I like to estimate the phosphatases activity in PGPR strain. So please give the standard method for quantification.
Hi
the simplest method (for me) is the MUF one.
4-Methylumbelliferyl phosphate phosphatase substrate | Sigma-Aldrich
There are referential article in the Sigma catalogue.
You can apply it quantitatively in liquid cultures or to see it qualitatively - active colonies on agar plates.
If you want to apply it in the nature, look for articles by Vrba, Štrojsová, Nedoma.
If you need to mark an active cell / organism, use ELF method
ELF 97 Endogenous Phosphatase Detection Kit - Thermo Fisher ...
but it was designed for us (working in the field) and it is probably useless for your purposes (pure cultures)
Cheers
Mirek Macek
Mirek Macek
Microorganisms on precipated calcium phosphate agar produces clear zones around their colonies if they are capable of solubilizing calcium phosphate.
This medium was used to isolate Pseudomonas . Pikovskaya’s. (1948). the medium constituent as follows: Composition of Pikovskaya’s medium g/L, Glucose 10.0 g ,Tricalcium phosphate 5.0 g, Ammonium sulphate 0.5 g, Nacl 0.2 g, MgSo4.7 H2O 2.5 m g ,MnSo4.2H20 2.5mg, FeSo4.7H2o 2.5mg, Yeast extract 5g, Agar 15.0 g, PH 7.0 ± 0.2
Pikovskayas Agar is normally used for the detection of phosphate-solubilizing bacteria.
Quantitative estimation of Phosphatase- culture filtrates were incubated with 2 ml of tris buffer pH 8.4 for 4 hrs. Further, 2 ml of the mixtures of reaction were transferred to the tubes containing 2 ml of 0.05 M EDTA and 2 ml of 0.5N NaOH. The activity of phosphatase was measured by spectrophotometrically at 420 nm against control. The quantity of released p-nitrophenol was estimated by using the standard graph. The unit of phosphatase activity is expressed as micro grams of p-nitrophenol released per minute per ml.
Dear All,
Thanks for the responses. Dr. Praveen can you share the reference for this methodology.
Two comments to Praveen recommendations:
(i) We were using p-nitrophenol many years ago when it was the best method. However, there are some limitations with it: lower sensitivity (comparing to fluorescence methods mentioned in my response) and the problem with colour, which is not negligible in cultivation media (and bog lakes containing humic substances, etc.): Th product is yellow, thus the blanc may be very high making even more troubles. As well, be careful with pH because both MUF and p-nitrophenol must be measured upon pretty high pH (in my lake it produced precipitation); MUF is fo acid-neutral waters.
(ii) Colleagues of mine (I was only using their methods) spent years to show that enzymes are "connected to microorganisms", i.e. produced / localized just above the membrane - ectoenzymes. I know, in the culture the concentration of enzymes in the filtrate would be very high but I disagree to standardize the method on such way. (For the theory look for Chrost and Vrba publications from 90'-00'; do not be surprised, it is not directed to phosphatases as the articles by Strojova do)
To use the whole culture, you will need to centrifuge the product, the process that result much more efficient for the fluorescence methods
If you have a fluorometer, use it. If not, photometric method is a good possibility but think twice if you want to see only soluble or total enzymatic activity. In a very dense culture it may be similar, in diluted one or waters it will be very different.
Cheers
Mirek Macek
Dear Dr. Jegan Sekar, did you manage to obtain the method for the quantification of phosphatases in bacterial cultures or a similar method, mentioned by Dr. Praveen? I also want to perform the same test in cultures of bacterial liquids. Please, you can share that information with me, I have searched for that information but I have not been successful, and the information I find is very scarce. Thank you very much for your reply.
Yes. You can follow the same method.
Quantitative estimation of Phosphatase- culture filtrates were incubated with 2 ml of tris buffer pH 8.4 for 4 hrs. Further, 2 ml of the mixtures of reaction were transferred to the tubes containing 2 ml of 0.05 M EDTA and 2 ml of 0.5N NaOH. The activity of phosphatase was measured by spectrophotometrically at 420 nm against control. The quantity of released p-nitrophenol was estimated by using the standard graph. The unit of phosphatase activity is expressed as micro grams of p-nitrophenol released per minute per ml.
Dr. Jegan, can you share the reference of this method?. Thanks you.
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