Aim

The aim of this protocol is to prepare slides for the Bacillus subtilis 168 cells which have yneA integrated into the chromosome and are induced by IPTG at various concentrations.

Materials

1. Flasks
2. Colonies of Bacillus subtilis 168 cells with different plasmid insertions.
3. Inoculation Loop
4. Shaking incubator
5. 1M IPTG stock solution
6. Appropriate antibiotic solutions
7. 1.2% agarose
8. 12 well glass slide
9. LB broth
10. Spectrophotometer

Protocol

1. Take 20 ml of LB broth with appropriate concentration of antibiotics in a flask and innoculate it with a particular colony of Bacillus subtilis 168 cells with a particular plasmid insertion.
2. Put it onto a shaking incubator at 37°C overnight.
3. Next morning, check the OD₆₀₀ by using spectrophotometer and record OD for future references and to check whether all the cells are in the same growth phase or not.
4. Now,in a flask add 10ml of LB broth with an appropriate concentration of antibiotics and add 100µl of the appropriate overnight culture in it.
5. Measure OD₆₀₀ after every 30 minutes and record it everytime.
6. In the mean time, prepare flasks with proper labels and also dilute 1M stock solution of IPTG to a range from 0.02mM, 0.2mM, 1mM and 2mM IPTG solutions.
7. When the OD of all the cell population comes to 0.1 then take 5ml of the sample from the flask and transfer it to a universal tube and add appropriate amount of the IPTG solution in it. In this way we will have 4 universal tubes for a particular sample having different concentrations of IPTG.
8. Incubate the cells at 37°C on a shaking incubator for 120 minutes and inbetween time, check the OD of the cell culture after every 30 minutes.
9. After 120 minutes put the cell cultures out on the bench at room temperature and start preparing the slide.
10. For slide preparation, first clean the slide with 100% ethanol.
11. Now make 10 ml of 1.2% agarose solution (agarose and water solution).
12. Now on the slide add 500µl of the agarose solution and but a long coverslip over it and be careful that all the wells are been filled up by the agarose solution.
13. After 5 minutes, remove the coverslip by sliding it horizontally so that we dont disturb the agarose layer.
14. Now we take 0.5µl of each sample and load it in each well separately and put the coverslip back on the slide.
15. Observe the slide using normal phase contrast or fluorescent microscope.