Hi, I should prapare a standard curve of IAA and I don't know the method is. For measuring of IAA in bacteria, I culture bacteria in TSB medium and mix the supernatant of inoculated medium with salkowski solution (1: 2). Should I mix the IAA with salkowski in standard curve? And how much IAA should I weigh and solve in NAOH?
Thanks
Preparing a standard curve for Indole-3-Acetic Acid (IAA) is crucial for quantifying IAA produced by bacteria. Here’s a comprehensive guide to the process:
Materials Needed
Step-by-Step Procedure
1. Preparation of IAA Stock Solution
- Weighing IAA: Weigh a precise amount of IAA (e.g., 10 mg).
- Dissolving IAA in NaOH: Dissolve the weighed IAA in a small volume of 1 N NaOH. Since IAA is more soluble in alkaline solutions, this will facilitate dissolution.Example Calculation: To make a 1 mg/mL stock solution, dissolve 10 mg of IAA in 10 mL of 1 N NaOH.
- Dilution: After complete dissolution, dilute the IAA solution with distilled water to a known final volume (e.g., 100 mL) to achieve the desired concentration.
2. Preparation of Standard Solutions
- Serial Dilutions: Prepare a series of standard IAA solutions by serial dilution. For example, prepare concentrations such as 0, 5, 10, 20, 40, 60, 80, 100 µg/mL. Procedure:Take 1 mL of the stock solution and add it to a 10 mL volumetric flask, then fill to the mark with distilled water to make a 100 µg/mL solution. From this 100 µg/mL solution, take 5 mL and dilute to 10 mL to make a 50 µg/mL solution. Repeat the process to prepare the other concentrations.
3. Mixing IAA Standard Solutions with Salkowski’s Reagent
- Reaction with Salkowski’s Reagent: Mix each standard solution with Salkowski’s reagent in a 1:2 ratio.Example: For a 1 mL standard solution, add 2 mL of Salkowski’s reagent.
- Incubation: Allow the mixture to incubate at room temperature for 30 minutes. This time is needed for the color to develop.
4. Measuring Absorbance
- Spectrophotometry: Measure the absorbance of each standard solution at 530 nm using a spectrophotometer.Zero the Spectrophotometer: Use a blank solution (1 mL distilled water mixed with 2 mL Salkowski’s reagent) to zero the spectrophotometer. Measure Samples: Measure the absorbance of the standard solutions.
5. Plotting the Standard Curve
- Plotting: Plot the absorbance values against the IAA concentrations to create the standard curve.X-axis: IAA concentration (µg/mL) Y-axis: Absorbance at 530 nm
- Linearity: Ensure that the plot shows a linear relationship between absorbance and IAA concentration. The standard curve should ideally be a straight line passing through the origin.
6. Using the Standard Curve
- Unknown Samples: For measuring IAA in bacterial supernatant:Preparation: Mix the supernatant with Salkowski’s reagent in the same 1:2 ratio. Incubation and Measurement: Follow the same incubation and absorbance measurement procedure. Quantification: Use the standard curve to determine the IAA concentration in the bacterial samples by finding the corresponding concentration for the measured absorbance.
Important Tips
By following these detailed steps, you can effectively prepare a standard curve for IAA and accurately measure IAA concentrations in your bacterial cultures.
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