Team:Newcastle/29 June 2010

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29 June 2010

Contents

Aims

The aim of this experiment is to prove that Bacillus subtilis 168 can take up arginine from the media into the cell.

Materials

  • pH strips
  • LB media consisting arginine
  • Autopipette
  • Gilson pipette
  • Autoclaved tips for gilson pipette
  • Burnsen Burner
  • Universal Tube

Protocol

  • Set up and label the universal tubes as indicated:
  1. Negative control- Media not containing Bacillus subtilis 168
  2. Test- Media inoculated with Bacillus subtilis 168 cells
  • Unscrew the caps of the universal tubes and after unscrewing, flame the mouth of the tube.
  • With the help of an automatic pipette, pipette out 4ml of LB broth from the stock solution and transfer it into the two universal tubes mentioned above.
  • Remove the universal tube consisting of an overnight culture of growing Bacillus subtilis 168 cells and unscrew the cap and flame the mouth of the tube.
  • Set the Gilson pipette to 1000 µl and attach a autoclaved tip to it.
  • Remove 1000 µl out of the tube consisting of overnight culture and put it in the universal tube consisting 4ml of LB media. Do this step twice so as to put 1000 µl in both the control and the test tubes.
  • Flame the mouth of the universal tubes and screw the cap (do not over tight the cap so as to allow oxygen to enter the tube)and shake gently so as to suspend the cells equally.
  • Put the universal tube on a shaker at 37° C and leave it for the cells to grow.
  • Record the pH change of the media after every 1 hour by removing 20 µl of the media from both control and test tubes with the help of Gilson pipette and putting it on the pH strip and note the change in the colour.

Result

Hours Negative control Test
0 pH 7 pH 7
1 pH 7 pH 7
2 pH 7 pH 7.5
3 pH 7 pH 7.5
4 pH 7 pH 8
5 pH 7 pH 8

Discussion

Arginine consists of a positive charge on it and thus is acidic in nature. As the cells take up the arginine from the media, the pH of the media increases. The tube labeled Negative control contains no cells and the pH of the media has remained constant for 5 hours which shows that the level of arginine in the media has remained constant, while the tube labeled Test which contains Bacillus subtilis 168 cells shows an increase in pH which shows that the level of arginine has decreased in the media.

Conclusion

It can be concluded that the Bacillus subtilis 168 cells are able to take up arginine from the media into the cells.


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