2018水质总大肠菌群、粪大肠菌群和大肠埃希氏菌的测定酶底物法

目 次前 言 ii1 适用范围 12 规范性引用文件 13 术语和定义 14 方法原理 25 干扰和消除 26 试剂和材料 27 仪器和设备 38 样品 39 分析步骤 410 结果计算与表示 6精密度和准确度 612 质量保证和质量控制 813 废弃物处理 8附录A（资料性附录）结果判定参考图片 9附录B（规范性附录）97孔定量盘法MPN表 12附录C（资料性附录）97孔定量盘法MPN表对应95%置信区间 20iPAGEPAGE1PAGEPAGE2水质 总大肠菌群、粪大肠菌群和大肠埃希氏菌的测定 酶底物法1 适用范围本标准规定了测定水中总大肠菌群、粪大肠菌群和大肠埃希氏菌的酶底物法。埃希氏菌的测定。本方法的检出限为10MPN/L。2 规范性引用文件标准。GB/T6682 分析实验用水规格和实验方法14581 湖泊和水库采样技术指导HJ494水质采样技术指导HJ/T91地表水和污水监测技术规范3 术语和定义下列术语和定义适用于本标准。3.1总大肠菌群 totalcoliforms37℃培养24h，能产生β-半乳糖苷酶（β-D-galactosidase），分解选择性培养基中的邻硝基苯-β-D-吡喃半乳糖苷（ONPG）生成黄色的邻硝基苯酚的肠杆菌科细菌。3.2粪大肠菌群 fecalcoliforms又称耐热大肠菌群（thermotolerant24h，能产生β-半乳糖苷酶色的邻硝基苯酚的肠杆菌科细菌。3.3大肠埃希氏菌 Escherichiacoli俗称大肠杆菌。37℃培养24培养基中的邻硝基苯-β-D-吡喃半乳糖苷（ONPG）生成黄色的邻硝基苯酚，同时产生β-葡萄糖醛酸酶（β-Glucuronidase），分解选择性培养基中的4-甲基伞形酮-β-D-葡萄糖醛酸苷（MUG）释放出荧光物质（4-甲基伞形酮）的肠杆菌科细菌。3.4最大可能数 mostprobablenumber（MPN）4 方法原理邻硝基苯酚（ONP）；大肠埃希氏菌同时又能产生β-葡萄糖醛酸酶，将选择性培养基中的4-甲基伞形酮-β-D-葡萄糖醛酸苷（MUG）分解为4-甲基伞形酮，在紫外灯照射下产生荧光。统计阳性反应出现数量，查MPN表，分别计算样品中总大肠菌群、粪大肠菌群、大肠埃希氏菌的浓度值。5 干扰和消除5.1 时加入硫代硫酸钠溶液（6.4）消除干扰。5.2 重金属离子具有细胞毒性，能破坏微生物细胞内的酶活性，导致细胞死亡，可在样品采集（8.1）时加入乙二胺四乙酸二钠溶液（6.5）消除干扰。6 试剂和材料除非另有说明，分析时均使用符合国家标准的分析纯试剂，实验用水应满足GB/T6682中三级水的要求。6.1 培养基。本标准采用MinimalMediumONPG-MUG培养基。每100ml样品需使用培养基粉末2.7g±0.5g，所含基本成分如下：硫酸铵[(NH4)2SO4] 0.5g硫酸锰（MnSO4） 0.05硫酸锌（ZnSO4） 0.05硫酸镁（MgSO4） 10mg氯化钠（NaCl） 1g氯化钙（CaCl2） 5mg亚硫酸钠（Na2SO3） 4mg两性霉素B（AmphotericinB） 0.1mg邻硝基苯-β-D-吡喃半乳糖苷（ONPG） 50mg4-甲基伞形酮-β-D-葡萄糖醛酸苷（MUG） 7.5mg茄属植物萃取物（Solanium萃取物） 50mgN-2-羟乙基哌嗪-N-2-乙磺酸钠盐（HEPES钠盐） 0.53gN-2-羟乙基哌嗪-N-2-乙磺酸（HEPES） 0.69g也可采用市售商品化培养基制品。6.2 6.3 6.4 硫代硫酸钠溶液：ρ（Na2S2O3）=0.10g/ml。称取15.7g硫代硫酸钠（6.2），溶于适量水中，定容至100ml，临用现配。6.5 乙二胺四乙酸二钠溶液：ρ（C10H14N2O8Na2·2H2O）=0.15g/ml。称取15g乙二胺四乙酸二钠（6.3），溶于适量水中，定容至100ml，此溶液保质期为30d。6.6 97孔定量盘：含49个大孔，48个小孔。其中，每个小孔可容纳0.186ml样品，大孔中48个大孔每个可容纳1.86ml样品，一个顶部大孔可容纳11ml样品。也可采用经环氧乙烷灭菌的市售商品化成品。6.7 标准阳性比色盘。6.8 无菌水：取适量实验用水，经121℃高压蒸汽灭菌20min，备用。7 仪器和设备7.1 采样瓶：具螺旋帽或磨口塞的100ml、250ml、500ml广口玻璃瓶。注：在采集不存在或不考虑余氧、金属离子干扰的样品时，可采用市售无菌采样瓶或无菌采样袋。7.2 高压蒸汽灭菌器：121℃可调。7.3 恒温培养箱：允许温度偏差37℃±1℃、44.5℃±0.5℃。7.4 程控定量封口机：用于97孔定量盘（6.6）的封口。7.5 紫外灯：365～366nm。7.6 移液管：1ml±0.01ml、10ml±0.1ml。也可采用计量合格的可调式移液器。7.7 三角瓶：100ml。7.8 量筒：100ml±1ml。7.9 一般实验室常用仪器和设备。注：三角瓶、移液管、采样瓶等玻璃器皿及采样器具要在试验前按无菌操作要求包扎，121℃高压蒸汽灭菌20min，烘干，备用。8 样品8.1 样品采集点位布设及采样频次按照GB/T14581、HJ/T494和HJ/T91的相关规定执行。采集微生物样品时，采样瓶（7.1）不得用样品洗涤，采集样品于灭菌的采样瓶中。面10～15cm处，瓶口朝水流方向，拔瓶塞，使样品灌入瓶内然后盖上瓶塞，将采样瓶从水品采集完毕后，迅速扎上无菌包装纸。3～5min，然后将龙头关闭，用火焰灼烧约3min灭菌或用70%～75%的酒精对龙头进行消毒，开足龙头，再放水1min，以充分除去水管中的滞留杂质。采样时控制水流速度，小心接入瓶内。采集地表水、废水样品及一定深度的样品时，也可使用灭菌过的专用采样装置采样。在同一采样点进行分层采样时，应自上而下进行，以免不同层次的搅扰。去活性氯对细菌的抑制作用（每125ml容积加入0.1ml干扰（每125ml容积加入0.3ml注：15.7mg硫代硫酸钠（6.2）可去除样品中1.5mg活性氯，硫代硫酸钠用量可根据样品实际活性氯量调整。8.2样品保存采样后应在2h内检测，否则，应10℃以下冷藏但不得超过6h。实验室接样后，不能立即开展检测的，将样品于4℃以下冷藏并在2h内检测。9分析步骤9.1样品稀释根据样品污染程度确定接种量（见表1），避免接种样品培养后97孔定量盘（6.6）出现全部阳性或全部阴性。接种量小于100ml时，应稀释样品后接种，接种量为10ml时，取10ml样品加入到盛有90ml1:10的稀释样品，其他接种量的稀释样品依次类推。对于未知样品，可选用多个接种量进行检测。表1 样品接种量参考表样品类型接种量（ml）1021010.110-2地表水水源水▲湖泊（水库）▲河流▲▲▲废水生活污水▲▲▲▲工业废水处理前▲▲▲▲处理后▲地下水▲9.2接种量取100ml样品或稀释样品于灭菌后的三角瓶，加入2.7g±0.5g培养基（6.1）粉末，充分混匀，完全溶解后，全部倒入97孔定量盘（6.6）内，以手抚平97孔定量盘背面，赶除孔内气泡，然后用程控定量封口机（7.4）封口。观察97孔定量盘颜色，若出现类似或深于标准阳性比色盘（6.7）的颜色，则需排查样品、培养基、无菌水等一系列因素后，终止试验或重新操作。注：9.1、9.2步骤在野外操作时应避开明显局部污染源，建议使用一次性手套、口罩、酒精灯等。9.3培养测定总大肠菌群和大肠埃希氏菌时，将封口后的97孔定量盘放入恒温培养箱（7.3）中37℃±1℃下培养24h。97孔定量盘放入的恒温培养箱中44.5℃±0.5℃下培养24h。9.4对照试验9.4.1空白对照每次试验都要用无菌水（6.8）按照步骤9.1～9.3进行实验室空白测定。培养后的97孔定量盘不得有任何颜色反应，否则，该次样品测定结果无效，应查明原因后重新测定。9.4.2阴性和阳性对照总大肠菌群、大肠埃希氏菌、粪大肠菌群的阴性、阳性菌株参考表2。