Hi, I am conducting plaque assay of MS2 phage now, and encountered a problem: host bacteria, which is E. coli K12k, didn't form bacterial lawn on plates.
The picture shows the plate condition of negative control in wich phage was not added.
White colonies were formed instead of cloudy lawn.
The following is the brief steps of the assay:
1. Add 1 mL of phage sample or LB liquid, as negative control, on 1.1% LB agar in Petri dishes
2. Add 40 mL enriched K12 in LB to 1 L of 0.8% LB agar medium
・ OD600 of K12 enrichment was 0.67
・ Temperature of the medium was under 42 C
3. Incubate them overnight at 37 C
Following the protocol, the bacteria won't form the lawn sometimes. That annoys me a lot.
I would like to know reasons why they won't form it, and tips to avoid the unexpected result.
Thank you for your kind help in advance!
Pls check the OD again as may be the cell lyse Or not able to grow properly. Or the nutrients are not Enough for the proper growth .
Hi!
When I prepared E. coli lawns, I used the same dilution (1/25=40 mL in 1L, or 100 uL in 2.5 mL), but with an overnigth culture of about 16 h. Is possible that the cell numbers are too low.
I think that if you want to count PFUs, the phage should be added with the E. coli in the soft agar.
Best,
Alejandro
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