyl alcohol saturated cloth
    2. __________ Before removal of any portion, thoroughly mix contents of each container
      1. __________ Shake raw and processed sample containers (approx ¾ full) 25 times in 7 sec with l ft movement
      2. __________ Invert filled retail container 25 times, each inversion a complete down and up motion
    3. __________ Remove test portion within 3 min of sample agitation
  6. __________ Sample Measurement, pipets
    1. __________ Use separate sterile pipets for the initial transfers from each container
      1. __________ Pipets in pipet container adjusted without touching the pipets
    2. __________ Pipet tip not dragged over exposed exterior of pipets in container
    3. __________ Pipet not dragged across lip or neck of sample container
    4. __________ Pipet not inserted more than 2.5 cm (1") below sample surface (foam avoided if possible)
    5. __________ Draw test portion above pipet graduation mark and remove pipet from liquid
      1. __________ Pipet aid used, mouth pipetting not permitted (_________________)
    6. __________ Adjust test volume to mark with lower side of pipet in contact with inside of sample container (above the sample surface)
    7. __________ Drainage complete, excess liquid not adhering to pipet
    8. __________ Release test portion to petri dish (tip in contact with plate, 45° angle) or dilution blank (with lower side of pipet in contact with neck of dilution blank, or dry area above buffer where appropriate) with column drain of 2–4 sec
    9. __________ Blow out last drop of undiluted sample from pipet using pipet aid
      1. __________ Blow out away from main part of sample in plate, do not make bubbles
    10. __________ Pipets discarded into disinfectant, or if disposable into biohazard bags or containers to be sterilized
  7. __________ Sample Measurement, pipettors (______________________)
    1. __________ Each day before use, vigorously depress plunger 10x to redistribute lubrication and assure smooth operation (mechanical pipettors)
    2. __________ Before each use examine pipettor to assure that no liquid is expelled from the pipettor nose-cone (contaminated), if fouling is detected do not use until cleaned as per manufacturer recommendation
    3. __________ Use separate sterile tip for the initial transfers from each container
    4. __________ Depress plunger to first stop (mechanical pipettors)
    5. __________ Tip/barrel not dragged across lip or neck of sample container, and pipettor barrel not allowed within sample container
    6. __________ Tip not inserted more than 1 cm below sample surface (foam avoided if possible)
    7. __________ With pipettor vertical slowly and completely release plunger (for electronic pipettors follow manufacturer instructions)
    8. __________ Remove tip from sample and depress plunger completely, re-insert tip into sample and repeat steps f and g, and then remove tip from liquid
    9. __________ Touch tip off to inside of sample container above the sample surface, excess liquid not adhering to tip (do not lay pipettor down once sample is drawn up, use vertical rack if necessary)
    10. __________ Release test portion to petri dish (tip in contact with plate) by slowly depressing plunger to first stop allowing about 1 or 2 seconds for complete drainage
    11. __________ Move tip to a dry spot on plate
      1. __________ If pipettor only has one (1) stop touch off
      2. __________ If pipettor has two (2) stops, depress plunger to second stop and touch off
    12. __________ Or, dispense test portion to dilution blank (tip in contact neck of dilution blank, or dry area above buffer where appropriate) by slowly depressing plunger to first stop
    13. __________ If pipettor has two (2) stops, depress plunger to second stop
    14. __________ Tips discarded into disinfectant, biohazard bags or containers to be sterilized
  8. __________ Dilution Agitation
    1. __________ Before removal of any portion, shake each dilution bottle 25 times in 7 sec with a 1 ft movement
    2. __________ Optionally, use approved mechanical shaker for 15 sec
    3. __________ Remove test portion within 3 min of dilution agitation
  9. __________ Dilution Measurement, pipets
    1. __________ Use separate sterile pipets for the initial transfers from each container
