水质 肠球菌的测定 固定底物技术酶底物法（报批稿）

ICSDB地方标准DB / 2019水质 肠球菌的测定 固定底物技术酶底物法Water qualityDetermination of EnterococciDefined substrate technology enzyme substrate method（报批稿）20- - 发布20- - 实施辽宁省市场监督管理局发布DB21/T *2018目次前言 II1 范围 12 规范性引用文件 13 术语和定义 14 方法原理 15 试剂和材料 26 仪器和设备 27 样品 38 分析步骤 39 结果计算与表示 410 精密度和准确度 411 质量保证和质量控制 512 废物处理 513 注意事项 5附录A 6前言本标准依据GB/T 1.1-2009标准化工作导则 第1部分：标准的结构和编写起草制订。本标准附录A为规范性附录。本标准由大连市市场监督管理局提出。本标准由辽宁省生态环境厅归口。本标准起草单位：大连市环境监测中心、国家海洋环境监测中心。本标准主要起草人：王中卫、明红霞、李翔宇、沈学崴、李玉璞、苏洁、曲翊、王琳琳、刘子成、孙沛雯、刘景泰、韩佳。7水质 肠球菌的测定 固定底物技术酶底物法1 范围本标准规定了测定水中肠球菌的固定底物技术酶底物法。本标准适用于地表水、地下水、饮用水、海水、生活废水和工业废水中肠球菌的测定。本标准的检出限：地表水、地下水、饮用水、生活废水、工业废水样品的检出限为1 CFU/100 ml，海水样品的检出限为10 CFU/100 ml。2 规范性引用文件下列文件对于本文件的应用是必不可少的。凡是注日期的引用文件，仅注日期的版本适用于本文件。凡是不注日期的引用文件，其最新版本（包括所有的修改单）适用于本文件。GB/T 5750.2 生活饮用水标准检验方法 样品的采集与保存GB/T 6682 分析实验室用水规格和试验方法GB 17378.7 海洋监测规范 第七部分：近海污染生态调查和生物监测HJ/T 164 地下水环境监测技术规范HJ/T 91 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 肠球菌 Enterococci41 培养24 h，能产生-半乳糖苷酶（-galactosidase），分解选择性培养基中的4-甲基伞形酮-葡萄糖醛酸苷（4-methyl-umbelliferyl-glucoside，MUG）释放出荧光物质（4-甲基伞形酮）的革兰氏阳性菌。3.2最大可能数 most probable number（MPN） 又称稀释培养计数，是一种基于泊松分布的间接计数法。利用统计学原理，根据一定体积不同稀释度样品经培养后产生的目标微生物阳性数，查表估算一定体积样品中目标微生物存在的数量（单位体积存在目标微生物的最大可能数）。4 方法原理在特定温度下培养特定的时间，肠球菌能产生-半乳糖苷酶，分解荧光底物释放出荧光产物，在366 nm紫外灯照射下产生特征性荧光。统计荧光反应出现数量，查MPN表，计算样品中肠球菌的浓度值。5 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，符合GB/T 6682 三级标准的蒸馏水或去离子水。5.1 培养基市售Enterolert培养基，也可以使用其他等效产品。5.2 硫代硫酸钠溶液：（Na2S2O35H2O）=0.10 g/ml称取硫代硫酸钠（Na2S2O35H2O）10.0 g，溶于适量实验用水，定容至100 ml，临用现配。5.3 乙二胺四乙酸二钠溶液：（C10H14N2O8Na22H2O）=0.15 g/ml称取乙二胺四乙酸二钠（C10H14N2O8Na22H2O）15.0 g，溶于适量实验用水，定容至100 ml，此溶液保质期为30 d。5.4 无菌水实验用水经121 高压蒸汽灭菌20 min，备用。6 仪器和设备6.1 恒温培养箱：能够恒温至41 ，控温精度0.5 。6.2 高压蒸汽灭菌器：能够加热至121 。6.3 紫外灯：台式，366 nm。6.4 封口机：用于定量盘的封口。6.5 采样瓶：具螺旋帽或磨口塞的250 ml玻璃瓶。6.6 三角瓶:100 ml。6.7 97孔定量盘。6.8 一般实验室常用仪器和设备。注：移液管、量筒、采样瓶、三角瓶等玻璃器皿和采样器具应按无菌操作要求包扎，于121 高压蒸汽灭菌20 min，烘干，备用。灭菌后的采样瓶，两周内未使用，需重新灭菌。7 样品7.1 样品采集与保存按照GB/T 5750.2、GB 17378.7、HJ/T 164和HJ/T 91 的相关规定进行样品采集。采样后水样应在4下冷藏保存，6 h内开始检测。7.2 干扰和消除7.2.1 含活性氯（余氯）的水样，在采样瓶灭菌前需加入0.2 ml硫代硫酸钠溶液。7.2.2 含重金属的水样，在采样瓶灭菌前需加入0.6 ml乙二胺四乙酸二钠溶液。8 分析步骤8.1 样品稀释根据水样污染程度确定稀释度，避免接种水样经培养后出现全部阳性或全部阴性。接种量小于100 ml时，水样应制成稀释样品后使用，接种量为10 ml时，以无菌操作方法取10 ml水样加入到装有90 ml无菌水的三角瓶（6.6）中混匀制成1:10（稀释度）稀释的样品，其它接种量的稀释样品依次类推。