A carbohydrate pulse experiment to demonstrate the sugar metabolization by S. mutans

Streptococcus mutans is a fast growing organism, of low cost and easily prepared culture medium. It has been  related  primarily to  an  elevated risk  of dental cavity development  in the host due  to the  acid-induced tooth demineralization. To prevent this disease, addition of fluoride can be required, promoting the mouth  hygiene. The  main  objective  of  this  experiment  is  to  show  the  influence  of  the  carbon  source  and fluoride on the acidogenic capacity of S.  mutans. The strain was cultivated in microaerophilia, at 37oC for 12  hours  in  complete  medium  (stationary  phase).  The  cells  were  harvested  by  centrifugation  at  room temperature,  washed  with  saline  solution  and  suspended  in  the  same  solution.  The  absorbance  was adjusted  to  1  and  the  pH  to  7.3  using  0,1  mol/L  KOH  solution.  To  10  mL  of  the  cell  suspension,  distinct carbohydrates  (glucose,  xilose,  sucrose,  fructose  or  maltose)  were  added,  enough  to  establish  a  50 mMol/L final concentration. Fluoride was added (1 mmol/L final concentration) and the pH was monitored during  2 hours. In this  incubation  period,  the  suspension  was  kept  at  room  temperature  with  slow  stirring and  the  pH  was  monitored  each  7  minutes.  In  the  20  initial  minutes  of  incubation  with  glucose,  fructose, maltose  and  sucrose,  an  intense  and  very  similar  pH  decrease  (2.5  units)  can  be  observed.  This acidification reflects both the sugar uptake and anaerobic metabolization. After this initial acid liberation, a phase of slow pH decrease is observed, continuing up to 120 minutes of incubation. In presence of xilose, the  acidification  is  less  intense  and  reaches  a  similar  value  to  that  of  the  control  without  carbohydrate addition (decreasing  1.4 units  of pH). The  initial  acidification  in the presence of xilose  may  occur  due  to the mechanism of sugar uptake by this organism, which involves the antiport with H+. In media without the addition  of  carbohydrate,  the  acidification  may  be  due  to  the  metabolization  of  intracellular  reserves  of sugars. Fluoride affects negatively the acidogenic capacity of S. mutans for all metabolized sugars.