      1. __________ Pipets in pipet container adjusted without touching the pipets
    2. __________ Pipet tip not dragged over exposed exterior of pipets in container
    3. __________ Pipet not dragged across lip or neck of dilution blank
    4. __________ Pipet not inserted more than 2.5 cm (1") below dilution surface
    5. __________ Draw dilution portion above pipet graduation mark and remove pipet from liquid
      1. __________ Pipet aid used, mouth pipetting not permitted (_______________)
    6. __________ Adjust dilution volume to mark with lower side of pipet in contact with inside of dilution blank neck
    7. __________ Drainage complete, excess liquid not adhering to pipet
    8. __________ Gently lift cover of petri dish just high enough to insert pipet
    9. __________ Hold pipet at 45° angle to dish with tip touching dish (or dilution blank neck)
    10. __________ Release dilution portion to dish (or dilution blank) with tip in contact with the bottom of the dish (or dilution blank neck, or dry area above buffer where appropriate) with column drain of 2–4 sec
    11. __________ Touch pipet tip once against dry spot on dish bottom (or dilution blank neck)
    12. __________ When measuring 0.1 mL, do not re-touch dry area
    13. __________ Pipets discarded into disinfectant, or if disposable into biohazard bags or containers to be sterilized
  10. __________ Dilution Measurement, pipettors (_____________________)
    1. __________ Use separate sterile tip for the initial transfers from each container
    2. __________ Depress plunger to first stop (mechanical pipettors)
    3. __________ Tip/barrel not dragged across lip or neck of dilution blank, and pipettor barrel not allowed within dilution blank
    4. __________ Tip not inserted more than 1 cm below dilution surface
    5. __________ With pipettor vertical slowly and completely release plunger (for electronic pipettors follow manufacturer instructions)
    6. __________ Remove tip from dilution and depress plunger completely, re-insert tip into dilution and repeat steps d and e, and then remove tip from liquid
    7. __________ Touch tip off to inside of dilution blank neck or dry area above buffer where appropriate, excess liquid not adhering to tip
    8. __________ Gently lift cover of petri dish just high enough to insert tip
    9. __________ Hold pipettor nearly vertical to dish with tip touching dish
    10. __________ Release test portion to petri dish (tip in contact with plate) by slowly depressing plunger to first stop
    11. __________ Move tip to a dry spot on plate
      1. __________ If pipettor only has one (1) stop touch off
      2. __________ If pipettor has two (2) stops, depress plunger to second stop and touch off
    12. __________ Tips discarded into disinfectant, biohazard bags/con- tainers or into spent dilution blanks to be sterilized
  11. __________ Samples Other than Milk
    1. __________ Weigh 11g aseptically into dilution blank
    2. __________ Use dilution blanks heated to 40–45C
  12. __________ Dry Milk Samples
    1. __________ Weigh 11g aseptically into dilution blank heated to 40–45C
      1. __________ Use standard dilution blank
      2. __________ Or, 2.0% sodium citrate blank (pH<8.0) for relatively insoluble sample (not to be used with Petrifilm)
    2. __________ Wet sample completely with gentle agitation (invert)
    3. __________ Let soak 2 min, then shake 25 times in 7 sec with l ft movement, use within 3 minutes of agitation
    PLATING
  13. __________ Plating
    1. __________ Melt agar quickly in boiling water, flowing steam not under pressure, or microwave oven (use extreme caution when microwaving)
    2. __________ Avoid prolonged exposure to high temperatures during and after melting, establish lab protocol
    3. __________ Do not melt more than will be used within 3 hours
    4. __________ Do not melt agar more than once
    5. __________ Promptly cool melted agar to 45±1C
      1. __________ Record temperature with other control information
    6. __________ Temperature control used for each test medium type
      1. __________ Contains medium identical to type being used
      2. __________ In container identical to that being used
      3. __________ Undergoes same heat treatment and cooling as test medium