注：海水样品至少经1:10稀释后再进行检测。8.2 接种以无菌操作方法量取100 ml原水样或稀释样品于三角瓶（6.6）中，加入1支培养基（5.1），充分混匀，完全溶解后，全部倒入97孔定量盘（6.7）内，以手抚平定量盘（6.7）背面，去除孔内气泡，然后用封口机（6.4）封口。8.3 培养将封口后的97孔定量盘（6.7）放入培养箱（6.1）中培养24 h。8.4 对照试验8.4.1 空白对照使用无菌水（5.4）进行实验室空白测定，培养后的97孔定量盘（6.7）不得有荧光产生，否则，该次样品测定结果无效，应重新测定。8.4.2 阴性及阳性对照阳性菌株参考使用粪肠球菌（Enterococcus faecalis）,阴性菌株参考使用粘质沙雷氏菌（Serratla marcescens）和绿脓杆菌（Aerococcus viridians）。将菌株配制成0.5麦氏浊度的菌悬液按照8.2和8.3要求操作，阳性菌株应呈阳性反应（有荧光），阴性菌株呈阴性反应（无荧光），否则该批次测定结果无效，应重新测定。8.5 结果判读与计数将培养24 h后的97孔定量盘（6.7）进行结果判读，水样在紫外灯（6.3）照射下有荧光，则判断为肠球菌阳性。若结果可疑，可延长培养时间到28 h进行结果判读，超过28 h后出现的荧光反应不作为阳性结果。9 结果计算与表示9.1 结果计算从97孔定量盘法MPN表（附录A）中查得每100 ml水样中肠球菌的MPN值，再根据样品不同的稀释度进行计算，每100 ml水样中肠球菌的计算公式如下：式中：C每100 ml水样中肠球菌浓度，CFU/100 ml； MPN值查表得到的MPN值，CFU/100 ml； f接种样品的稀释度。9.2 结果表示测定结果保留两位有效数字，大于等于100时以科学计数法表示，结果单位为CFU/100 ml。10 精密度和准确度10.1 精密度多家实验室分别对肠球菌有证标准样品（22137 CFU/100 ml）和低浓度（5CFU/100 ml）、中浓度（980CFU/100 ml）、高浓度(114500 CFU/100 ml)实际样品进行了测定，实验室内的相对标准偏差范围分别为：4.3%5.9%、14%22%、2.4%4.0%、1.1%1.4%；实验室间的相对标准偏差分别为：0.81%、11.2%、0.92%、0.34%；重复性限为0.32、0.42、0.25、0.15；再现性限为0.32、0.46、0.25、0.14。10.2 准确度多家实验室对肠球菌有证标准样品（22137 CFU/100 ml）进行测定，实验室内相对误差的范围是-1.4%-2.9%，相对误差的最终值为-2.0%1.6%。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。11 质量保证和质量控制每批次样品均需进行空白对照试验。每批次实验至少取待测样品数量的10 %，进行平行双样分析,其检测所得数值全部经对数（以10为底）转换后数值的相对标准偏差25%。不同批次培养基需用有证标准菌株/标准样品进行培养基质量检验，其测定值应在保证值范围内。12 废物处理实验产生的废物经121 高压蒸汽灭菌20 min后，作为一般废物处理。13 注意事项在进行结果判读时，实验人员应带手套在黑暗环境下进行，确保荧光对准定量盘，避免直射眼睛和皮肤。附录A（规范性附录）97孔定量盘法MPN表（CFU/100 ml）大孔阳性格数小孔阳性格数0123456789101112131415161718192021222324011.02.03.04.05.06.07.08.09.010.011.012.013.014.115.116.117.118.119.120.221.222.223.324.311.02.03.04.05.06.07.18.19.110.111.112.113.214.215.216.217.318.319.320.421.422.423.524.525.622.03.04.15.16.17.18.19.210.211.212.213.314.315.416.417.418.519.520.621.622.723.724.825.826.933.14.15.16.17.28.29.210.311.312.413.414.515.516.517.618.619.720.821.822.923.925.026.127.128.244.15.26.27.28.39.310.411.412.513.514.615.616.717.818.819.921.022.023.124.225.326.327.428.529.655.26.37.38.49.410.511.512.613.714.715.816.917.919.020.121.222.223.324.425.526.627.728.829.931.066.37.48.49.510.611.612.713.814.916.017.018.119.220.321.422.523.624.725.826.928.029.130.231.332.477.58.59.610.711.812.813.915.016.117.218.319.420.521.622