Dierengezondheidszorg Vlaanderen (Animal Healthcare Flanders)
Question
Asked 28th Aug, 2020
What is the best way to perform a serial dilution of E.coli, when the culture is starting on a solid media (LB)?
I have an LB growth media plate with E.coli on it. I want to dilute the culture. I am working with 3M Coliform detection plates, and I want to get a solution of E.coli diluted enough to grow single colonies, rather than a blob or solid streak of E.coli (on the Colliform plates). Should I measure out LB agar (with E.coli growth on it) and add it to a liquid buffer like phosphate buffer solution to make a 1:10 dilution (example .5ml E.coli media : 4.5ml PBS) and blend? Or should I transfer the E.coli culture to a liquid growth medium, incubate, and then start the dilution from that? Maybe I am thinking about this all wrong, if anyone can provide some advice that would be a great help.
Most recent answer
I must agree with the advise of Michael J. Benedik that there is an even simpler way of proceeding if you do not need to count.
All Answers (4)
Dierengezondheidszorg Vlaanderen (Animal Healthcare Flanders)
Sam John Rice Donato The easiest way to handle this is to swab your original plate and dip (and mix) it in PBS. Then make a serial dilution in PBS and pipet a certain volume (+spread it out) of every tube of your serial dilution onto a plate. Atleast one of your plates will have single colonies. Good luck with your experiment and make sure your materials used are sterile before use.
I added a picture so you can visually see how it works.
Hamad bin Khalifa University
Sam John Rice Donato if you want to count colonies then the dilution protocol that Laurent Lanckman provided will work great. However if you only need to get a few single colonies you can just streak isolate from the original plate to get single colonies with a sterile innoculating loop.
Dierengezondheidszorg Vlaanderen (Animal Healthcare Flanders)
I must agree with the advise of Michael J. Benedik that there is an even simpler way of proceeding if you do not need to count.
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