    7. __________ Select number of samples in any series so that all will be plated within 20 min (pref £ 10) after diluting first sample
    8. __________ After depositing test portions, promptly pour 10 12 mL medium into each plate of series, or 15 20 mL for > 1 mL portion/plate or where agar weight loss is a problem that can not be corrected by other actions (documentation must be kept to indicate that this is a routine practice)
    9. __________ Lift cover of petri dish just high enough to pour medium
    10. __________ As each plate is poured thoroughly and evenly mix medium and test portion in petri dish
      1. __________ Multiple plates may be poured and mixed, however, plates may not be stacked prior to mixing
    11. __________ Allow to solidify within 10 min on level surface
    12. __________ For dry milk sample, overlay plate with 3 5 mL PCA
    13. __________ For coliform count, overlay plate with 3 4 mL VRB
    14. __________ Invert and incubate within 10 min of medium solidification
    CONTROLS
  14. __________ Controls
    1. __________ Check sterility of dilution blanks, medium, petri dishes, and pipets used for each group of samples (AM and PM)
    2. __________ Expose a poured plate with cover completely removed or pre-hydrated Petrifilm Aerobic Count (PAC) film (both wet surfaces completely exposed) to air for 15 min during plating, AM and PM
      1. __________ The air control plate must be the first plate poured immediately before samples are shaken and must be located such that it is in the area of the plating activity (not off to the side)
      2. __________ If count > 15, take and note corrective actions
      3. __________ For PAC films see item 45b7
    3. __________ Records maintained
    4. __________ Include information on bench, work sheet or report sheet(s)
    INCUBATION
  15. __________ Incubation
    1. __________ Incubate SPC plates at 32±1C for 48±3 hours (dry milk for 72±3 hours) and incubate coliform plates at 32±1C for 24±2 hours
    2. __________ Stack plates no more than 6 high
    3. __________ Arrange stacks so each is at least 1" from adjacent stacks and from incubator surfaces
    4. __________ Place stacks directly over each other on successive shelves
    COUNTING COLONIES
  16. __________ Counting Aids
    1. __________ Count colonies with aid of magnification under uniform and properly controlled artificial illumination with a hand tally
  17. __________ Recording Standard Plate Count
    1. __________ After incubating plates, promptly count all colonies on selected plates
    2. __________ Where impossible to count at once, store plates at 0–4.4C for not longer than 24 hr (avoid as a routine practice)
    3. __________ Record dilutions used and number of colonies on each plate counted
    4. __________ Record results of sterility and control tests
    5. __________ When possible, select spreader free plates with 25–250 colonies and count all colonies including those of pinpoint size
      1. __________ Use higher magnification if necessary to distinguish colonies from foreign matter
      2. __________ Examine edge of petri plates for colonies
    6. __________ If consecutive plates yield 25–250 colonies, count all colonies on plate(s) from both dilutions
    7. __________ Spreaders
      1. __________ Count colonies on representative portion only when colonies are well distributed and area covered or repressed does not exceed 25% of plate
      2. __________ Do not count if repressed growth area > 25% of plate area
      3. __________ When spreaders must be counted, count each as a single colony
      4. __________ Count chains/spreaders from separate sources as separate colonies
      5. __________ If 5% of plates are more than ¼ covered by spreaders, take immediate steps to eliminate and resolve problem
    8. __________ If there is no 25–250 colony plate, use plate having nearest to 250 colonies
    9. __________ If plates from all dilutions exceed 250 colonies, estimate counts as follows
      1. __________ Count colonies in portions representative of distribution and estimate total
      2. __________ Where there are < 10 colonies/sq cm, count colonies in 12 squares, selecting 6 consecutive squares horizontally across the plate and six consecutive squares at right angles