表2 阴性、阳性菌株参考表检测指标阳性菌种阴性菌种总大肠菌群大肠埃希氏菌、产气肠杆菌（Enterobacteraerogenes）假单胞菌属（Pseudomonassp.）大肠埃希氏菌大肠埃希氏菌金黄色葡萄球菌、假单胞菌属粪大肠菌群产气肠杆菌、粪链球菌（Streptococcus假单胞菌属将标准菌株制成300～3000个/ml操作，阳性菌株应呈现阳性反应；阴性菌株呈现阴性反应，否则，该次样品测定结果无效，应重新测定。况用无菌水（6.8）稀释至300～3000个/ml。9.4.3 结果判读与计数将培养24h后的97孔定量盘进行结果判读，样品变黄色判断为总大肠菌群或粪大肠菌群阳性；样品变黄色且在紫外灯（7.5）照射下有蓝色荧光，判断为大肠埃希氏菌阳性。如果结果可疑，可延长培养至28h进行结果判读，超过28h后出现的颜色反应不作为阳性结果。可使用保质期内的标准阳性比色盘以辅助判读，参见附录A。分别记录97孔定量盘中大孔和小孔的阳性孔数量。10 结果计算与表示10.1 结果计算从97孔定量盘法MPN表（见附录B）中查得每100ml样品中总大肠菌群、粪大肠菌群数或大肠埃希氏菌的MPN值（置信区间参见附录C）后，再根据样品不同的稀释度，按照公式（1）换算样品中总大肠菌群、粪大肠菌群数或大肠埃希氏菌浓度（MPN/L）：CMPN值×1000f

（1）式中：C——样品中总大肠菌群、粪大肠菌群数或大肠埃希氏菌浓度，MPN/L；MPN值——每100ml样品中总大肠菌群、粪大肠菌群数或大肠埃希氏菌浓度，MPN/100ml；1000——将C单位由MPN/ml转换为MPN/L；f——最大接种量，ml。10.2 结果表示测定结果保留两位有效数字，当测定结果≥100MPN/L时，以科学计数法表示；若97孔均为阴性，可报告为总大肠菌群、粪大肠菌群数或大肠埃希氏菌未检出或＜10MPN/L。11 精密度和准确度11.1 精密度6个实验室分别对低浓度（地下水，浓度均值为6.0×102MPN/L）、中浓度（地表水，浓度均值为4.0×104MPN/L）和高浓度（生活污水，浓度均值为9.0×107MPN/L）三个不同浓度细菌总数的样品及有证标准样品（浓度为2000MPN/L）进行了6次重复测定：实验室内相对标准偏差范围分别为0.26%～1.3%、0.65%～2.0%、1.3%～3.7%和0.68%～2.8%；实验室间相对标准偏差分别为和0.18；再现性限为1.8、0.23、0.38和0.20；实验室间置信区间见表3。表3 总大肠菌群实验室间95%置信区间表低浓度（MPN/L）中浓度（MPN/L）高浓度（MPN/L）均值95%置信区间均值95%置信区间均值95%置信区间均值95%置信区间6.4×1025.5×102～7.4×1024.0×1043.5×104～4.6×1044.1×1063.7×106～4.6×1061.4×1031.8×103～2.3×1036个实验室分别对低浓度（地下水，浓度均值为70MPN/L）、中浓度（地表水，浓度均值为9.0×103MPN/L）和高浓度（生活污水，浓度均值为2.0×106MPN/L）三个不同浓度细菌总数的样品及有证标准样品（浓度为2000MPN/L）进行了6次重复测定：实验室内相对标准偏差范围分别为0.69%～1.9%、0.93%～2.9%、5.4%～21%和1.8%～3.0%；实验室间相对标准偏差分别为9.6%、1.6%、15%和3.7%；重复性限为0.23、0.21、0.58和0.19；再现性限为1.7、0.26、0.90和0.35；实验室间置信区间见表4。表4 大肠埃希氏菌实验室间置信区间表低浓度（MPN/L）中浓度（MPN/L）高浓度（MPN/L）有证标准样品（MPN/L）均值95%置信区间均值95%置信区间均值95%置信区间均值95%置信区间5939～908.5×1037.3×103～9.9×1031.5×1061.3×106～1.7×1067.7×1021.7×103～2.3×1036个实验室分别对低浓度（地下水，浓度均值为1.0×102MPN/L）、中浓度（地表水，浓度均值为1.3×104MPN/L）和高浓度（生活污水，浓度均值为3.0×106MPN/L）三个不同浓度细菌总数的样品及有证标准样品（浓度为3700MPN/L）进行了6次重复测定：实验室内相对标准偏差范围分别为0.40%～2.4%、1.3%～2.9%、3.4%～17%和2.4%～9.2%；实验室间相对标准偏差分别为8.9%、6.4%、6.6%、6.4%；重复性限为0.22、0.26、0.58和0.55；再现性限为1.6、0.76、0.64和0.75；实验室间置信区间见表5。表5 粪大肠菌群实验室间置信区间表低浓度（MPN/L）中浓度（MPN/L）高浓度（MPN/L）有证标准样品（MPN/L）均值95%置信区间均值95%置信区间均值95%置信区间均值95%置信区间9262～1.3×1021.1×1049.5×103～1.4×1041.8×1061.6×106～2.1×1061.4×1032.5×103～5.4×10311.2 准确度6家实验室对总大肠菌群有证标准样品（20006的相对误差范围为-6.4%～-3.5%；相对误差的最终值为-4.7%±1.2%。6家实验室对大肠埃希氏菌群标准样品（20006的相对误差范围为-15%～-6.3%；相对误差的最终值为-13%±3.3%。6家实验室对粪大肠菌群的有证标准样品（37006内的相对误差范围为-17%～-0.81%；相对误差的最终值为-13%±6.1%。注：微生物检测数据为偏态分布，其测定结果全部经以10为底对数转换后进行计算。12 质量保证和质量控制12.1 每批样品按对照试验（9.4）进行空白对照测定，定期使用有证标准菌株进行阳性和阴性对照试验。12.2 每20个样品或每批次样品（≤20个/批）测定一个平行双样。12.3 对每批次培养基须使用有证标准菌株进行培养基质量检验。12.4 定期使用有证标准菌株/标准样品进行质量控制。13 废物处理实验中产生的废物经121℃高压蒸汽灭菌20min后，作为一般废物处理。附录A（资料性附录）结果判定参考图片12图A.1 总大肠菌群、粪大肠菌群酶底物法阴性、阳性结果参考图PAGEPAGE13PAGEPAGE1012图A.2 大肠埃希氏菌酶底物法阴性、阳性结果参考图图A.3 标准阳性比色盘附录B（规范性附录）97孔定量盘法MPN表表B.1 97孔定量盘法MPN表（MPN/100ml）大孔阳性格数小孔阳性格数0123456789100<1.01.02.03.04.05.06.07.08.09.010.011.011.02.03.04.05.06.07.18.19.110.111.112.122.03.04.15.16.17.18.19.210.211.212.213.333.14.15.16.17.28.29.210.311.312.413.414.544.15.26.27.28.39.310.411.412.513.514.615.655.26.37.38.49.410.511.512.613.714.715.816.966.37.48.49.510.611.612.713.814.916.017.018.177.58.59.610.711.812.813.915.016.117.218.319.488.69.710.811.913.014.115.216.317.418.519.620.799.810.912.013.114.215.316.417.618.719.820.922.01011.012.113.214.415.516.617.718.920.021.122.323.412.213.414.515.616.817.919.120.221.422.523.724.81213.514.615.816.918.119.320.421.622.823.925.126.31314.816.017.118.319.520.621.823.024