水質(zhì)　腸球菌的測(cè)定　酶底物法

水質(zhì)腸球菌的測(cè)定酶底物法警告：腸球菌為致病微生物，應(yīng)該在相應(yīng)級(jí)別的生物安全實(shí)驗(yàn)室中檢測(cè)，操作時(shí)應(yīng)按要求佩戴生物防護(hù)用品，注意廢棄物的生物消毒。范圍本標(biāo)準(zhǔn)規(guī)定了測(cè)定生活飲用水及其水源水中腸球菌的酶底物法。本標(biāo)準(zhǔn)適用于生活飲用水及其水源水中腸球菌的測(cè)定。當(dāng)水樣體積為100?mL時(shí)，本方法的檢測(cè)限為1?MPN/100?mL。規(guī)范性引用文件下列文件對(duì)于本文件的應(yīng)用是必不可少的。凡是注日期的引用文件，僅所注日期的版本適用于本文件。凡是不注日期的引用文件，其最新版本（包括所有的修改單）適用于本文件。GB/T5750.2—2006生活飲用水標(biāo)準(zhǔn)檢驗(yàn)方法水樣的采集與保存GB/T6682分析實(shí)驗(yàn)室用水規(guī)格和試驗(yàn)方法方法原理腸球菌在41?℃溫度條件下培養(yǎng)24?h，能夠產(chǎn)生β-D-葡糖苷酶，將選擇性培養(yǎng)基中熒光底物4-甲基傘形酮β-D-葡糖苷（4-MUG）分解，分解后的熒光底物在366?nm紫外光下產(chǎn)生熒光，統(tǒng)計(jì)陽(yáng)性反應(yīng)數(shù)量，通過(guò)查詢MPN表，計(jì)算樣品中的腸球菌濃度。試劑和材料市售Enterolert培養(yǎng)基，也可以使用其他等效產(chǎn)品。生理鹽水：氯化鈉濃度為0.85?%。標(biāo)準(zhǔn)菌株：糞腸球菌有證標(biāo)準(zhǔn)菌株，大腸埃希氏菌有證標(biāo)準(zhǔn)菌株。無(wú)菌水：實(shí)驗(yàn)用水滿足GB/T6682二級(jí)及以上分析實(shí)驗(yàn)室用水要求，經(jīng)121?℃高壓蒸汽滅菌20?min，備用。硫代硫酸鈉：純度≥98?%。硫代硫酸鈉溶液（5.0?g/L）：稱取0.5?g硫代硫酸鈉（4.5），精確至0.1?g，溶于適量水中，定容至100?mL，121?℃高壓滅菌20?min，備用。磷酸鹽緩沖液（PBS）：氯化鈉8.01?g氯化鉀0.2?g磷酸氫二鈉1.44?g磷酸二氫鉀0.24?g上述成分溶解于950?mL無(wú)菌水（4.4）中，用鹽酸調(diào)節(jié)溶液pH值到7.4后加無(wú)菌水（4.4）定容至1?000?mL，121?℃高壓滅菌20?min，備用。儀器和設(shè)備采樣瓶：具螺旋蓋或磨口塞的100?mL、500?mL玻璃瓶。恒溫培養(yǎng)箱：允許溫度偏差41?℃±1?℃。高壓蒸汽滅菌器：121?℃可調(diào)。程控定量封口機(jī)：用于定量盤的封口。紫外燈及配套燈箱：366?nm。定量盤：51孔或97孔。天平：精確至0.001?g。容量瓶：10?mL和100?mL。樣品使用滅菌處理的玻璃瓶（也可選用有效期內(nèi)市售無(wú)菌采樣瓶和采樣袋）采集水樣，采樣量不少于200?mL。加入硫代硫酸鈉溶液（4.6）去除殘留余氯（每125?mL容積加入20?μL的硫代硫酸鈉溶液，水源水腸球菌測(cè)定忽略此步），樣品采集與保存按照GB/T5750.2—2006中微生物指標(biāo)要求執(zhí)行。分析步驟定性分析用滅菌處理過(guò)的100?mL采樣瓶（5.1）量取100?mL水樣，加入一包培養(yǎng)基（4.1），混搖均勻使之完全溶解，放入恒溫培養(yǎng)箱（5.2）中41?℃±1?℃培養(yǎng)24?h。定量分析用滅菌處理過(guò)的100?mL采樣瓶（5.1）量取100?mL水樣，加入一包培養(yǎng)基（4.1），混搖均勻使之完全溶解。將上述100?mL水樣全部倒入51孔無(wú)菌定量盤（5.6）內(nèi)，撫XX量盤背面以趕除孔穴內(nèi)氣泡，然后用程控定量封口機(jī)（5.4）封口，放入恒溫培養(yǎng)箱（5.2）中41?℃±1?℃培養(yǎng)24?h。空白對(duì)照選用無(wú)菌水（4.4）按照7.1～7.2步驟進(jìn)行空白對(duì)照測(cè)定。結(jié)果計(jì)算與報(bào)告定性結(jié)果定性分析（7.1）的樣品，在紫外燈（5.5）下觀察，產(chǎn)生熒光，則判定為陽(yáng)性反應(yīng)；不產(chǎn)生熒光，則判定為陰性反應(yīng)。定性反應(yīng)結(jié)果以腸球菌“陽(yáng)性（+）”或“陰性（-）”報(bào)告。定量結(jié)果定量分析（7.2）的樣品，在紫外燈（5.5）下觀察培養(yǎng)24h的定量盤，計(jì)數(shù)有熒光反應(yīng)的孔數(shù)，參照附錄A表A.1計(jì)數(shù)定量，結(jié)果以MPN/100?mL表示。如所有孔穴均未產(chǎn)生熒光，則報(bào)告腸球菌“未檢出”。若熒光不明顯，可以XX培養(yǎng)時(shí)間至28h進(jìn)行結(jié)果判讀，超過(guò)28h后出現(xiàn)的顏色反應(yīng)，不作為陽(yáng)性結(jié)果。對(duì)于腸球菌濃度較高的樣品，可選用97孔定量盤或采用無(wú)菌生理鹽水（4.2）對(duì)樣品1:10梯度稀釋后進(jìn)行多梯度檢測(cè)，其他操作步驟同51孔定量盤法一致，結(jié)果檢索表見(jiàn)附錄B表B.1～表B.4，乘以稀釋倍數(shù)報(bào)告。