.723.824.926.027.128.329.430.531.632.833.988.69.710.811.913.014.115.216.317.418.519.620.721.822.924.125.226.327.428.629.730.832.033.134.335.499.810.912.013.114.215.316.417.618.719.820.922.023.224.325.426.627.728.930.031.232.333.534.635.837.01011.012.113.214.415.516.617.718.920.021.122.323.424.625.726.928.029.230.331.532.733.835.036.237.438.61112.213.414.515.616.817.919.120.221.422.523.724.826.027.228.329.530.731.933.034.235.436.637.839.040.21213.514.615.816.918.119.320.421.622.823.925.126.327.528.629.831.032.233.434.635.837.038.239.540.741.91314.816.017.118.319.520.621.823.024.225.426.627.829.030.231.432.633.835.036.237.538.739.941.242.443.61416.117.318.519.720.922.123.324.525.726.928.129.330.531.733.034.235.436.737.939.140.441.642.944.245.41517.518.719.921.122.323.524.725.927.228.429.630.932.133.334.635.837.138.439.640.942.243.444.746.047.31618.920.121.322.623.825.026.227.528.730.031.232.533.735.036.337.538.840.141.442.744.045.346.647.949.21720.321.622.824.125.326.627.829.130.331.632.934.135.436.738.039.340.641.943.244.545.947.248.549.851.21821.823.124.325.626.928.129.430.732.033.334.635.937.238.539.841.142.443.845.146.547.849.250.551.953.21923.324.625.927.228.529.831.132.433.735.036.337.639.040.341.643.044.345.747.148.449.851.252.654.055.42024.926.227.528.830.131.532.834.135.436.838.139.540.842.243.644.946.347.749.150.551.953.354.756.157.62126.527.929.230.531.833.234.535.937.338.640.041.442.844.145.546.948.449.851.252.654.155.556.958.459.92228.229.530.932.333.635.036.437.739.140.541.943.344.846.247.649.050.551.953.454.856.357.859.360.862.32329.931.332.734.135.536.838.339.741.142.543.945.446.848.349.751.252.754.255.657.158.660.261.763.264.72431.733.134.535.937.338.840.241.743.144.646.047.549.050.552.053.555.056.558.059.561.162.664.265.867.32533.635.036.437.939.340.842.243.745.246.748.249.751.252.754.355.857.358.960.562.063.665.266.868.470.02635.536.938.439.941.442.844.345.947.448.950.452.053.555.156.758.259.861.463.064.766.367.969.671.272.92737.438.940.442.043.545.046.548.149.651.252.854.456.057.659.260.862.464.165.767.469.170.872.574.275.92839.541.042.644.145.747.348.850.452.053.655.256.958.560.261.863.565.266.968.670.372.073.775.577.379.02941.743.244.846.448.049.651.252.854.556.157