Preservation of Leptospiras by Liquid-Nitrogen Refrigeration

Alexander, A. D.; Lessel, Erwin F.; Evans, L. B.; Franck, Eva B.; Green, S. S.

doi:10.1099/00207713-22-3-165

Cited by 17 publications

(5 citation statements)

References 10 publications

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“…Bacterial strains, media, and plasmids. Virulent and culture-attenuated L. kirschneri RM52 organisms (1,14) were passaged in Johnson-Harris bovine serum albumin Tween 80 medium (Bovuminar PLM-5 Microbiological Media; Intergen) (19). E. coli DH5␣ (supE44 ⌬lacU169 [80 lacZ ⌬M15] hsdR17 recA1 endA1 gyrA96 thi-1 relA1) was used as the host strain for transformations of recombinant DNA.…”

Section: Methodsmentioning

confidence: 99%

Characterization of Leptospiral Outer Membrane Lipoprotein LipL36: Downregulation Associated with Late-Log-Phase Growth and Mammalian Infection

et al. 1998

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We report the cloning of the gene encoding a 36-kDa leptospiral outer membrane lipoprotein, designated LipL36. We obtained the N-terminal amino acid sequence of a staphylococcal V8 proteolytic-digest fragment in order to design an oligonucleotide probe. A Lambda-Zap II library containing EcoRI fragments of Leptospira kirschneri DNA was screened, and a 2.3-kb DNA fragment which contained the entire structural lipL36 gene was identified. Several lines of evidence indicate that LipL36 is lipid modified in a manner similar to that of LipL41, a leptospiral outer membrane lipoprotein we described in a previous study (E. S. Shang, T. A. Summers, and D. A. Haake, Infect. Immun. 64:2322–2330, 1996). The deduced amino acid sequence of LipL36 would constitute a 364-amino-acid polypeptide with a 20-amino-acid signal peptide, followed by an L-X-Y-C lipoprotein signal peptidase cleavage site. LipL36 is solubilized by Triton X-114 extraction of L. kirschneri; phase separation results in partitioning of LipL36 exclusively into the hydrophobic, detergent phase. LipL36 is intrinsically labeled during incubation of L. kirschneri in media containing [3H]palmitate. Processing of LipL36 is inhibited by globomycin, a selective inhibitor of lipoprotein signal peptidase. After processing, LipL36 is exported to the outer membrane along with LipL41 and lipopolysaccharide. Unlike LipL41, there appears to be differential expression of LipL36. In early-log-phase cultures, LipL36 is one of the most abundant L. kirschneri proteins. However, LipL36 levels drop considerably beginning in mid-log phase. LipL36 expression in vivo was evaluated by examining the humoral immune response to leptospiral antigens in the hamster model of leptospirosis. Hamsters surviving challenge with culture-adapted virulent L. kirschneri generate a strong antibody response to LipL36. In contrast, sera from hamsters surviving challenge with host-adaptedL. kirschneri do not recognize LipL36. These findings suggest that LipL36 expression is downregulated during mammalian infection, providing a marker for studying the mechanisms by which pathogenic Leptospira species adapt to the host environment.

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Section: Methodsmentioning

confidence: 99%

Characterization of Leptospiral Outer Membrane Lipoprotein LipL36: Downregulation Associated with Late-Log-Phase Growth and Mammalian Infection

et al. 1998

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“…Los medios con cada cepa se incubaron a 30°C de 1 a 3 semanas. La concentración de leptospiras viables se determinó por conteo microscópico en cámara, antes y luego sobre los cultivos crioconservados a intervalos de 30, 90, 180, 270 y 360 días; sólo se contaron organismos móviles (Alexander et al, 1972).…”

Section: Prueba De Viabilidadunclassified

“…Sin embargo, varios investigadores (Stalheim, 1971;Alexander et al, 1972;Palit et al, 1986y Reed et al, 2000 crioconservaron leptospiras en nitrógeno líquido (-196°C), por un largo periodo, manteniendo la viabilidad y las características genéticas de estos microorganismos.…”

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Comparación de tres tratamientos para la crioconservación de serovares de Leptospira en nitrógeno líquido

