In my current project, I am dealing with bacterial strains collected from an abandoned gold mine. I want to determine the amount of cyanide produced by these isolates.
I have done the qualitative analysis experiment which confirms the ability of the studied isolates to produce cyanide (by change in color of filter papers).
But I want to know how to estimate the amount quantitatively since I don't have any specific kits or electrodes to do so.
In one article, the use of Kings B agar was mentioned (the lids of petri plates were fitted with Whatman No. 1 filter papers) which after incubation (48 hrs, 28 deg. C temperature) has to be extracted with 5 ml, 1 M NaOH and then titrated with 4.25 ml acetic acid. After that, the cyanide in NaOH solution was quantified by absorbance spectrophotometry at 575 nm after allowing the NaOH solution to react with barbituric acid-pyridine reagent.
My question is, how do I get the quantitative value after having the absorbance readings at 575 nm? What would be the concentration, or unit of the produced cyanide?
Thanks beforehand for your time on this and for your valuable contributions as well towards my question.
Horseradish peroxidase (HRP) is reversibly inhibited by cyanide and sulfide at a concentration of 10-5 M. You can employ a peroxidase inhibition assay where you make and titrate an HRP solution using the standard OPD substrate methodology for colour development. Once you have established the dilution of HRP which produces an absorbance of ~1.8 using OPD substrate ( http://www.piercenet.com/product/opd-substrates ) you preform the inhibition assay on culture supernatants using a cyanide standard curve (ng/ml - ug/ml). Incubate cyanide standard and unknowns for 30 minutes with Pre determined HRP dilution add substrate and measure absorbance at 492 nm. You must maintain culture pH > 7.0.
I carried out the determination of cyanides using method described by Bradbury. You can buy the test kit or prepare your own picrate papers and other components by yourself but please not that picric acid that is used as one of the substrates for the preparation of picrate papers is highly explosive. You can find the description of the protocol in ''PROTOCOL A: DETERMINATION OF TOTAL CYANIDE IN
CASSAVA ROOTS''. This method will allow you to avoid using potassium cyanide and preparing calibration ciurve.
You just expand your horizon a little and you will get the answer to your question. Here to help you, I recommend that you search for cyanide production by Sorghum sorghi. It is very well known. There are methods which can estimate this cyanide produced. Use these references (may be you will need to modify these methods a little) and apply on your bacterial isolates. Try it and let me know of your success.
Horseradish peroxidase (HRP) is reversibly inhibited by cyanide and sulfide at a concentration of 10-5 M. The results of qualitative test of HCN production show low percent of isolated bacteria are capable of producing HCN (about .6%).Pseudomonas florescent. 10 Pseudomonas strains could produce HCN in different levels . The production of cyanide is detected 48h after inoculation, using picrate/Na2Co3 paper fixed to the under side of the Petri-dish lids which where scaled with parafilm before incubation at 28৹c. A change from yellow to orange, red, brown, or reddish brown is recorded as an indication of weak, moderate, or strongly cyanogenic potential, respectively.
kindly explain the function of picric acid solution used in HCN production by rhizobacteria
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