Hi,
I prepared the medium by adding peptone, NaCl, CaCl2•2H2O, agar and Tween-20 for lipolytic activity assay. And cultures are spotted into the medium. Finally I hoped clear zones around bacteria, but didn't see. Why this problem occurs?
1st of all you need to be more specific.. ok you ve added the compounds, but this is not a cooking recipe.. define how much of which compound, v/v, concentrations etc.. otherwise how you expect us to help you.
a good approach would be Gopinath S. C. B., Anbu P., Hilda A. 2005. Mycoscience 46: 119–126.
- peptone: 10 g/L
- NaCl: 5 g/L
- CaCl2. 2H2O: 0.1 g/L
- agar: 20 g/L
- tween 20: 10 mL, v/v
what you ve used? and what was the temperature you chose for the application of the assay?
Try using a positive control - a culture you are sure is lipolytic and seeing how does it behave under your experimental conditions.
thank you for answers.
as you say Evangelos Petropoulos: I prepared components according to Bettache, et.al. (2012) (From this article : Isolation and Identification of Lactic Acid Bacteria from Dhan, a Traditional Butter and Their Major Technological Traits)
And cultures were incubated at 32 oC 24 h. Bacteria grew under this conditions but i didn't see clear zones.
Indeed a positive control would help you distinguish whether the issue is the lipases or the substrate you are using for the assay. to accelerate things maybe increase the temperature a bit, around 37oC.
The use of a positive control is a must, to ensure that assay works correctly, but another possibility is that the lipolytic activity may be intracellular and therefore may not have access to the Tween substrate in healthy growing cells. It might be worth attenuating cells and checking these with live cells to see if you get a zone of clearing.
try with 0.5% Tributyrin.
It is best substrate to screen the lipase.
Good luck
Actually, the choice of substrate is also important. It should mimic the "natural" system where your bacterial samples come from - it doesn't necessarily have to be tributyrin.
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