DB21-T 3111-2019水質(zhì) 腸球菌的測定 固定底物技術酶底物法

ICS13.060

B21

備案號：62033-2019DB21

遼寧省地方標準

DB21/T3111—2019

水質(zhì)腸球菌的測定

固定底物技術酶底物法

Waterquality—DeterminationofEnterococci—Definedsubstratetechnology

enzymesubstratemethod

DB21/T3111—2019

前言

本標準依據(jù)GB/T1.1-2009《標準化工作導則第1部分：標準的結構和編寫》起草制訂。

本標準附錄A為規(guī)范性附錄。

本標準由大連市市場監(jiān)督管理局提出。

本標準由遼寧省生態(tài)環(huán)境廳歸口。

本標準起草單位：大連市環(huán)境監(jiān)測中心、國家海洋環(huán)境監(jiān)測中心。

本標準主要起草人：王中衛(wèi)、明紅霞、李翔宇、沈?qū)W崴、李玉璞、蘇潔、曲翊、王琳琳、劉子成、

孫沛雯、劉景泰、韓佳。

II

DB21/T3111—2019

水質(zhì)腸球菌的測定固定底物技術酶底物法

1范圍

本標準規(guī)定了測定水中腸球菌的固定底物技術酶底物法。

本標準適用于地表水、地下水、飲用水、海水、生活廢水和工業(yè)廢水中腸球菌的測定。

本標準的檢出限：地表水、地下水、飲用水、生活廢水、工業(yè)廢水樣品的檢出限為1CFU/100ml，

海水樣品的檢出限為10CFU/100ml。

2規(guī)范性引用文件

下列文件對于本文件的應用是必不可少的。凡是注日期的引用文件，僅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生活飲用水標準檢驗方法樣品的采集與保存