.225.426.627.81416.117.318.519.720.922.123.324.525.726.928.129.31517.518.719.921.122.323.524.725.927.228.429.630.91618.920.121.322.623.825.026.227.528.730.031.232.51720.321.622.824.125.326.627.829.130.331.632.934.11821.823.124.325.626.928.129.430.732.033.334.635.91923.324.625.927.228.529.831.132.433.735.036.337.62024.926.227.528.830.131.532.834.135.436.838.139.52126.527.929.230.531.833.234.535.937.338.640.041.42228.229.530.932.333.635.036.437.739.140.541.943.3续表续表续表续表大孔阳性格数小孔阳性格数0123456789102329.931.332.734.135.536.838.339.741.142.543.945.42431.733.134.535.937.338.840.241.743.144.646.047.52533.635.036.437.939.340.842.243.745.246.748.249.72635.536.938.439.941.442.844.345.947.448.950.452.02737.438.940.442.043.545.046.548.149.651.252.854.42839.541.042.644.145.747.348.850.452.053.655.256.92941.743.244.846.448.049.651.252.854.556.157.859.53043.945.547.148.750.452.053.755.457.158.860.562.23146.247.949.551.252.954.656.358.159.861.663.365.13248.750.452.153.855.657.359.160.962.764.566.368.23351.253.054.856.558.360.262.063.865.767.669.571.43453.955.757.659.461.363.165.067.068.970.872.874.83556.858.660.562.464.466.368.370.372.374.376.378.43659.861.763.765.767.769.771.773.875.978.080.182.33762.965.067.069.171.273.375.477.679.882.084.286.53866.368.470.672.774.977.179.481.683.986.288.691.03970.072.274.476.778.981.383.686.088.490.993.495.94073.876.278.580.983.385.788.290.893.395.998.5101.24178.080.583.085.588.090.693.395.998.7101.4104.3107.14282.685.287.890.593.296.098.8101.7104.6107.6110.6113.74387.690.493.296.099.0101.9105.0108.1114.5117.8121.14493.196.199.1102.2105.4108.6115.3118.7122.3125.9129.64599.3102.5105.8109.2112.6116.2119.8123.6127.4131.4135.4139.646106.3109.8113.4117.2121.0125.0129.1133.3137.6142.1146.7151.547114.3118.3122.4126.6130.9135.4140.1145.0150.0155.3160.7166.448123.9128.4133.1137.9143.0148.3153.9159.7165.8172.2178.9186.049135.5140.8146.4152.3158.5165.0172.0179.3187.2195.6204.6214.3大孔阳性格数小孔阳性格数121314151617181920212223012.013.014.115.116.117.118.119.120.221.222.223.3113.214.215.216.217.318.319.320.421.422.423.524.5214.315.416.417.418.519.520.621.622.723.724.825.8315.516.517.618.619.720.821.822.923.925.026.127.1416.717.818.819.921.022.023.124.225.326.327.428.5517.919.020.121.222.223.324.425.526.627.728.829.9619.220.321.422.523.624.725.826.928.029.130.231.3720.521.622.723.824.926.027.128.329.430.531.632.8821.822.924.125.226.327.428.629.730.832.033.134.3923.224.325.426.627.728.930.031.232.333.534.635.81024.625.726.928.029.230.331.532.733.835.036.237.426.027.228.329.530.731.933.034.235.436.637.839.01227.528.629.831.032.233.434.635.837.038.239.540.71329.030.231.432.633.835.036.237.538.739.941.242.41430.531.733.034.235.436.737.939.140.441.642.944.21532.133.334.635.837.138.439.640.942.243.444.746.01633.735.036.337.538.840.141.442.744.045.346.647.91735.436.738.039.340.641.943.244.545.947.248.549.81837.238.539.841.142.443.845.146.547.849.250.551.91939.040.341.643.044.345.747.148.449.851.252.654.02040.842.243.644.946.347.749.150.551.953.354.756.12142.844.145.546.948.449.851.252.654.155.556.958.42244.846.247.649.050.551.953.454.856.357.859.360.82346.848.349.751.252.754.255.657.158.660.261.763.22449.050.552.053.555.056.558.059.561.162.664.265.8大孔阳性格数小孔阳性格数1213141516171819202122232551.252.754.355.857.358.960.562.063.665.266.868.42653.555.156.758.259.861.463.064.766.367.969.671.22756.057.659.260.862.464