When I grow E,coli stain on solid support( i.e Agarose) for time lapse microscopy Experiment , I found fast in growth relative to batch culture in broth media. I tried to maintain all condition like temperature, nutrient and others like in Batch culture. please, Is there any suggestion?
Hi; That is because it is a aerobic bacteria and Agare or solid medium support the bacteria with more Oxygen because of the structure of bacteria growth in the surface
Are you using same conditions?
Molarity of both mediums?
Are you inoculating both mediums with the same concentration of bacteria, how are you measuring its growth?
Hi
Subculture the ones grew on agar into broth then incubate it with shaking at the optimum temp.
Hope this helps,
How are you comparing growth, which unit, how are you measuring it?
How do you know that one has more groth than the other?
It does not help if they do not grow in the same conditions from the same stock at the same inoculation concentration and in a medium with same concentration of nutrients.
Hi Jose
First, I grow the bacteria in 5ml broth Media overnight and follow up for several generation by diluting with fresh media and measure the OD as measurement for Generation time. When the cell reach in the Steady state, I transferred 2 ul of the steady state cell to 3%Agarose pad ( which is prepared in the same media) and take image with 2.5min by Time Lapse Microscopy and follow up for 3-4 generation. Finally, I used imagej to measure cell length or cell area to calculate the Generation time of the cell on the Agarose pad. The measured value indicates that the growth on Agarose pad is 2x faster than in broth culture under the same condition. Why?
Because you change the conditions of growth.
Why should a bacteria in agitated liquid culture grow to the same rate than bacteria on a solid media with a very very different contact with air, and probably with other cells ?.
I suppose that you agarose pad is covered by a coverslip that should highly limit O2 availability.
On the other way, measuring the OD is not the same as measuring the cell surface or lenght.
What about, when culturing your bacteria in broth medium, harvesting samples at selected time points to take (rapidly) images to analyse with imageJ the same parameters you use when performing your agar pad assay.
Hope this help
You can't compare data in different units... it has no sense, and you can not extract any conclusions like the one you said.
Hi
Agarose is polysaccharide that contains galactose residue, I doubt that it will support the growth.
Hope this help
In addition to mentioned above, when bacteria have ''no swimming conditions" they loose less energy.
It's very difficult to compare growth on solid culture vs growth in a liquid broth. It's just different in many ways. For the most part, I agree with most answers that oxygenation is richer when shaking in liquid rather than sitting on a matrix of agar at the same temperature. Plus, by changing the rpm at which the liquid broth is shaking, you could, to some extent, manipulate the rate of growth. Within limits, faster shaking equals faster growth.
I don´t agree with the first answer. E. coli is not strict aerobic like Pseudomonas. But I think you should use a really big Erlenmeyer-glassbottle. It should be filled very low( 1/3) and it should stand on a bottle-shaker. But what I think is the problem. E. coli is very fast growing and the PH of the Media is than too low. You should read something about generation-time of E.coli.
Nice greatings from Bad Vöslau
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