Genetic stability of strains preserved on LENTICULE discs and by freeze-drying: a comparison using fluorescent amplified fragment length polymorphism analysis

Abstract

Fluorescent amplified fragment length polymorphism (FAFLP) analysis, a high-resolution genome fingerprinting method, was used to ascertain the DNA integrity of bacterial strains during preservation by lenticulation and by traditional freeze-drying into glass ampoules. This was achieved by comparing FAFLP genotypes of a range of paired bacterial isolates recovered from LENTICULE discs (preserved between 1995 and 2004) and from freeze-dried (FD) cultures in glass ampoules (preserved between 1966 and 2000). A choice of two endonuclease combinations EcoRI/MseI or HindIII/HhaI was used for FAFLP analysis of the five different bacterial genera comprising of 10 strains. Each of these 10 strains exhibited unique FAFLP profiles. However, there were no detectable differences between the FAFLP profiles for each of the individual strains, irrespective of their preservation format or their year of preservation. Thus, the FAFLP data suggests that LENTICULE production does not result in any detectable genetic changes during drying onto LENTICULE discs and storage for at least 5 years. The provision of such FD reference cultures on LENTICULE discs rather than FD glass ampoules will provide a cost-effective format that is easier to use.

Assessment of the stability of cell-surface components of microorganisms by MALDI-TOF-MS following preservation on lenticule discs

“…The results of this study demonstrate unequivocally that cells preserved on lenticule discs remain structurally and physiologically similar to those preserved by the traditional freeze-drying method in accord with data derived by genotypic analysis (Desai et al, 2006). Because lenticule discs are less cumbersome to prepare and revival of cell cultures is simpler, this method of preservation and transport between laboratories may supersede traditional freeze-drying for many purposes and will find application across broad areas of microbiology.…”

“…Bacterial cells preserved on lenticule discs were shown recently to have comparable whole genome DNA fingerprinting patterns (fluorescent amplified fragment length polymorphism, FAFLP) to those preserved by standard freezedrying procedures (Desai et al, 2006). However, while this establishes that the genomes have remained stable, changes in proteins (e.g.…”

Examining the genetic variation of reference microbial cultures used within food and environmental laboratories using fluorescent amplified fragment length polymorphism analysis

“…However, it is essential to maintain the integrity of the cultures with respect to growth characteristics, cell viability and genetic stability. Previous studies have highlighted the genetic stability of cultures following lenticulation and freeze-drying using FAFLP (Desai et al, 2006). In this study, we used FAFLP to examine potential gross chromosomal changes associated with subculturing of working cultures and the potential of cross-contamination of the working culture with a different strain.…”

“…DNA was eluted in 100-mL volumes and stored at À 20 1C. FAFLP was performed with genomic DNA using the endonucleases HindIII and HhaI (New England BioLabs, UK) for all strains, as described previously (Lawson et al, 2004;Desai et al, 2006). Briefly, 500 ng of DNA was sequentially digested with the two endonucleases followed by ligation to endonuclease-specific adaptors (MWG Eurofins, Germany).…”

“…A strain-characteristic FAFLP profile varying in the number and sizes of the fragments is obtained. A recent paper by Desai et al (2006) examined the genetic stability of bacterial strains preserved by two different methods, lenticulation and freeze-drying, using FAFLP. No detectable genetic changes were found between the two approaches to preservation, or as a result of storage of isolates over a 5-year period.…”