精密度和準(zhǔn)確度精密度6個(gè)實(shí)驗(yàn)室對(duì)腸球菌濃度＜30?MPN/100?mL的樣品定性結(jié)果一致，對(duì)濃度為20.4?MPN/100?mL～46.3?MPN/100?mL、73.1?MPN/100?mL～146.0?MPN/100?m/L的樣品7次重復(fù)測(cè)定，相對(duì)標(biāo)準(zhǔn)偏差分別為1.4?%～11.2?%和1.4?%～6.4?%。準(zhǔn)確度6個(gè)實(shí)驗(yàn)室分別測(cè)定濃度為166±32?MPN/100?mL的腸球菌質(zhì)控樣品，測(cè)XX果與認(rèn)證值的相對(duì)誤差在-4.8?%～0.8?%范圍內(nèi)。微生物檢測(cè)數(shù)據(jù)為偏態(tài)分布，上述精密度、準(zhǔn)確度均為對(duì)測(cè)XX果以10取對(duì)數(shù)后進(jìn)行計(jì)算。質(zhì)量保證和質(zhì)量控制空白對(duì)照每批樣品進(jìn)行空白對(duì)照測(cè)定，選用無(wú)菌水（4.4）按照7.3步驟進(jìn)行空白對(duì)照測(cè)定，培養(yǎng)后的取樣瓶或定量盤應(yīng)為陰性。否則，該批次樣品測(cè)XX果無(wú)效，應(yīng)查明原因后重新測(cè)定。陽(yáng)性對(duì)照和陰性對(duì)照定期使用糞腸球菌標(biāo)準(zhǔn)菌株（4.3）和大腸埃希氏菌標(biāo)準(zhǔn)菌株（4.3）作為陽(yáng)性和陰性對(duì)照菌株，對(duì)檢測(cè)質(zhì)量進(jìn)行控制。將標(biāo)準(zhǔn)菌株制備成菌濃度＞10?MPN/100?mL的菌懸液，按照7.1～7.2步驟進(jìn)行分析測(cè)定，陽(yáng)性菌株呈陽(yáng)性反應(yīng)，陰性菌株呈陰性反應(yīng)。否則，該批次樣品測(cè)XX果無(wú)效，應(yīng)查明原因后重新測(cè)定。培養(yǎng)基質(zhì)量控制對(duì)每批次培養(yǎng)基須選用有證標(biāo)準(zhǔn)菌株或標(biāo)準(zhǔn)樣品進(jìn)行培養(yǎng)基質(zhì)量檢驗(yàn)。廢物處理實(shí)驗(yàn)中產(chǎn)生的廢棄物經(jīng)高壓蒸汽滅菌器（5.3）121?℃滅菌20?min后，作為一般廢物處理。

（規(guī)范性附錄）

51孔定量盤法不同陽(yáng)性結(jié)果的最可能數(shù)（MPN）及95?%可信范圍51孔定量盤法不同陽(yáng)性結(jié)果的最可能數(shù)（MPN）及95?%可信范圍見(jiàn)表A.1。51孔定量盤法不同陽(yáng)性結(jié)果的最可能數(shù)（MPN）及95?%可信范圍陽(yáng)性數(shù)腸球菌MPN/100?mL95?%置信范圍MPN/100?mL下限上限0＜10.03.711.00.35.622.00.67.333.11.19.044.21.710.755.32.312.366.43.013.977.53.715.588.74.517.199.95.318.81011.16.120.51112.47.022.11213.77.923.91315.08.825.71416.49.827.51517.810.829.41619.211.931.31720.713.033.31822.214.135.21923.815.337.32025.416.539.42127.117.741.62228.819.043.92330.620.446.32432.421.848.72534.423.351.22636.424.753.92738.426.456.62840.628.059.5表A.151孔定量盤法不同陽(yáng)性結(jié)果的最可能數(shù)（MPN）及95%可信范圍（續(xù)）陽(yáng)性數(shù)腸球菌MPN/100?mL95?%置信范圍MPN/100?mL下限上限2942.929.762.53045.331.565.63147.833.469.03250.435.472.53353.137.576.23456.039.780.13559.142.084.43662.444.688.83765.947.293.73869.750.099.03973.853.1104.84078.256.4111.24183.159.9118.34288.563.9126.24394.568.2135.444101.373.1146.045109.178.6158.746118.485.0174.547129.892.7195.048144.5102.3224.149165.2115.2272.250200.5135.8387.651＞200.5146.1—

（規(guī)范性附錄）

97孔定量盤法MPN表97孔定量盤法MPN表見(jiàn)表B.1～表B.4。