      3. __________ When there are 10 or more colonies/sq cm, count 4 representative squares
      4. __________ Multiply average number colonies/sq cm by area of plate in sq cm
    10. __________ If plates yield < 25 colonies each, record actual number in lowest dilution
    11. __________ If all plates from a sample show no colonies, record count as 0
  18. __________ Coliform Count
    1. __________ After incubating plates, promptly count colonies
    2. __________ Where impossible to count at once, store plates at 0–4.4C for not longer than 24 hr (avoid as a routine practice)
    3. __________ Dark red colonies measuring 0.5 mm or more in diameter on agar plates are considered coliforms in plates containing £ 154 colonies
    4. __________ On crowded plates, coliform colonies may be atypical; count and confirm presence of lactose fermentors
    5. __________ Confirmation of colonies
      1. __________ Pick 10% up to 10 representative colonies per plate with relative percentages of each colony type and inoculate into brilliant green lactose bile broth; incubate 24–48 hr at 32±1C as appropriate
      2. __________ Presence of any gas in a BGB tube constitutes a confirmed test
      3. __________ Record the number of picked colonies and the number of colonies that produced gas (if necessary calculate % confirmed and multiply by total number of colonies)
    6. __________ If no colonies appear on plate(s), record count as 0
    7. __________ If there are 1–154 colonies on a plate, record number counted
    8. __________ If > 154 colonies develop in highest dilution plate, record number as > 150
    9. __________ When multiple plates of a dilution are used, sum counts of plates
  19. __________ Personal Errors
    1. __________ Avoid inaccurate counting due to carelessness, fatigue, or impaired vision
    2. __________ Discover cause and correct if unable to duplicate your own counts on the same plate
    3. __________ Perform monthly counting
      1. __________ If 3 or more analysts use the RpSm method, see current SMEDP, records maintained
      2. __________ If less than three analysts, comparative counts agree £ 8% for the same analyst and £ 10% between two analysts, records maintained
    REPORTS
  20. __________ Computing and Reporting Counts
    1. __________ Multiply number of colonies (or estimated number if necessary) by the reciprocal of the dilution
    2. __________ If consecutive dilutions yield 25–250 colonies, compute count using formula below (see current SMEDP)
      N = åC/[(1 x n1) + (0.1 x n2)]d

      Where, N = number of colonies per milliliter or gram
      åC = sum of all colonies on all plates counted
      n1 = number of plates in lower dilution counted
      n2 = number of plates in next highest dilution counted
      d = dilution from which the first counts were obtained

      Example: 1:100 = 244 colonies 1:1,000 = 28 colonies
      N = (244 + 28)/ [(1 x 1) + (0.1 x 1)]0.01
      = 272/[1.1]0.01
      = 272/0.011
      = 24,727 [25,000 (reported)]

      Note: In the NCIMS Program the denominator will always be 0.11 for 1:10 dilutions and 0.011 for 1:100 dilutions
    3. __________ Report SPC and coliform counts only if inhibitors are not detected
    4. __________ Report computed count as Standard Plate Count/mL or /g (SPC/mL or SPC/g) when taken from plate(s) in the 25–250 range
    5. __________ Report count as Coliform Count (confirmed)/mL or /g when taken from plate(s) in the l l54 range
    6. __________ If no colonies appear on SPC plates, report as < 25 times the reciprocal of the dilution and report as estimated
    7. __________ If no colonies appear on coliform plates, report as < 1 times the reciprocal of the dilution and report as estimated
    8. __________ Report SPC plate counts of 0 to 24 as < 25 times the reciprocal of the dilution and report as estimated
    9. __________ When colonies on SPC plates exceed 100/sq cm, compute count by multiplying 100 x dilution factor x area of plate in sq cm and report as > computed count estimated
    10. __________ Computed counts from SPC plates outside the 25-250 range are reported as Estimated SPC (ESPC)
    11. __________ Counts from coliform plates > 154 are reported as > 150 Estimated Coliform Count (ECC)
