I tied transforming B. subtilis with a plasmid containing ermR and plated them on erythromycin agar plates. After incubation at 37 overnight, I found the bacteria are spreading over whole plates, resulting in a smear and cloudy bacterial lawn on plates. Although I saw some colonies, they are embed in the smear bacterial lawn. What is the problem for that?
In fact I would say it has got nothing to with transformation procedure. It is just a bad habit of nearly all Bacillus species to give spreaded growth. Well the way out of it is that instead of incubating overnight (which may mean anything from 12 to 18 hours) just incubate it for 8 hours and you may get isolated colonies.
reduce plating amount and also you can keep plates for shorter incubation time... that might help... or try to use bacillus strain which lack ability to form biofilm, that will give nice colonies...
Reduce the volume of culture used for plating and also incubate the plates for less duration 7-8 h instead of overnight incubation
In addition to limiting time you grow Bacillus you might want to be sure your plates don't have too much moisture in them. After you pour the plates dry them in a laminator flow hood to a point that the excess moisture has been removed. This worked for us with B. subtilis.
It is either too much bacteria plated on the plate or too long incubation for growth. Try reducing the two components to check any growth of of bacteria.
My answer would go in the same way with others,
1.Reduce the incubation time to get a smaller single colony (so that the colony won't spread much)
2.Dilute the cell suspension more so that the colonies won't stick together
Nothing is wrong. Typical bacillus behavior. Sometimes they even look like tiny clouds taking up an entire plate. I've worked with hundreds of bacillus species, not to worry.
Try a shorter incubation period and when you inoculate, be sure to leave enough room for isolated colonies to show up- that is, away from your heavy inoculation point. Remember, some love to grow at room temperature, so try not to leave your plates out at room temp for too long (i.e. overnight).
I can recommend similar to our colleagues that you should reduce the volume of culture used for plating and also incubate the plates for less duration 7-8 h instead of overnight incubation. It should be visible individual colonies. not only bacterial lawn.
I can recommend similar to our colleagues that you should reduce the volume of culture used for plating and also incubate the plates for less duration 7-8 h instead of overnight incubation. It should be visible individual colonies, not only bacterial lawn.
I agree with D. Collins : this is typical of motile bacteria behavior. Possible solutions :
-add a few drops of ethyl alcool in the cover plate. This has been found to inhibit motility in some bacteria;
- before spreading, dilute the suspension with molten 1% agar. The viscosity will stop any possible colony spreading.
Erm is also a pretty terrible selection marker in Bacillus because there are spontaneous mutations that make them resistant. In fact, these are the first colonies to come up and will be bigger than your real colonies.
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