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I've got petri dishes with a base agar layer ready for E. coli culturing. However, I do not have soft agar in which to dilute the E. coli before pouring it on to the plate.

Any suggestions on what substance I can use to substitute for soft agar, or how I might be able to make it? I have the following at my disposal from the lab at school:

  • Tryptone base agar
  • Nutrient broth
  • MacConkey agar

The original soft agar medium had the following composition:

  • 10 g Bacto™ tryptone
  • 9 g Bacto™ agar
  • 5 g KCl
  • 1000 mL water
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  • $\begingroup$ You want to take up the cells in a medium containing agar and then plate them? $\endgroup$ – Chris Feb 23 '16 at 13:35
  • $\begingroup$ @Chris Yes! I was originally using a soft agar solution composed of 10 g Bacto™ tryptone, 9 g Bacto™ agar, 5 g KCl, 1000 mL water. $\endgroup$ – Samuel Spencer Feb 23 '16 at 13:54
  • $\begingroup$ And I guess that your standard plates are made with your media + agar? $\endgroup$ – Chris Feb 23 '16 at 13:59
  • $\begingroup$ @Chris Correct, the E. coli in the medium is poured over the base agar so the standard plates have the media and the agar. $\endgroup$ – Samuel Spencer Feb 23 '16 at 14:02
  • $\begingroup$ Ah, I prepare the plates with medium + agar. $\endgroup$ – Chris Feb 23 '16 at 14:05
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The good news is: You don't need any agar in your plating media, since the bacteria grow on the surface anyway and the plate can take up some additional moisture from your plating media.

What I usually do is to dry the plates for a while (and also let them warm up, since they are stored in the fridge) with the plate upside down and opened a bit. Contamination is usually not a problem here.

Then I dilute the cells in an appropriate volume of media, then plate about 50 to 100µl on the agar and spread it with a Drygalski spatula. Put the plate in the incubator and everything is fine.

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