GB/T6682分析實驗室用水規(guī)格和試驗方法

GB17378.7海洋監(jiān)測規(guī)范第七部分：近海污染生態(tài)調(diào)查和生物監(jiān)測

HJ/T164地下水環(huán)境監(jiān)測技術規(guī)范

HJ/T91地表水和污水監(jiān)測技術規(guī)范

3術語和定義

下列術語和定義適用于本標準。

3.1腸球菌Enterococci

41℃培養(yǎng)24h，能產(chǎn)生β-半乳糖苷酶（β-Ｄ-galactosidase），分解選擇性培養(yǎng)基中的4-甲基

傘形酮-β-Ｄ-葡萄糖醛酸苷（4-methyl-umbelliferyl-β-Ｄ-glucoside，MUG）釋放出熒光物質(zhì)（4-

甲基傘形酮）的革蘭氏陽性菌。

3.2最大可能數(shù)mostprobablenumber（MPN）

又稱稀釋培養(yǎng)計數(shù)，是一種基于泊松分布的間接計數(shù)法。利用統(tǒng)計學原理，根據(jù)一定體積不同稀釋

度樣品經(jīng)培養(yǎng)后產(chǎn)生的目標微生物陽性數(shù)，查表估算一定體積樣品中目標微生物存在的數(shù)量（單位體積

存在目標微生物的最大可能數(shù)）。

4方法原理

1

DB21/T3111—2019

在特定溫度下培養(yǎng)特定的時間，腸球菌能產(chǎn)生β-半乳糖苷酶，分解熒光底物釋放出熒光產(chǎn)物，在

366nm紫外燈照射下產(chǎn)生特征性熒光。統(tǒng)計熒光反應出現(xiàn)數(shù)量，查MPN表，計算樣品中腸球菌的濃度值。

5試劑和材料

除非另有說明，分析時均使用符合國家標準的分析純試劑，符合GB/T6682三級標準的蒸餾水或去

離子水。

5.1培養(yǎng)基

市售Enterolert培養(yǎng)基，也可以使用其他等效產(chǎn)品。

5.2硫代硫酸鈉溶液：ρ（Na2S2O3·5H2O）=0.10g/ml

稱取硫代硫酸鈉（Na2S2O3·5H2O）10.0g，溶于適量實驗用水，定容至100ml，臨用現(xiàn)配。

5.3乙二胺四乙酸二鈉溶液：ρ（C10H14N2O8Na2·2H2O）=0.15g/ml

稱取乙二胺四乙酸二鈉（C10H14N2O8Na2·2H2O）15.0g，溶于適量實驗用水，定容至100ml，此溶液

保質(zhì)期為30d。

5.4無菌水

實驗用水經(jīng)121℃高壓蒸汽滅菌20min，備用。

6儀器和設備

6.1恒溫培養(yǎng)箱：能夠恒溫至41℃，控溫精度0.5℃。

6.2高壓蒸汽滅菌器：能夠加熱至121℃。

6.3紫外燈：臺式，366nm。

6.4封口機：用于定量盤的封口。

6.5采樣瓶：具螺旋帽或磨口塞的250ml玻璃瓶。

6.6三角瓶:100ml。

6.797孔定量盤。

6.8一般實驗室常用儀器和設備。

2

DB21/T3111—2019

注：移液管、量筒、采樣瓶、三角瓶等玻璃器皿和采樣器具應按無菌操作要求包扎，于121℃高壓蒸汽滅菌20min，

烘干，備用。滅菌后的采樣瓶，兩周內(nèi)未使用，需重新滅菌。

7樣品

7.1樣品采集與保存

按照GB/T5750.2、GB17378.7、HJ/T164和HJ/T91的相關規(guī)定進行樣品采集。采樣后水樣應

在4℃下冷藏保存，6h內(nèi)開始檢測。

7.2干擾和消除

7.2.1含活性氯（余氯）的水樣，在采樣瓶滅菌前需加入0.2ml硫代硫酸鈉溶液。

7.2.2含重金屬的水樣，在采樣瓶滅菌前需加入0.6ml乙二胺四乙酸二鈉溶液。

8分析步驟

8.1樣品稀釋

根據(jù)水樣污染程度確定稀釋度，避免接種水樣經(jīng)培養(yǎng)后出現(xiàn)全部陽性或全部陰性。接種量小于100

ml時，水樣應制成稀釋樣品后使用，接種量為10ml時，以無菌操作方法取10ml水樣加入到裝有90ml

無菌水的三角瓶（6.6）中混勻制成1:10（稀釋度）稀釋的樣品，其它接種量的稀釋樣品依次類推。

注：海水樣品至少經(jīng)1:10稀釋后再進行檢測。

8.2接種

以無菌操作方法量取100ml原水樣或稀釋樣品于三角瓶（6.6）中，加入1支培養(yǎng)基（5.1），充分

混勻，完全溶解后，全部倒入97孔定量盤（6.7）內(nèi)，以手撫平定量盤（6.7）背面，去除孔內(nèi)氣泡，然

后用封口機（6.4）封口。

8.3培養(yǎng)

將封口后的97孔定量盤（6.7）放入培養(yǎng)箱（6.1）中培養(yǎng)24h。

8.4對照試驗

8.4.1空白對照

使用無菌水（5.4）進行實驗室空白測定，培養(yǎng)后的97孔定量盤（6.7）不得有熒光產(chǎn)生，否則，該

次樣品測定結果無效，應重新測定。

8.4.2陰性及陽性對照

3

DB21/T3111—2019

陽性菌株參考使用糞腸球菌（Enterococcusfaecalis）,陰性菌株參考使用粘質(zhì)沙雷氏菌（Serratla

marcescens）和綠膿桿菌（Aerococcusviridians）。將菌株配制成0.5麥氏濁度的菌懸液按照8.2

和8.3要求操作，陽性菌株應呈陽性反應（有熒光），陰性菌株呈陰性反應（無熒光），否則該批次測

定結果無效，應重新測定。

8.5結果判讀與計數(shù)

