PMID- 8919803
OWN - NLM
STAT- MEDLINE
DCOM- 19970102
LR  - 20210526
IS  - 0099-2240 (Print)
IS  - 1098-5336 (Electronic)
IS  - 0099-2240 (Linking)
VI  - 62
IP  - 4
DP  - 1996 Apr
TI  - Development and field application of a quantitative method for examining natural 
      assemblages of protists with oligonucleotide probes.
PG  - 1416-23
AB  - A fluorescent in situ hybridization method that uses rRNA-targeted 
      oligonucleotide probes for counting protists in cultures and environmental water 
      samples is described. Filtration, hybridization, and enumeration of fixed cells 
      with biotinylated eukaryote-specific probes and fluorescein 
      isothiocyanate-conjugated avidin were performed directly on 0.4-microns-pore-size 
      polycarbonate filters of Transwell cell culture inserts (Costar Corp., Cambridge, 
      Mass.). Counts of various species of cultured protists by this probe 
      hybridization method were not significantly different from counts obtained by the 
      4',6-diamidino-2-phenylindole (DAPI) and acridine orange (AO) staining methods. 
      However, counts of total nanoplankton (TNAN) based on probe hybridizations in 
      several field samples and in samples collected from a mesocosm experiment were 
      frequently higher than TNAN counts obtained by staining with DAPI or AO. On the 
      basis of these results, 25 to 70% of the TNAN determined with probes were not 
      detectable by DAPI or AO staining. The underestimation of TNAN abundances in 
      samples stained with DAPI or AO was attributed to the existence of small 
      nanoplanktonic cells which could be detected with probes but not DAPI or AO and 
      the difficulty associated with distinguishing DAPI- or AO-stained protists 
      attached to or embedded in aggregates. We conclude from samples examined in this 
      study that enumeration of TNAN with oligonucleotide probes provides estimates of 
      natural TNAN abundances that are at least as high as (and in some cases higher 
      than) counts obtained with commonly employed fluorochrome stains. The 
      quantitative in situ hybridization method we have described here enables the 
      direct enumeration of free-living protists in water samples with oligonucleotide 
      probes. When combined with species-specific probes, this method will enable 
      quantitative studies of the abundance and distribution of specific protistan 
      taxa.
FAU - Lim, E L
AU  - Lim EL
AD  - Biology Department, Woods Hole Oceanographic Institution, Massachusetts 02543, 
      USA.
FAU - Caron, D A
AU  - Caron DA
FAU - Delong, E F
AU  - Delong EF
LA  - eng
PT  - Comparative Study
PT  - Journal Article
PT  - Research Support, U.S. Gov't, Non-P.H.S.
PL  - United States
TA  - Appl Environ Microbiol
JT  - Applied and environmental microbiology
JID - 7605801
RN  - 0 (DNA, Bacterial)
RN  - 0 (Fluorescent Dyes)
RN  - 0 (Indoles)
RN  - 0 (Oligonucleotide Probes)
RN  - 0 (RNA, Bacterial)
RN  - 0 (RNA, Ribosomal, 16S)
RN  - 47165-04-8 (DAPI)
RN  - F30N4O6XVV (Acridine Orange)
SB  - IM
MH  - Acridine Orange
MH  - Animals
MH  - Colony Count, Microbial/methods
MH  - DNA, Bacterial/genetics
MH  - Ecosystem
MH  - Escherichia coli/genetics
MH  - Evaluation Studies as Topic
MH  - Fluorescent Dyes
MH  - In Situ Hybridization, Fluorescence/*methods
MH  - Indoles
MH  - Molecular Probe Techniques
MH  - Oligonucleotide Probes/*genetics
MH  - Plankton/*genetics/*isolation & purification
MH  - RNA, Bacterial/genetics
MH  - RNA, Ribosomal, 16S/genetics
MH  - *Water Microbiology
PMC - PMC167908
EDAT- 1996/04/01 00:00
MHDA- 1996/04/01 00:01
PMCR- 1996/04/01
CRDT- 1996/04/01 00:00
PHST- 1996/04/01 00:00 [pubmed]
PHST- 1996/04/01 00:01 [medline]
PHST- 1996/04/01 00:00 [entrez]
PHST- 1996/04/01 00:00 [pmc-release]
AID - 10.1128/aem.62.4.1416-1423.1996 [doi]
PST - ppublish
SO  - Appl Environ Microbiol. 1996 Apr;62(4):1416-23. doi: 
      10.1128/aem.62.4.1416-1423.1996.