PMID- 15676195
OWN - NLM
STAT- MEDLINE
DCOM- 20050405
LR  - 20061115
IS  - 0167-7012 (Print)
IS  - 0167-7012 (Linking)
VI  - 61
IP  - 1
DP  - 2005 Apr
TI  - Improved permeabilization protocols for fluorescence in situ hybridization (FISH) 
      of mycolic-acid-containing bacteria found in foams.
PG  - 47-54
AB  - Formation of thick, stable foams and scums on activated sludge wastewater 
      treatment plants is a worldwide problem, and to better understand what causes 
      this foam and to cure it, there is a need to identify and quantify the bacteria 
      present there. Fluorescence in situ hybridisation (FISH) overcomes the 
      difficulties experienced with microscopic methods of identification for the 
      mycolic-acid-containing actinomycetes (the mycolata), which are present in foams, 
      where many share the morphotype of right-angled branching filaments. However, the 
      presence of hydrophobic mycolic acids in their cell wall makes this group of 
      bacteria particularly difficult to permeabilise, which greatly reduces the 
      usefulness of FISH. While several permeabilisation treatments have been 
      described, none appear to adequately permeabilise all genera of the mycolata. In 
      this study several protocols for permeabilisation were assessed with both pure 
      cultures of selected genera of the mycolata and foam samples. Combining mild acid 
      hydrolysis with enzyme treatments (either mutanolysin/lysozyme or 
      lipase/proteinase K) was found to be the most effective method, although other 
      evidence presented here suggests that negative FISH results can not always be 
      explained in terms of cell permeability to the probes.
FAU - Carr, Emma L
AU  - Carr EL
AD  - Biotechnology Research Centre, La Trobe University, Bendigo, Victoria, 3550, 
      Australia.
FAU - Eales, Kathryn
AU  - Eales K
FAU - Soddell, Jacques
AU  - Soddell J
FAU - Seviour, Robert J
AU  - Seviour RJ
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - Netherlands
TA  - J Microbiol Methods
JT  - Journal of microbiological methods
JID - 8306883
RN  - 0 (DNA, Bacterial)
RN  - 0 (Mycolic Acids)
RN  - 0 (RNA, Ribosomal, 16S)
RN  - 0 (Sewage)
RN  - EC 3.1.1.3 (Lipase)
RN  - EC 3.2.1.17 (Muramidase)
RN  - EC 3.4.- (Endopeptidases)
RN  - EC 3.4.21.64 (Endopeptidase K)
RN  - EC 3.4.99.- (mutanolysin)
SB  - IM
MH  - Actinobacteria/genetics/*isolation & purification/*metabolism
MH  - Cell Membrane Permeability/drug effects
MH  - Cell Wall/metabolism
MH  - DNA, Bacterial/genetics
MH  - Endopeptidase K/pharmacology
MH  - Endopeptidases/pharmacology
MH  - In Situ Hybridization, Fluorescence/*methods
MH  - Lipase/pharmacology
MH  - Muramidase/pharmacology
MH  - Mycolic Acids/*metabolism
MH  - RNA, Ribosomal, 16S/genetics
MH  - Sewage/*microbiology
EDAT- 2005/01/29 09:00
MHDA- 2005/04/06 09:00
CRDT- 2005/01/29 09:00
PHST- 2004/04/23 00:00 [received]
PHST- 2004/10/28 00:00 [revised]
PHST- 2004/10/28 00:00 [accepted]
PHST- 2005/01/29 09:00 [pubmed]
PHST- 2005/04/06 09:00 [medline]
PHST- 2005/01/29 09:00 [entrez]
AID - S0167-7012(04)00306-9 [pii]
AID - 10.1016/j.mimet.2004.10.023 [doi]
PST - ppublish
SO  - J Microbiol Methods. 2005 Apr;61(1):47-54. doi: 10.1016/j.mimet.2004.10.023.