PMID- 17442439
OWN - NLM
STAT- MEDLINE
DCOM- 20070719
LR  - 20191210
IS  - 0167-7012 (Print)
IS  - 0167-7012 (Linking)
VI  - 69
IP  - 3
DP  - 2007 Jun
TI  - Routine fluorescence in situ hybridization in soil.
PG  - 451-60
AB  - The use of fluorescence in situ hybridization (FISH) to identify and enumerate 
      soil bacteria has long been hampered by the autofluorescence of soil particles 
      masking the bacterial signals and because the need of counting hundreds of 
      bacteria in order to achieve statistically reliable data is time consuming. 
      Recently, it was demonstrated that Nycodenz facilitates FISH in soil by 
      concentrating bacteria on membrane filters and avoiding autofluorescent soil 
      particles. We present a routine protocol for FISH in soil including the use of 
      Nycodenz. The protocol allows fast and easy enumeration of hundreds of bacteria. 
      We propose the use of silicon grease coated slides to treat in parallel seven 
      samples per hybridization. Further, we developed a semi-automated approach for 
      the enumeration of bacteria by implementing macros concatenating all steps of the 
      image analyzes in the Image J software. Using Nycodenz, software-assisted 
      bacterial counts statistically matched eye-counts of the same images and it was 
      possible to count 880 DAPI stained bacteria per ten images. Fifty-five percent of 
      these bacteria were co-labelled with the FISH probe specific for the Domain 
      Bacteria, in accordance with recent FISH studies of bacterial populations in bulk 
      soil. With a soil slurry protocol used for comparison, soil particles impaired 
      automatic counts of the bacteria and FISH analysis, and only 88 DAPI stained 
      bacteria per ten images could be counted by eye. With the Nycodenz protocol, 5 mM 
      Na(2)EDTA used as an extractant increased the number of bacteria observed by 49%. 
      In contrast, Tween 20 (1% or 5%) had no significant effect and increased the 
      variability between the samples. Overall, the proposed procedure allows to 
      process a high number of samples and to achieve a time efficient FISH 
      characterization of soil bacterial communities.
FAU - Bertaux, J
AU  - Bertaux J
AD  - Institute of Zoology, Darmstadt University of Technology, Schnittspahnstrasse 3, 
      D-64287 Darmstadt, Germany. bertaux@bio.tu-darmstadt.de
FAU - Gloger, U
AU  - Gloger U
FAU - Schmid, M
AU  - Schmid M
FAU - Hartmann, A
AU  - Hartmann A
FAU - Scheu, S
AU  - Scheu S
LA  - eng
PT  - Evaluation Study
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20070303
PL  - Netherlands
TA  - J Microbiol Methods
JT  - Journal of microbiological methods
JID - 8306883
RN  - 0 (Indoles)
RN  - 4419T9MX03 (Iohexol)
RN  - 47165-04-8 (DAPI)
SB  - IM
MH  - Bacteria/*isolation & purification
MH  - Bacteriological Techniques
MH  - Colony Count, Microbial
MH  - Filtration/methods
MH  - Image Processing, Computer-Assisted
MH  - In Situ Hybridization, Fluorescence/*methods
MH  - Indoles
MH  - Iohexol/*pharmacology
MH  - Micropore Filters
MH  - *Soil Microbiology
EDAT- 2007/04/20 09:00
MHDA- 2007/07/20 09:00
CRDT- 2007/04/20 09:00
PHST- 2007/01/18 00:00 [received]
PHST- 2007/02/22 00:00 [revised]
PHST- 2007/02/22 00:00 [accepted]
PHST- 2007/04/20 09:00 [pubmed]
PHST- 2007/07/20 09:00 [medline]
PHST- 2007/04/20 09:00 [entrez]
AID - S0167-7012(07)00066-8 [pii]
AID - 10.1016/j.mimet.2007.02.012 [doi]
PST - ppublish
SO  - J Microbiol Methods. 2007 Jun;69(3):451-60. doi: 10.1016/j.mimet.2007.02.012. 
      Epub 2007 Mar 3.