.859.561.262.964.666.368.069.871.573.375.176.978.780.582.43043.945.547.148.750.452.053.755.457.158.860.562.264.065.767.569.371.072.974.776.578.380.282.184.085.93146.247.949.551.252.954.656.358.159.861.663.365.166.968.770.572.474.276.178.079.981.883.785.787.689.63248.750.452.153.855.657.359.160.962.764.566.368.270.071.973.875.777.679.581.583.585.487.589.591.593.63351.253.054.856.558.360.262.063.865.767.669.571.473.375.277.279.281.283.285.287.389.391.493.695.797.83453.955.757.659.461.363.165.067.068.970.872.874.876.878.880.882.985.087.189.291.493.595.797.9100.2102.43556.858.660.562.464.466.368.370.372.374.376.378.480.582.684.786.989.191.393.595.798.0100.3102.6105.0107.33659.861.763.765.767.769.771.773.875.978.080.182.384.586.788.991.293.595.898.1100.5102.9105.3107.7110.2112.73762.965.067.069.171.273.375.477.679.882.084.286.588.891.193.495.898.2100.6103.1105.6108.1110.7113.3115.9118.63866.368.470.672.774.977.179.481.683.986.288.691.093.495.898.3100.8103.4105.9108.6111.2113.9116.6119.4122.2125.03970.072.274.476.778.981.383.686.088.490.993.495.998.4101.0103.6106.3109.0111.8114.6117.4120.3123.2126.1129.2132.24073.876.278.580.983.385.788.290.893.395.998.5101.2103.9106.7109.5112.4115.3118.2121.2124.3127.4130.5133.7137.0140.34178.080.583.085.588.090.693.395.998.7101.4104.3107.1110.0113.0116.0119.1122.2125.4128.7132.0135.4138.8142.3145.9149.54282.685.287.890.593.296.098.8101.7104.6107.6110.6113.7116.9120.1123.4126.7130.1133.6137.2140.8144.5148.3152.2156.1160.24387.690.493.296.099.0101.9105.0108.1111.2114.5117.8121.1124.6128.1131.7135.4139.1143.0147.0151.0155.2159.4163.8168.2172.84493.196.199.1102.2105.4108.6111.9115.3118.7122.3125.9129.6133.4137.4141.4145.5149.7154.1158.5163.1167.9172.7177.7182.9188.24599.3102.5105.8109.2112.6116.2119.8123.6127.4131.4135.4139.6143.9148.3152.9157.6162.4167.4172.6178.0183.5189.2195.1201.2207.546106.3109.8113.4117.2121.0125.0129.1133.3137.6142.1146.7151.5156.5161.6167.0172.5178.2184.2190.4196.8203.5210.5217.8225.4233.347114.3118.3122.4126.6130.9135.4140.1145.0150.0155.3160.7166.4172.3178.5185.0191.8198.9206.4214.2222.4231.0240.0249.5259.5270.048123.9128.4133.1137.9143.0148.3153.9159.7165.8172.2178.9186.0193.5201.4209.8218.7228.2238.2248.9260.3272.3285.1298.7313.0328.249135.5140.8146.4152.3158.5165.0172.0179.3187.2195.6204.6214.3224.7235.9248.1261.3275.5290.9307.6325.5344.8365.4387.3410.6435.2续表大孔阳性格数小孔阳性格数252627282930313233343536373839404142434445464748025.3 