Strange result on Agar Plate - (Jul/23/2009 )
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?
Were your uninoculated control plates clean after incubation?
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.
Is the incubator shelf level? Maybe the liquid collected on one side of the plate?
GeorgeWolff on Jul 23 2009, 05:02 PM said:
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.
Penguin on Jul 24 2009, 07:41 AM said:
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.