Characteristics of the viability and accordance with the taxonomic status of the lyophilized samples of museum strains of Escherichia coli isolated in 1946-1959 years

Abstract

Effective microorganisms’ conservation with the aim of long-term storage of the strains in the collection without changes in the morphological, physiological and genetical properties is provided by methods that allow the shift of the vegetative forms of bacterial cells into the anabiosis state. The most widespread among them is the lyophilization method. The museum of microorganisms of the State Establishment “Mechnikov Institute of microbiology and immunology of the National Academy of Medical Sciences of Ukraine” has one of the oldest bacterial collections in Europe that consists of more than 4000 lyophilized samples of microorganisms strains. The aim of the study was the testing of viability and species specific properties of the museum strains of E. coli, that were preserved in lyophilized state long-term. Materials and methods. The objects of the study were the 30 lyophilized samples of the 22 strains of E. coli. Lyophilized cultures were restored through dilution of the ampule content in the 1,0 ml nutritive broth and seeding of the microbial suspension from the 100-times dilutions onto the agar-based media (blood agar, Endo medium); the viability was determined based on the quantity of the colony forming units (CFU/ml). Re-identification of the microbial cultures was carried out with the use of API system produced by «Bio-Mеrieux», France (ID 32 GN – for enterobacteria identification). The phenotypic intra-strains heterogeneity of the population was evaluated by dissociation index that reflects the ratio (%) of the certain colony forms (S-, R-, D-, M- forms) compared to the total count. The statistical analysis was carried out based on the parametric statistic methods with the use of Microsoft Excel 2007 and STATISTICA 6.0 computer programs. Results and discussion. There were (66,7±8,6) % of the total amount of E. сoli strains participating in the experiment that were able to be restored, and therefore those samples were selected for further studies. It was established that the quantity of the colonies on the solid nutritive media in the restored strains varied from 104 to 109 CFU/ml, the average survivability parameter was (26,7±4,6) %. During the statistical analysis of the results no correlations between the CFU count and the storage term were established (r=0). It was established that the majority of the re-cultivated strains (90,0±3,8) % was characterized by dissociation into different colonial and morphological variants. The dissociation index (ID) values in the microbial populations of the studied Escherichia strains varied in the range of 10,0 % to 90,0 %. The statistical analysis of the data has established the presence of correlation between the dissociation index and term of storage of the sample in the lyophilized state (r>0,95). The established colonial polymorphism of the studied strains of E. coli, in our opinion, is caused by adaptation to the stressful conditions and leads to the increase of the survivability of the bacterial population in course of the long-term storage in the lyophilized state. According to the re-identification results, the majority (90,0±3,5) % of the samples corresponded to the data indicated in the strains passport, except two strains that did not correspond to their initial identification based on the total of biochemical tests. Conclusions. The restoring of the lyophilized cultures of E. coli from 1946-1959 yy. of isolation the majority of samples was found to be viable and the viability varied in the range of 0,001 % to 100,0 %. It was established that the populations (90,0±3,8) % of restored strains were characterized by dissociation into different colonial and morphological variants. Based on the re-identification results of E. coli the correct identification of strains was carried out and corrections were put in their passports. Further studies perspectives. It is planned to study the sensitivity to antibiotics of the collection strains of E. coli, isolated in the different periods of antibiotic use.