I ran a resazurin microtiter plate assay for Mtb. The compounds used were rifampicin and one other compound who is known from our previous data that has activity at the highest concentration only. When the results was read, rifampicin wells all turned blue which indicates no growth. The sterilization(media + solvent) wells and media only wells also had no growth. The other compound with supposed activity only in the higher concentrations turned blue at high conc and turned pink at lower concentrations. At the lower conc of this compound, there is supposed to be growth and that was manifested by the pink color. Everything else is correct except that the growth control, where the inoculum + solvent was added, did not turn pink indicating no growth. This cancels out the whole plate. It really has been boggling me for months. Why does the growth control does not turn pink?
For more details on the protocol, please see my prev question below. Thank you!
"I'm pretty sure that my cultures are growing well since my cultures look good and the growth in the broth is also fine. When I perform the assay, I adjust the absorbance to a Mcfarland 1 which is around 0.25. And then I dilute my culture further to a 1:49::culture:broth. For each well in my 96 well plate, the contents are 100uL inoculum + 98uL m7h9 broth media + 2uL of the test compound (2uL solvent for the growth control). I leave it for a week in a shaking incubator at 37°C and 150rpm. I add 20uL 0.02% resazurin after a week and read the absorbance after 24hrs, 48hrs and 72hrs incubation. The growth controls are supposed to turn pink but they're not. My whole plate is compromised because of this. I don't really know where I'm doing something wrong. Any thoughts? Thank you!"
https://www.researchgate.net/post/Im_performing_the_resazurin_microtiter_plate_assay_for_Mtb_and_Ive_been_having_problems_with_the_growth_control_not_turning_pink_What_should_I_do#58106ddf96b7e4316e334d36
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