.165.767.469.170.872.574.22858.560.261.863.565.266.968.670.372.073.775.577.32961.262.964.666.368.069.871.573.375.176.978.780.53064.065.767.569.371.072.974.776.578.380.282.184.03166.968.770.572.474.276.178.079.981.883.785.787.63270.071.973.875.777.679.581.583.585.487.589.591.53373.375.277.279.281.283.285.287.389.391.493.695.73476.878.880.882.985.087.189.291.493.595.797.9100.23580.582.684.786.989.191.393.595.798.0100.3102.6105.03684.586.788.991.293.595.898.1100.5102.9105.3107.7110.23788.891.193.495.898.2100.6103.1105.6108.1110.7113.3115.93893.495.898.3100.8103.4105.9108.6113.9116.6119.4122.23998.4101.0103.6106.3109.0114.6117.4120.3123.2126.1129.240103.9106.7109.5112.4115.3118.2121.2124.3127.4130.5133.7137.041110.0113.0116.0119.1122.2125.4128.7132.0135.4138.8142.3145.942116.9120.1123.4126.7130.1133.6137.2140.8144.5148.3152.2156.143124.6128.1131.7135.4139.1143.0147.0151.0155.2159.4163.8168.244133.4137.4141.4145.5149.7154.1158.5163.1167.9172.7177.7182.945143.9148.3152.9157.6162.4167.4172.6178.0183.5189.2195.1201.246156.5161.6167.0172.5178.2184.2190.4196.8203.5210.5217.8225.447172.3178.5185.0191.8198.9206.4214.2222.4231.0240.0249.5259.548193.5201.4209.8218.7228.2238.2248.9260.3272.3285.1298.7313.049224.7235.9248.1261.3275.5290.9307.6325.5344.8365.4387.3410.6大孔阳性格数小孔阳性格数242526272829303132333435024.325.326.427.428.429.530.531.532.633.634.735.7125.626.627.728.729.830.831.932.934.035.036.137.2226.927.929.030.031.132.233.234.335.436.537.538.6328.229.330.431.432.533.634.735.836.837.939.040.1429.630.731.832.833.935.036.137.238.339.440.541.6531.032.133.234.335.436.537.638.739.941.042.143.2632.433.534.735.836.938.039.240.341.442.643.744.8733.935.036.237.338.439.640.741.943.044.245.346.5835.436.637.738.940.041.242.343.544.745.947.048.2937.038.139.340.541.642.844.045.246.447.648.850.0.1038.639.740.942.143.344.545.746.948.149.350.651.840.241.442.643.845.046.347.548.749.951.252.453.71241.943.144.345.646.848.149.350.651.853.154.355.61343.644.946.147.448.649.951.252.553.755.056.357.61445.446.748.049.350.551.853.154.455.757.058.359.61547.348.649.951.252.553.855.156.457.859.160.461.81649.250.551.853.254.555.857.258.559.961.262.664.01751.252.553.955.256.658.059.360.762.163.564.966.31853.254.656.057.458.860.261.663.064.465.867.268.61955.456.858.259.661.062.463.965.366.868.269.771.12057.659.060.461.963.364.866.367.769.270.772.273.72159.961.362.864.365.867.368.870.371.873.374.976.42262.363.865.366.868.369.871.472.974.576.177.679.22364.766.367.869.471.072.574.175.777.378.980.582.22467.368.970.572.173.775.377.078.680.381.983.685.2大孔阳性格数小孔阳性格数2425262728293031323334352570.071.773.375.076.678.380.081.783.385.186.888.52672.974.676.378.079.781.483.184.886.688.490.191.92775.977.679.481.182.984.686.488.290.091.993.795.52879.080.882.684.486.388.189.991.893.795.697.599.42982.484.286.187.989.891.793.795.697.599.5101.5103.53085.987.889.791.793.695.697.699.6101.6103.7105.7107.83189.691.693.695.697.799.7101.8103.9106.0108.2110.3112.53293.695.797.899.9102.0104.2106.3108.5110.7113.0115.2117.53397.8100.0102.2104.4106.6108.9113.5115.8118.2120.5122.934102.4104.7107.0109.3114.0116.4118.9121.3123.8126.3128.835107.3109.7112.2114.6117.1119.6122.2124.7127.3129.9132.6135.336112.7115.2117.8120.4123.0125.7128.4131.1133.9136.7139.5142.437118.6121.3124.0126.8129.6132.4135.3138.2141.2144.2147.3150.338125.0127.9130.8133.8136.8139.9143.0146.2149.4152