Chapter 11 Chemical, Biological, and Physical Methods Most of the methods for detecting and characterizing microorganisms covered in this chapter have been developed since 1960 Many can be used to estimate numbers of cells or quantity of cellular byproducts Unlike direct microscopic counts, most of those that follow are based on metabolic activity of microorganisms on given substrates, measurements of growth response, measurements of some part of cells including nucleic acids, or combinations of these CHEMICAL METHODS The methods covered in this section are used primarily to detect, enumerate, or identify foodborne organisms or their products: Thermostable nuclease (for Staphylococcus aureus) Limulus amoebocyte lysate (LAL) assay (for Gram-negative bacteria) ATP assay (for live cells) Radiometry Fluorogenic/chromogenic substrates (to identify/differentiate microbial species or strains) The relative sensitivity of these methods compared to others in this chapter can be seen from Table 11–1 Thermostable Nuclease The presence of S aureus in significant numbers in a food can be determined by examining the food for the presence of thermostable nuclease (DNase) This is possible because of the high correlation between the production of coagulase and thermostable nuclease by S aureus strains, especially enterotoxin producers For example, in one study, 232 of 250 (93%) enterotoxigenic strains produced coagulase, and 242 or 95% produced thermostable nuclease.118 Non-S aureus species that produce DNAse are discussed in Chapter 23 The examination of foods for this enzyme was first carried out by Chesbro and Auborn32 employing a spectrophotometric method for nuclease determination They showed that as the numbers of cells 241 242 Modern Food Microbiology Table 11–1 Reported Minimum Detectable Levels of Toxins or Organisms by Biological, Chemical, and Physical Methods of Analysis Methods Toxin or Organism Flow cytometry S Typhimurium in milk Impedance Coliforms in meats Coliforms in culture media S aureus cells S aureus Beef carcass Frozen orange juice flora Coliforms in water Salmonellae Staph enterotoxin B From S aureus From S aureus Gram-negative endotoxins Staph enterotoxins A, B, C, D, and E in foods Staph enterotoxin B in nonfat dry milk Staph enterotoxins A and B Staph enterotoxin C2 E coli STa enterotoxin Aflatoxin M1 in milk Ochratoxin A Bacterial cells Aflatoxin B1 in corn, wheat, peanut butter Deoxynivalenol in corn, wheat C perfringens enterotoxin Botulinal toxins S aureus enterotoxins A and B C perfringens type A toxin Listeria monocytogenes E coli LT enterotoxin B cereus enterotoxin Microcalorimetry ATP measurement Radiometry Fluorescent antibody Thermostable nuclease Limulus lysate test Radioimmunoassay Electroimmunodiffusion Micro-Ouchterlony Lux-phage Passive immune hemolysis Aggregatehemagglutination Latex agglutination Single radial immunodiffusion Hemagglutination– inhibition Reverse passive hemagglutination Sensitivity 103 /ml within 40 minutes, 10/ml after hours nonselective enrichment 103 /g in 6.5 hours 10 in 3.8 hours cells in 12–13 hours Minimum HPR∗ ∼104 cells/ml 102 /cm2 in ∼5 minutes 104 cells/g in 6–10 hours 1–10 cells in hours 106 cells/ml ∼50ng/ml 10 ng/g 2.5–5 ng 2–6 pg of E coli LPS 0.5–1.0 ng/g 2.2 ng/ml 0.1 ng/ml for A; 0.5 ng/ml for B 100 pg 50–500 pg/tube 0.5 ng/ml 20 ppb 500–1,000 cells in 8–10 minutes ng/g 20 ng/g 10 ng 3.7–5.6 mouse LD50 /0.1 ml 10–100 ng/ml 500 ng/ml