97孔定量盤法MPN表（MPN/100mL）（小孔0～11）大孔陽(yáng)性數(shù)小孔陽(yáng)性數(shù)012345678910110＜1.01.02.03.04.05.06.07.08.09.010.011.011.02.03.04.05.06.07.18.19.110.111.112.122.03.04.15.16.17.18.19.210.211.212.213.333.14.15.16.17.28.29.210.311.312.413.414.544.15.26.27.28.39.310.411.412.513.514.615.655.26.37.38.49.410.511.512.613.714.715.816.966.37.48.49.510.611.612.713.814.916.017.018.177.58.59.610.711.812.813.915.016.117.218.319.488.69.710.811.913.014.115.216.317.418.519.620.799.810.912.013.114.215.316.417.618.719.820.922.01011.012.113.214.415.516.617.718.920.021.122.323.41112.213.414.515.616.817.919.120.221.422.523.724.81213.514.615.816.918.119.320.421.622.823.925.126.31314.816.017.118.319.520.621.823.024.225.426.627.81416.117.318.519.720.922.123.324.525.726.928.129.31517.518.719.921.122.323.524.725.927.228.429.630.91618.920.121.322.623.825.26.227.528.730.031.232.51720.321.622.824.125.326.627.829.130.331.632.934.11821.823.124.325.626.928.129.430.732.033.334.635.91923.324.625.927.228.529.831.132.433.735.036.337.62024.926.227.528.830.131.532.834.135.436.838.139.52126.527.929.230.531.833.234.535.937.338.640.041.42228.229.530.932.333.635.036.437.739.140.541.943.32329.931.332.734.135.536.838.339.741.142.543.945.42431.733.134.535.937.338.840.241.743.144.646.047.52533.635.036.437.939.340.842.243.745.246.748.249.72635.536.938.439.941.442.844.345.947.448.950.452.02737.438.940.442.043.545.046.548.149.651.252.854.42839.541.042.644.145.747.348.850.452.053.655.256.9表B.197孔定量盤法MPN表（MPN/100mL）（小孔0～11）（續(xù)）大孔陽(yáng)性數(shù)小孔陽(yáng)性數(shù)012345678910112941.743.244.846.448.049.651.252.854.556.157.859.53043.945.547.148.750.452.053.755.457.158.860.562.23146.247.949.551.252.954.656.358.159.861.663.365.13248.750.452.153.855.657.359.160.962.764.566.368.23351.253.054.856.558.360.262.063.865.767.669.571.43453.955.757.659.461.363.165.067.068.970.872.874.83556.858.660.562.464.466.368.370.372.374.376.378.43659.861.763.765.767.769.771.773.875.978.080.182.33762.965.067.069.171.273.375.477.679.882.084.286.53866.368.470.672.774.977.179.481.683.986.288.691.03970.072.274.476.778.981.383.686.088.490.993.495.94073.876.278.580.983.385.788.290.893.395.998.5101.24178.080.583.085.588.090.693.395.998.71001.4104.3107.14282.685.287.890.593.296.098.8101.7104.6107.6110.6113.74387.690.493.296.099.0101.9105.0108.1111.2114.5117.8121.14493.196.199.1102.2105.4108.6111.9115.3118.7122.3125.9129.64599.3102.5105.8109.2112.6116.2119.8123.6127.4131.4135.4139.646106.3109.8113.4117.2121.0125.0129.1133.3137.6142.1146.7151.547114.3118.3122.4126.6130.9135.4140.1145.0150.0155.3160.7166.448123.9128.4133.1137.9143.0148.3153.9159.7165.8172.2178.9186.049135.5140.8146.4152.3158.5165.0172.0179.3187.2195.6204.6214.397孔定量盤法MPN表（MPN/100mL）（小孔12～23）大孔陽(yáng)性數(shù)小孔陽(yáng)性數(shù)121314151617181920212223012.013.014