Extreme Base Resistance Test

Aim

The aim of this experiment is to test that the chassis is able to acclimatise to the increase in the environmental pH.

Materials Required

1. Bacillus subtilis 168 colony plates
2. Potassium modified Luria broth (LBK)
3. HEPES (a dibasic compound which would act as a buffer)
4. Conical flasks

Procedure

Day 1

1. Grow B. subtilis 168 cultures in LBK buffered with 100mM HEPES at pH 7 and incubate them overnight at 37 °C for 16 hours.

Day 2

1. Make 2 different LBK solutions buffered with 50mM HEPES at either pH 7 and pH 9.
2. Dilute the overnight cultures by 100 fold in the LBK solution mentioned above.
3. Let the bacteria grow until it reach at the OD600 of 0.3.
4. Prepare a LBK solution buffered with 50mM HEPES at pH 10.
5. Dilute the cultures by 200 fold in the LBK broth mentioned in the above step and incubate them for 2 hours at 37 °C.
6. Plate serial dilutions of the culture mentioned in the above step on a LBK plate and also plate the original culture in the medium at pH 7.
7. Let the cells to grow and see the growth of the colony next morning.

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