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Learning about microbes

Learning about microbes. Counting Identification. The good, the bad, and the ugly. Good microbes are used for food production (controlled fermentation) Bad microbes cause food spoilage (anaerobic or fermentative respiration) Ugly microbes cause disease (damage tissues).

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Learning about microbes

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  1. Learning about microbes Counting Identification

  2. The good, the bad, and the ugly • Good microbes are used for food production (controlled fermentation) • Bad microbes cause food spoilage (anaerobic or fermentative respiration) • Ugly microbes cause disease (damage tissues)

  3. How do microbes get in food? • Environment (innate) • Handling • Processing • Grinding • Packaging • Improper sanitation Determining levels of microbes is important for evaluating quality of food

  4. Sample preparation • Aseptic technique • Analyze sample as quickly as possible • If sample must be held, prevent further growth • SOPs for preparation • Homogenization • Standard sample size • Standard dilution and counting protocol

  5. Remember this? Conventional/traditional techniques

  6. Advantages and disadvantages of the different methods • Large numbers of microbes vs small • Rapid vs time consuming • Viable vs total counts • Technology needs

  7. Direct microscopic count sciencelane.com Rapid but can be tricky Viability stains available

  8. Some devices count bacteria automatically • Coulter counter allows cells to be counted individually • Flow cytometer can distinguish viable and nonviable cells

  9. (Aerobic) plate count requires some technique and math

  10. Membrane filtration concentrates dilute samples

  11. Most probable number (MPN) is an estimate

  12. Most probable number (MPN) is an estimate

  13. Turbidity changes over time if culture is viable

  14. Metabolic tests are important for identification of specific microbes Openwetware.org Many have been miniaturized and combined

  15. Rapid surface testing Sticky tape RODAC (replicate organism detection and counting) Sterile swab test

  16. Summary of “conventional” techniques • Sample collection must be aseptic • Food samples must be prepared properly to maximize accuracy • Preparation environment must be tested also • Conventional methods have been modified or updated for more rapid identification

  17. Special problems with counting microbes in food • Food is complex- different nutrients, different states • There are naturally lots of different types of microbes present- and the microbe of interest is present in small numbers • Sometimes you’re looking for a molecule, like a toxin • You need to consider VNC organisms • There are more foodborne pathogens or spoilage organisms to look for! • Rapid identification is critical

  18. What do we mean by a rapid assay? • Conventional assays may take days • Rapid assay “should” be completed within an 8-hr period- maybe a few minutes • Sample collection may still be time consuming • Enrichment may still be required

  19. Requirements for rapid (or any) assays • Sensitivity • Ability to detect low numbers • Target <1 cell/25 g of food • Limits currently ca 102-105 CFU/g of food • Accuracy • Minimize false positives and false negatives • Specificity • Ability to distinguish one microbe from another • Reproducibility- so many people can use the test reliably • Ease of use

  20. Strategies • Automation • Alternatives to agar media (Petrifilms- just add water) • Often developed to detect specific microbes • Immunological methods are extremely sensitive and may not require additional microbial growth • May test for products like toxins

  21. Polymerase chain reaction Distinguishes cells at the DNA level Can identify single cells Strict controls are required Sensitive detection methods are being developed (biosensors) Ease of use, reliability are key (and cost)

  22. Indicators and microbiological criteria • Remember: foods are very rarely sterile • Food spoilage causes economic loss and, possibly, food shortage • Microbiological criteria help determine quality of food and potential shelf life • Potential health risks • GMPs (good manufacturing practices) • Many professional/scientific organizations involved in this (national and international)

  23. What’s in a criterion? • What is the food? • What is the contaminant? • How was it found and enumerated? • What is the sampling plan? • What are the acceptable limits that can be determined with the plan? • Standard- mandatory • Guideline-advisory

  24. Sampling plans • Small amount is sampled; must be consistent with the total mass • Single attribute: lot is sampled once to look for a specific item • Multiple attributes: test again if initial sample is suspect • Three-class attribute: categories would be acceptable, marginal, unacceptable • Collection, storage, enumeration methods standard

  25. How do you know there is a problem? Specific microbes associated with foods Various enumeration methods

  26. Sensory tests may be unhelpful or misleading • Odor, texture, flavor • Too late • Volatile substances may be released • Nonmicrobial causes of change • May be effective when combined with other tests (microbiological and chemical)

  27. anaerobe psychrotroph acidophile

  28. Table 6.4 hazardous organisms • Category I severe hazard • Zero tolerance • Category II- moderate, potential to spread • Category III- moderate, little potential to spread

  29. Enumeration is effective but time consuming • Bacterial structures (LPS) • Microscopy • Metabolic by products • Sulfides • Ammonium ions • Acids • Gases • pH changes • Enzymes (heat-stable proteinases, lipases)

  30. Indicator organisms • Easy to measure • Associated with pathogens • Present when pathogens are present • Behave like pathogens in life and death • May spoil food but are not necessarily pathogenic • Examples: • Bacillus in bread dough • Byssochlamys (mold) in canned fruit) • Lactic acid bacteria in beer and wine • Flat-sour spores in canned vegetables

  31. Fecal coliforms and E. coli • Gram-negative, gas-producing lactose fermenters • Suggestive of fecal contamination • Easily destroyed by cooking • E. coli vs other coliforms: E. coli is the most authentic indicator of fecal contamination

  32. There are no universal criteria for all foods • Guideliness for categories of foods • Standards for certain microbes and many foods • Food safety objectives (acceptable level of hazard) • GMPs (good manufacturing processes) • HACCPs (hazard analysis critical control programs) • May refer to microbes or toxins • May be international in scope

  33. Summary • Measuring microbial activity is essential but has its limitations • Emphasis on rapidity and sensitivity • Many agencies have collaborated on standards, guidelines, and criteria • Properties of different kinds of food (and microbes that inhabit them) must be considered • Indicator microbes and metabolites are of great value

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