.115.116.117.118.119.120..221.222.224.3113.214.215.216.217.318.319.320.421.422.423.524.5214.315.416.417.418.519.520.621.622.723.724.825.8315.516.517.618.619.720.821.822.923.925.026.127.1416.717.818.819.921.022.023.124.225.326.327.428.5517.919.020.121.222.223.324.425.526.627.728.829.9619.220.321.422.523.624.725.826.928.029.130.231.3720.521.622.723.824.926.027.128.329.430.531.632.8821.822.924.125.226.327.428.629.730.832.033.134.3923.224.325.426.627.728.930.031.232.333.534.635.81024.625.726.928.029.230.331.532.733.835.036.237.41126.027.228.329.530.731.933.034.235.436.637.839.0表B.297孔定量盤法MPN表（MPN/100mL）（小孔12～23）（續(xù)）大孔陽(yáng)性數(shù)小孔陽(yáng)性數(shù)1213141516171819202122231227.528.629.831.032.233.434.635.837.038.239.540.71329.030.231.432.633.835.036.237.538.739.941.242.41430.531.733.034.235.436.737.939.140.441.642.944.21532.133.334.635.837.138.439.640.942.243.444.746.01633.735.036.337.538.840.141.442.744.045.346.647.91735.436.738.039.340.641.943.244.545.947.248.549.81837.238.539.841.142.443.845.146.547.849.250.551.91939.040.341.643.044.345.747.148.449.851.252.654.02040.842.243.644.946.347.749.150.551.953.354.756.12142.844.145.546.948.449.851.252.654.155.556.958.42244.846.247.649.050.551.953.454.856.357.859.360.82346.848.349.751.252.754.255.657.158.660.261.763.22449.050.552.053.555.056.558.059.561.162.664.265.82551.252.754.355.857.358.960.562.063.665.266.868.42653.555.156.758.259.861.463.064.766..367.969.671.22756.057.659.260.862.464.165.767.469.170.872.574.22858.560.261.863.565.266.968.670.372.073.775.577.32961.262.964.666.368.069.871.573.375.176.978.780.53064.065.767.569.371.072.974.776.578.380.282.184.03166.968.770.572.474.276.178.079.981.883.785.787.63270.071.973.875.777.679.581.583.585.487.589.591.53373.375.277.279.281.283.285.287.389.391.493.695.73476.878.880.882.985.087.189.291.493.595.797.9100.23580.582.684.786.989.191.393.595.798.0100.3102.6105.03684.586.788.991.293.595.898.1100.5102.9105.3107.7110.23788.891.193.495.898.2100.6103.1105.6108.1110.7113.3115.93893.495.898.3100.8103.4105.9108.6111.2113.9116.6119.4122.23998.4101.0103.6106.3109.0111.8114.6117.4120.3123.2126.1129.240103.9106.7109.5112.4115.3118.2121.2124.3127.4130.5133.7137.041110.0113.0116.0119.1122.2125.4128.7132.0135.4138.8142.3145.942116.9120.1123.4126.7130.1133.6137.2140.8144.5148.3152.2156.143124.6128.1131.7135.4139.1143.0147.0151.0155.2159.4163.8168.244133.4137.4141.4145.5149.7154.1158.5163.1167.9172.7177.7182.945143.9148.3152.9157.6162