將培養(yǎng)24h后的97孔定量盤（6.7）進行結果判讀，水樣在紫外燈（6.3）照射下有熒光，則判斷為

腸球菌陽性。若結果可疑，可延長培養(yǎng)時間到28h進行結果判讀，超過28h后出現(xiàn)的熒光反應不作為陽

性結果。

9結果計算與表示

9.1結果計算

從97孔定量盤法MPN表（附錄A）中查得每100ml水樣中腸球菌的MPN值，再根據(jù)樣品不同的稀釋度

進行計算，每100ml水樣中腸球菌的計算公式如下：

式中：C——每100ml水樣中腸球菌濃度，CFU/100ml；

MPN值——查表得到的MPN值，CFU/100ml；

f——接種樣品的稀釋度。

9.2結果表示

測定結果保留兩位有效數(shù)字，大于等于100時以科學計數(shù)法表示，結果單位為CFU/100ml。

10精密度和準確度

10.1精密度

多家實驗室分別對腸球菌有證標準樣品（221±37CFU/100ml）和低濃度（5CFU/100ml）、中濃

度（980CFU/100ml）、高濃度(114500CFU/100ml)實際樣品進行了測定，實驗室內(nèi)的相對標準偏差范

圍分別為：4.3%～5.9%、14%～22%、2.4%～4.0%、1.1%～1.4%；實驗室間的相對標準偏差分別為：0.81%、

11.2%、0.92%、0.34%；重復性限為0.32、0.42、0.25、0.15；再現(xiàn)性限為0.32、0.46、0.25、0.14。

10.2準確度

多家實驗室對腸球菌有證標準樣品（221±37CFU/100ml）進行測定，實驗室內(nèi)相對誤差的范圍是

-1.4%～-2.9%，相對誤差的最終值為-2.0%±1.6%。

注：微生物檢測數(shù)據(jù)為偏態(tài)分布，其測定結果全部經(jīng)以10為底對數(shù)轉(zhuǎn)換后進行計算。

4

DB21/T3111—2019

11質(zhì)量保證和質(zhì)量控制

每批次樣品均需進行空白對照試驗。每批次實驗至少取待測樣品數(shù)量的10%，進行平行雙樣分析,

其檢測所得數(shù)值全部經(jīng)對數(shù)（以10為底）轉(zhuǎn)換后數(shù)值的相對標準偏差≤25%。不同批次培養(yǎng)基需用有證

標準菌株/標準樣品進行培養(yǎng)基質(zhì)量檢驗，其測定值應在保證值范圍內(nèi)。

12廢物處理

實驗產(chǎn)生的廢物經(jīng)121℃高壓蒸汽滅菌20min后，作為一般廢物處理。

13注意事項

在進行結果判讀時，實驗人員應帶手套在黑暗環(huán)境下進行，確保熒光對準定量盤，避免直射眼睛和

皮膚。

5

DB21/T3111—2019

附錄A

（規(guī)范性附錄）

97孔定量盤法MPN表（CFU/100ml）

大孔陽小孔陽性格數(shù)

性格數(shù)0123456789101112131415161718192021222324

0<11.02.03.04.05.06.07.08.09.010.011.012.013.014.115.116.117.118.119.120.221.222.223.324.3

11.02.03.04.05.06.07.18.19.110.111.112.113.214.215.216.217.318.319.320.421.422.423.524.525.6

22.03.04.15.16.17.18.19.210.211.212.213.314.315.416.417.418.519.520.621.622.723.724.825.826.9

33.14.15.16.17.28.29.210.311.312.413.414.515.516.517.618.619.720.821.822.923.925.026.127.128.2

44.15.26.27.28.39.310.411.412.513.514.615.616.717.818.819.921.022.023.124.225.326.327.428.529.6

55.26.37.38.49.410.511.512.613.714.715.816.917.919.020.121.222.223.324.425.526.627.728.829.931.0

66.37.48.49.510.611.612.713.814.916.017.018.119.220.321.422.523.624.725.826.928.029.130.231.332.4

77.58.59.610.711.812.813.915.016.117.218.319.420.521.622.723.824.926.027.128.329.430.531.632.833.9

88.69.710.811.913.014.115.216.317.418.519.620.721.822.924.125.226.327.428.629.730.832.033.134.335.4

99.810.912.013.114.215.316.417.618.719.820.922.023.224.325.426.627.728.930.031.232.333.534.635.837.0

1011.012.113.214.415.516.617.718.920.021.122.323.424.625.726.928.029.230.331.532.733.835.036.237.438.6

1112.213.414.515.616.817.919.120.221.422.523.724.826.027.228.329.530.731.933.034.235.436.637.839.040.2

1213.514.615.816.918.119.320.421.622.823.925.126.327.528.629.831.032.233.434.635.837.038.239.540.741.9

1314.816.017.118.319.520.621.823.024.225.426.627.829.030.231.432.633.835.036.237.538.739.941.242.443.6

1416.117.318.519.720.922.123.324.525.726.928.129.330.531.733.034.235.436.737.939.140.441.642.944.245.4

1517.518.719.921.122.323.524.725.927.228.429.630.932.133.334.635.837.138.439.640.942.243.444.746.047.3

