1. Introduction to detection methods

4. 21 CFR

6. Definitions Enrichment broth Differential agar Selective agar Issues;
Target present at too low of levels;
Target is injured.

7. Indicator organisms Easily and rapidly detectable;
Easy to distinguish from other microorganisms;
Typically associated with pathogen of interest;
Numbers correlate with those of the pathogen;
Possess growth requirements and rate equivalent to the pathogen;
Die-off parallels pathogen;
Absent from foods that are free of the pathogen.
From Doyle and Beuchat, Food Microbiology 3rd edn

9. Coliforms
By definition: organisms capable of fermenting lactose at 37 oC
Theory: presence of coliforms indicates possible fecal contamination, and therefore potential for pathogens transmitted through fecal contamination (Salmonella, Shigella, certain Escherichia coli, viruses)
Organisms are part of family Enterobacteriaceae;
Citrobacter, Edwardsiella, Enterobacter, Erwinia, Escherichia, Hafnia, Klebsiella, Proteus, Salmonella, Serratia, Shigella, Yersinia
Does it hold up?

10. Not really straight forward…
Test
Basis
shortcoming
Coliforms test
Organisms capable of fermenting lactose at 37 oC in presence of bile salts
Organisms not of fecal origin (Citrobacter, Enterobacter) can grow; Salmonella and Shigella are lactose negative
Fecal coliform test
Same as above, but growth at 44 oC to “specifically” grow coliforms of fecal origin
Certain pathogenic E. coli do not grow well at 44 oC; not all recoverable organisms are of human fecal origin
Total Enterobacteriaceae (violet red bile glucose agar)
Recover all organisms within the genus, including lactose negative pathogens
Many enterobacteriaceae (Erwinia, Serratia, Hafnia, etc, are not of fecal origin)
None of these tests are good indicators of presence of viruses or parasites

11. From Adams and Moss, Food Microbiology, 3rd edn

13. Injury (of E. coli O157:H7) Before After
Black et al., J. Appl. Microbiol. 108:1352

14. Comparison of agars
Sorbitol MacConkey agar

15. Effect of oxygen on recovery
(Heat treated 62 C for 10 minutes)
CA = catalase: H2O2  H2O + O2
SOD = superoxide dismutase: O2-  either O2 or H2O2
Knabel et al., 1990

16. Detection – 4 styles Traditional agar based ATP-based Immunological
DNA
“Petrifilm”

17. ATP determination
luciferin
Detection limit, ~ cells