.4167.4172.6178.0183.5189.2195.1201.246156.5161.6167.0172.5178.2184.2190.4196.8203.5210.5217.8225.447172.3178.5185.0191.8198.9206.4214.2222.4231.0240.0249.5259.548193.5201.4209.8218.7228.2238.2248.9260.3272.3285.1298.7313.049224.7235.9248.1261.3275.5290.9307.6325.5344.8365.4387.3410.697孔定量盤法MPN表（MPN/100mL）（小孔24～35）大孔陽(yáng)性數(shù)小孔陽(yáng)性數(shù)242526272829303132333435024.325.326.427.428.429.530.531.532.633.634.735.7125.626.627.728.729.830.831.932.934.035.036.137.2226.927.929.030.031.132.233.234.335.436.537.538.6328.229.330.431.432.533.634.735.836.839.739.040.1429.630.731.832.833.935.036.137.238.339.440.541.6531.032.133.234.335.436.537.638.739.941.042.143.2632.433.534.735.836.938.039.240.341.442.643.744.8733.935.036.237.338.439.640.741.943.044.245.346.5835.436.637.738.940.041.242.343.544.745.947.048.2937.038.139.340.541.642.844.045.246.447.648.850.01038.639.740.942.143.344.545.746.948.149.350.651.81140.241.442.643.845.046.347.548.749.951.252.453.71241.943.144.345.646.848.149.350.651.853.154.355.61343.644.946.147.448.649.951.252.553.755.056.357.61445.446.748.049.350.551.853.154.455.757.058.359.61547.348.649.951.252.553.855.156.457.859.160.461.81649.250.551.853.254.555.857.258.559.961.232.664.01751.252.553.955.256.658.059.360.762.163.564.966.31853.264.656.057.458.860.261.663.064.465.867.268.61955.456.858.259.661.062.463.965.366.868.269.771.12057.659.060.461.963.364.866.367.769.270.772.273.72159.961.332.834.365.867.368.870.371.873.374.976.42262.363.865.366.868.369.871.472.974.576.177.679.22364.766.367.869.471.072.574.175.777.378.980.582.22467.368.970.572.173.775.377.078.680.381.983.685.22570.071.773.375.076.678.380.081.783.385.186.888.52672.974.676.378.079.781.483.184.886.688.490.191.92775.977.679.481.182.984.686.488.290.091.993.795.52879.080.882.684.486.388.189.991.893.795.697.599.42982.484.286.187.989.891.793.795.697.599.5101.5103.53085.987.889.791.793.695.697.699.6101.6103.7105.7107.83189.691.693.695.697.799.7101.8103.9106.0108.2110.3112.53293.695.797.899.9102.0104.2106.3108.5110.7113.0115.2117.53397.8100.0102.2104.4106.6108.9111.2113.5115.8118.2120.5122.934102.4104.7107.0109.3111.7114.0116.4118.9121.3123.8126.3128.835107.3109.7112.2114.6117.1119.6122.2124.7127.3129.9132.6135.3表B.397孔定量盤法MPN表（MPN/100mL）（小孔24～35）（續(xù)）大孔陽(yáng)性數(shù)小孔陽(yáng)性數(shù)24252627282930313233343536112.7115.2117.8120.4123.0125.7128.4131.1133.9136.7139.5142.437118.6121.324.0126.8129.6132.4135.3138.2141.2144.2147.3150.338125.0127