Link to this post | posted 22 Sep, 2016 16:54

clfleischacker

My original plating of mSmeg from the stock sent to me yielded colonies with a yellowish color and looked dry and had irregular borders. Now, my smeg on the same type of agar plate (taken from the same stock tube) looks white with very small colonies. I am worried that I am growing the wrong type of bacteria! Does msmeg look different over time? These small white colonies did not appear until about 3 days after inoculation. We are also having trouble seeing any plaques: none from direct isolation and none so far from the enriched isolation (2 days incubation with 1 day post- spot test). Any suggestions as to how to proceed? Should I restart the msmeg culture from the yellow cultures or the white cultures? Christine Fleischacker Univ. of Mary

Link to this post | posted 22 Sep, 2016 21:09

viknesh

Hi Christine, It's going to be hard to tell what you have without photos of your plates with colonies. I've attached a figure with smeg on streak plates over time when incubated at 37C. You'll see from the figure that small specks become visible at day 2, and they gradually grow into larger well-defined colonies with a dry and irregular morphology by day 4 - 5. They are white to tan, depending on the age of the colonies and the properties of the plate. An important test for you to perform is a plaque assay (or a spot test) with D29. If you see plaques or a cleared spot on your bacterial lawn, then you can be confident that you are working with smeg. Vic 686Kb

Link to this post | posted 23 Sep, 2016 02:15

clfleischacker

Ok, we are trouble shooting this week since we are not getting plaques. If we do indeed have smeg then we are thinking that our smeg cultures are not dense enough to support plaques. We set up cultures with D29 but did not see any growth of the bacteria and therefore no plaques. hopefully we will know soon if our bacteria is good as we are further growing it up this week to increase its density. Then we will test it with the D29 again. Do people often see contamination with smeg when they grow it on plates with antibiotics? Also, if we see small white pin prick dots on our smeg and phage lysate plates cultured in soft agar, is that the Smeg? As you can see my concerns are mostly about my ability to recognize true growth of smeg. I guess I will just wait to test the more dense smeg cultures with the D29 and see what happens. I will let you know if we have any more problems, hopefully we can solve this one soon! Thanks for your speedy reply! Christine

Link to this post | posted 27 Sep, 2016 18:42
viknesh	Christine, While you are doing your tests, it may be worth asking for smeg from your buddy school. That way, you'll at the very least have a strain to compare to. Vic

Link to this post | posted 29 Sep, 2016 18:22

dsronderos

Vic, I see in our plates: 1. a smooth, yellowish lawn of what looks like the smeg to me. 2. random clearings at times (as though the agar solidified too soon maybe, before thoroughly mixing?) 3. small, whiter-colored "colonies" 4. Rough, translucent fibrous-looking debris (maybe from agarose?) Please see attached photos in this post, and the next few: Sincerely, David Ronderos U Mary, North Dakota Edited 29 Sep, 2016 18:48 1.50Mb

Link to this post | posted 29 Sep, 2016 18:22
dsronderos	Plate pic 2 1.40Mb

Link to this post | posted 29 Sep, 2016 18:23
dsronderos	Plate pic 3 1.08Mb

Link to this post | posted 29 Sep, 2016 18:23
dsronderos	Plate pic 4 1.36Mb

Link to this post | posted 29 Sep, 2016 18:23
dsronderos	Plate pic 5 1.14Mb