Revista argentina de microbiología
versión On-line ISSN 1851-7617
Rev. argent. microbiol. v.40 n.2 Ciudad Autónoma de Buenos Aires abr./jun. 2008
Long-term preservation of leptospiras by liquid nitrogen
C. A. Rossetti*, C. D. Auteri
Area de Bacteriología, Instituto de Patobiología, CICVyA - CNIA, INTA, CC25 (1712) Castelar, Prov. Buenos Aires, Argentina
*Correspondence. E-mail: firstname.lastname@example.org
Liquid nitrogen freezing is recommended for long-term preservation of Leptospira serovars. However, there is no standard protocol to follow for this methodology. We herein report a simple procedure to preserve well-characterized Leptospira serovars unaltered for long-term storage in liquid nitrogen. Forty-three (43) leptospira strains, cryoprotected with 10% (v/v) glycerol were rapidly frozen in a dry-ice methanol bath and immediately submerged in liquid-nitrogen. Viability was retained in 100%, 93% and 83% of the frozen cultures after 6, 18 and 54 months, following freezing and storage in liquid nitrogen, respectively. Motility and agglutinability were not altered. These results demonstrate the usefulness of this protocol for long-term storage of genus Leptospira in liquid nitrogen.
Key words: Leptospira; Conservation; Liquid nitrogen
Conservación de leptospiras en nitrógeno líquido. Se recomienda la congelación en nitrógeno líquido para el mantenimiento de cepas de leptospiras a largo plazo. Sin embargo, no existe para ello una metodología de trabajo estandarizada. En este trabajo se presenta y evalúa un protocolo simple para conservar inalteradas cepas de leptospiras en nitrógeno líquido durante largo tiempo. Cuarenta y tres (43) cepas de leptospiras crioprotegidas con glicerol al 10% (v/v) fueron rápidamente congeladas en un baño de metanol y hielo seco, e inmediatamente sumergidas en nitrógeno líquido. Fue posible recuperar el 100%, 93% y 83% de los cultivos congelados a los 6, 18 y 54 meses poscongelación, respectivamente, sin observarse alteración en la movilidad ni en la aglutinabilidad de las cepas recuperadas. Estos resultados demuestran la utilidad del protocolo presentado para conservar cepas del género Leptospira en nitrógeno líquido durante largos períodos de tiempo.
Palabras clave: Leptospira; Conservación; Nitrógeno líquido
Leptospira spp. are the etiological agents of leptospirosis, a worldwide zoonotic infectious disease that causes jaundice, hemorrhages and renal failure with high mortality in susceptible animal species (7). The isolation of leptospiras from infected animals is difficult due to the slow growth rate and the stringent and fastidious in vitro culture requirements. Moreover, the maintenance of the isolated strains is cumbersome and laborious. Leptospiras are usually maintained at room temperature (RT) in semisolid media by periodic subculture into fresh media at intervals not exceeding 2 months (3). However, this preservation method has important drawbacks: it is time-consuming (especially for those laboratories with large Leptospira collections) and the regular handling of live cultures increases the operators' risk of infection. There is also a higher risk of contamination with faster-growing bacteria or cross-contamination between sub-cultures and the possible loss of the strain or extra labor for purification. In addition, the virulence of pathogenic leptospiras is gradually lost after several passages in culture media (7). Conventional methodologies used for long-term preservation of other microorganisms, such as freezing at -20 or -70 °C and freeze-drying have reported inconsistent results for storing leptospiras (4, 5, 8, 11, 15), but previous studies using liquid nitrogen (LN2) have shown promising results (1, 12, 13, 14). This methodology has been strongly recommended for long-term preservation of leptospiras (2), however a unique protocol has not been established yet. Basically, the differences between protocols are: the amount and type of cryoprotectants used, the initial concentration of the inoculum and the freezing method. Furthermore, reported results are based on a few strains or short periods of storage time (1, 5, 12, 13, 14, 15). In this study, we evaluate a simple procedure to preserve, unaltered, a large number of well-characterized Leptospira serovars for long periods of time in an attempt to standardize a method to safely store large Leptospira collections.
