title: Helicobacter pylori is a fragile bacteria when stored at low and ultra-low temperatures.
abstract: OBJECTIVE
  Usually, bacteria are cryopreserved for short-term storage at low and ultra-low temperatures. There are no reports as to whether Helicobacter pylori is a fragile bacteria when stored at low and ultra-low temperatures as compared with other intestinal bacteria. A study was done on seven H. pylori strains and other intestinal bacteria to compare different temperatures for storage of organisms in saline solution.
  
  
  METHODS
  Seven H. pylori strains, specifically American Type Culture Collection (ATCC) strains 43504 and TN2GF4, and five strains isolated from the present patients were grown on a modified Skirrow's agar for H. pylori. Escherichia coli and Bacteroides distasonis, both representing isolates from the present patients, were grown on trypticase soy blood agar for E. coli, and EG agar for B. distasonis. Culture was for 4-5 days under microaerobic, aerobic and anaerobic conditions at 37 degrees C. Cells were harvested by scraping growth from the solid medium and into sterile saline. The cells were adjusted to concentrations of 109 viable cells/mL in saline and preserved at 4 degrees C, -20 degrees C, or -80 degrees C for 3 weeks before reculture under microaerobic, aerobic and anaerobic conditions at 37 degrees C for 7 days. After incubation, morphologically distinct colonies were counted, isolated, and identified by standard bacteriologic techniques. The H. pylori were morphologically analyzed by electronic microscopy before and after preservation. Mongolian gerbils were inoculated with the cryopreserved H. pylori to evaluate the bacterial infectivity.
  
  
  RESULTS
  Six of the seven H. pylori strains failed to culture after being preserved at 4 degrees C, -20 degrees C, or -80 degrees C. Only ATCC 43504 could be cultured after freezing at -80 degrees C. The number of H. pylori ATCC 43504 before preservation was 9.0 +/- 0.5 (log10 no. organisms/mL) and decreased to 5.7 +/- 0.6 after preservation. Morphologically, all H. pylori except ATCC 43504 strains transformed from a bacillary to a coccoid form after preservation. In addition, none of the H. pylori strains could infect Mongolian gerbils after preservation. Escherichia coli and B. distasonis were recovered. Titers before and after 4 degrees C, -20 degrees C, and -80 degrees C, respectively, were 9.1 +/- 0.2, 8.9 +/- 0.5, 8.6 +/- 0.3, and 8.7 +/- 0.3 for E. coli and 9.1 +/- 0.4, 8.7 +/- 0.6, 8.6 +/- 0.5, and 8.8 +/- 0.3 for B. distasonis.
  
  
  CONCLUSIONS
  Helicobacter pylori is a fragile bacteria for storage at low and ultra-low temperatures in comparison with other intestinal bacteria.
authors.affiliation: Department of Gastroenterology, Juntendo University School of Medicine, Division of Animal Center, Tokyo Medical and Dental University, Tokyo, Japan.
authors.forename: Toshifumi
authors.initials: T
authors.lastname: Ohkusa
authors.forename: Hiroto
authors.initials: H
authors.lastname: Miwa
authors.forename: Shu
authors.initials: S
authors.lastname: Endo
authors.forename: Isao
authors.initials: I
authors.lastname: Okayasu
authors.forename: Nobuhiro
authors.initials: N
authors.lastname: Sato
mesh_terms: D000818:Animals; D001431:Bacteriological Techniques; D001439:Bacteroides / Q000254:growth & development; D015169:Colony Count, Microbial; D015925:Cryopreservation; D004926:Escherichia coli / Q000254:growth & development; D005849:Gerbillinae; D016480:Helicobacter pylori / Q000254:growth & development* / Q000472:pathogenicity / Q000648:ultrastructure; D008297:Male; D014774:Virulence