In this method, the first layer containing active bacterial culture agar is spotted. After 48 hours when the bacterial colony has grown, the second layer of soft agar containing indicator bacteria is slowly poured... However, the bacterial colony that It was spotted, unfortunately it is spread... what is the solution?
You can make the lower agar layer stiffer and drier. Colonies tend to smear less with dry plates. Colonies that have been given a few days to establish in a fridge also are stickier so have fewer cells washed away by pouring the new layer. Also pour the second layer very slowly and maybe even let the poured agar flow along a pipette tip in a corner away from the colonies to reduce the sheer forces applied. Still it is very hard to not get smearing when using an overlay method. Have you considered plating both bacteria in the same agar?
@Robert Adolf bronze
Thank you for your reply, the first layer contained MRS with 1.3% agar and the second layer contained MHB with 0.6% agar, what is the appropriate percentage for the first layer?
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