Forty-three leptospira strains (Table 1) were separately grown in EMJH medium (Difco, Detroit, MI) at 28 °C for 7 days. Well-developed cultures were centrifuged at 7,500X g for 20 minutes at RT and each pellet was re-suspended in 1 ml of EMJH supplemented with 10% of inactivated sterile rabbit serum. Viable (motile) number of leptospiras was determined by counting in the Petroff-Hausser counting chamber (Clay-Adams, Inc., New York, NY) and samples with at least 109 total motile leptospiras were considered suitable for freezing. The purity of each culture was confirmed by the absence of growth on blood agar. Before freezing, 10% (v/v) glycerol (sterilized by autoclaving at 121 °C for 15 min) was added to each leptospira suspension, well-mixed and dispensed in 1 ml volumes in 1.8 ml screw-capped cryovials. After 30 min of stabilization at RT, the leptospiras were rapidly frozen (< 3 min) in a dry ice-methanol bath and immediately submerged in LN2. The viability of stored cultures was determined on day 1, and 6, 18 and 54 months after freezing, by thawing the vials in a water bath at 37 °C, initially observing the culture using dark-field microscopy and culture in EMJH medium at 1:10 supplemented with 1% of rabbit serum. Cultures were incubated at 28 °C and examined for the presence of viable leptospiras using a dark field micro- scope once a week for at least a month. Microscopic agglutination tests were performed according to standardized procedures (6).
Table 1. Leptospira strains used in this study and their viability after storage in LN2
Viable leptospiras with similar morphology and motility to those in non-frozen cultures were observed in all samples by dark-field microscopy, immediately after thawing. All 43 frozen strains retained their viability on day 1 and 6 months after freezing. Forty (93%) of the 43 frozen cultures grew in the EMJH medium supplemented with 1% rabbit serum after 18 month-storage while contamination was observed in the other 3 cultures (sv. Javanica, Manhao and Andaman). At 54 month-storage, 34 (83%) of 41 frozen cultures were recovered, 5 strains (sv. Pyrogenes, Fortbragg, Celledoni, Cynoptery and Manhao) did not grow and 2 (sv. Andaman and Pomona Bayur) were contaminated (Table 1).
To determine whether the storage time had modified some antigenic property in the agent, serum agglutinin titers of hyperimmune rabbit antiserum to sv. Canicola Hond Utrecht, sv. Icterohaemorrhagiae Ictero I, sv. Pomona Pomona and sv. Wolffi 3705, were measured with homologous 54 month-storage Leptospira strains and the results were compared with the agglutinin titers with homologous non-frozen strains. Results showed that LN2- preserved strains had serum agglutination profiles similar to non-frozen strains.
The protocol herein described was optimum for maintaining, unaltered and for a long period of time, a broad number of well-characterized Leptospira serovars in LN2. The effect of age and concentration of the cultures was considered an important factor for the successful recovery of leptospiras. Only one week-old cultures were stored in LN2, based on previous reports that showed that after freeze-drying, leptospira cultures at the log-phase of growth had higher survival rate than cultures at the stationary- phase (11). Furthermore, the total number of viable leptospiras was considered critical for successful recovery after LN2 storage. Our preliminary experiments indicated that frozen cultures with a total bacterial number lower than 5x108 were inconsistently recovered (unpublished data). In this study, without considering the 5 contaminated cultures, viability was retained in 160 of 165 frozen cultures throughout the experiment. As regards the five cultures not recovered after 54 months of storage in LN2, it is possible that the number of viable leptospiras initially frozen had been lower than initially determined or that these particular serovars were more sensitive to longterm preservation. Alexander et al. (1972) did not recover 9 of 103 frozen Leptospira cultures after 38 month-storage, but the authors did not mention which strains had not been recovered (1).