    12. __________ If for any reason, an entire plate is not counted, the computed count is reported as Estimated
    13. __________ Report only first two left hand digits
      1. __________ If the third digit is 5 round the second number using the following rules
        1. __________ When the second digit is odd round up (odd up, 235 to 240)
        2. __________ When the second digit is even round down (even down, 225 to 220)
    14. __________ If all plates from a sample have excessive spreader growth, report as spreaders (SPR), or are known to contain inhibitor(s) report as growth inhibitors (GI)
    15. __________ If a laboratory accident renders a plate uncountable, report as laboratory accident (LA)
    PLATE LOOP COUNT METHOD
    APPARATUS
  21. __________ Loop 0.001 mL
    1. __________ True circle, welded I.D. 1.45±0.06 mm, calibrated to contain 0.001 mL, made of appropriate wire
    2. __________ Loop fits over a No. 54 but not a No. 53 twist drill bits (lab must have set), checked monthly, records maintained
    3. __________ Modified by making a 30° bend 3–4 mm from loop, compare to template before use
    4. __________ Opposite end of wire kinked in several places
  22. __________ Hypodermic Needle, Luer-Lok
    1. __________ 13 gauge (sawed off 24–36 mm from the point where the barrel enters the hub)
    2. Kinked end of loop wire shank inserted into needle until bend is 12–14 mm from end of barrel, compare to template before use
  23. __________ Cornwall Continuous Pipetting Outfit
    1. __________ Consisting of metal holder, Cornwall Luer Lok syringe and filling outfit
    2. __________ Syringe, 2 mL capacity, adjusted to deliver 1.0 mL
      1. __________ Calibrated by checking ten 1 mL discharges (10 mL) using a 10 mL Class A graduate cylinder each day of use, records maintained
    3. __________ With Luer-Lok of needle attached to Luer Lok fitting of syringe
    PREPARATION
  24. __________ Heat Treatment of Pipetting Equipment
    1. __________ Sterilize assembled outfit wrapped in kraft paper or in a closed container by autoclaving at 120±1C for 15 min
  25. __________ Assembly of Complete Apparatus for Use
    1. __________ Place end of rubber supply tube (attached to syringe) in sterile dilution buffer blank and depress syringe plunger several times to pump buffer into syringe
    2. __________ Briefly flame loop and allow to cool 15 sec
    3. __________ Discharge several 1 mL portions to waste, then discharge 1 mL portion of buffer into instrument control plate
    PROCEDURE
  26. __________ Comparative Test with SPC
    1. __________ Comparisons done by each analyst performing test
      1. __________ Comparison is valid only if done using similar plate count methods, i.e. SPC agar with pipets (or pipettors) to SPC agar with the PLC device or Petrifilm with pipets (or pipettors) to Petrifilm with the PLC device. Mixing methods is not permissible
      2. __________ Results must be shown to be acceptable prior to official use of test in laboratory
    2. __________ Copy of comparison and results in QC record (or easily accessible file in laboratory)
  27. __________ Identifying Plates (as item 4)
  28. __________ Sample Agitation (as item 5)
  29. __________ Inoculating Plates
    1. __________ Dip loop into each sample (avoiding foam) to bend in shank and withdraw vertically from surface three times in 3 sec with uniform movement of 2.5 cm
    2. __________ Raise cover of petri dish (just high enough to insert loop), insert loop and depress plunger causing sterile dilution buffer to flow across charged loop washing measured 0.001 mL of sample into dish
    3. __________ Do not depress plunger so rapidly that buffer fails to flow across loop
  30. __________ Plating
    1. __________ As described in item 13 or 44
    2. __________ Pour plates with 12–15 mL agar
    CONTROLS
  31. __________ Controls
    1. __________ See item 14
    2. __________ Initial rinse control, see item 25c and 29c
    3. __________ Determine if loop is free rinsing by preparing a rinse control plate after every 20 samples plated
    4. __________ After all samples have been run discharge a final rinse to a control plate
    INCUBATION
  32. __________ Incubation (see item 15)
    1. __________ 48±3 hr at 32±1C
    COUNTING COLONIES
