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dc.contributor.authorJiao NZzh_CN
dc.contributor.authorZhang Yzh_CN
dc.contributor.authorChen Yzh_CN
dc.date.accessioned2011-04-26T08:01:33Z
dc.date.available2011-04-26T08:01:33Z
dc.date.issued2006-01zh_CN
dc.identifier.citationJOURNAL OF MICROBIOLOGICAL METHODS 65 (3): 442-452 JUN 2006zh_CN
dc.identifier.issn0167-7012zh_CN
dc.identifier.urihttps://dspace.xmu.edu.cn/handle/2288/1346
dc.description.abstractBacteriochlorophyll a Containing Microbes (BCM) are a unique group of microorganisms in the marine environment. Accurate determination of their abundance is critical for understanding their role in energy flow and carbon cycle in the ecosystem. The InfraRed Epifluorescence Microscopy (IREM) method, using infrared fluorescence as the diagnostic signal of BCM, is the most convenient means to date for enumeration of BCM in seawater, but IREM methodology suffers from serious errors introduced by cyanobacteria, which also can emit infrared fluorescence and whose abundance is of the same order of magnitude as BCM. In the present study, an advanced "Time-series observation based cyanobacteria-calibrated InfraRed Epifluorescence Microscopy (TIREM)" approach is established for accurate enumeration of BCM in marine environments. The protocol is distinguished by its use of time series observation, auto-imaging and digital analysis. In principle, the correct count of BCM can be obtained by subtracting the cyanobacterial count from the total infrared positive count. The challenge, however, is that Prochlorococcus, the most abundant cyanobacterium in the sea, is readily visible in infrared images but not visible in the initial cyanobacterial images obtained by epifluorescence microscopy because its emission signals are masked by brighter fluorescence from larger cells like Synechococcus coexisting in seawater samples. Prochlorococcus cells become gradually visible when the fluorescence from Synechococcus cells declines after a period of exposure to excitation light. Therefore the plateau (maximum) count of the cyanobacterial cells in time series images rather than in the initial ones, as previously believed, represents the correct count for the total number of cyanobacteria (Synechococcus plus Prochlorococcus cells). Thus, the accurate estimation of BCM abundance can only be calculated from the formula: [BCM cells] = [plateau count of infrared positive cells] - [plateau count of cyanobacterial cells]. The conceptual advance of the TIREM protocol is that in classical epifluorescence microscopy or in IREM protocols, quick observation is recommended to avoid quenching the fluorescence, but in the TIREM protocol, instead, time series observation is the key for obtaining reliable data. The TIREM protocol is validated by studies using BCM and cyanobacterial pure cultures as well as by examination of samples from various marine environments. (c) 2005 Elsevier B.V. All rights reserved.zh_CN
dc.language.isoenzh_CN
dc.publisherELSEVIER SCIENCE BV, PO BOX 211, 1000 AE AMSTERDAM, NETHERLANDSzh_CN
dc.subjectauto-photographyzh_CN
dc.subjectbacteriochlorophyll a containing microbeszh_CN
dc.subjectcalibrationzh_CN
dc.subjectdigitizationzh_CN
dc.titleTime series observation based InfraRed Epifluorescence Microscopic (TIREM) approach for accurate enumeration of bacteriochlorophyll-containing microbes in marine environmentszh_CN
dc.typeArticlezh_CN


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