Total Collaborative Study Protocol_Solus One Salmonella v1 1

calculate the MPN value with 95% Confidence interval levels (7). 

Prior to inoculation of shredded cheddar cheese and pasteurized liquid egg matrixes, the respective 

broth culture inoculums were heat stressed for 10 ± 1 min at 50 ± 1°C in a water bath. The heat stressed 

Salmonella  starter cultures were subsequently diluted in BHI broth, added to the respective matrixes 

and subsequently mixed. Following inoculation, a bulk lot of the matrix was homogenized by hand and 

held for 48–72 h at 2–8°C prior to analysis, thereby allowing time for the target organism to equilibrate 

within the sample. At these low and high inoculum levels, fractional positives or consistently positive 

results at the time of sampling were anticipated. 

The degree of injury of the culture was estimated by plating an aliquot of diluted culture onto xylose 

lysine deoxycholate agar (XLD) and Tryptic Soy agar (TSA). The agars were incubated for 18–24 h at 35 ± 

1°C before colonies were enumerated. The degree of injury was estimated as:  ൬1 െ ௦௘௟௘௖௧ ௡௢௡௦௘௟௘௖௧ ൰ 100 Where  = number of colonies on selective agar 

 = number of colonies on non‐selective agar 

For the inoculation of a non‐fat dry milk (NFDM) powder matrix, a lyophilized culture was used. The 

lyophilized culture was prepared by transferring a single  Salmonella  colony from SBA into BHI broth for 

18–24 h at 35 ± 2°C. The culture was then diluted in a sterile cryo‐protectant, reconstituted NFDM blend 

and freeze dried for 48–72 h. The lyophilized  Salmonella  culture was subsequently diluted in powdered 

NFDM and used to inoculate a bulk lot of the matrix at levels that would yield fractional positives or 

consistently positive results at the time of sampling. Following inoculation, a bulk lot of the NFDM 

powder matrix was homogenized by hand and held for two weeks at room temperature (15–25°C) prior 

to analysis, thereby allowing time for the target organism to equilibrate within the sample. 

For stainless steel environmental surfaces, 4” x 4” areas were co‐inoculated with 0.25 mL diluted

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