Team:Groningen/Template/MODULE/PP/collaborations/methods

We performed a halo experiment to test if S. aureus has sensitivity to subtilin, produced by B. subtilis. This was performed in the ML-II lab of the MolGen group at the University of Groningen and supervised by Renske van Raaphorst. Below we report what steps were taken and give an overview of the results. Also we provide you with some references of the strains we used.

- Inoculated three different strains of S. aureus (see below) in 3 ml Nutrient Broth (Difco). Also two different B. subtilis strains were inoculated under different conditions (see below). The ATCC6633 strain was inoculated in 3 ml LB and in 3 ml of the defined medium SMM. Also the 168 strain was inoculated in 3 ml LB. These samples were grown overnight at 37ºC in a shaking incubator.

Used strains S. aureus: (All strains were provided by Auke van Heel)

Used strains B. subtilus:

- Of all the S. aureus strains two different blood agar plates were made. The first set was named CAL1, NWZ1, CECT1, which meant that per plate 200 μl of sheep blood was added to 10 ml Colorado Blood agar. These were poured in different plates and afterwards 120 μl of each S. aureus strain was streaked out on top of it.

- The second set was named CAL2, NWZ2, CECT2. In these plates 200 μl of sheep blood was added to 10 ml Colorado Blood agar per plate. These were poured on top of 120 μl of each S. aureus strain on each plate separately. This was mixed thoroughly so that the agar was mixed with the strain.

- All the plates were dried under the fumehold.

- On these six plates 6 μl of the three different sets of B. subtilis were spotted and put at 37 ºC for 24h.

- All the diameters of the halo's were measured and the plates were discarded. (See results below).