Introduction to detection methods

21 CFR 101.95

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

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

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?

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

From Adams and Moss, Food Microbiology, 3rd edn

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

Comparison of agars Sorbitol MacConkey agar

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

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

ATP determination luciferin Detection limit, ~100-1000 cells