PMID- 12732531
OWN - NLM
STAT- MEDLINE
DCOM- 20030805
LR  - 20210526
IS  - 0099-2240 (Print)
IS  - 1098-5336 (Electronic)
IS  - 0099-2240 (Linking)
VI  - 69
IP  - 5
DP  - 2003 May
TI  - Automated enumeration of groups of marine picoplankton after fluorescence in situ 
      hybridization.
PG  - 2631-7
AB  - We describe here an automated system for the counting of multiple samples of 
      double-stained microbial cells on sections of membrane filters. The application 
      integrates an epifluorescence microscope equipped with motorized z-axis drive, 
      shutters, and filter wheels with a scanning stage, a digital camera, and image 
      analysis software. The relative abundances of specific microbial taxa are 
      quantified in samples of marine picoplankton, as detected by fluorescence in situ 
      hybridization (FISH) and catalyzed reporter deposition. Pairs of microscopic 
      images are automatically acquired from numerous positions at two wavelengths, and 
      microbial cells with both general DNA and FISH staining are counted after object 
      edge detection and signal-to-background ratio thresholding. Microscopic fields 
      that are inappropriate for cell counting are automatically excluded prior to 
      measurements. Two nested walk paths guide the device across a series of 
      triangular preparations until a user-defined number of total cells has been 
      analyzed per sample. A backup autofocusing routine at incident light allows 
      automated refocusing between individual samples and can reestablish the focal 
      plane after fatal focusing errors at epifluorescence illumination. The system was 
      calibrated to produce relative abundances of FISH-stained cells in North Sea 
      samples that were comparable to results obtained by manual evaluation. Up to 28 
      preparations could be analyzed within 4 h without operator interference. The 
      device was subsequently applied for the counting of different microbial 
      populations in incubation series of North Sea waters. Automated digital 
      microscopy greatly facilitates the processing of numerous FISH-stained samples 
      and might thus open new perspectives for bacterioplankton population ecology.
FAU - Pernthaler, Jakob
AU  - Pernthaler J
AD  - Max Planck Institute for Marine Microbiology, Celsiusstrasse 1, D-28359 Bremen, 
      Germany. jperntha@mpi-bremen.de
FAU - Pernthaler, Annelie
AU  - Pernthaler A
FAU - Amann, Rudolf
AU  - Amann R
LA  - eng
PT  - Comparative Study
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - United States
TA  - Appl Environ Microbiol
JT  - Applied and environmental microbiology
JID - 7605801
SB  - IM
MH  - Automation
MH  - Bacteria/*genetics/*isolation & purification
MH  - Colony Count, Microbial/instrumentation/*methods/statistics & numerical data
MH  - Ecosystem
MH  - Germany
MH  - *In Situ Hybridization, Fluorescence
MH  - Plankton/*genetics/*isolation & purification
MH  - Seawater/*microbiology
PMC - PMC154506
EDAT- 2003/05/07 05:00
MHDA- 2003/08/06 05:00
PMCR- 2003/05/01
CRDT- 2003/05/07 05:00
PHST- 2003/05/07 05:00 [pubmed]
PHST- 2003/08/06 05:00 [medline]
PHST- 2003/05/07 05:00 [entrez]
PHST- 2003/05/01 00:00 [pmc-release]
AID - 1567 [pii]
AID - 10.1128/AEM.69.5.2631-2637.2003 [doi]
PST - ppublish
SO  - Appl Environ Microbiol. 2003 May;69(5):2631-7. doi: 
      10.1128/AEM.69.5.2631-2637.2003.