Reactivation of a ceparium of Yersinia enterocolitica that had been conserved by two different methods for more than three decades

Abstract

The cepariums are genetic resources that preserve microorganisms, guaranteeing the availability of biological material for teaching 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, without phenotypic variations or mutations with respect to the original conditions. The World Federation of Culture Collections recommends creating a duplicate of the collection and storing it in a different location, in case such collections may be lost due to exposure to hazards such as fire, flood, earthquake or war. In addition, it states that they must be preserved using two different methods to ensure conservation. Criteria for method selection are feasibility, purity, cost, amount of culture, and frequency of use. Ultrafreezed and liofilized are long-term preservation methods, also known as methods of choice. Regardless of 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 the first batch, as well as after certain periods of time. The objective of this work was to reactivate 20 strains Yersinia enterocolitica cepariums 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 soy broth (TSB) + 0.6% yeast extract (STBY) and finally in brain heart broth (BHB). The strains conserved in SS medium were reactivated in STBY, picked up at BHB and finally at TSB at 25 ºC for 24 h, respectively. All strains, after reactivated, were seeded 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 because the strains were very weak, and it was necessary to carry out the replicate cultures in nutritionally rich culture medium in order to 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 the importance of establishing periodic reactivation protocols and control of viability, in order to preserve every strain from the collection. In our study it was shown that conservation in SS medium gives better results than LIO, for long periods of conservation of Y. enterocolitica strains.