Helicobacter pylori in Liquid Culture: Evaluation of Growth Rates and Ultrastructure

Abstract.

This study investigated the growth of Helicobacter (H.) pylori in Brucella broth supplemented with either IsoVitaleX (1% vol/vol), hemin (0.1% wt/vol), agar (0.3% wt/vol), or blood agar blocks (1.5% wt/vol agar). IsoVitaleX was found to significantly shorten the lag phase, while hemin inhibited the growth within the first 24 hours but later acted as a growth stimulant. There was a tendency toward stronger growth when blood agar blocks were added to the medium. Subsequent electron microscopic evaluation revealed that cells of H. pylori were attached to blood agar block surfaces. In contrast, the supplementation of Brucella broth with agar did not significantly increase the cell density. When H. pylori was grown in the presence of IsoVitaleX, strongly stainable electron-dense bodies (140–200 nm) were seen in the cytoplasms. Incubation of cultures on rotary shakers at 120 rpm significantly enhanced growth. The addition of glycerol (15% vol/vol) or fetal bovine serum (15% vol/vol) showed good ultrastructural preservation of bacteria with undamaged cell walls and cytoplasmic membranes, and the cytoplasms were ribosome-dense. Cell counts revealed that cultures stored in glycerol or fetal bovine serum had a significantly lower loss in viability when compared with cultures stored without cryopreservatives. Unprotected cells of H. pylori showed on electron micrographs clumping, cell lysis, and flagellar damage. Finally, the survival rates of H. pylori after multiple thawing from storage at −80°C were best in Brucella broth/glycerol, Brucella broth/fetal bovine serum, and Brucella broth without cryopreservative (in descending order).