I tried to carry out cellulase plate screening for some fungi using congo red dye. But I find it difficult to measure the halo zone because the fungi has scattered on the plate despite the incubation period was 72hrs. How do I ensure a circular growth of the fungi.
Secondly, how can i measure the enzymatic index EI. In the article below http://www.hindawi.com/journals/er/2012/793708/
EI =hydrolysis zone/colony diameter. Is the hydrolysis zone the measurement of colony+zone or zone alone?
Since your colony is expectedly at the centre of the zone, you will still measure the whole zone, recording your diameters. I think if you report exactly what you have measured and how, and you did the same for your controls as the experimental, it should be acceptable. The scattering of your fungi may be peculiar to the manner of growth of fungus. If your profile pic as seen is a sample, then you clearly have circular zones that are measurable and jut measure your diameters as accurately as you could.
Hi there,
To be sure to obtain a circular growth you have to ensure that the drop of fungus suspension you spotted on the agar surface has remained circular when drying. It means that first the spotting is made carefully to preserve the drop shape and that second the plate is strictly horizontal during the drying step. The agar has to be sufficiently dry to avoid any visible moisture trace at its surface and has not to be overdried to avoid any visible wrinkles at its surface. Moisture and wrinkles are the main reasons for not getting proper spots on agar plate. Hope it helps...
My dear Friend Kaazeem: Your question is very typical not only for cellulases, but also for amylase and some time certain phospholipase like lecithinase. You problem can be solved by growing the fungi in presence of cellulose in a liquid medium. Then separate the biomass and precipitate the enzyme preferably using cold acetone or by ammonium sulfate. Dialyze it against the buffer and now do the assay by estimating glucose by DNSA method. You should be able to calculate the EI of your enzyme. Agar zone methods do not always work especially if you are using a spreading type of microorganims like fungi.
Hi
This method it's just Qualitative, you can't calculate how many enzyme hydrolyzed the culture media. I suggest you prepare a dilution spores and put a standard amount of spores in the plate, do this with a micropipette and sterile tips ( such as 10,000 spores in max volume of 10 microliter), wait for the growth and then put the congo red and add a little amount of diluted acetic acid to down the pH and see a better contrast. Check what is the "limit" of growth of the fungus on the plate ( see inside the agar plate and find where are vegetative mycelium and use that as reference) and check the relation of growth/hydrolysis.
I send you an image of my work.
1.- Growth of the fungus (after staining of course)
2.-Limit of the growth
3.- relation between the growth and the hidrolysis
I quite agree with the suggestion that plate or qualitative screening generally for fungal enzymes, or at least those which are associated with glycosyl hydrolysis including but not limited to cellulases, is not usually a reflection of the cellulolytic ability of the organisms. Rate of enzyme diffusion in the solid media have been suggested to be affected by; 1-molecular weight of the enzyme, 2-enviromental temp, 3- the intensity of the agar and, among other factors, the inoculum sizes of the organisms. The use of fermentative technique (SSF or SMF) is, despite its challenges, a better sustitute. Plate screening can however
You may check this link for details:
http://www.ajol.info/index.php/ajb/article/viewIhttp://www.ajol.info/index.php/ajb/article/view/94129
In addition to the factors above and as you earlier observed; there are some fast sporulating organisms that, regardless of the period of incuation would spread faster and cover the plate, thus making the process (plate screening) ineffective for proper screening.
On EI, the previous suggestions are to me very good.
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