REACTIVATION OF A CEPARIUM OF Yersinia enterocolitica THAT HAD BEEN CONSERVED BY TWO DIFFERENT METHODS FOR MORE THAN THREE DECADES

IRIARTE, HEBE J.; FAVIER, GABRIELA; ESCUDERO, MARÍA ESTHER; LUCERO ESTRADA, CECILIA

Mendoza

Congreso; REUNIÓN CONJUNTA SAIB-SAMIGE 2021; 2021

SAIB-SAMIGE

REACTIVATION OF A CEPARIUM OF Yersinia enterocolitica THAT HAD BEENCONSERVED BY TWO DIFFERENT METHODS FOR MORE THAN THREE DECADESIriarte HJ1,2, Favier GI2, Escudero ME2, Lucero Estrada CSM1,21IMIBIO-SL-CONICET. 2Área de Microbiología e Inmunología (FQBYF-UNSL).E-mail: hebeirimicro@gmail.comThe cepariums are genetic resources that preserve microorganisms, guaranteeing the availability of biological material forteaching and scientific research activities. Preservation must guarantee its viability in an inactive pure homogeneous state,under conditions that ensure microscopic, macroscopic, biochemical, physiological, and genetic stability, that is, withoutphenotypic variations or mutations with respect to the original conditions. The World Federation of Culture Collectionsrecommends creating a duplicate of the collection and storing it in a different location, in case such collections may be lostdue to exposure to hazards such as fire, flood, earthquake or war. In addition, it states that they must be preserved using twodifferent methods to ensure conservation. Criteria for method selection are feasibility, purity, cost, amount of culture, andfrequency of use. Ultrafreezed and liofilized are long-term preservation methods, also known as methods of choice. Regardlessof the preservation techniques used, a quality control must be carried out that includes the evaluation of viability, purity,biochemical and molecular properties. These evaluations must be carried out at the beginning, after the conservation of thefirst batch, as well as after certain periods of time. The objective of this work was to reactivate 20 strains Yersinia enterocoliticacepariums conserved in the 1980s under two different preservation methods: lyophilization (LIO) and semi-solid medium (SS).The LIO strains were reactivated in tindalized skim milk and cultured at 25 ºC for 24 h, then were replicated in tryptic soybroth (TSB) + 0.6% yeast extract (STBY) and finally in brain heart broth (BHB). The strains conserved in SS medium werereactivated in STBY, picked up at BHB and finally at TSB at 25 ºC for 24 h, respectively. All strains, after reactivated, wereseeded on Mac Conkey agar and biochemically identified to corroborate purity. Subsequently, they were ultrafreezed at -80 °C in TSB + 20% glycerol in duplicate, and in tryptic soy SS medium in duplicate at 4 °C. Counting was not performed becausethe strains were very weak, and it was necessary to carry out the replicate cultures in nutritionally rich culture medium in orderto prioritize survival over quantification. Of the 20 LIO strains, 5 were reactivated, representing 25% survival; meanwhile,from the strains conserved in SS medium, 14 strains could be reactivated, representing 70% survival. This demonstrates theimportance of establishing periodic reactivation protocols and control of viability, in order to preserve every strain from thecollection. In our study it was shown that conservation in SS medium gives better results than LIO, for long periods ofconservation of Y. enterocolitica strains.