.9130.8133.8136.8139.9143.0146.2149.4152.6155.9159.239132.2135.3138.5141.7145.0148.3151.7155.1158.6162.1165.7169.440140.3143.7147.1150.6154.2157.8161.5165.3169.1173.0177.0181.141149.5153.2157.0160.9164.8168.9173.0177.2181.5185.8190.3194.842160.2164.3168.6172.9177.3181.9186.5191.3196.1201.1206.2211.443172.8177.5182.3187.3192.4197.6202.9208.4214.0219.8225.8231.844188.2193.6199.3205.1211.0217.2223.5230.0236.7243.6250.8258.145207.5214.1220.9227.9235.2242.7250.4258.4266.7275.3284.1293.346233.3241.5250.0258.9268.2277.8287.8298.1308.8319.9331.4343.347270.0280.9292.4304.4316.9330.0343.6357.8372.5387.740.4419.848328.2344.1360.9378.4396.8416.0436.0456.9478.6501.2524.7549.349435.2461.1488.4517.2547.5579.4613.1648.8686.7727.0770.1816.497孔定量盤法MPN表（MPN/100mL）（小孔36～48）大孔陽(yáng)性數(shù)小孔陽(yáng)性數(shù)36373839404142434445464748036.837.838.940.041.042.143.144.245.346.347.448.549.5138.239.340.441.442.543.544.745.746.847.949.050.151.2239.740.841.943.044.045.146.247.348.449.550.651.752.8341.242.343.444.545.646.747.848.950.051.252.353.454.5442.843.945.046.147.248.349.550.651.752.954.055.156.3544.445.546.647.748.950.051.252.353.554.655.856.958.1646.047.148.349.450.651.752.954.155.256.457.658.759.9747.748.850.051.252.353.554.755.957.158.359.460.661.8849.450.651.853.054.155.356.557.759.060.261.462.663.8951.252.453.654.856.057.258.459.760..962.163.464.665.81053.054.255.556.757.959.260.461.762.964.265.466.767.91154.956.157.458.659.961.262.463.765.066.367.568.870.11256.858.159.460.762.063.264.565.867.168.169.771.072.41358.960.261.562.864.165.466.768.069.370.772.073.374.71460.962.363.664.966.367.668.970.371.673.074.475.777.11563.164.565.867.268.569.971.372.674.075.476.878.279.61665.366.768.169.570.972.373.775.176.577.979.380.882.2表B.497孔定量盤法MPN表（MPN/100mL）（小孔36～48）（續(xù)）大孔陽(yáng)性數(shù)小孔陽(yáng)性數(shù)363738394041424344454647481767.769.170.571.973.374.876.277.679.180.582.083.384.91870.171.573.074.475.977.378.880.381.883.384.886.387.81972.674.175.577.078.580.081.583.184.686.187.689.290.72075.276.778.279.881.382.884.485.987.589.190.792.293.82177.979.581.182.684.285.887.489.090.692.293.895.497.12280.882.484.085.687.288.990.592.193.895.597.198.8100.52383.885.487.188.790.492.193.895.597.298.9100.6102.4104.12486.988.690.392.093.895.597.299.0100.7102.5104.3106.1107.92590.292.093.795.597.399.9100.9102.7104.5106.3108.2110.0111.92693.795.597.399.2101.0102.9104.7106.6108.5110.4112.3114.2116.22797.499.3101.2103.1105.0106.9108.8110.8112.7114.7116