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Survival or revival: long-term preservation induces a reversible viable but non-culturable state in methane-oxidizing bacteria

Sven Hoefman UGent, Koenraad Van Hoorde UGent, Nico Boon UGent, Peter Vandamme UGent, Paul De Vos UGent and Kim Heylen UGent (2012) PLOS ONE. 7(4).
abstract
Knowledge on long-term preservation of micro-organisms is limited and research in the field is scarce despite its importance for microbial biodiversity and biotechnological innovation. Preservation of fastidious organisms such as methane-oxidizing bacteria (MOB) has proven difficult. Most MOB do not survive lyophilization and only some can be cryopreserved successfully for short periods. A large-scale study was designed for a diverse set of MOB applying fifteen cryopreservation or lyophilization conditions. After three, six and twelve months of preservation, the viability (via live-dead flow cytometry) and culturability (via most-probable number analysis and plating) of the cells were assessed. All strains could be cryopreserved without a significant loss in culturability using 1% trehalose in 10-fold diluted TSB (TT) as preservation medium and 5% DMSO as cryoprotectant. Several other cryopreservation and lyophilization conditions, all of which involved the use of TT medium, also allowed successful preservation but showed a considerable loss in culturability. We demonstrate here that most of these non-culturables survived preservation according to viability assessment indicating that preservation induces a viable but non-culturable (VBNC) state in a significant fraction of cells. Since this state is reversible, these findings have major implications shifting the emphasis from survival to revival of cells in a preservation protocol. We showed that MOB cells could be significantly resuscitated from the VBNC state using the TT preservation medium.
Please use this url to cite or link to this publication:
author
organization
year
type
journalArticle (original)
publication status
published
subject
keyword
SACCHAROMYCES-CEREVISIAE, NONCULTURABLE STATE, METHANOTROPHIC BACTERIA, FLUORESCENT-PROBES, ESCHERICHIA-COLI, MICROBIOLOGY, CRYOPROTECTANTS, MICROORGANISMS, RESUSCITATION, DIVERSITY
journal title
PLOS ONE
PLoS One
volume
7
issue
4
article_number
e34196
pages
9 pages
Web of Science type
Article
Web of Science id
000305341000008
JCR category
MULTIDISCIPLINARY SCIENCES
JCR impact factor
3.73 (2012)
JCR rank
7/56 (2012)
JCR quartile
1 (2012)
ISSN
1932-6203
DOI
10.1371/journal.pone.0034196
language
English
UGent publication?
yes
classification
A1
copyright statement
I have retained and own the full copyright for this publication
id
3047493
handle
http://hdl.handle.net/1854/LU-3047493
date created
2012-11-05 15:54:42
date last changed
2012-11-05 16:35:13
@article{3047493,
  abstract     = {Knowledge on long-term preservation of micro-organisms is limited and research in the field is scarce despite its importance for microbial biodiversity and biotechnological innovation. Preservation of fastidious organisms such as methane-oxidizing bacteria (MOB) has proven difficult. Most MOB do not survive lyophilization and only some can be cryopreserved successfully for short periods. A large-scale study was designed for a diverse set of MOB applying fifteen cryopreservation or lyophilization conditions. After three, six and twelve months of preservation, the viability (via live-dead flow cytometry) and culturability (via most-probable number analysis and plating) of the cells were assessed. All strains could be cryopreserved without a significant loss in culturability using 1\% trehalose in 10-fold diluted TSB (TT) as preservation medium and 5\% DMSO as cryoprotectant. Several other cryopreservation and lyophilization conditions, all of which involved the use of TT medium, also allowed successful preservation but showed a considerable loss in culturability. We demonstrate here that most of these non-culturables survived preservation according to viability assessment indicating that preservation induces a viable but non-culturable (VBNC) state in a significant fraction of cells. Since this state is reversible, these findings have major implications shifting the emphasis from survival to revival of cells in a preservation protocol. We showed that MOB cells could be significantly resuscitated from the VBNC state using the TT preservation medium.},
  articleno    = {e34196},
  author       = {Hoefman, Sven and Van Hoorde, Koenraad and Boon, Nico and Vandamme, Peter and De Vos, Paul and Heylen, Kim},
  issn         = {1932-6203},
  journal      = {PLOS ONE},
  keyword      = {SACCHAROMYCES-CEREVISIAE,NONCULTURABLE STATE,METHANOTROPHIC BACTERIA,FLUORESCENT-PROBES,ESCHERICHIA-COLI,MICROBIOLOGY,CRYOPROTECTANTS,MICROORGANISMS,RESUSCITATION,DIVERSITY},
  language     = {eng},
  number       = {4},
  pages        = {9},
  title        = {Survival or revival: long-term preservation induces a reversible viable but non-culturable state in methane-oxidizing bacteria},
  url          = {http://dx.doi.org/10.1371/journal.pone.0034196},
  volume       = {7},
  year         = {2012},
}

Chicago
Hoefman, Sven, Koenraad Van Hoorde, Nico Boon, Peter Vandamme, Paul De Vos, and Kim Heylen. 2012. “Survival or Revival: Long-term Preservation Induces a Reversible Viable but Non-culturable State in Methane-oxidizing Bacteria.” Plos One 7 (4).
APA
Hoefman, S., Van Hoorde, K., Boon, N., Vandamme, P., De Vos, P., & Heylen, K. (2012). Survival or revival: long-term preservation induces a reversible viable but non-culturable state in methane-oxidizing bacteria. PLOS ONE, 7(4).
Vancouver
1.
Hoefman S, Van Hoorde K, Boon N, Vandamme P, De Vos P, Heylen K. Survival or revival: long-term preservation induces a reversible viable but non-culturable state in methane-oxidizing bacteria. PLOS ONE. 2012;7(4).
MLA
Hoefman, Sven, Koenraad Van Hoorde, Nico Boon, et al. “Survival or Revival: Long-term Preservation Induces a Reversible Viable but Non-culturable State in Methane-oxidizing Bacteria.” PLOS ONE 7.4 (2012): n. pag. Print.