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Characterization of cryptic prophages (monocins) in Listeria and sequence analysis of a holin/endolysin gene Microbiology 141:2577–2584 Chapter 12 Bioassay and Related Methods After establishing the presence of pathogens or toxins in foods or food products, the next important concern is whether the organisms/toxins are biologically active For this purpose, experimental animals are employed where feasible When it is not feasible to use whole animals or animal systems, a variety of tissue culture systems have been developed that, by a variety of responses, provide information on the biological activity of pathogens or their toxic products These bioassay and related tests are the methods of choice for some foodborne pathogens, and some of the principal ones are listed in Table 12–1 WHOLE-ANIMAL ASSAYS Mouse Lethality This method was first employed for foodborne pathogens around 1920 and continues to be an important bioassay method To test for botulinal toxins in foods, appropriate extracts are made and portions are treated with trypsin (for toxins of nonproteolytic Clostridium botulinum strains) Pairs of mice are injected intraperitoneally (IP) with 0.5 ml of trypsin-treated and untreated preparations Untreated preparations that have been heated for 10 minutes at 100◦ C are injected into a pair of mice All injected mice are observed for 72 hours for symptoms of botulism or death Mice injected with the heat preparations should not die because the botulinal toxins are heat labile Specificity in this test can be achieved by protecting mice with known botulinal antitoxin, and in a similar manner, the specific serologic type of botulinal toxin can be determined (see Chapter 24 for toxin types) Mouse lethality may be employed for other toxins Stark and Duncan45 used the method for Clostridium perfringens enterotoxin Mice were injected IP with enterotoxin preparations and observed for up to 72 hours for lethality The mouse-lethal dose was expressed as the reciprocal of the highest dilution that was lethal to the mice within 72 hours Genigeorgis et al.18 employed the method by use of intravenous (IV) injections C perfringens enterotoxin preparations were diluted in phosphate buffer, pH 6.7, to achieve a concentration of 5–12 µg/ml From each dilution prepared, 0.25 ml was injected IV into six male mice weighing 12–20 g, the number of deaths were recorded, and the LD50 was calculated The mouse is the most widely used animal for virulence assessment of Listeria spp The LD50 for L monocytogenes in normal adult mice is 105 –106 , and for 15-g infant mice, as few as 50 cells may be lethal (see Chapter 25) 285 ... monocytogenes in food products J Food Protect 66:1 658? ??1665 193 Sperber, W.H., and R.H Deibel 1969 Accelerated procedure for Salmonella detection in dried foods and feeds involving only broth cultures... mice are injected intraperitoneally (IP) with 0.5 ml of trypsin-treated and untreated preparations Untreated preparations that have been heated for 10 minutes at 100◦ C are injected into a pair... Selected Microbial Toxins in Foods and Feeds, ed J.L Richard, 225–238 New York: Plenum 224 Wolber, P.K 1993 Bacterial ice nucleation Adv Microbiol Physiol 34:203–237 225 Wolcott, M.J 1991 DNA-based