1. © 2007 Her Majesty the Queen in Right of Canada (Canadian Food Inspection Agency), all rights reserved. Use without permission is prohibited. Results Assessment for Microbiology April 30,2009 Katie Eloranta CFIA Burnaby Laboratory- Microbiology

2. 2 Items for consideration We’ll Examine… The Regulations 2 Class vs 3 Class Plans Understanding the Lingo Examples Additional Commodity Specific Policies Results Assessment for Microbiology

3. 3 So the testing is done…now what does this mean? First Step- The Regulations For Most Commodities Health Canada- Provides an Interpretive Summary of Current Regulatory Standards Available at http://www.hc-sc.gc.ca/fn-an/res-rech/analy-meth/microbio/volume1/intsum-somexp-eng.php ( Search the Compendium of Analytical Methods- Volume I)

4. 4 For Fish Products Canadian Food Inspection Agency Appendix 2 Bacteriological Guidelines for Fish and Fish Product Available at www.inspection.gc.ca/english/fssa/fispoi/man/samnem/app2e.shtml (Search on inspection.gc.ca for Bacteriological Guidelines)

5. 5 For certain foods, microbiological standards have been established : These standards have legal status and are defined in the Food and Drugs Act Examples: Table 1 b. from Interpretive Summary

6. 6 For other foods, while no specific legal standard has been created, they still fall under the general clauses of the Food and Drugs Act: Section 4: No person shall sell an article of food that has in or upon it any poisonous or harmful substance; is unfit for human consumption; consists in whole or in part of any filthy, putrid, disgusting, rotten, decomposed or diseased animal or vegetable substance; is adulterated; or was manufactured, prepared, preserved, packaged or stored under unsanitary conditions.

7. 7 To aid in assessing these foods, the Health Products and Food Branch develops microbiological guidelines While these guidelines are not defined in the Regulations of the Food and Drugs Act, Used to judge compliance under : Section 4, 5, and 7 of the Food and Drug Act

8. 8 2 Class vs 3 Class Plans 2 Class Plans: 2 outcomes, either acceptable or unacceptable. 2 Class Plans are used when no living cells of a specific organism is tolerated (Presence/ Absence Testing) Example: Tolerance of Salmonella in any food is zero Finding any isolates of Salmonella would result in an unsatisfactory assessment

9. 9 3 Class Plan: 3 possible outcomes; acceptable, marginally acceptable, and unacceptable. Used when some cells of the organism in question are tolerated. (Enumeration used to determine whether cell levels exceed tolerance). Example: The presence of some E. coli in Cheese would be acceptable, depending on just how much

10. 10 Finding 100 cfu/g in one sub-sample would result in a satisfactory assessment But…finding 10,000 cfu/g in one sub-sample would result in an unsatisfactory assessment For 3 Class Plans, the assessment depends on the levels, not just the presence or absence of an organism

11. 11 As a very general rule of thumb, testing of pathogens is usually 2- Class, while testing of indicator organisms is generally 3-Class. Indicator organisms, while at low levels are not considered harmful, can at higher levels indicate sanitation or spoilage concerns.

12. 12 The Tables…Making Sense of the Jargon

13. 13 When assessing microbiology results, the following parameters are considered…. n : number of samples of the product lot Did the testing include enough samples? If not, then assessment is not possible and an assessment of No decision is made. (The one exception: If a 2 class test is done on only 1 sample, and you detect the pathogen…it would still be appropriate to assess as Unsatisfactory!)

14. 14 When assessing microbiology results, the following parameters are considered…. m : acceptable concentration of microbes (usually expressed as CFU/g or mL of the food). For a presence/absence test…m is 0 For a 3- class plan m is based on levels achievable under GMP and is used to distinguish between samples of acceptable quality and those with marginal quality.

15. 15 When assessing microbiology results, the following parameters are considered…. c : The maximum allowable number of marginally acceptable samples (How many of my 5 samples are allowed to have levels before I have a problem?)

16. 16 When assessing microbiology results, the following parameters are considered…. M: (For 3 class plans only) Is the concentration of the microbe that indicate a (potential) hazard, imminent spoilage, or gross insanitation. If any of my samples have levels in excess of M, the lot is unsatisfactory.

17. 17 Some Practical Help…Examples!!! I submitted 5 pints of my latest lot of Double Chocolate Swirl Ice Cream to the laboratory. The lab tested for a total aerobic colony count, and provided me with the following results: Sample 1 : 400 CFU/g Sample 2: 200 CFU/g Sample 3: 1,200,000 CFU/g Sample 4: 1000 CFU/g Sample 5: 3,000 CFU/g How would I assess this Ice Cream?

18. 18 Example 1: How’s My Ice Cream? Step 1: Did I submit enough samples? Yes…n=5 Step 2: Did any samples exceed m? Yes, Sample 3 is 1,200,000, while m is only 100,000. Step 3: Did any samples exceed M? Yes, Sample 3 is 1,200,000, while M is 1,000,000. You can stop right here! Since I’ve exceeded M its clear to me that this Ice Cream is Unsatisfactory. Time to go back and find out what went wrong!

19. 19 Now, I decided to get out of the Ice Cream business, and became an oyster farmer! I submitted 5 samples from a lot of Raw Oysters. The lab tested for E. coli and gave me back the following results: Sample 1: 200 MPN/100g Sample 2: 78 MPN/100g Sample 3: < 18 MPN/100g Sample 4: 250 MPN/100g Sample 5: 20 MPN/100g How would I assess this lot of oysters?

