Sep 12, 2025
Siderophore Detection assay
- Md Sahadat Ali1,
- Jonathan D. Eisenback1,
- Fatima Tuz Zohora Mony1
- 1Virginia Tech
Protocol Citation: Md Sahadat Ali, Jonathan D. Eisenback, Fatima Tuz Zohora Mony 2025. Siderophore Detection assay. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg3184el25/v1
Manuscript citation:
Ali, M. S., Mony, F. T. Z., Evans, M., Rideout, S., Haak, D., & Eisenback, J. D. (2025). Unveiling the antagonistic activity of ‘Candidatus Pseudomonas auctus’ JDE115 against Agroathelia rolfsii: A soybean nodule endophyte with biocontrol potential. (In preparation)
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 10, 2025
Last Modified: September 12, 2025
Protocol Integer ID: 226968
Keywords: Siderophore, CAS assay, Pseudomonas auctus JDE115, Chrome Azurol S, HDTMA, microbial antagonism, iron chelation, plant growth-promoting rhizobacteria (PGPR), quantification of siderophore production, siderophore detection, siderophore production, siderophore production index, biocontrol bacteria, siderophore activity, bacterial isolate, bacterial suspension, surrounding bacterial colony, bacterial colony, agar assay
Disclaimer
This protocol is provided for research and educational purposes only. The procedures described have been successfully applied in our laboratory; however, results may vary depending on strains, media, and laboratory conditions. Users are responsible for ensuring compliance with institutional biosafety, chemical safety, and ethical regulations. The authors and Virginia Tech assume no liability for misuse or unintended applications of this protocol.
Abstract
This protocol describes the detection and quantification of siderophore production by 'Biocontrol bacteria' using the Chrome Azurol S (CAS) agar assay, adapted from Schwyn and Neilands (1987). CAS agar was prepared by incorporating a dye complex of CAS, HDTMA, and FeCl₃ into LB agar. Bacterial suspensions were spotted onto CAS agar plates at different dilutions and incubated at 28 °C. Siderophore activity was visualized as yellow-orange halos surrounding bacterial colonies, and the siderophore production index (SPI) was calculated. This method enables reproducible, semi-quantitative screening of siderophore production by bacterial isolates.
Attachments
Image Attribution
All experimental images and figures (e.g., CAS assay plates, halo measurements) were generated by the authors at Virginia Tech. Images are original and may be used for academic and research purposes with proper citation of this protocol.
Guidelines
- Use a freshly prepared FeCl₃ solution for optimal complex formation in the CAS reagent.
- Prepare CAS reagent and LB agar separately and mix only after cooling to ~50 °C to prevent dye degradation.
- Ensure all glassware and solutions are free from iron contamination by rinsing with acid-washed water if possible.
- Maintain aseptic conditions throughout the experiment to avoid false positives from contaminant microbes.
- Include both positive and negative controls to validate results and calculate the Siderophore Production Index (SPI) accurately.
- Perform at least three independent experimental repetitions to ensure reproducibility and statistical validity.
Materials
- Biocontrol-Bacteria
- 2000 mL Beaker
- 1000 mL Graduated Cylinder
- 250 mL Graduated Cylinder
- 125 mL Flask
- 2x 100 mL Beaker
- 15 mL Falcon Tube
- Fume Hood
- Biological Safety Cabinet (BSC)
- Spatulae
- Stir Bar
- Stir Plate
- Weigh Boat
- Weigh Paper
- Precise Scale (?)
- Non-Precise Scale (?)
- Aluminum Foil
- Autoclave Tape
- Prepared 1 mM FeCl3
- HDTMA (Hexadecyltrimethylammonium bromide)
- Chrome Azurol S (CAS) Dye (in powder form)
- DI Water
Troubleshooting
Safety warnings
- Handle FeCl₃ with care; it is corrosive. Wear gloves and eye protection.
- CAS and HDTMA dyes can stain; handle with protective clothing.
- Dispose of CAS-containing waste as chemical waste.
Ethics statement
This protocol involves microbial cultures only and does not require human or animal ethics approval. All work was conducted under BSL-2 safety regulations at Virginia Tech.
Before start
- Ensure that all glassware is acid-washed to remove trace iron contamination.
- Prepare fresh FeCl₃ solution before use.
- Autoclave media and solutions separately; mix only after cooling to ~50 °C.
Preparation of CAS agar (Day 1)
- Prepare 900 mL LB agar: dissolve 5 g yeast extract, 10 g NaCl, and 15 g agar in deionized water; autoclave at 121 °C for 15 min.
- In separate beakers, dissolve:
- 72.9 mg HDTMA in 40 mL dH₂O
- 60.5 mg CAS dye in 50 mL dH₂O
- Prepare fresh 1 mM FeCl₃ solution in dH₂O.
- Mix FeCl₃ solution with HDTMA-CAS dye complex, adjust to 100 mL final CAS reagent.
- Cool LB agar to ~50 °C.
- Aseptically add CAS reagent at a 1:10 ratio (100 mL CAS reagent + 900 mL LB agar).
- Pour plates and allow to solidify.
Preparation of bacterial inoculum (Day 2)
- Inoculate a single colony of JDE115 into 10 mL LB broth.
- Incubate at 28 °C, 150 rpm until OD₆₀₀ ≈ 0.6 (log phase)
Spot inoculation on CAS agar
- Prepare treatments:
LB broth only (negative control)
D1: 1:1,000 dilution of JDE115
D2: 1:100 dilution of JDE115
D3: 1:10 dilution of JDE115
JDE115: undiluted inoculum
Positive control (known siderophore producer)
- Spot 10 µL of each inoculum onto CAS agar plates in sector-wise layout.
- Incubate plates at 28 °C for 5–7 days.
Observation and data collection
Examine plates daily for halo formation.
Record the presence and size of yellow-orange zones around colonies.
Measure colony diameter and halo diameter.
Calculate SPI (Siderophore Production Index):
Protocol references
Schwyn, B., & Neilands, J. B. (1987). Universal chemical assay for the detection and determination of siderophores. Analytical Biochemistry, 160(1), 47–56. https://doi.org/10.1016/0003-2697(87)90612-9
Arora, N. K., & Verma, M. (2017). Modified microplate method for rapid and efficient estimation of siderophore production. 3 Biotech, 7(6), 381. https://doi.org/10.1007/s13205-017-1008-y
Acknowledgements
We acknowledge the Nematode Diagnostic Lab at Virginia Tech for providing facilities and resources essential for conducting this work.