Document Detail

Time series observation based InfraRed Epifluorescence Microscopic (TIREM) approach for accurate enumeration of bacteriochlorophyll-containing microbes in marine environments.
MedLine Citation:
PMID:  16213618     Owner:  NLM     Status:  MEDLINE    
Bacteriochlorophyll 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.
Nianzhi Jiao; Yao Zhang; Yao Chen
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Publication Detail:
Type:  Evaluation Studies; Journal Article; Research Support, Non-U.S. Gov't     Date:  2005-10-06
Journal Detail:
Title:  Journal of microbiological methods     Volume:  65     ISSN:  0167-7012     ISO Abbreviation:  J. Microbiol. Methods     Publication Date:  2006 Jun 
Date Detail:
Created Date:  2006-05-15     Completed Date:  2006-07-12     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  8306883     Medline TA:  J Microbiol Methods     Country:  Netherlands    
Other Details:
Languages:  eng     Pagination:  442-52     Citation Subset:  IM    
National Key Laboratory for Marine Environmental Science, Xiamen University, PR China.
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MeSH Terms
Bacteriochlorophylls / metabolism*
Image Processing, Computer-Assisted / methods*
Infrared Rays*
Microscopy, Fluorescence / instrumentation,  methods
Prochlorococcus / isolation & purification*
Reproducibility of Results
Roseobacter / isolation & purification
Seawater / microbiology*
Sphingomonadaceae / isolation & purification
Synechococcus / isolation & purification*
Reg. No./Substance:

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