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Troubleshooting Micro:

Troubleshooting Micro:. A discussion of some common problems and solutions Robin Cook. FSEA Fall Meeting October 17, 2007. Methods on the agenda:. Total and Fecal MF P/A methods Some on MTF (a.k.a MPN) All of these are both selective and differential. Pros and Cons. Pros and Cons cont. .

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Troubleshooting Micro:

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  1. Troubleshooting Micro: A discussion of some common problems and solutions Robin Cook FSEA Fall Meeting October 17, 2007

  2. Methods on the agenda: • Total and Fecal MF • P/A methods • Some on MTF (a.k.a MPN) • All of these are both selective and differential

  3. Pros and Cons

  4. Pros and Cons cont.

  5. Injured Organisms • Not really dead • Present under normal conditions • Can revive given the opportunity such as ingestion • Do not grow well in selective medias • Pathogens can also retain potential virulence and then recover with ingestion

  6. Good Practices • For MF use dilutions that will give the big picture: • Know the clients/utilities limits if possible. • TNTC is could lead to reporting >20,000 cfu/100 ml. • Do everything possible to get an actual number • Use good technique and when in doubt make the extra effort

  7. Some thoughts on Cultures • Used to see appropriate biochemical reactions • Blank, Pos, and Neg are required for each lot of media when applicable • Method specific requirements apply • Verify purity and viability appropriate to your use. • NOT A REQUIREMENT: A pos with each batch can verify that everything is working properly especially when all others are “clean”. Just an extra piece of evidence. Example: hurricanes

  8. Culture Failures • Usually improper rehydration or suspension • Use good aseptic technique and follow directions • Use media that has already been verified, not too many variables • How about in your lab?

  9. Media Does Not Work • Most likely the carbohydrate has broken down due to carmelization • Watch the autoclave times and temps carefully- Timers and thermometers need to function properly to do this • Don’t forget your max/min thermometer • pH not in range is a very good initial indicator for both lab-made and pre-made.

  10. Thermometers • The temps we use are both selective and differential • Need to maintain temps as close as possible so that reactions happen as expected • For Standard Methods, verify bi-annually. This applies to all thermometer for micro: incubators, autoclave, sample receipt (IR), etc. Have a procedure in place and document it. Can you think of any others?

  11. Blanks • Purpose is to verify that equipment will not contaminate a sample • NELAC has a bit of a misnomer in chp 5 app D.3.1.a.2: • Sterility Check to verify sterility • Method blank is to check technique and method • These are not the same but they have been combined in the standard

  12. Blanks continued • Multiple port manifolds- Under current NELAC each funnel in the manifold must be checked. Extra blanks • Good Science? • We trust something we cannot control over something we can? • What is the intent of this rule? • Is there another way?

  13. Hold Times • Easiest thing to miss and easiest thing to fix • What happens when we miss it? • Reject Sample? • Qualify Data? • What does your QM say? Who, when, how and why.

  14. Everyone’s Favorite: Chlorine • 5.5.8.3.1.a.3 There are 3 conditions that must be met • Cl2 residual must be measured in the field • Cl2 concentration must be recorded on the COC • Lab has verified that the bottles used will neutralize 5 mg/L for DW and 15 mg/L for non-potable water • Lab provides the bottles-Are you sure they are your bottles? What about containers from other sources? • Be careful which you use, some are pH dependent • Document the removal check- How would you do this?

  15. More on Chlorine • If no Cl2 is recorded, check it • Even if you know the source does not have Cl2, still have to check it, either in the field and record, or in the lab. • NELAC chp 5 only applies to labs. If you have another organizational arrangement, address it in your QA manual. • Captive labs probably do not have issue as the bac-t sheet is also the COC.

  16. A Special Circumstance • All 3 stipulations for Cl2 have been met but you still get the “blue flash” from media. Now what? • Manufacturer says to invalidate • “Flash” happens at about 700 mg/L Cl2 • Who should be notified? • What do you think?

  17. Conclusions • Ask the questions • Do not read too much into things • Read your QA manual and edit to suit your needs • Read SM section 9020, all the QC is here for the SM certifications • Use your resources-this info is no good unless it is shared

  18. More Questions • Any other concerns? • Anything to share? • Reach me: cookr@codb.us or 386-671-8856

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