  33. __________ Counting Aids (see item 16 or 47b)
  34. __________ Recording Plate Loop Counts (see item 17 or 48)
  35. __________ Personal Errors (see item 19 or 49)
    REPORTS
  36. __________ Reporting Counts
    1. __________ See item 20 or 50
    2. __________ If 0 to 24 colonies on plate report as < 25,000 Estimated Plate Loop Count/mL (EPLC/mL) or if Petrifilm used, EPPLC/mL
    3. __________ If count is between 25 and 250, report count as PLC/mL or PPLC/mL
    4. __________ If colony count is > 250, report as EPLC/mL or EPPLC/mL
    5. __________ When colonies exceed 100/sq cm, compute count by multiplying 100 x dilution factor x area of plate in sq cm and report as > computed count estimated
    PETRIFILM AEROBIC COUNT METHOD
    APPARATUS
  37. __________ Petrifilm Aerobic Count (PAC) Films
  38. __________ Plastic Spreader
    1. __________ Provided with Petrifilm films, concave (ridge) side used
  39. __________ Identifying Films (as item 4)
  40. __________ Sample Agitation (as item 5)
  41. __________ Sample Measurement (as item 6&7)
  42. __________ Dilution Agitation (as item 8)
  43. __________ Dilution Measurement (as item 9&10)
  44. __________ Procedure
    1. __________ Place the film on a level surface
    2. __________ Lift the top film and deposit 1 mL of sample or dilution onto the center of the base film, touching off the last drop
      1. __________ Deposit samples with pipet (since only 1 mL samples can be used; 10 fold dilution will have to be made)
      2. __________ Or, deposit samples with pipettor (capable of making a 1:10 dilution in the tip)
      3. __________ Or, deposit sample with PLC apparatus (item 29)
    3. __________ Carefully drop the top film onto the inoculum
    4. __________ Place the plastic spreader with the ridge side down (item 38) on the top film over the sample and press down gently on the center of the spreader to distribute inoculum to the circular ridge of the spreader
    5. __________ Leave film undisturbed for 1 min for gel solidification
    6. __________ Incubate within 10 min of solidification
  45. __________ Controls
    1. __________ See item 14 above except for air plates
    2. __________ Air plates
      1. __________ Inoculate PAC film with dilution buffer (1 mL)
      2. __________ Drop film down onto the dilution buffer and spread as described in item 44d above
      3. __________ Leave film undisturbed for 1 minute for solidification of gel
      4. __________ The film must be the first one prepared immediately before samples are shaken and must be located such that it is in the area of the plating activity (not off to the side)
      5. __________ Roll top film back and away from bottom film and expose film for 15 min
      6. __________ After the 15 min roll top film back down and incubate with other films as usual
      7. __________ Incubated, exposed films should contain £ 10 colonies, if count > 10, take and note corrective actions
    INCUBATION
  46. __________ Incubation
    1. __________ Place films in horizontal position, clear side up
    2. __________ Stack films no more than 20 high
    3. __________ Incubate 48±3 hr at 32±1C
    COUNTING COLONIES
  47. __________ Counting PAC Films
    1. __________ See item 16, or
    2. __________ Optionally, count using an approved Petrifilm reader
      1. __________ Refer to manufacturer's instructions for set-up and operation information
      2. __________ 3M Petrifilm Information Management System (PIMS)
        1. __________ Store control cards in a clean, dry and enclosed container
        2. __________ Scan and record control card result prior to the start of and at the end of each operation period
        3. __________ Scan and record control card result hourly with continuous operation
        4. __________ Control card result must fall in the 92 to 108 range, if outside of this range an alarm will sound to alert the operator of a failure
          1. __________ If alarm sounds, inspect card for defects, if defect(s) are observed replace control card, scan and report result of new card
          2. __________ Do not proceed unless control card gives acceptable result, seek technical assistance
      3. __________ 3M Petrifilm Plate Reader
        1. __________ Store System Verification Cards in a clean, dry and enclosed container