26.4 27.4 28.4 29.5 30.5 31.5 32.6 33.6 34.7 35.7 36.8 37.8 38.9 40.0 41.0 42.1 43.1 44.2 45.3 46.3 47.4 48.5 49.5 126.6 27.7 28.7 29.8 30.8 31.9 32.9 34.0 35.0 36.1 37.2 38.2 39.3 40.4 41.4 42.5 43.6 44.7 45.7 46.8 47.9 49.0 50.1 51.2 227.9 29.0 30.0 31.1 32.2 33.2 34.3 35.4 36.5 37.5 38.6 39.7 40.8 41.9 43.0 44.0 45.1 46.2 47.3 48.4 49.5 50.6 51.7 52.8 329.3 30.4 31.4 32.5 33.6 34.7 35.8 36.8 37.9 39.0 40.1 41.2 42.3 43.4 44.5 45.6 46.7 47.8 48.9 50.0 51.2 52.3 53.4 54.5 430.7 31.8 32.8 33.9 35.0 36.1 37.2 38.3 39.4 40.5 41.6 42.8 43.9 45.0 46.1 47.2 48.3 49.5 50.6 51.7 52.9 54.0 55.1 56.3 532.1 33.2 34.3 35.4 36.5 37.6 38.7 39.9 41.0 42.1 43.2 44.4 45.5 46.6 47.7 48.9 50.0 51.2 52.3 53.5 54.6 55.8 56.9 58.1 633.5 34.7 35.8 36.9 38.0 39.2 40.3 41.4 42.6 43.7 44.8 46.0 47.1 48.3 49.4 50.6 51.7 52.9 54.1 55.2 56.4 57.6 58.7 59.9 735.0 36.2 37.3 38.4 39.6 40.7 41.9 43.0 44.2 45.3 46.5 47.7 48.8 50.0 51.2 52.3 53.5 54.7 55.9 57.1 58.3 59.4 60.6 61.8 836.6 37.7 38.9 40.0 41.2 42.3 43.5 44.7 45.9 47.0 48.2 49.4 50.6 51.8 53.0 54.1 55.3 56.5 57.7 59.0 60.2 61.4 62.6 63.8 938.1 39.3 40.5 41.6 42.8 44.0 45.2 46.4 47.6 48.8 50.0 51.2 52.4 53.6 54.8 56.0 57.2 58.4 59.7 60.9 62.1 63.4 64.6 65.8 1039.7 40.9 42.1 43.3 44.5 45.7 46.9 48.1 49.3 50.6 51.8 53.0 54.2 55.5 56.7 57.9 59.2 60.4 61.7 62.9 64.2 65.4 66.7 67.9 1141.4 42.6 43.8 45.0 46.3 47.5 48.7 49.9 51.2 52.4 53.7 54.9 56.1 57.4 58.6 59.9 61.2 62.4 63.7 65.0 66.3 67.5 68.8 70.1 1243.1 44.3 45.6 46.8 48.1 49.3 50.6 51.8 53.1 54.3 55.6 56.8 58.1 59.4 60.7 62.0 63.2 64.5 65.8 67.1 68.4 69.7 71.0 72.4 1344.9 46.1 47.4 48.6 49.9 51.2 52.5 53.7 55.0 56.3 57.6 58.9 60.2 61.5 62.8 64.1 65.4 66.7 68.0 69.3 70.7 72.0 73.3 74.7 1446.7 48.0 49.3 50.5 51.8 53.1 54.4 55.7 57.0 58.3 59.6 60.9 62.3 63.6 64.9 66.3 67.6 68.9 70.3 71.6 73.0 74.4 75.7 77.1 1548.6 49.9 51.2 52.5 53.8 55.1 56.4 57.8 59.1 60.4 61.8 63.1 64.5 65.8 67.2 68.5 69.9 71.3 72.6 74.0 75.4 76.8 78.2 79.6 1650.5 51.8 53.2 54.5 55.8 57.2 58.5 59.9 61.2 62.6 64.0 65.3 66.7 68.1 69.5 70.9 72.3 