PMID- 10103277
OWN - NLM
STAT- MEDLINE
DCOM- 19991005
LR  - 20200724
IS  - 0099-2240 (Print)
IS  - 1098-5336 (Electronic)
IS  - 0099-2240 (Linking)
VI  - 65
IP  - 4
DP  - 1999 Apr
TI  - Counting and size classification of active soil bacteria by fluorescence in situ 
      hybridization with an rRNA oligonucleotide probe.
PG  - 1753-61
AB  - A fluorescence in situ hybridization (FISH) technique based on binding of a 
      rhodamine-labelled oligonucleotide probe to 16S rRNA was used to estimate the 
      numbers of ribosome-rich bacteria in soil samples. Such bacteria, which have high 
      cellular rRNA contents, were assumed to be active (and growing) in the soil. 
      Hybridization to an rRNA probe, EUB338, for the domain Bacteria was performed 
      with a soil slurry, and this was followed by collection of the bacteria by 
      membrane filtration (pore size, 0.2 micrometer). A nonsense probe, NONEUB338 
      (which has a nucleotide sequence complementary to the nucleotide sequence of 
      probe EUB338), was used as a control for nonspecific staining. Counting and size 
      classification into groups of small, medium, and large bacteria were performed by 
      fluorescence microscopy. To compensate for a difference in the relative staining 
      intensities of the probes and for binding by the rhodamine part of the probe, 
      control experiments in which excess unlabelled probe was added were performed. 
      This resulted in lower counts with EUB338 but not with NONEUB338, indicating that 
      nonspecific staining was due to binding of rhodamine to the bacteria. A value of 
      4.8 x 10(8) active bacteria per g of dry soil was obtained for bulk soil 
      incubated for 2 days with 0.3% glucose. In comparison, a value of 3.8 x 10(8) 
      active bacteria per g of dry soil was obtained for soil which had been air dried 
      and subsequently rewetted. In both soils, the majority (68 to 77%) of actively 
      growing bacteria were members of the smallest size class (cell width, 0.25 to 0.5 
      micrometer), but the active (and growing) bacteria still represented only 
      approximately 5% of the total bacterial population determined by DAPI (4', 
      6-diamidino-2-phenylindole) staining. The FISH technique in which slurry 
      hybridization is used holds great promise for use with phylogenetic probes and 
      for automatic counting of soil bacteria.
FAU - Christensen, H
AU  - Christensen H
AD  - Department of Veterinary Microbiology, Royal Veterinary and Agricultural 
      University, 1870 Frederiksberg C, Denmark. kvlhc@unidhp.uni-c.dk
FAU - Hansen, M
AU  - Hansen M
FAU - Sorensen, J
AU  - Sorensen J
LA  - eng
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
RN  - 0 (Oligonucleotide Probes)
RN  - 0 (RNA, Bacterial)
RN  - 0 (RNA, Ribosomal, 16S)
RN  - 0 (Rhodamines)
SB  - IM
MH  - Bacteria/*genetics/*isolation & purification
MH  - Colony Count, Microbial
MH  - *In Situ Hybridization, Fluorescence
MH  - Microscopy, Fluorescence
MH  - Oligonucleotide Probes
MH  - RNA, Bacterial/genetics
MH  - RNA, Ribosomal, 16S/*genetics
MH  - Rhodamines
MH  - *Soil Microbiology
PMC - PMC91247
EDAT- 1999/04/02 00:00
MHDA- 1999/04/02 00:01
PMCR- 1999/04/01
CRDT- 1999/04/02 00:00
PHST- 1999/04/02 00:00 [pubmed]
PHST- 1999/04/02 00:01 [medline]
PHST- 1999/04/02 00:00 [entrez]
PHST- 1999/04/01 00:00 [pmc-release]
AID - 1580 [pii]
AID - 10.1128/AEM.65.4.1753-1761.1999 [doi]
PST - ppublish
SO  - Appl Environ Microbiol. 1999 Apr;65(4):1753-61. doi: 
      10.1128/AEM.65.4.1753-1761.1999.