        2. __________ Scan and record System Verification Card result prior to the start of and at the end of each operation period
          1. __________ Use Log File feature to automatically save electronic pass/fail result
        3. __________ Scan and record System Verification Card result hourly with continuous operation
          1. __________ Use Log File feature to automatically save electronic pass/fail result
        4. __________ System Verification Cards used to check the function of the Petrifilm Plate Reader prior to reading test films (red, green and blue top lights, and backlights flash)
          1. __________ If inserting the System Verification Card results in an error message, remove and re-insert card
          2. __________ If an error occurs a second time, inspect card for visible dirt or defects, clean and re-insert card
          3. __________ If card gives a third error replace card, scan and report result of new card
          4. __________ Do not proceed unless System Verification Card gives an acceptable result, seek technical assistance
      4. __________ Advanced Instruments PetriScan Reader
        1. __________ Inspect scanner glass for spots and if necessary clean using a soft, lint-free cloth with a mild glass cleaner
        2. __________ Place templates 1 and 2, and two films with no growth in the PetriScan grid and scan
        3. __________ Count and record all results prior to the start of and at the end of each operation period
        4. __________ Scan, count and record template and no growth film results hourly with continuous operation
        5. __________ Template 1 gives count between 27 and 33
        6. __________ Template 2 gives count between 190 and 210
        7. __________ No growth films give a count of zero
        8. __________ If any results out of range
          1. __________ Inspect templates and films for defects and scanner glass for spots
          2. __________ If defect(s) found replace template or film and scan, count and record new result(s)
          3. __________ Do not proceed until template and no growth films give acceptable results, seek technical assistance
      5. __________ Maintain records
    3. __________ Examine each test film visually prior to placing into the reader
      1. __________ For films with no growth, assure reader count is zero
      2. __________ For atypical films, spreader, confluent growth, excessive growth around edge of film, etc., do not count with reader, record as appropriate using items 17 & 48
  48. __________ Petrifilm Count
    1. __________ Count all colonies stained various shades of red, even those outside the circular indentation left by the spreader
    2. __________ See item 17
    3. __________ Select spreader free films with 25–250 colonies and count all red colonies
    4. __________ If films from all dilutions yield < 25 colonies each, record actual number in lowest dilution
    5. __________ If all films from a sample show no colonies, record count as 0
    6. __________ If films from all dilutions exceed 250 colonies, estimate (as per manufacturer specification)
  49. __________ Personal Errors
    1. __________ See item 19, or
    2. __________ If using an approved film reader (item 47b) analysts must perform monthly visual counts comparing to reader results (reader = one analyst)
      1. __________ If one analyst, count must be £ 10% between visual and the reader result
      2. __________ If two or more analysts, use RpSm method (see current SMEDP) using the reader result as an analyst count
    REPORTS
  50. __________ Reporting Counts
    1. __________ See item 20
    2. __________ If the count is between 25 and 250, report count as Petrifilm Aerobic count/mL (PAC/mL)
    3. __________ If count is 0 to 24, report as < 25x reciprocal of the dilution as Estimated PAC/mL (EPAC/mL)
    4. __________ If count is > 250, report as EPAC/mL
    5. __________ When colonies exceed 100/sq cm, compute count by multiplying 100 x dilution factor x 20 sq cm and report as > computed count estimated
    PETRIFILM COLIFORM COUNT METHOD APPARATUS
  51. __________ Petrifilm Coliform Count (PCC) Films
  52. __________ Plastic Spreader
    1. __________ Provided with Petrifilm films, smooth, flat side used
    PROCEDURE
