HELICOBACTER-PYLORI IN LIQUID CULTURE - EVALUATION OF GROWTH-RATES AND ULTRASTRUCTURE

This study investigated the growth of Helicobacter (H.) pylori in Bruc ella 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 a s a growth stimulant. There was a tendency toward stronger growth when blood agar blocks were added to the medium. Subsequent electron micro scopic evaluation revealed that cells of H, pylori were attached to bl ood 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. Incuba tion of cultures on rotary shakers at 120 rpm significantly enhanced g rowth. The addition of glycerol (15% vol/vol) or fetal bovine serum (1 5% vol/vol) showed good ultrastructural preservation of bacteria with undamaged cell walls and cytoplasmic membranes, and the cytoplasms wer e ribosome-dense. Cell counts revealed that cultures stored in glycero l or fetal bovine serum had a significantly lower loss in viability wh en compared with cultures stored without cryopreservatives. Unprotecte d cells of H. pylori showed on electron micrographs clumping, cell lys is, and flagellar damage. Finally, the survival rates of H. pylori aft er multiple thawing from storage at -80 degrees C were best in Brucell a broth/glycerol, Brucella broth/fetal bovine serum, and Brucella brot h without cryopreservative (in descending order).