.6155.9159.239132.2135.3138.5141.7145.0148.3151.7155.1158.6162.1165.7169.440140.3143.7147.1150.6154.2157.8161.5165.3169.1173.0177.0181.141149.5153.2157.0160.9164.8168.9173.0177.2181.5185.8190.3194.842160.2164.3168.6172.9177.3181.9186.5191.3196.1201.1206.2211.443172.8177.5182.3187.3192.4197.6202.9208.4214.0219.8225.8231.844188.2193.6199.3205.1211.0217.2223.5230.0236.7243.6250.8258.145207.5214.1220.9227.9235.2242.7250.4258.4266.7275.3284.1293.346233.3241.5250.0258.9268.2277.8287.8298.1308.8319.9331.4343.347270.0280.9292.4304.4316.9330.0343.6357.8372.5387.7403.4419.848328.2344.1360.9378.4396.8416.0436.0456.9478.6501.2524.7549.349435.2461.1488.4517.2547.5579.4613.1648.8686.7727.0770.1816.4大孔阳性格数小孔阳性格数36373839404142434445464748036.837.838.940.041.042.143.144.245.346.347.448.549.5138.239.340.441.442.543.644.745.746.847.949.050.151.2239.740.841.943.044.045.146.247.348.449.550.651.752.8341.242.343.444.545.646.747.848.950.051.252.353.454.5442.843.945.046.147.248.349.550.651.752.954.055.156.3544.445.546.647.748.950.051.252.353.554.655.856.958.1646.047.148.349.450.651.752.954.155.256.457.658.759.9747.748.850.051.252.353.554.755.957.158.359.460.661.8849.450.651.853.054.155.356.557.759.060.261.462.663.8951.252.453.654.856.057.258.459.760.962.163.464.665.81053.054.255.556.757.959.260.461.762.964.265.466.767.954.956.157.458.659.961.262.463.765.066.367.568.870.11256.858.159.460.762.063.264.565.867.168.469.771.072.41358.960.261.562.864.165.466.768.069.370.772.073.374.71460.962.363.664.966.367.668.970.371.673.074.475.777.11563.164.565.867.268.569.971.372.674.075.476.878.279.61665.366.768.169.570.972.373.775.176.577.979.380.882.21767.769.170.571.973.374.876.277.679.180.582.083.584.91870.171.573.074.475.977.378.880.381.883.384.886.387.81972.674.175.577.078.580.081.583.184.686.187.689.290.72075.276.778.279.881.382.884.485.987.589.190.792.293.82177.979.581.182.684.285.887.489.090.692.293.895.497.12280.882.484.085.687.288.990.592.193.895.597.198.8100.52383.885.487.188.790.492.193.895.597.298.9100.6102.4104.12486.988.690.392.093.895.597.299.0100.7102.5104.3106.1107.9大孔阳性格数小孔阳性格数363738394041424344454647482590.292.093.795.597.399.1100.9102.7104.5106.3108.2110.02693.795.597.399.2101.0102.9104.7106.6108.5110.4112.3114.2116.22797.499.3101.2103.1105.0106.9108.8110.8112.7114.7116.7118.7120.728101.3103.3105.2107.2109.2113.2115.2117.3119.3121.4123.5125.629105.5107.5109.5113.7115.7117.8120.0122.1124.2126.4128.6130.830109.9112.0114.2116.3118.5120.6122.8125.1127.3129.5131.8134.1136.431114.7116.9119.1121.4123.6125.9128.2130.5132.9135.3137.7140.1142.532119.8122.1124.5126.8129.2131.6134.0136.5139.0141.5144.0146.6149.133125.4127.8130.3132.8135.3137.8140.4143.0145.6148.3150.9153.7156.434131.4134.0136.6139.2141.9144.6147.4150.1152.9155.7158.6161.5164.435138.0140.8143.6146.4149.2152.1155.0158.0161.0164.0167.1170.2173.336145.3148.3151.3154.3157.3160.5163.6166.8170.0173.3176.6179.9183.337153.5156.7159.9163.1166.5169.8173.2176.7180.2183.7187.3191.0194.738162.6166.1169.6173.2176.8180.4184.2188.0191.8195.7199.7203.7207.739173.1176.9180.7184.7188.7192.7196.8201.0205.3209.6214.0218.5223.040185.2189.4193.7198.1202.5207.1211.7216.4221.1226.0231.0236.0241.141199.5204.2209.1214.0219.1224.2229.4234.8240.2245.8251.5257.2263.142216.7222.2227.7233.4239.2245.2251.3257.5263.8270.3276.9283.6290.543238.1244.5251.0257.7264.6271.7278.9286.3293.8301.5309.4317.4325.744265.6273.3281.2289.4297.8306.3315.1324.1333.3342.8352.4362.3372.445302.6312.3322.3332.5343.0353.8364.9376.2387.9399.8412.0424.5437.446355.5368.1381.1394.5408.3422.5437.1452.0467.4483.3499.6516.3533.547436.6454.1472.1490.7509.9529.8550.4571.7593.8616.7640.5665.3691.048574.8601.5629.4658.6689.3721.5755.6791.5829.7870.4913.9960.61011.249866.4920.8980.41046.21203.31299.71413.61553.11732.91986.32419.6＞2419.6PAGEPAGE21PAGEPAGE20附录C（资料性附录）97孔定量盘法MPN表对应95%置信区间表C.1 