20. 20 Example 2: Oh those Oysters! Step 1: Did I submit enough samples? Yes…n=5 Step 2: Did any samples exceed m? Yes, Sample 4 is 250, while m is 230. Step 3: Did any samples exceed M? No, all samples were less than 330. Step 4: Did the number of samples exceeding m, exceed c? (Are too many of my samples at the marginal line?) No…c= 1 Therefore, this product can be assessed as Satisfactory for E. coli

21. 21 Example 3: What about these Oysters? What if instead of 200 MPN/100g, Sample 1 had 300 MPN/100g? Sample 1: 300 MPN/100g Sample 2: 78 MPN/100g Sample 3: < 18 MPN/100g Sample 4: 250 MPN/100g Sample 5: 20 MPN/100g In this case you now have 2 samples with marginally acceptable levels, which exceeds c=1. This product would now be assessed as Unsatisfactory for E.coli levels.

22. 22 What about composite testing??? To help save time and resources it is quite common for laboratories to pool multiple samples into one composite sample. This can change how a final lab result is assessed. When 3 –class plan sample are pooled, determining m and M for the individual sub-sample is no longer possible. For these samples, a new set of assessment criteria are applied. AnalysisAssessment (per g) SatisfactoryInvestigativeUnsatisfactory ACC≤ 6 x10 4 6 x10 4 <x ≤ 4.6 x10 5 >4.6 x10 5

23. 23 Example 4 : More Ice Cream! In the previous case of Ice Cream for ACC testing, Let’s say I submitted a second lot, this time for composite testing. When the 5 pints of Ice Cream were tested all together as a composite, the laboratory gave me the following result: Pooled Sample: 72,000 CFU/g How do I apply the table to a pooled result?

24. 24 Example 4 : More Ice Cream continued There are multiple scenarios to arrive at 72,000 cfu/g pooled: Possibility A: Sample 1: 0 CFU/g Sample 2: 0 CFU/g Sample 3: 360,000 CFU/g Sample 4: 0 CFU/g Sample 5: 0 CFU/g Possibility B: Sample 1: 0 CFU/g Sample 2: 120,000 CFU/g Sample 3: 120,000 CFU/g Sample 4: 0 CFU/g Sample 5: 120,000 CFU/g

25. 25 Example 4 : More Ice Cream continued For Possibility A: Looking back at my original assessment criteria of n=5, c=2, m=100,000, M=1,000,000, this sample would be Satisfactory. While m is exceeded, c and M are not. For Possibility B: Again looking at the original assessment criteria,this lot would be Unsatisfactory. This is because m is exceeded in 3 samples, while c=2.

26. 26 Example 4 : More Ice Cream continued As both scenarios are possible for my pooled sample, I cannot make this determination without re-testing the separate samples, and the composite result is Investigative.

27. 27 Assessing Presence /Absence Testing Luckily, assessing a 2- Class plan is much simpler! It really comes down to only two items to consider… Did I test the right amount of samples? and Did we find the target organism or not?

28. 28 Example 5: Froglegs For my next food production venture, I’ve decided to get more exotic and I’m now selling Froglegs. But, since they are so expensive, for my first lab submission I only submitted 3 packages for testing. The good news is that the tests came back negative for Salmonella, but the lab wouldn’t give me the Satisfactory assessment I was after? What gives?

29. 29 Example 5: Froglegs Continued Step 1: Did I submit enough samples? No…n=5, but I only submitted 3 packages. Right away I understand why my sample was assessed as No Decision. Without testing the full amount of samples, the product cannot be assessed as satisfactory.

30. 30 Example 5: Froglegs Continued For my second lab submission, I made sure to send in the full 5 samples. This time the lab found low levels of Salmonella. Once again I didn’t get the Satisfactory decision I was after, in fact its even worse; they called it Unsatisfactory. Why? Step 1: Did I submit enough samples? Yes…n=5 Step 2: What the product free of the target organism? No

31. 31 More to consider…. In addition to the standards and guidelines outlined by the Health Canada Health Products and Food Branch Interpretive Summary, Additional Polices are listed for the following commodities: Sprouts Raw Ground Beef Fermented Sausages Unpasteurized Fruit Juice/Cider Ready to Eat Foods You need to be aware of their details if you are assessing results for these food products

32. 32 Policy on Listeria monocytogenes in RTE Foods RTE Foods are divided into 3 Categories as they relate to their risk for L. mono: Category 1: RTE foods causally linked to documented outbreaks of listeriosis and or to any RTE food that is rated as “high risk”. These foods require 50 g samples tested as a 2- Class plan ( Presence/Absence).

33. 33 Policy on Listeria monocytogenes in RTE Foods Category 2: All other RTE foods supporting growth of L. mono with refrigerated shelf life greater than 10 days These foods require 25 g samples tested as a 2- Class plan ( Presence/Absence).

34. 34 Policy on Listeria monocytogenes in RTE Foods Category 3: RTE foods supporting the growth of L. mono with refrigerated shelf life of fewer than 10 days, and all RTE foods not supporting growth. Assessment is based on counts <= 100 cfu/g is satisfactory > 100 cfu/g is unsatisfactory

35. 35 So to Summarize….. When assessing a microbiology laboratory sample Check that the sample size was appropriate Verify that the appropriate test was conducted Use m, M, and c from Health Canada and CFIA websites Remember that the rules change for composite samples Be aware of any additional policies for the foods you are assessing

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