This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Nsabimana, E. Articles by Jouany, J. P. Search for Related Content PubMed PubMed Citation Articles by Nsabimana, E. Articles by Jouany, J. P. Agricola Articles by Nsabimana, E. Articles by Jouany, J. P.

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, July 2003, p. 3826-3832, Vol. 69, No. 7
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.7.3826-3832.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Two-Step Freezing Procedure for Cryopreservation of Rumen Ciliates, an Effective Tool for Creation of a Frozen Rumen Protozoa Bank

E. Nsabimana,¹ S. Kiidayová,² D. Macheboeuf,¹ C. J. Newbold,³ and J. P. Jouany^1*

Centre de Recherches de Clermont-Ferrand/Theix, Institut National de la Recherche Agronomique, 63122 Saint-Genès Champanelle, France,¹ Slovak Academy of Sciences, Soltesova, O4001 Kosice, Slovakia,² Rowett Research Institute, Aberdeen AB21 9SB, United Kingdom³

Received 13 January 2003/ Accepted 16 April 2003

The present study aimed at the long-term storage of rumen protozoa as living cells in liquid nitrogen. The two-step or interrupted slow freezing procedure was used to cryopreserve six of the dominant species of rumen ciliates isolated from monofaunated animals, Dasytricha ruminantium, Entodinium caudatum, Epidinium ecaudatum caudatum, Eudiplodinium maggii, Isotricha prostoma, and Polyplastron multivesiculatum. We optimized the first step in the interrupted slow freezing procedure, from the extracellular ice nucleation temperature to the holding temperature, and studied the effects of the cooling rates on survival. In addition to the nature of the cryoprotectant (dimethyl sulfoxide), the equilibration temperature and equilibration time (25°C and 5 min, respectively), and the holding time at subzero temperature (45 min) recommended previously (S. Kiidayová, J. Microbiol. Methods 22:185-192, 1995), we found that a holding temperature of -30°C, a cooling rate from extracellular ice nucleation temperature to holding temperature of between 1.2°C/min and 2.5°C/min, depending on the ciliate, and rumen juice as the freezing and thawing medium markedly improved the survival rate. Survival rates determined after 2 weeks in liquid nitrogen were 100% for Isotricha, 98% for Dasytricha, 85% for Epidinium, 79% for Polyplastron, 63% for Eudiplodinium, and 60% for Entodinium. They were not significantly modified after a period of 1 year in liquid nitrogen. Four of the five ciliate species cryopreserved for 8 months in liquid nitrogen successfully colonized the rumen when inoculated into defaunated animals. These results have made it possible to set up a bank of cryopreserved rumen protozoa.

---

* Corresponding author. Mailing address: Centre de Recherches de Clermont-Ferrand/Theix, Institut National de la Recherche Agronomique, 63122 Saint-Genès Champanelle, France. Phone: 33 (0)4 73 62 40 54. Fax: 33 (0)4 73 62 46 59. E-mail: jouany{at}clermont.inra.fr.

---

Applied and Environmental Microbiology, July 2003, p. 3826-3832, Vol. 69, No. 7
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.7.3826-3832.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Dehority, B. A. (2008). Improved in vitro procedure for maintaining stock cultures of three genera of rumen protozoa. J ANIM SCI 86: 1395-1401 [Abstract] [Full Text]