Higuera¹,

Ortega²,

Rodríguez-Bautista³

et al. 2009

CTA

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La preservación de bacterias pertenecientes al género Leptospira por métodos tradicionales -repiques frecuentes del cultivo- es costosa, dispendiosa y puede generar pérdidas de las características genéticas del cultivo. En el presente estudio se estandarizó una técnica de crioconservación en nitrógeno líquido para seis serovares de Leptospira -Pomona, Hardjoprajitno, Canicola, Icterohaemorrhagiae, Grippotyphosa y Bratislava-, usando como agente crioprotector el dimetil sulfóxido (DMSO) a una concentración final de 2% y una tasa de enfriamiento de 1°C/min y se comparó con el uso de glicerol a concentraciones finales de 0,2% y 10%. Los tres métodos se evaluaron mediante la determinación de la viabilidad bacteriana antes y después de la congelación (a 0, 30, 90, 180, 270 y 360 días) por recuento bacteriano en cámara. Los datos se sometieron a análisis de varianza y comparación por parejas usando la prueba de Bonferroni. La presencia de glicerol al 10% (concentración final) en el medio crioconservante disminuyó la viabilidad de los serovares. La técnica de criopreservación en nitrógeno líquido con DMSO al 2% y glicerol al 0,2% (concentraciones finales) permitió la exitosa conservación de los seis serovares de Leptospira. El método de criopreservación aseguró una alta viabilidad, lo que disminuyó costos y tiempo en su ejecución y demostró ser un mejor método de conservación a largo plazo en relación con el mantenimiento tradicional por subcultivos periódicos. Comparison of three methods for the cryopreservation of Leptospira strains in Liquid NitrogenTraditional methods for preservation of bacteria belonging to the genus Leptospira, by frequent passages in culture media, are expensive, time consuming and may cause losses in genetic characteristics of the strains. In the present study a cryoprotective technique in liquid nitrogen was standardized for six serovars of Leptospira (Pomona, Hardjoprajitno, Canicola, Icterohaemorrhagiae, Grippotyphosa and Bratislava), by using as agent cryopreservative a 2% Dimethyl Sulfoxide (final concentration) solution in a 1°C/min rate of cooling, which was compared with 0.2% and 10% glycerol solutions (final concentrations). All three cryopreservative methods were evaluated by the determination of the bacterial viability pre and post freezing. In 0, 30, 90, 180, 270 and 360 days bacterial counting was done using a counting chamber. Analysis of variance (ANOVA) and the Bonferroni method were used for the analysis of the results. A final concentration of 10% Glycerol in the cryopreservative reduced the viability of the serovars. The liquid nitrogen cryopreservation technique either with 2% Dimethyl Sulfoxide or 0.2% (final concentrations) glycerol, allowed the successful preservation of all six serovars of Leptospira during a year. The cryopreservative method ensured the conservation of the bacterial viability and was less expensive and less time consuming. Additionally, this method is a better method of long term conservation when compared with the traditional maintenance by serial subcultivation.

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“…Long-term preservation of cultures is accomplished by maintaining them at liquid nitrogen temperature in the presence of a cryoprotective agent such as glycerol (10%) or dimethylsulfoxide (Alexander et al, 1972). The viability and virulence of the cultures initially decreased 10-to 100-fold but remained unchanged thereafter over a 5-year observation period.…”

Section: Maintenancementioning

confidence: 99%

Aerobic Spirochetes: The Genus Leptospira

Johnson¹

1981

The Prokaryotes

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Preservation of Leptospiras by Liquid-Nitrogen Refrigeration

Cited by 17 publications

References 10 publications

Characterization of Leptospiral Outer Membrane Lipoprotein LipL36: Downregulation Associated with Late-Log-Phase Growth and Mammalian Infection

Characterization of Leptospiral Outer Membrane Lipoprotein LipL36: Downregulation Associated with Late-Log-Phase Growth and Mammalian Infection

Comparación de tres tratamientos para la crioconservación de serovares de Leptospira en nitrógeno líquido

Aerobic Spirochetes: The Genus Leptospira

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