Effects of Sediments on Estimates of Bacterial Density

R. L. Kepner, J. Pratt
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Mean differences attributable to choice of stain may be as great as differences attributable to potential masking effects of sediments. Despite culture variability, DAPI and AO methods yielded count estimates agreeing within an order of magnitude. Greater correspondence between counts was observed for turbid natural water samples spanning a more limited range of bacterial concentrations. Identifying and interpreting the biotic and abiotic factors responsible for the regulation of bacterial numbers is a major goal of microbial ecology. Bacteria have been enumerated in several ways, with many of the same techniques being applied in a variety of systems. If reported usage is indicative, methods of direct microscopic visualization are increasingly favored over standard platecount techniques which, by necessity, exert a selective effect leading to underestimates of total bacterial numbers. A serious and recognized drawback of plate counts is that no single medium will culture all bacteria (Buck, 1979), yet the presence of large numbers of bacteria (regardless of culturability) may be of great significance. Recently, epifluorescent direct-count techniques have become the methods of choice for enumeration of total bacteria (Fry, 1988). However, clear differences in numbers of bacteria observed depend upon both staining technique and physico-chemical characteristics of the sample. Two fluorochrome stains are most often used in direct-count methods. DAPI (4',6-diamidino-2-phenylindole) is a DNA-specific stain that fluoresces blue or bluish-white when bound to DNA and excited with light at a wavelength of 365 nm. When unbound or bound to non-DNA material, it may fluoresce over a range of yellow colors. As with the other most commonly used strain, AO [3,6-bis(dimethylamino)acridinium chloride or acridine orange], bacteria are identified based not only upon color but also upon size and shape. AO binds to both DNA and RNA, and the excitation maximum for AO is approximately 470 nm. Both DAPI and AO stain bacteria and other fine particulate organic matter (FPOM) differentially. Two experiments were performed to assess differences in bacterial directcount methods. In particular, we were interested in potential differences in counts obtained using DAPI and AO. At the same time, we examined differences TRANS. AM. MICROSC. SOC., 112(4): 316-330. 1993. ? Copyright, 1993, by the American Microscopical Society, Inc. This content downloaded from 157.55.39.163 on Wed, 21 Sep 2016 05:28:09 UTC All use subject to http://about.jstor.org/terms VOL. 112, NO. 4, OCTOBER 1993 in our ability to enumerate bacteria in the presence or absence of fine inorganic sediments (primarily silts and clays) and whether this ability was influenced by choice of stain. Environmental samples also were collected and bacterial counts obtained using the two stains were compared. Particle-attached bacteria pose special problems because of masking effects and the need for dilution of high-turbidity samples. Turbid samples are encountered frequently in many types of studies (e.g., studies examining stormflow, estuarine or saltmarsh water, pore water, sediments, and soil water). The frequent occurrence of sediment-laden samples and common usage of DAPI and AO direct-count (AODC) methods in the enumeration of bacteria have created the need for further methodological comparisons of the type presented here. MATERIALS AND METHODS","PeriodicalId":23957,"journal":{"name":"Transactions of the American Microscopical Society","volume":"6 1","pages":"316-330"},"PeriodicalIF":0.0000,"publicationDate":"1993-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"9","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Transactions of the American Microscopical Society","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.2307/3226566","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}
引用次数: 9

