I am using LB Broth (25 gr in 1L) and Incubating the samples for 24 hours in 25 degrees Celsius. I put the culture tubes on a shaker with 300 RPM for the first twelve hours but still, what I see under microscope is that the motile cells only make about 10% of the total number. Is there something that I am missing here?
Dear Kiarash,
P. putida KT2440 grows perfectly on LB medium (on agar plates, in shake flasks and bioreactors). Composition per liter is: 10 g tryptone, 5 g yeast extract and 10 g NaCl. You may add 2 g/L glucose to accelerate growth. Try 230 rpm, 30°C and baffled shake flasks. And it has been shown that flagellar motion is not crucial for survival under lab conditions as chemotaxis is usually not necessary in rich medium.
Best
Michael
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Dear Michael
Thanks for the papers and your helpful response. I am studying the motility of P. putida so I would like a good percentage of my bacteria to have flagellar motilty.
From what I read in the papers you sent, I guess I can try using a less nutrient medium/ or not using the shaker for flasks. Do you have any other suggestions?
Try E2 or M9 minimal medium in your cultivations and see how this goes. Be sure you're investigating vital cells in the exponential growth phase. 24 hours at 25°C might not be optimal.
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