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  1. Laboratory Methods (Food)

Immunocompromised Mouse Pathogenicity Test

About 4 mg of carrageenan (Sigma type II) dissolved in distilled water (40 mg/ml) is injected, i.p., into 18-20 g mice 24 h before the Listeria challenge. The exact volume of carrageenan solution injected, about 0.1 ml, depends on the mouse body weight. The target dose is 200 mg carrageenan per kg bodyweight. Grow isolate for 24 h at 35° C in TSBye. Transfer to 2 tubes of TSBye for another 24 h at 35° C. Place a total of 10 ml culture broth from both tubes into a 16 × 125 mm tube and centrifuge at 1600 g for 30 min. Discard supernatant and resuspend pellet in 1 ml of phosphate buffered saline. This suspension will contain approximately 1010 bacteria/ml; dilute to 105 bacteria per ml and determine actual concentration by a pour or spread-plate count. Inject (i.p.) 16 to 18 g immunocompromised Swiss white mice (5 mice/culture) with 0.1 ml of the concentrated suspension, i.e. approximately 104 bacteria per mouse. Observe for death over 5-day period. Nonpathogenic strains will not kill, but 104 pathogenic cells will kill, usually within 3 days. Use known pathogenic and nonpathogenic strains and carrageenan-treated, uninoculated mice as controls. Use 5 mice per control group. Carrageenan controls should be challenged with 0.1 ml PBS.

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