Salter Robert S, Durbin Gregory W, Bird Patrick, Fisher Kiel, Crowley Erin, Chen Yi, Clark Dorn, Ziemer Wayne
Charm Sciences, Inc., 659 Andover St, Lawrence, MA 01843, USA.
J AOAC Int. 2016 Jan-Feb;99(1):153-69. doi: 10.5740/jaoacint.15-0254. Epub 2016 Feb 6.
Peel Plate™ EC is a low-profile plastic, 47 mm culture dish with an adhesive top that contains a dried medium with Gram-negative selective agents and with enzyme substrate indicators for β-galactosidase (coliform) and β-glucuronidase (Escherichia coli). The method provides a conventional quantitative coliform (red) and E. coli (blue/purple/black) count with simple rehydration and incubation for 24 ± 2 h at 35 ± 1°C, while providing a total coliform result, sum of E. coli, and coliform without color differential in dairy products at 32 ± 1°C for 24 ± 2 h. Dairy matrixes claimed and supported with total coliform data are whole milk, skim milk, chocolate milk (2% fat), heavy cream (35% fat), pasteurized whole goat milk, ultra-high-temperature pasteurized milk, powdered milk, lactose-reduced milk, strawberry milk, shredded cheddar cheese, raw cow milk, raw goat milk, raw sheep milk, sour cream, condensed milk, eggnog, vanilla ice cream, condensed whey, yogurt, and cottage cheese. Matrixes claimed for E. coli and total coliform detection are raw ground beef, mixed cellulose 0.45 μm filtered bottled water, environmental sponge of stainless steel, raw ground turkey, dry dog food, liquid whole pasteurized eggs, milk chocolate, leafy green (mixed greens) rinse/flume water, irrigation water, poultry carcass rinse, and large animal carcass sponge. The method has been independently evaluated for total coliform in whole milk, skim milk, chocolate milk, and heavy cream. The method was also independently evaluated for E. coli and coliform in ground beef, filtered bottled water, and sponge rinse from stainless steel surfaces. In inclusivity and exclusivity studies, the method detected 57 of 58 different strains of coliform and E. coli at 32 ± 1°C and 35 ± 1°C in and excluded 31 of 32 different noncoliform strains consisting of Gram-negative and Gram-positive bacteria. In the matrix study, each matrix was assessed separately at each contamination level in comparison to an appropriate reference method. Colony counts were determined for each level and then log10 transformed. The transformed data were evaluated for repeatability, log-mean comparison between methods with 95% confidence interval, and r(2). A 95% confidence interval range of -0.5 to 0.5 on the mean difference was used as the acceptance criterion to establish significant statistical difference between methods. The evaluations demonstrate that the Peel Plate EC method provides no statistical differences across most of the matrixes. The coliform r(2) values were greater than 0.9 except in the case of skim milk (r(2) = 0.77 and 0.69), sheep milk (0.84), and chocolate (0.81). In the case of skim milk, the three highest concentrations were significantly biased low compared with the reference method, whereas in the case of chocolate, the highest concentration was significantly biased high. The E. coli r(2) values were greater than 0.9 except in the case of hog rinse (0.89), flume water (0.82), and chocolate (0.77). The lower values were generally from only a 1 log difference between highest and lowest concentrations except in the case of chocolate, in which the highest concentration was biased high compared with the reference method. Within-method repeatability of Peel Plate EC was similar to the reference method, with relative SDs generally less than 5% when log10 means were ≥1.5. QC data support that the Peel Plate EC is stable for 1 year when refrigerated. Incubation temperature ranges, 30-36°C, and times, 22-26 and 48 h for yogurt, were not significantly different in paired t-test comparison. The method is selective without the need for confirmation, although confirmation of coliform and E. coli was performed as part of the validation work.
Peel Plate™ EC是一种外形低矮的塑料材质、直径47毫米的培养皿,顶部带有粘性,含有添加革兰氏阴性选择剂的干燥培养基以及用于β-半乳糖苷酶(大肠菌群)和β-葡萄糖醛酸酶(大肠杆菌)的酶底物指示剂。该方法通过简单复水并在35±1°C下培养24±2小时,可对常规大肠菌群(红色)和大肠杆菌(蓝色/紫色/黑色)进行计数,同时在32±1°C下培养24±2小时,可得出乳制品中总大肠菌群结果、大肠杆菌总和以及无颜色差异的大肠菌群结果。宣称并得到总大肠菌群数据支持的乳制品基质包括全脂牛奶、脱脂牛奶、巧克力牛奶(2%脂肪)、重奶油(35%脂肪)、巴氏杀菌全脂羊奶、超高温杀菌牛奶、奶粉、低乳糖牛奶、草莓牛奶、切达干酪丝、生牛奶、生羊奶、生绵羊奶、酸奶油、炼乳、蛋奶酒、香草冰淇淋、浓缩乳清、酸奶和农家干酪。宣称用于大肠杆菌和总大肠菌群检测的基质包括生牛肉末、混合纤维素0.45μm过滤瓶装水、不锈钢环境海绵块、生火鸡肉末、干狗粮、液态全脂巴氏杀菌鸡蛋、牛奶巧克力、绿叶蔬菜(混合蔬菜)冲洗/水槽水、灌溉水、家禽胴体冲洗液和大型动物胴体海绵块。该方法已针对全脂牛奶、脱脂牛奶、巧克力牛奶和重奶油中的总大肠菌群进行了独立评估。该方法还针对牛肉末、过滤瓶装水和不锈钢表面海绵冲洗液中的大肠杆菌和大肠菌群进行了独立评估。在包容性和排他性研究中,该方法在32±1°C和35±1°C下检测出58种不同大肠菌群和大肠杆菌菌株中的57种,并排除了32种不同非大肠菌群菌株(包括革兰氏阴性菌和革兰氏阳性菌)中的31种。在基质研究中,与适当的参考方法相比,每种基质在每个污染水平下分别进行评估。确定每个水平的菌落计数,然后进行log10转换。对转换后的数据进行重复性评估、方法间log均值比较(95%置信区间)和r(2)评估。以平均差异的95%置信区间范围为-0.5至0.5作为接受标准,以确定方法间的显著统计差异。评估表明,Peel Plate EC方法在大多数基质中无统计学差异。大肠菌群的r(2)值除脱脂牛奶(r(2)=0.77和0.69)、羊奶(0.84)和巧克力(0.81)外均大于0.9。在脱脂牛奶中情况是,与参考方法相比,三个最高浓度显著偏低,而在巧克力中,最高浓度显著偏高。大肠杆菌的r(2)值除猪冲洗液(0.89)、水槽水(0.82)和巧克力(0.77)外均大于0.9。较低的值通常仅来自最高和最低浓度之间1个对数的差异,巧克力除外,与参考方法相比,其最高浓度偏高。Peel Plate EC方法的方法内重复性与参考方法相似,当log10均值≥1.5时,相对标准差通常小于5%。质量控制数据表明,Peel Plate EC在冷藏条件下1年内稳定。在配对t检验比较中,培养温度范围30 - 36°C以及酸奶培养时间22 - 26小时和48小时无显著差异。该方法具有选择性,无需进行确认,不过作为验证工作的一部分,对大肠菌群和大肠杆菌进行了确认。