Development of a sensitive and rapid method for the measurement of total microbial activity using fluorescein diacetate (FDA) in a range of soils
Development of a sensitive and rapid method for the measurement of total microbial activity using fluorescein diacetate (FDA) in a range of soils
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DOI：
10.1016/s0038-0717(00)00244-3
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发表时间：
2001-06-01
影响因子：
9.7
通讯作者：
Duncan, H
中科院分区：
文献类型：
--
作者：
Adam, G;Duncan, H
双乙酸黄绿素（FDA）水解被广泛认为是一种准确而简单的方法，用于测量包括土壤在内的一系列环境样品中的总微生物活性。无色荧光素二乙酸酯被游离酶和膜结合酶水解，释放出有色终产物荧光素，其可通过分光光度法测量。目前测定土壤中FDA水解的方法在应用上受到限制。FDA活性在桑迪和粘性土壤中很低。由于原作者选择了终止水解反应的溶剂，因此难以测量这些土壤类型中观察到的低活性。发现丙酮（50%v/v）在停止水解反应方面最有效。在本研究期间，发现丙酮（50%v/v）导致测量的土壤样品产生的荧光素吸光度降低约37%。尽管这种颜色损失与初始荧光素浓度无关，但它使得在具有低微生物活性的土壤即桑迪和/或粘性土壤中测量FDA水解活性极其困难。氯仿/甲醇（2：1 v/v）被发现成功地停止水解反应长达50分钟，在一系列的土壤样品，而不会导致与丙酮观察到的颜色损失。通过改变用于终止水解反应的溶剂，可以成功地测量低活性土壤。水解反应的其他参数进行了优化，包括pH值的影响，最佳温育温度，土壤的量，温育时间，底物的量和制备合适的标准。提出了一种新的，更灵敏的方法，从原来的方法，它提供了一个更准确的测定FDA水解在广泛的土壤。(C)2001爱思唯尔科技有限公司版权所有。
Fluorescein diacetate (FDA) hydrolysis is widely accepted as an accurate and simple method for measuring total microbial activity in a range of environmental samples, including soils. Colourless fluorescein diacetate is hydrolysed by both free and membrane bound enzymes, releasing a coloured end product fluorescein which can be measured by spectrophotometry. The current method for measuring FDA hydrolysis in soils is limited in its application. FDA activity was very low in sandy and clayey soils. The low activity observed for these soil types was made difficult to measure by the original authors choice of solvent for terminating the hydrolysis reaction. Acetone (50% v/v) was found to be most efficient at stopping the hydrolysis reaction. During this study acetone (50% v/v) was found to cause a decrease of approximately 37% in the absorbance of fluorescein produced by the soil samples measured. Although this colour loss is independent of initial fluorescein concentration, it makes the measurement of FDA hydrolytic activity extremely difficult in soils with low microbial activity i.e. sandy and/or clayey soils. Chloroform/methanol (2:1 v/v) was found to successfully stop the hydrolysis reaction for up to 50 min in a range of soil samples without causing the loss of colour observed with acetone. By changing the solvent used for terminating the hydrolysis reaction, low activity soils could be measured successfully. Other parameters of the hydrolysis reaction were optimised for the measurement of soil samples including effect of pH. optimum temperature of incubation, amount of soil, time of incubation, amount of substrate and preparation of suitable standards. A new, more sensitive method is proposed adapted from the original method, which provides a more accurate determination of FDA hydrolysis in a wide range of soils. (C) 2001 Elsevier Science Ltd. All rights reserved.