Mirror Image Phage Display - "special" contamination (Apr/20/2011 )

Hello everybody,

I started a Mirror Image Phage Display with all three available peptide libraries from NEB (C7C, 7, 12) and except of the "normally" occuring wildtype phages in later panning rounds, we found interesting surface structures on the IPTG/Xgal plates for out- and input-titering:

I serially dilute the eluted and the amplificated phages to 10E-2 to 10E-8 (elutions) and 10E-8 to 10E-13 (amplificated phages) and we have an adequate titer dilution by counting blue plaques.
In the low dilutions we have a bacterial lawn and also a shade of blue all over it (so far so good) BUT the surface area of this bacterial lawn is not even (regarding to the characteristics of the phages, one might consider a lot of small "wells" within the lawn due to reduced baterial growth) The surface looks like, what I would call, "goosebump-like".
In every titering round I have negative controls consisting of only bacteria in TOP-agarose on IPTG/Xgal plates and in these controls, the surface is even and looks like "normal" E.coli lawn and also no blue staining occurs.

Can someone tell me what these "goosebumps" might be?


Thanks in advance,

sr

I have some experience with the PhD libraries. Can you post a picture so I would know better what you are talking about.

It will take a few days because we have easter holidays here now but when I am back, I will send you pictures from the control plate and at least one of the strange looking plates asap!

Thanks a lot!

Hy there!

I added a picture of our control plate (Bild0269)and two pictures of the "goosebumps" (Bil0270 = overview picture and Bild0247 for a closer look). Unfortunately I took the pictures only with my cell phone camera so the quality is not that good.

After talking to my supervisor, we came to the conclusion, that maybe the TOP-agar was too hot. My additional idea was a stress response of E.coli due to the high phage titer (as I wrote before, these "goosebumps" do not occur on plates with higher dilution and also not on the control plate) or the TOP-agar temperature.

I´m looking forward for your opinion!

Best regards!

not too hot, too cool. those appear to be lumps of agar. it's been a long time, but i have seen that before, when we over cooled our top agar.

Oh! Thank you, I didn´t thought in that direction.
We have our TOP-Agar at 57°C to keep it liquid and after filling up the volume I need into a 50ml tube, I kept this tube in a glass filled with hot water and used it immediately.

I gave the idea a thought and have one question left:

I made the control plate during the other plates and no agar lumps occur here, but if it is because of the TOP-agar temperature, there should also be these agar lumps, shouldn´t they?
And also I plate out one serial dilution after another and then start with the next approach and plate out these dilutions, so there should be an increase in agar lumps from approach to approach as the TOP-agar cools down while palting, but the agar lumps appear in every approach in the low dilutions but never in the higher ones.


I´ll show you a scheme here, plated out from left to right top to bottom:
(letters = the different approaches, numbers = dilutions, (-) = negative control and underlined means that one can see "lumps" or whatever they are on these plates):

A1 A2 A3 A4 A5 A6
B1 B2 B3 B4 B5 B6 (-)
C1 C2 C3 C4 C5 C6
D1 D2 D3 D4 D5 D6

the top agar is further cooled by the addition of the sample and then by the plate agar.

look at the top agar after adding the sample and mixing, before plating. does it appear homogenous or can you see small "floaters" or "bubbles".

I'd agree with mdfenko, those are lumps of top agarose. You either didn't melt it thoroughly or it solidified when you poured it over the plate. You have to prewarm the plates to 37 deg C for at least an hour, otherwise the agarose solidifies when it touches the cold agar plate.

Okay, I will try this!

Thanks a lot to both of you. I´ll report if your suggestions solve the problem (actually it is not really a problem because I was still able to count my plaques, but I was afraid of a contamination).

Have a nice weekend.