97孔定量盘法MPN表对应95%置信区间（MPN/100ml）大孔阳性格数小孔阳性格数012345600.0～3.70.0～3.70.3～5.60.6～7.31.1～8.91.7～10.52.3～12.110.1～5.50.3～5.90.6～7.31.1～8.91.7～10.52.3～12.13.0～13.720.3～7.10.7～7.41.2～9.01.6～10.62.3～12.13.0～13.73.7～15.330.7～8.91.2～9.11.7～10.62.3～12.13.0～13.73.6～15.34.4～16.941.7～9.51.8～10.82.4～12.22.9～13.73.6～15.34.5～16.95.2～18.552.3～11.92.5～12.72.9～13.93.7～15.34.3～16.95.2～18.56.0～20.162.9～13.73.2～14.43.7～15.34.4～16.95.3～18.56.0～20.16.8～21.773.6～14.93.9～15.64.4～16.95.1～18.56.1～20.16.9～21.77.8～22.984.5～16.94.5～17.25.2～18.65.9～20.26.7～21.87.8～23.48.7～24.694.7～18.45.6～19.56.0～20.36.8～21.87.6～23.48.5～25.19.5～26.4105.7～20.16.5～21.17.1～22.07.7～23.68.6～25.19.6～26.810.6～27.56.8～21.47.4～22.37.8～23.68.7～25.39.7～26.810.7～28.211.4～29.3127.8～23.48.2～24.68.8～25.79.4～27.010.4～28.611.5～30.012.6～31.2138.5～25.19.2～26.49.9～27.410.5～28.811.6～30.412.7～31.813.4～33.1149.3～26.810.3～28.211.0～29.211.7～30.712.4～32.313.6～33.714.8～35.01510.1～28.610.8～30.011.9～30.912.6～32.613.7～34.114.9～35.215.7～36.51611.3～30.412.4～31.812.7～32.613.9～34.514.6～35.815.9～37.117.2～38.51712.1～32.212.9～33.714.0～35.014.8～36.516.1～37.716.9～39.218.2～40.61813.4～33.914.2～35.715.4～37.115.7～38.417.1～39.818.4～41.319.2～42.71914.4～36.115.1～37.616.4～39.117.3～40.218.1～41.819.5～43.420.9～44.92015.8～38.116.6～39.717.5～41.218.3～42.719.7～44.220.6～45.722.1～47.22116.8～40.118.2～41.318.5～43.119.4～44.820.8～46.421.7～47.923.3～49.52217.9～42.318.8～44.019.6～45.521.2～47.222.0～48.823.6～50.324.5～51.3续表续表续表续表大孔阳性格数小孔阳性格数01234562319.0～44.420.5～46.021.4～47.722.3～49.223.9～51.024.8～52.225.8～53.82420.7～46.621.7～48.123.3～50.124.2～51.925.2～53.326.1～54.727.1～56.42522.0～48.922.9～51.223.8～52.625.5～54.026.5～55.928.3～57.329.3～59.02623.2～52.024.9～53.725.9～55.426.9～56.527.9～58.629.7～60.130.7～61.82724.5～54.526.2～55.927.3～57.428.3～59.729.3～60.831.2～63.032.3～64.72825.9～57.027.7～58.828.7～60.730.6～62.530.8～63.832.8～65.933.9～67.52928.1～59.829.1～61.430.2～63.431.3～65.433.3～67.234.4～68.535.5～70.73029.6～62.630.7～64.531.8～66.633.8～68.034.9～70.136.1～72.238.3～74.03131.1～65.532.3～67.534.4～69.335.5～71.736.7～73.737.9～75.639.1～77.63232.8～69.033.9～70.936.1～73.037.3～75.038.5～77.239.7～79.142.1～81.23334.5～72.336.7～74.338.0～76.439.2～78.640.5～80.642.9～83.044.2～85.13437.4～75.838.6～77.939.9～80.041.2～82.243.7～84.645.0～86.946.4～89.23539.4～79.540.7～81.542.0～83.743.3～86.244.6～88.647.3～90.448.7～93.03641.4～83.442.8～85.444.1～87.845.5～90.346.9～92.749.7～95.351.1～97.53743.6～87.445.0～89.646.5～92.047.9～95.650.7～98.352.2～100.153.8～102.63846.0～91.747.5～96.248.9～98.551.9～101.053.4～102.955.0～105.756.6～107.73948.5～98.550.0～101.153.0～103.654.6～106.256.3～108.857.9～111.459.6～113.84051.2～103.652.8～106.256.0～109.057.7～111.759.4～114.661.1～117.262.9～120.24154.1～109.055.8～111.857.5～114.960.9～118.062.8～121.064.6～124.166.5～126.34257.3～115.459.1～118.360.9～121.962.8～125.066.5～128.068.4～131.770.4～135.34360.7～123.162.7～126.064.6～129.168.5～132.170.5～136.772.7～140.474.8～143.94464.6～130.666.6～135