Isolation Medium for Iron Bacteria

Intended Use

Recommended for the isolation of iron bacteria, especially those belonging to Sphaerotilus - Leptothrix group.

Composition

Formula adjusted, standardized to suit performance parameters

Directions

Suspend 10.88 grams (the equivalent weight of dehydrated medium prer litre) in 1000 ml purified / distilled water. Heat just to boiling. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes. Cool to 45-50°C. Mix well and dispense into sterile test tubes. Note: Due to the presence of calcium carbonate, the prepared medium forms opalescent solution with white precipitate.

Principle And Interpretation

Sphaerotilus-Leptothrix group are filamentous bacteria that form sheath. The sheathed bacteria have the ability to deposit ferric hydroxide and sometimes manganese dioxide on their sheaths (2). The specific deposition of ferric ions on the sheath of S. discophorus (also Leptothrix species) was demonstrated by Rogers and Anderson (5,6). Iron bacteria obtain energy by the oxidation of iron from the ferrous to ferric state. Some bacteria that do not oxidize ferrous ions may dissolve or deposit it indirectly. During their growth, they either liberate iron by utilizing organic radicals to which the iron is attached, or alter environmental conditions to permit the deposition of iron. Isolation Medium for Iron Bacteria is recommended by APHA (1) for the isolation of iron bacteria, especially those belonging to the Sphaerotilus-Leptothrix group. The medium has been proven helpful for identifying various groups of filamentous organisms including iron bacteria (7).

Magnesium sulphate, ammonium sulphate, potassium chloride and calcium nitrate are sources of ions that stimulate metabolism. Glucose acts as the carbon source. Dipotassium phosphate buffers the medium. The bacteria of both genera, Sphaerotilus and Leptothrix require vitamin B12 as an essential growth factor. A number of Leptothrix strains have been found to require additionally thiamine as growth factor.

Type of specimen

Water samples

Specimen Collection and Handling:

Prepare agar slants of these media and aseptically pipette 3 ml test water sample on to surface of slants. Inoculate the tubes and incubate at room temperature until turbid growth develops in the liquid layer (1).

After use, contaminated materials must be sterilized by autoclaving before discarding.

Warning and Precautions :

Read the label before opening the container. Wear protective gloves/protective clothing/eye protection/ face protection. Follow good microbiological lab practices while handling specimens and culture. Standard precautions as per established guidelines should be followed while handling specimens. Safety guidelines may be referred in individual safety data sheets.

Limitations :

• Further biochemical and serological tests must be carried out for further identification.

Performance and Evaluation

Performance of the medium is expected when used as per the direction on the label within the expiry period when stored at recommended temperature.

Quality Control

Appearance: Cream to beige homogeneous free flowing powder

Gelling: Firm, comparable with 1.0% Agar gel.

Colour and Clarity of prepared medium: Medium yellow coloured, clear to slightly opalescent gel forms in tubes as slants

Cultural Response: Cultural characteristics observed after an incubation at 22-25°C upto 5 days.

• Organism: Leptothrix discophora ATCC 43182, Growth: good-luxuriant
• Organism: Sphaerotilus natans ATCC 13338, Growth: good-luxuriant
• Organism: Ferrobacillus ferrooxidans, Growth: good-luxuriant

Storage and Shelf Life

Store between 10-30°C in a tightly closed container and the prepared medium at 20-30°C. Use before expiry date on the label. On opening, product should be properly stored dry, after tightly capping the bottle in order to prevent lump formation due to the hygroscopic nature of the product. Improper storage of the product may lead to lump formation. Store in dry ventilated area protected from extremes of temperature and sources of ignition. Seal the container tightly after use. Product performance is best if used within stated expiry period.

Disposal

User must ensure safe disposal by autoclaving and/or incineration of used or unusable preparations of this product. Follow established laboratory procedures in disposing of infectious materials and material that comes into contact with sample must be decontaminated and disposed of in accordance with current laboratory techniques (3,4).

Reference

1. Baird R.B., Eaton A.D., and Rice E.W., (Eds.), 2015, Standard Methods for the Examination of Water and Wastewater, 23rd ed., APHA, Washington, D.C.
2. Balows A., Truper H. G., Dworkin M., Harder W., Schleifer K. H., (Eds.), The Prokaryotes, 2nd Edition, Vol. III, Springer-Verlag.
3. Isenberg, H.D. Clinical Microbiology Procedures Handbook 2nd Edition.
4. Jorgensen, J.H., Pfaller, M.A., Carroll, K.C., Funke, G., Landry, M.L., Richter, S.S and Warnock., D.W. (2015) Manual of Clinical Microbiology, 11th Edition. Vol. 1.
5. Rogers S. R., Anderson J. J., 1976, J. Bacteriol., 126: 257-263.
6. Rogers S. R., Anderson J. J., 1976, J. Bacteriol., 126: 264-271.
7. VanVeen W. L., 1973, Antonie Van Leeuwenhoek (Holland), 39:189.