Materials and Methods:

1
Materials and Methods

• Preparation of Bacillus isolate
• Roots from strawberry plants inoculated with a
  biocontrol bacterium, Bacillus subtilis FZB24 WG,
  were pasteurised for 10 minutes at 80ºC, serially
  diluted and plated onto tryptic soy agar (TSA)
  (Traver et al 1987).
• Colonies were selected, restreaked and subjected
  to MALDI analysis.

2
Materials and Methods

• Matrix selection
• Matrix selection is important in MALDI analysis
  as it is the matrix which absorbs the photon
  energy from the laser.
• The most commonly used matrices in whole cell
  MALDI analysis are 3,5-dimethoxy-4-hydroxycinnamin
  ic acid (sinapinic acid, SA) and
  a-cyano-4-hydroxycinnaminic acid (HCCA) (Lay,
  200).
• For Bacillus species, sinapinic acid gives poor
  spectra with few peaks, while HCCA gave a good
  range of peaks. Similarly with Pseudomonas
  species, HCCA was found to give a better range of
  peaks.

3
Materials and Methods

• Preparation of Bacillus Material for Analysis
• Bacillus species were grown on TSA plates and
  Pseudomonas on Nutrient Agar for 24 hours prior
  to analysis.
• Cultures stored at 40oC were allowed at least two
  passages on appropriate medium to obtain isolated
  colonies.