.368.7～138.770.9～142.275.1～145.477.4～150.079.8～154.04568.8～140.471.1～144.273.4～148.975.7～152.678.1～156.680.6～160.783.1～165.24671.6～151.776.1～155.978.7～159.981.3～163.983.9～168.386.7～176.889.5～181.94779.3～163.782.0～168.084.8～177.085.3～181.988.2～187.291.3～192.797.2～198.84883.5～181.986.5～187.289.7～192.992.9～199.099.2～206.3102.8～212.4106.7～222.44988.7～203.492.1～209.398.7～219.3102.6～228.4106.8～235.5111.2～243.4115.9～260.5大孔阳性格数小孔阳性格数78910121302.9～13.73.7～15.34.5～15.85.2～16.95.9～18.56.9～20.17.8～21.213.7～15.34.3～16.25.2～17.26.0～18.56.8～20.17.6～21.78.7～22.224.4～16.95.3～17.86.0～18.66.8～20.17.7～21.78.5～22.99.4～24.035.1～18.36.1～18.86.9～20.27.7～21.88.6～23.69.5～24.110.2～25.345.9～19.56.9～20.57.8～21.88.7～23.49.6～24.610.3～25.710.9～26.956.8～21.27.6～21.98.5～23.49.4～25.110.4～26.411.0～27.011.7～28.667.7～22.08.6～23.69.5～25.110.5～26.811.1～27.511.8～28.812.5～30.478.7～24.09.6～25.310.6～26.911.6～28.212.3～29.312.6～30.513.7～31.989.7～25.7104～26.911.4～28.612.4～30.013.1～30.713.9～32.314.6～33.8910.5～27.011.5～28.612.6～30.413.3～31.214.0～32.614.7～34.215.9～35.21011.6～28.812.7～30.413.4～31.814.1～33.114.9～34.516.1～35.716.8～37.112.4～30.713.6～32.314.3～33.815.5～35.015.8～36.117.0～37.718.3～39.11213.7～32.614.9～34.215.7～35.215.9～36.517.2～38.118.0～39.219.3～40.61314.6～34.515.8～35.716.6～37.117.4～38.518.2～39.919.5～41.320.3～42.71416.0～36.116.8～37.717.6～39.118.4～40.619.7～41.820.6～43.422.0～44.41517.0～38.117.8～39.718.6～41.219.4～42.320.8～44.021.6～44.923.1～46.51618.0～40.218.8～41.619.6～42.721.0～44.421.9～45.723.4～47.224.3～48.81719.0～42.020.4～43.421.3～44.922.2～46.527.3～48.024.6～49.525.5～51.01820.7～44.221.6～45.722.4～47.223.3～48.824.9～50.325.8～51.226.7～52.81921.8～46.522.7～48.023.6～49.525.2～51.026.1～52.027.0～53.727.9～55.42023.0～48.823.9～50.325.5～51.226.4～52.827.4～54.528.3～56.230.1～57.62124.2～51.025.8～52.026.8～53.727.7～55.428.7～57.130.5～58.631.5～60.12226.2～52.927.1～54.528.1～56.229.1～58.030.9～59.731.9～60.832.9～62.52326.7～55.428.5～57.129.5～58.930.5～60.232.3～61.833.4～63.634.4～65.42428.9～58.029.9～59.830.9～61.332.8～63.033.9～64.734.9～66.136.0～67.8大孔阳性格数小孔阳性格数7891012132530.3～60.731.3～62.533.3～64.234.4～66.035.4～67.336.5～69.037.6～70.72631.8～63.532.8～65.434.9～66.636.0～68.537.1～70.438.2～72.240.4～73.72733.3～66.135.4～67.836.5～69.737.6～71.738.8～73.441.0～75.042.2～77.22835.0～69.137.1～71.038.2～73.039.4～74.441.7～76.441.7～77.944.1～80.02936.6～72.338.8～74.140.0～75.941.2～77.842.4～79.544.8～81.446.0～83.33039.5～75.840.7～77.641.9～79.543.1～81.445.6～83.346.8～85.348.1～87.33141.4～79.542.6～81.443.9～83.346.4～85.347.7～87.349.0～89.550.3～91.63243.4～83.344.7～85.346.0～87.348.6～89.549.9～91.651.3～93.952.6～94.63345.5～87.246.8～89.249.5～91.650.9～93.952.3～94.653.7～96.255.1～98.53447.7～91.550.5～93.850.5～94.651.9～96.254.8～98.556.2～101.057.7～103.63550.1～94.651.5～96.453.0～98.855.9～101.257.4～103.659.0～106.260.5～109.03652.6～99.554.1～101.455.6～103.858.7～106.460.3～109.161.9～111.863.5～114.73755.3～104.556.9～107.060.0～109.461.7～112.263.3～114.965.0～117.566.7～120.23858.2～110.359.8～112.863.2～115.464.9～118.366.6～121.068.4～123.170.2～126.03961.3～115.563.0～119.266.6～121.668.4～124.270.2～127.672.1～130.674.0～133.74066.5～123.166.5～125.470.2～128.972.2～132.174.1～135.076.1～137.078.2～140.44168.4～130.572.3～133.774.3～136.176.4～139.378.5～142.780.6～146.282.8～149.14272.5～138.274.6～142.178.8～145.481.0～148.883.3～151.785.6～155.987.9～159.84377.0～147.279.3～151.781.6～155.586.3～158.288.7～163.391.2～167.893.8～172.14482.2～158.184.7～162.787.2～