  53. __________ Selecting Dilutions
    1. __________ For milk samples, 1 mL direct and/or decimal dilutions
    2. __________ For other milk products use 1/10 dilution, must plate 10 mL, i.e., use 10 PCC films or see 64d
  54. __________ Identifying Films (as item 4)
  55. __________ Sample Agitation (as item 5)
  56. __________ Sample Measurement (as item 6 & 7)
  57. __________ Procedure
    1. __________ Place films on level surface
    2. __________ Lift top film and deposit 1 mL of sample above the center of the base film, touching off the last drop
    3. __________ Carefully roll the top film into the inoculum, avoid trapping air bubbles
    4. __________ Place the plastic spreader with the flat side down (item 52) on the top film and press down gently on the center of the spreader to distribute inoculum over growth area
    5. __________ Leave films undisturbed for 1 min for gel solidification
    6. __________ Incubate films within 10 min of solidification
    INCUBATION
  58. __________ Incubation
    1. __________ Place films in horizontal position, clear side up
    2. __________ Stack films no more than 20 high
    3. __________ Incubate 24±2 hr at 32±1C
    COUNTING COLONIES
  59. __________ Counting Aids (see item 16)
  60. __________ Petrifilm Count
    1. __________ Count only red colonies having 1 or more gas bubbles within 1 colony diameter
    2. __________ Colonies with gas bubbles are confirmed, no other testing is required
    REPORTS
  61. __________ Reporting Counts
    1. __________ See item 20
    2. __________ If the count is between 1 and 154, report count as Petrifilm Coliform count/mL (PCC/mL)
    3. __________ If count is 0, report as < 1 Estimated PCC/mL (EPCC/mL)
    4. __________ If count is > 154, report as > 150 EPCC/mL
    PETRIFILM HIGH-SENSITIVITY COLIFORM COUNT METHOD APPARATUS
  62. __________ Petrifilm High-Sensitivity Coliform Count (HSCC) Films
  63. __________ Plastic Spreader for HSCC Films

    PROCEDURE
  64. __________ Selecting Dilutions
    1. __________ For milk samples, apply 5 mL direct and/or make decimal dilutions
    2. __________ 1:5 minimum dilution required for: chocolate milk, cottage cheese, dip, evaporated milk, frozen yogurt, heavy and light cream, ice cream, sour cream, sweetened condensed milk and/or decimal dilutions
    3. __________ 1:10 minimum dilution required for: butter, buttermilk, cheese, dry dairy products, yogurt and/or decimal dilutions
    4. __________ 1:10 dilutions of milk or milk products test 10 mL (5 mL on two films)
  65. __________ Identifying Films (as item 4)
  66. __________ Sample Agitation (as item 5)
  67. __________ Sample Measurement (as item 6 & 7)
  68. __________ Procedure
    1. __________ Place film on level surface
    2. __________ Lift top film and deposit 5 mL of sample or dilution just above the center of the bottom film, touching off the last drop
    3. __________ Carefully roll the top film onto the sample gently to prevent pushing the inoculum off the film and to avoid trapping air bubbles
    4. __________ Place the plastic spreader (item 63) on the top film over the inoculum
    5. __________ Distribute sample with a gentle downward pressure on the handle of the spreader to distribute inoculum to the circular ridge of the spreader .
    6. __________ Leave film undisturbed for 2–5 min for gel to solidify
    7. __________ Incubate within 10 min of solidification
    INCUBATION
  69. __________ Incubation (see item 58)
    1. __________ Stack films no more than 10 high
    COUNTING COLONIES
  70. __________ Counting Aids (see item 16)
  71. __________ Petrifilm Count (see items 18 & 60)

    REPORTS
  72. __________ Reporting Counts
    1. __________ See items 20 and 61
    2. __________ On 5 mL direct films report:
      1. __________ 1 to 4 colonies as < 1 coliform/mL or gm
      2. __________ 5 colonies as 1 coliform/mL or gm
      3. __________ > 5 colonies as 1 coliform for every 5 colonies counted, rounding up to the next number if not even multiples of 5 (ex. 11=3 coliforms/mL or gm)
    3. __________ 5 mL of 1:5 dilution provides a 1:1 sensitivity
    4. __________ 5 mL of 1:10 dilution provides a sensitivity of 2 coliforms/mL or gm, run 1:10 dilutions in duplicate to get a sensitivity of 1 coliform/mL or gm as required by the PMO