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Strange result on Agar Plate


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5 replies to this topic

#1 chp298

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Posted 23 July 2009 - 11:28 AM

Hello,

I run a number of experiments on mouse stool, plating it on a variety of different agars. Lately, I have been plating mice infected with a pathogen that has a kanamycin resistant gene, so I've been using MacConkey Agar with kanamycin sterilely filtered in.

Ran it this week, and have had a couple of the plates come back with growth only on the very edge of the plate. Take a circle, draw a straight line through it so you're splitting the plate 15 percent one side, and 85 on the other, and I've got growth only on the 15 percent side. It's like a perfect line stopping the growth.

When I add the kanamycin to the bottle of agar, I mix it thoroughly before pipetting 5 mLs of agar into 14 mL "pour plate" tubes, then after adding stool in PBS to the agar tube, I vortex it properly before pour plating into a plate. I also mix the plate around delicately in order to get an even mix in the plate. I cannot understand why I am getting growth like that.

We have had issues with our non-CO2 incubator, wherein we incubate these plates, but I keep a close on the temperature constantly. Might this have something to do with the plates being near the walls of the incubator rather than in the middle?

Thanks!

#2 GeorgeWolff

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Posted 23 July 2009 - 01:02 PM

Were your uninoculated control plates clean after incubation?

#3 miraclestrain

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Posted 23 July 2009 - 10:53 PM

Hi there,

Why dont you try doing a broth and also try to find the density of growth (turbidity) using a spectrophotometer. Hope this might help you.

MS

#4 Penguin

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Posted 24 July 2009 - 03:41 AM

Is the incubator shelf level? Maybe the liquid collected on one side of the plate?

#5 chp298

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Posted 24 July 2009 - 06:30 AM

Were your uninoculated control plates clean after incubation?


Yes they came up totally blank, no growth of any sort.

Quick update, I left the plates in overnight, and there was further growth... so you take that line I spoke of in the original post, and push it along.

#6 chp298

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Posted 24 July 2009 - 06:44 AM

Is the incubator shelf level? Maybe the liquid collected on one side of the plate?



Yes, we keep the incubator up on our tabletop. Original I put the plates on the bottom level, there were a load, so some were against the wall, others were in the middle.




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