73.7 75.1 76.5 77.9 79.3 80.8 82.2 1752.5 53.9 55.2 56.6 58.0 59.3 60.7 62.1 63.5 64.9 66.3 67.7 69.1 70.5 71.9 73.3 74.8 76.2 77.6 79.1 80.5 82.0 83.5 84.9 1854.6 56.0 57.4 58.8 60.2 61.6 63.0 64.4 65.8 67.2 68.6 70.1 71.5 73.0 74.4 75.9 77.3 78.8 80.3 81.8 83.3 84.8 86.3 87.8 1956.8 58.2 59.6 61.0 62.4 63.9 65.3 66.8 68.2 69.7 71.1 72.6 74.1 75.5 77.0 78.5 80.0 81.5 83.1 84.6 86.1 87.6 89.2 90.7 2059.0 60.4 61.9 63.3 64.8 66.3 67.7 69.2 70.7 72.2 73.7 75.2 76.7 78.2 79.8 81.3 82.8 84.4 85.9 87.5 89.1 90.7 92.2 93.8 2161.3 62.8 64.3 65.8 67.3 68.8 70.3 71.8 73.3 74.9 76.4 77.9 79.5 81.1 82.6 84.2 85.8 87.4 89.0 90.6 92.2 93.8 95.4 97.1 2263.8 65.3 66.8 68.3 69.8 71.4 72.9 74.5 76.1 77.6 79.2 80.8 82.4 84.0 85.6 87.2 88.9 90.5 92.1 93.8 95.5 97.1 98.8 100.5 2366.3 67.8 69.4 71.0 72.5 74.1 75.7 77.3 78.9 80.5 82.2 83.8 85.4 87.1 88.7 90.4 92.1 93.8 95.5 97.2 98.9 100.6 102.4 104.1 2468.9 70.5 72.1 73.7 75.3 77.0 78.6 80.3 81.9 83.6 85.2 86.9 88.6 90.3 92.0 93.8 95.5 97.2 99.0 100.7 102.5 104.3 106.1 107.9 2571.7 73.3 75.0 76.6 78.3 80.0 81.7 83.3 85.1 86.8 88.5 90.2 92.0 93.7 95.5 97.3 99.1 100.9 102.7 104.5 106.3 108.2 110.0 111.9 2674.6 76.3 78.0 79.7 81.4 83.1 84.8 86.6 88.4 90.1 91.9 93.7 95.5 97.3 99.2 101.0 102.9 104.7 106.6 108.5 110.4 112.3 114.2 116.2 2777.6 79.4 81.1 82.9 84.6 86.4 88.2 90.0 91.9 93.7 95.5 97.4 99.3 101.2 103.1 105.0 106.9 108.8 110.8 112.7 114.7 116.7 118.7 120.7 2880.8 82.6 84.4 86.3 88.1 89.9 91.8 93.7 95.6 97.5 99.4 101.3 103.3 105.2 107.2 109.2 111.2 113.2 115.2 117.3 119.3 121.4 123.5 125.6 2984.2 86.1 87.9 89.8 91.7 93.7 95.6 97.5 99.5 101.5 103.5 105.5 107.5 109.5 111.6 113.7 115.7 117.8 120.0 122.1 124.2 126.4 128.6 130.8 3087.8 89.7 91.7 93.6 95.6 97.6 99.6 101.6 103.7 105.7 107.8 109.9 112.0 114.2 116.3 118.5 120.6 122.8 125.1 127.3 129.5 131.8 134.1 136.4 3191.6 93.6 95.6 97.7 99.7 101.8 103.9 106.0 108.2 110.3 112.5 114.7 116.9 119.1 121.4 123.6 125.9 128.2 130.5 132.9 135.3 137.7 140.1 142.5 3295.7 97.8 99.9 102.0 104.2 106.3 108.5 110.7 113.0 115.2 117.5 119.8 122.1 124.5 126.8 129.2 131.6 134.0 136.5 139.0 141.5 144.0 146.6 149.1 33100.0 102.2 104.4 106.6 108.9 111.2 113.5 115.8 118.2 120.5 122.9 125.4 127.8 130.3 132.8 135.3 137.8 140.4 143.0 14