Glycerol is widely used to cryopreserve microbes (9), but its toxicity to Leptospira cultures has been reported (10, 12, 13) and its use has often been discarded (2). In concordance with Alexander et al. (1972) (1) and Stalheim (1971) (14), in this study, 10% (v/v) glycerol not only successfully preserved but also did not interfere with the growth of the Leptospira serovars tested in fresh medium after preservation in LN2. Another critical element for successful leptospira preservation is the cooling rate. Previous descriptions of leptospira cryopreservation in LN2 have used gradual or quick cooling rates (1, 5, 10, 12, 13, 14). In our study, rapid rather than gradual freezing was employed because of its simplicity and convenience and for not interfering with the recovery rate.
In summary, we developed a successful, simple procedure for the unaltered long-term preservation of large Leptospira collections in LN2.
Acknowledgements: We wish to thank to Dr. Luis E. Samartino for his support.
1. Alexander AD, Lessel EF, Evans LB, Franck E, Green SS. Preservation of leptospiras by liquid-nitrogen refrigeration. Int J Syst Bacteriol 1972; 22: 165-9. [ Links ]
2. Anonymous. International Committee on Systematic Bacteriology, Subcommittee on the Taxonomy of Leptospira. Minutes of the Meetings, 3 to 6 September, 1978, Munich, Germany. Int J Syst Bacteriol 1982; 32: 245-7. [ Links ]
3. Alexander AD. Leptospira. In: Ballows A, Hausler WJ, Herrmann KL, Isenberg HD, Shadomy HJ, editors. Manual of Clinical Microbiology. Washington D.C., ASM Press, 1991, p. 554-9. [ Links ]
4. Brihuega B, Fernández E. La liofilización como método de conservación para las leptospiras. Vet Arg 1996; 13: 268- 71. [ Links ]
5. Coghlan JD, Lumsden WHR, McNeillage GJC. Low-temperature preservation of Leptospira, preliminary communication. J Hyg Camb 1967; 65: 373-9. [ Links ]
6. Faine S. Guidelines for the control of leptospirosis. WHO offset publication Nº 67, World Health Organization, Geneva, 1982, 161 pp. [ Links ]
7. Faine S, Adler B, Bolin C, Perolat P. Leptospira and leptospirosis. Melbourne, MediSci, 1999, 272 pp. [ Links ]
8. Gastinel P, Hamelin A, Vaisman A, Dunoyer F. Recherches sur la lyophilisation et la conservation par le froid de quelques spirochetes. Annales de L'Institut Pasteur 1958; 94: 249-55. [ Links ]
9. Kirsop BE, Snell JJS. Maintenance of microorganisms. London, Academic Press, Inc., 1984, 207 pp. [ Links ]
10. Linscott WD, Boak RA. Protective action of glycerol in the freezing of leptospirae. J Bacteriol 1960; 80: 573-4. [ Links ]
11. Otsuka S, Manako K. Studies on the preservation of leptospirae by freeze-drying. Jap J Microbiol 1961; 5: 141-8. [ Links ]
12. Palit A, Haylock LM, Cox JC. Storage of pathogenic leptospires in liquid nitrogen. J Applied Bacteriol 1986; 61: 407-11. [ Links ]
13. Reed NE, Varney WC, Goddard RC, Wyeth PJ. The maintenance of challenge strains used in the potency test for canine leptospira vaccines. Biologicals 2000; 28: 25-8. [ Links ]
14. Stalheim OHV. Viable, avirulent Leptospira interrogans serotype pomona vaccine: preservation in liquid nitrogen. Applied Microbiol 1971; 22: 726-7. [ Links ]
15. Stavitsky AB. Preservation of L. icterohaemorrhagiae in vitro. J Bacteriol 1945; 50: 118-9. [ Links ]
Recibido: 4/9/07 - Aceptado: 26/2/08