Micro December 2018

allowed to solidify. To avoid spreading colonies and surface growth, an overlay of 5 mL of VRB 1 with 4-methyl-umbelliferyl-β-D-glucuronide (MUG) was added. All plates were inverted, 2 incubated for 18 - 24 hours at 35 ± 1 ºC and enumerated. Purple-red colonies that were 0.5 mm 3 or larger in diameter and surrounded by a zone of precipitated bile acids were enumerated as 4 coliform colonies. E. coli colonies were determined by observing bluish fluorescence when 5 viewed under a longwave UV light. Counts of 25 – 250 CFU/plate were considered countable, 6 while counts outside that range were considered estimates. Coliform colonies were confirmed by 7 picking typical colonies to tubes containing BGLB broth and incubating at 35 ± 1°C. Tubes were 8 examined at 24 and 48 hours for gas production. E. coli colonies were confirmed by transferring 9 presumptive colonies to EC-MUG broth and incubating at 35 ± 1°C for 48 ± 2 hours. Tubes were 10 then examined for fluorescence. In addition, for each positive sample, a single colony was 11 confirmed using API 20E, AOAC OMA 978.24 [7]. 12 3M Petrifilm Rapid E. coli/Coliform Count Plate & ISO 13 14 A 10 g test portion was diluted with 90 mL of PSS, allowed to sit for 20 minutes to soften the 15 dry dog kibble, and homogenized with a paddle blender for 2 minutes ± 10 sec. Ten-fold serial 16 dilutions of each sample were prepared in PSS and a 1.0 mL aliquot of each dilution was plated 17 onto duplicate 3M Petrifilm Rapid E. coli /Coliform Count Plates for each dilution. One plate 18 was incubated at 37 ± 1 o C for 18-24 hours and the other plate was incubated at 42 ± 1 o C for 18- 19 24 hours. After incubation, plates were enumerated for total coliform (37 o C) and E. coli (37 o C & 20 42 o C). Plates containing greater than 100 colonies were recorded as too numerous to count 21 (TNTC). Final results were determined by multiplying the counts by the dilution factor for that 22 plate. 23 Each test portion analyzed by the candidate method was also analyzed with the ISO 4382 and 24 ISO16649-2 reference methods in a paired study design. For ISO 4832, serial dilutions for each 25 sample were plated in sterile Petri dishes followed by the addition of violet red bile agar with 26 lactose (VRBL). To avoid spreading colonies and surface growth, an overlay of 5 mL of VRBL 27 was added. Agar plates were incubated for 24 ± 2 h at 37 ± 1 o C. Typical colonies in the 28 countable range (10-150) were enumerated using a standard colony counter. If atypical colonies 29 were present, further confirmation was conducted by transferring colonies to brilliant green 30 lactose bile broth (BGLB). For ISO 16649-2, serial dilutions for each sample were plated in 31 singular using tryptone bile X-glucuronide medium (TBX). Agar plates were incubated for 18-24 32 h at 44 ± 1 o C. Typical colonies in the countable range (10-150) were enumerated using a 33 standard colony counter. 36 37 Each collaborating laboratory recorded the CFU/g results for the reference methods and the 38 candidate method on the electronic spreadsheet provided. The data sheets were submitted to the 39 study director at the end of the study for analysis. A logarithmic transformation [CFU/g +0.1f, 40 where f is the reported CFU/g corresponding to the smallest reportable result]. A Youden plot 41 was prepared to identify discrepancies between test replicates. Outliers were identified using the 42 Cochran and Grubbs’ tests. The differences of means, including 95% upper and lower 43 confidence limits, were determined for each contamination level [8]. If the difference of means 44 between the two methods was < 0.5 Log10, it was considered that no statistical difference 45 existed between the two methods [9]. The repeatability (s r ) and reproducibility (s R ) of the 46 methods were also determined [10]. 47 34 35 Statistical Analysis

4