4. AOACRIMicroMethods-2018Awards

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a test portion for each matrix labeled as “temperature control”. Participants were instructed to record the temperature of this portion upon receipt of the shipment, document results on the Sample Receipt Confirmation form provided and fax to the study director.

of a method. In addition, the difference of means and reverse transformed difference of means for each contamination level was determined (3). AOAC Official Method 2014.05 Enumeration of Yeast and Mold in Food 3M™ Petrifilm™ Rapid Yeast and Mold Count Plate First Action 2014 [Applicable to the enumeration of yeast and mold in the following high-water activity matrices: yogurt, frozen bread dough, fermented salami, sour cream, ready-made pie, raw frozen ground beef patties (77% lean), ready-to-eat deli sandwiches, sliced apples, and the following low-water activity matrixes: raw almonds and dehydrated soup.] See Tables 2014.05A and 2014.05B for a summary of results of the collaborative study. The result for each collaborating laboratory’s aerobic plate count analysis for each matrix is shown in Table 2014.05C . See Tables 2–9 for detailed results of the collaborative study. A. Principle The 3M Petrifilm Rapid Yeast and Mold Count (RYM) Plate is a sample-ready culture medium system, which contains nutrients supplemented with antibiotics, a cold-water-soluble gelling agent, and an indicator system that facilitates yeast and mold enumeration. 3M Petrifilm RYM Count Plates are used for the enumeration of yeast and mold in as little as 48 h in the food and beverage industries. 3M Food Safety is certified to ISO (International Organization for Standardization) 9001 for design and manufacturing. B. Apparatus and Reagents ( a ) 3M Petrifilm RYM Count Plate. —25 plates/pouch; two pouches/box (3M Food Safety, St. Paul, MN). ( b ) Sterile diluents.— 0.1% peptone water. ( c ) Pipets.— Capable of 1000 µL or a serological pipet. ( d ) Sterile pipet tips.— Capable of 1000 µL. ( e ) Stomacher.— Seward or equivalent. ( f ) Filter stomacher bags. —Seward or equivalent. ( g ) 3M Petrifilm Flat Spreader. ( h ) Incubators.— Capable of maintaining 25 ± 1°C and 28 ± 1°C and having a solid front to maintain a dark interior. ( i ) Refrigerator.— Capable of maintaining 2–8°C, for storing the 3M Petrifilm RYM Plates. ( j ) L-shaped spreaders. ( k ) Standard colony counter or illuminated magnifier. C. General Instructions ( a ) Store unopened 3M Petrifilm RYM Plate pouches refrigerated or frozen (–20 to 8°C/–4 to 46°F). Just prior to use, allow unopened pouches to come to room temperature before opening (20–25°C/<60% RH). Return unused 3M Petrifilm RYM Plates to the pouch. Seal by folding the end of the pouch over and applying adhesive tape. To prevent exposure to moisture, do not refrigerate opened pouches. Store resealed pouches in a cool dry place (20–25°C/<60% RH) for no longer than 4 weeks. It is recommended that resealed pouches of 3M

Test Portion Analysis

Collaborators followed the appropriate preparation and analysis protocol according to the method specified for each matrix. For both matrixes, each collaborator received eight test portions (two high, two medium, two low, and two uninoculated replicates). For the analysis of the each matrix by the 3M Petrifilm RYM Count Plate method, a 25 g test portion was diluted with 225 mL of 0.1% peptone water and homogenized for 2 min. Ten-fold serial dilutions of each sample were prepared and a 1.0 mL aliquot of each dilution was plated onto four separate 3M Petrifilm RYM Count Plates. For each dilution, two of the plates were incubated at 25±1°C and two of the plates were incubated at 28±1°C. Plates were removed from incubation after 48±2 h and typical yeast and mold colonies in the countable range (plates that contain up to 150 colonies) were enumerated using a standard colony counter. Plates containing greater than 150 colonies were either estimated or recorded as too numerous to count (TNTC). All plates were re-incubated at the appropriate temperatures for an additional 10–14 h (to reach a total incubation time of 60 h) and colonies were enumerated a second time in the same manner as the 48 h time point. Both matrixes analyzed by the 3M Petrifilm RYM Count Plate method were also analyzed using the BAM and ISO reference methods in a paired study design. Serial dilutions for each sample were spread plated in triplicate onto Dichloran-rose Bengal Chloramphenicol (DRBC) agar (raw frozen ground beef patties) or Dichloran 18% Glycerol (DG18) agar (raw almonds). Agar plates were incubated for 5 days at 25±1°C and typical colonies in the countable range (10–150) were enumerated using a standard colony counter. Plates containing no colonies were re-incubated for 2 days and observed for typical growth. Each collaborating laboratory recorded the CFU/g results for the reference methods and the 3M Petrifilm RYM Count Plate method on the electronic spreadsheet provided in the collaborator study outline. The data sheets were submitted to the study director at the end of each week of testing for analysis. The data from each duplicate plates (3M Petrifilm RYM Count Plate) or triplicate plates (BAM and ISO) were averaged. The averaged counts were converted into logarithms for data analysis. Outliers were identified using the Cochran and Grubbs’ tests. A paired t -test was conducted to determine if the mean of replicate samples at each contamination level for each matrix was different between the 3M Petrifilm RYM Count Plate method and the reference methods. (3). A P -value greater than the standard alpha value of 0.05 indicated no statistical difference between the two methods. The repeatability (s r ), reproducibility (s R ), RSD r , and RSD R of the 3M Petrifilm RYM Count Plate method and reference methods were determined by the mean of the logarithm transformations of the counts for each contamination level of each matrix (7). A lower standard deviation value indicated a greater propensity for repeatability Statistical Analysis

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