167.292.2～172.095.0～174.495.1～177.997.9～182.94585.7～170.190.8～176.493.6～180.496.5～184.099.5～190.0102.6～195.0105.7～199.94692.4～186.998.1～192.3101.3～196.8104.6～201.1108.0～207.8111.6～215.4115.2～220.647100.5～204.8104.0～211.8107.7～218.7111.4～226.4115.4～234.0119.5～242.2123.8～250.548110.7～229.7114.9～238.0119.4～245.0124.0～257.8125.3～268.8130.4～279.5135.7～284.049120.8～270.3126.1～281.0128.0～292.9137.9～306.9140.2～320.9147.0～343.5159.0～360.0大孔阳性格数小孔阳性格数1415161718192008.6～21.99.0～23.49.6～24.910.5～25.711.5～26.912.5～28.613.2～29.319.4～23.69.7～25.110.6～26.411.6～27.012.6～28.613.3～30.014.0～30.729.8～25.110.4～26.811.4～27.512.4～28.813.5～30.414.1～31.214.8～32.6310.5～26.911.5～28.212.5～29.213.6～30.414.3～31.815.0～32.715.7～34.2411.6～28.612.6～29.313.7～30.714.8～32.215.1～33.115.8～34.517.0～35.7512.4～30.013.4～31.214.6～32.315.7～33.816.0～35.017.2～36.117.9～37.5613.6～31.814.7～32.615.9～34.216.6～35.217.4～36.518.1～37.819.4～39.1714.9～33.115.6～34.516.8～35.717.5～37.118.3～38.119.6～39.720.4～40.6815.7～35.017.0～36.117.7～37.718.5～38.519.2～40.220.6～41.321.4～42.7917.1～36.517.9～38.118.7～39.219.5～40.620.8～41.821.6～43.222.4～44.41018.1～38.518.9～39.719.7～41.221.0～42.321.8～43.423.3～44.923.5～46.519.1～40.219.9～41.620.7～42.722.1～44.223.6～45.723.7～46.825.2～48.01220.1～42.021.5～43.422.3～44.923.2～46.224.7～47.224.8～48.826.4～50.31321.8～44.222.6～45.623.4～46.625.0～48.025.8～49.526.7～51.027.6～52.01422.9～45.823.7～47.224.6～48.826.1～50.327.0～51.227.9～52.828.8～54.51524.0～48.024.9～49.526.4～51.027.3～52.028.2～53.729.1～55.230.9～56.21625.2～50.326.8～51.227.7～52.828.6～54.529.5～55.930.4～57.132.2～58.61726.4～52.028.0～53.728.9～55.429.9～56.430.8～58.032.6～59.733.6～60.81827.6～54.529.3～56.130.3～57.431.2～58.933.0～60.234.0～61.835.0～63.41929.7～57.030.7～58.031.6～59.832.6～61.334.5～62.635.5～64.236.5～66.02031.1～59.032.0～60.733.0～62.534.9～63.636.0～65.437.0～66.638.0～68.52132.5～61.633.5～63.035.4～64.736.5～66.137.5～67.838.5～69.239.6～70.92233.9～64.235.0～66.037.0～67.338.0～69.039.1～70.440.2～72.241.2～73.72335.5～66.637.5～68.538.6～70.339.7～71.740.8～73.441.9～75.043.0～76.42438.1～69.739.2～71.040.3～73.041.4～74.442.5～75.944.8～77.845.9～79.5大孔阳性格数小孔阳性格数141516171819202539.7～72.440.9～74.042.0～75.943.1～77.645.5～79.546.7～81.247.8～83.02641.5～75.742.7～77.643.8～79.245.0～80.846.2～82.748.6～84.249.8～85.42743.3～78.644.5～80.645.7～82.346.9～83.749.4～85.350.7～87.351.9～89.52845.3～81.546.5～83.447.7～85.350.3～87.351.6～89.552.8～91.654.1～93.92947.3～85.348.6～87.351.2～89.551.1～91.653.8～93.955.1～94.656.5～96.23049.4～89.550.7～91.652.0～93.954.8～94.656.1～96.257.5～98.658.9～101.03151.7～93.953.0～94.655.8～96.257.2～98.558.6～101.060.1～103.661.5～105.73254.0～96.256.9～98.558.4～101.059.8～103.661.3～106.262.8～108.064.3～109.43358.1～101.059.5～103.661.0～106.262.6～108.364.1～110.365.6～112.267.2～114.93460.8～106.262.3～108.863.9～111.165.5～112.867.1～115.168.7～118.070.3～120.43562.1～111.465.3～113.867.0～115.468.6～118.370.3～121.272.0～123.173.7～126.03666.9～116.468.6～119.270.3～121.872.0～124.273.8～127.175.5～129.177.3～132.13770.3～121.972.0～125.073.8～127.675.7～130.677.5～133.779.4～135.383.4～138.73873.9～128.975.8～132.177.7～135.079.7～137.081.6～140.483.6～143.085.6～146.23975.9～135.377.9～138.782.0～142.284.0～145.486.1～148.288.3～151.792.8～154.74080.2～144.084.5～147.986.7～150.088.9～154.091.1～156.695.9～159.995.8～163.74187.2～152.689.6～156.191.9～159.894.3～163.794.2～168.099.2～172.4101.8～174.44292.8～163.795.3～168.095.3～172.497.8～174.4103.1～177.0105.9～182.0108.7～187.24396.5～17