1618.920.121.322.623.825.026.227.528.730.031.232.533.735.036.337.538.840.141.442.744.045.346.647.949.2

1720.321.622.824.125.326.627.829.130.331.632.934.135.436.738.039.340.641.943.244.545.947.248.549.851.2

1821.823.124.325.626.928.129.430.732.033.334.635.937.238.539.841.142.443.845.146.547.849.250.551.953.2

1923.324.625.927.228.529.831.132.433.735.036.337.639.040.341.643.044.345.747.148.449.851.252.654.055.4

2024.926.227.528.830.131.532.834.135.436.838.139.540.842.243.644.946.347.749.150.551.953.354.756.157.6

2126.527.929.230.531.833.234.535.937.338.640.041.442.844.145.546.948.449.851.252.654.155.556.958.459.9

2228.229.530.932.333.635.036.437.739.140.541.943.344.846.247.649.050.551.953.454.856.357.859.360.862.3

2329.931.332.734.135.536.838.339.741.142.543.945.446.848.349.751.252.754.255.657.158.660.261.763.264.7

2431.733.134.535.937.338.840.241.743.144.646.047.549.050.552.053.555.056.558.059.561.162.664.265.867.3

2533.635.036.437.939.340.842.243.745.246.748.249.751.252.754.355.857.358.960.562.063.665.266.868.470.0

2635.536.938.439.941.442.844.345.947.448.950.452.053.555.156.758.259.861.463.064.766.367.969.671.272.9

2737.438.940.442.043.545.046.548.149.651.252.854.456.057.659.260.862.464.165.767.469.170.872.574.275.9

2839.541.042.644.145.747.348.850.452.053.655.256.958.560.261.863.565.266.968.670.372.073.775.577.379.0

2941.743.244.846.448.049.651.252.854.556.157.859.561.262.964.666.368.069.871.573.375.176.978.780.582.4

3043.945.547.148.750.452.053.755.457.158.860.562.264.065.767.569.371.072.974.776.578.380.282.184.085.9

3146.247.949.551.252.954.656.358.159.861.663.365.166.968.770.572.474.276.178.079.981.883.785.787.689.6

3248.750.452.153.855.657.359.160.962.764.566.368.270.071.973.875.777.679.581.583.585.487.589.591.593.6

3351.253.054.856.558.360.262.063.865.767.669.571.473.375.277.279.281.283.285.287.389.391.493.695.797.8

3453.955.757.659.461.363.165.067.068.970.872.874.876.878.880.882.985.087.189.291.493.595.797.9100.2102.4

3556.858.660.562.464.466.368.370.372.374.376.378.480.582.684.786.989.191.393.595.798.0100.3102.6105.0107.3

3659.861.763.765.767.769.771.773.875.978.080.182.384.586.788.991.293.595.898.1100.5102.9105.3107.7110.2112.7

3762.965.067.069.171.273.375.477.679.882.084.286.588.891.193.495.898.2100.6103.1105.6108.1110.7113.3115.9118.6

3866.368.470.672.774.977.179.481.683.986.288.691.093.495.898.3100.8103.4105.9108.6111.2113.9116.6119.4122.2125.0

3970.072.274.476.778.981.383.686.088.490.993.495.998.4101.0103.6106.3109.0111.8114.6117.4120.3123.2126.1129.2132.2

4073.876.278.580.983.385.788.290.893.395.998.5101.2103.9106.7109.5112.4115.3118.2121.2124.3127.4130.5133.7137.0140.3

4178.080.583.085.588.090.693.395.998.7101.4104.3107.1110.0113.0116.0119.1122.2125.4128.7132.0135.4138.8142.3145.9149.5

4282.685.287.890.593.296.098.8101.7104.6107.6110.6113.7116.9120.1123.4126.7130.1133.6137.2140.8144.5148.3152.2156.1160.2

4387.690.493.296.099.0101.9105.0108.1111.2114.5117.8121.1124.6128.1131.7135.4139.1143.0147.0151.0155.2159.4163.8168.2172.8

4493.196.199.1102.2105.4108.6111.9115.3118.7122.3125.9129.6133.4137.4141.4145.5149.7154.1158.5163.1167.9172.7177.7182.9188.2

4599.3102.5105.8109.2112.6116.2119.8123.6127.4131.4135.4139.6143.9148.3152.9157.6162.4167.4172.6178.0183.5189.2195.1201.2207.5