Abstract

We compared direct epifluorescent microscopic bacterial counts using either acridine orange [AO; 3,6-bis(dimethylamino)acridinium chloride] or DAPI (4',6-diamidino2-phenylindole) in the presence and absence of fine sediments. Differences in a time-series of population estimates obtained using either AO or DAPI direct-count methods were greater in sediment-amended cultures. In the presence of sediments, DAPI yielded significantly lower estimates of bacterial densities than AO. A sediment-addition experiment indicated that DAPI counts may be more sensitive to sediment presence than AO counts, although interaction between treatments (stain, sediment) clouds statistical interpretation. Variation between replicate cultures was greater than between subsamples of preserved combined samples. Mean differences attributable to choice of stain may be as great as differences attributable to potential masking effects of sediments. Despite culture variability, DAPI and AO methods yielded count estimates agreeing within an order of magnitude. Greater correspondence between counts was observed for turbid natural water samples spanning a more limited range of bacterial concentrations. Identifying and interpreting the biotic and abiotic factors responsible for the regulation of bacterial numbers is a major goal of microbial ecology. Bacteria have been enumerated in several ways, with many of the same techniques being applied in a variety of systems. If reported usage is indicative, methods of direct microscopic visualization are increasingly favored over standard platecount techniques which, by necessity, exert a selective effect leading to underestimates of total bacterial numbers. A serious and recognized drawback of plate counts is that no single medium will culture all bacteria (Buck, 1979), yet the presence of large numbers of bacteria (regardless of culturability) may be of great significance. Recently, epifluorescent direct-count techniques have become the methods of choice for enumeration of total bacteria (Fry, 1988). However, clear differences in numbers of bacteria observed depend upon both staining technique and physico-chemical characteristics of the sample. Two fluorochrome stains are most often used in direct-count methods. DAPI (4',6-diamidino-2-phenylindole) is a DNA-specific stain that fluoresces blue or bluish-white when bound to DNA and excited with light at a wavelength of 365 nm. When unbound or bound to non-DNA material, it may fluoresce over a range of yellow colors. As with the other most commonly used strain, AO [3,6-bis(dimethylamino)acridinium chloride or acridine orange], bacteria are identified based not only upon color but also upon size and shape. AO binds to both DNA and RNA, and the excitation maximum for AO is approximately 470 nm. Both DAPI and AO stain bacteria and other fine particulate organic matter (FPOM) differentially. Two experiments were performed to assess differences in bacterial directcount methods. In particular, we were interested in potential differences in counts obtained using DAPI and AO. At the same time, we examined differences TRANS. AM. MICROSC. SOC., 112(4): 316-330. 1993. ? Copyright, 1993, by the American Microscopical Society, Inc. This content downloaded from 157.55.39.163 on Wed, 21 Sep 2016 05:28:09 UTC All use subject to http://about.jstor.org/terms VOL. 112, NO. 4, OCTOBER 1993 in our ability to enumerate bacteria in the presence or absence of fine inorganic sediments (primarily silts and clays) and whether this ability was influenced by choice of stain. Environmental samples also were collected and bacterial counts obtained using the two stains were compared. Particle-attached bacteria pose special problems because of masking effects and the need for dilution of high-turbidity samples. Turbid samples are encountered frequently in many types of studies (e.g., studies examining stormflow, estuarine or saltmarsh water, pore water, sediments, and soil water). The frequent occurrence of sediment-laden samples and common usage of DAPI and AO direct-count (AODC) methods in the enumeration of bacteria have created the need for further methodological comparisons of the type presented here. MATERIALS AND METHODS
沉积物对细菌密度估计的影响
我们比较了吖啶橙[AO;3,6-二(二甲氨基)氯化吖啶]或DAPI(4',6-二氨基-苯基吲哚)。使用AO或DAPI直接计数方法获得的种群估计的时间序列差异在沉积物修正培养中更大。在沉积物存在的情况下,DAPI产生的细菌密度估计值明显低于AO。一项沉积物添加实验表明,DAPI计数可能比AO计数对沉积物的存在更敏感,尽管处理(染色、沉积物)之间的相互作用会影响统计解释。重复培养物之间的差异大于保存组合样品的亚样品之间的差异。可归因于染色剂选择的平均差异可能与可归因于沉积物潜在遮蔽效应的差异一样大。尽管文化差异,DAPI和AO方法产生的计数估计在一个数量级内一致。在浑浊的天然水样中,观察到细菌浓度范围更有限,计数之间的对应关系更大。识别和解释负责调节细菌数量的生物和非生物因素是微生物生态学的主要目标。细菌已经以几种方式被列举出来,其中许多相同的技术被应用于各种系统中。如果报告的使用是指示性的,那么直接显微镜可视化方法越来越受到标准平板计数技术的青睐,因为标准平板计数技术必然会产生选择性效应,导致细菌总数的低估。平板计数的一个严重和公认的缺点是,没有一种培养基可以培养所有的细菌(Buck, 1979),然而大量细菌的存在(不管可培养性如何)可能具有重要意义。最近,表观荧光直接计数技术已成为枚举总细菌的首选方法(Fry, 1988)。然而,观察到的细菌数量的明显差异取决于染色技术和样品的物理化学特性。两种荧光染色剂最常用于直接计数法。DAPI(4',6-二氨基-2-苯基吲哚)是一种DNA特异性染色剂,当与DNA结合并在365 nm波长的光激发下发出蓝色或蓝白色荧光。当与非dna材料分离或结合时,它可能发出一系列黄色的荧光。与另一种最常用的菌株AO[3,6-二(二甲胺)吖啶酸氯或吖啶橙]一样,细菌的识别不仅基于颜色,还基于大小和形状。AO能同时与DNA和RNA结合,最大激发波长约为470 nm。DAPI和AO对细菌和其他细颗粒有机物(FPOM)的染色差异较大。进行了两个实验来评估细菌直接计数方法的差异。我们特别感兴趣的是使用DAPI和AO获得的计数的潜在差异。同时,我们检查了TRANS的差异。点。MICROSC。SOC。生物医学工程学报,12(4):316-330。1993. ? 版权所有,1993年,美国显微学会,Inc。此内容于2016年9月21日(星期三)05:28:09 UTC下载于157.55.39.163。我们在细无机沉积物(主要是泥沙和粘土)存在或不存在的情况下枚举细菌的能力,以及这种能力是否受到染色选择的影响。同时采集环境样本,比较两种染色剂获得的细菌数量。颗粒附着的细菌由于遮蔽效应和需要稀释高浊度样品而造成特殊问题。混浊样品在许多类型的研究中经常遇到(例如,研究风暴流、河口或盐沼水、孔隙水、沉积物和土壤水)。经常出现的含沙样品和DAPI和AO直接计数(AODC)方法在细菌枚举中的普遍使用,创造了需要对这里提出的类型进行进一步的方法学比较。材料与方法
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