46106.3109.8113.4117.2121.0125.0129.1133.3137.6142.1146.7151.5156.5161.6167.0172.5178.2184.2190.4196.8203.5210.5217.8225.4233.3

47114.3118.3122.4126.6130.9135.4140.1145.0150.0155.3160.7166.4172.3178.5185.0191.8198.9206.4214.2222.4231.0240.0249.5259.5270.0

48123.9128.4133.1137.9143.0148.3153.9159.7165.8172.2178.9186.0193.5201.4209.8218.7228.2238.2248.9260.3272.3285.1298.7313.0328.2

49135.5140.8146.4152.3158.5165.0172.0179.3187.2195.6204.6214.3224.7235.9248.1261.3275.5290.9307.6325.5344.8365.4387.3410.6435.2

6

DB21/T3111—2019

續(xù)表

大孔陽小孔陽性格數(shù)

性格數(shù)252627282930313233343536373839404142434445464748

025.326.427.428.429.530.531.532.633.634.735.736.837.838.940.041.042.143.144.245.346.347.448.549.5

126.627.728.729.830.831.932.934.035.036.137.238.239.340.441.442.543.644.745.746.847.949.050.151.2

227.929.030.031.132.233.234.335.436.537.538.639.740.841.943.044.045.146.247.348.449.550.651.752.8

329.330.431.432.533.634.735.836.837.939.040.141.242.343.444.545.646.747.848.950.051.252.353.454.5

430.731.832.833.935.036.137.238.339.440.541.642.843.945.046.147.248.349.550.651.752.954.055.156.3

532.133.234.335.436.537.638.739.941.042.143.244.445.546.647.748.950.051.252.353.554.655.856.958.1

633.534.735.836.938.039.240.341.442.643.744.846.047.148.349.450.651.752.954.155.256.457.658.759.9

735.036.237.338.439.640.741.943.044.245.346.547.748.850.051.252.353.554.755.957.158.359.460.661.8

836.637.738.940.041.242.343.544.745.947.048.249.450.651.853.054.155.356.557.759.060.261.462.663.8

938.139.340.541.642.844.045.246.447.648.850.051.252.453.654.856.057.258.459.760.962.163.464.665.8

1039.740.942.143.344.545.746.948.149.350.651.853.054.255.556.757.959.260.461.762.964.265.466.767.9

1141.442.643.845.046.347.548.749.951.252.453.754.956.157.458.659.961.262.463.765.066.367.568.870.1

1243.144.345.646.848.149.350.651.853.154.355.656.858.159.460.762.063.264.565.867.168.469.771.072.4

1344.946.147.448.649.951.252.553.755.056.357.658.960.261.562.864.165.466.768.069.370.772.073.374.7

1446.748.049.350.551.853.154.455.757.058.359.660.962.363.664.966.367.668.970.371.673.074.475.777.1

1548.649.951.252.553.855.156.457.859.160.461.863.164.565.867.268.569.971.372.674.075.476.878.279.6

1650.551.853.254.555.857.258.559.961.262.664.065.366.768.169.570.972.373.775.176.577.979.380.882.2

1752.553.955.256.658.059.360.762.163.564.966.367.769.170.571.973.374.876.277.679.180.582.083.584.9

1854.656.057.458.860.261.663.064.465.867.268.670.171.573.074.475.977.378.880.381.883.384.886.387.8

1956.858.259.661.062.463.965.366.868.269.771.172.674.175.577.078.580.081.583.184.686.187.689.290.7

2059.060.461.963.364.866.367.769.270.772.273.775.276.778.279.881.382.884.485.987.589.190.792.293.8

2161.362.864.365.867.368.870.371.873.374.976.477.979.581.182.684.285.887.489.090.692.293.895.497.1

2263.865.366.868.369.871.472.974.576.177.679.280.882.484.085.687.288.990.592.193.895.597.198.8100.5

2366.367.869.471.072.574.175.777.378.980.582.283.885.487.188.790.492.193.895.597.298.9100.6102.4104.1

2468.970.572.173.775.377.078.680.381.983.685.286.988.690.392.093.895.597.299.0100.7102.5104.3106.1107.9

2571.773.375.076.678.380.081.783.385.186.888.590.292.093.795.597.399.1100.9102.7104.5106.3108.2110.0111.9

2674.676.378.079.781.483.184.886.6