Utilization of carbohydrates (standard mineral base)
From Bugwoodwiki
Purpose
Used for identification of bacteria in general, but particularly useful for pseudomonads.
Ingredients
| Ingredient | 1 L | 500 ml | 250 ml |
|---|---|---|---|
| Part 2 | |||
| Distilled water | 900 ml | 450 ml | 225 ml |
| Na2HPO4 (dibasic anhydrous) | 3.79 g | 1.89 g | 0.95 g |
| KH2PO4 | 4.53 g | 2.27 g | 1.13 g |
| NH4Cl | 1 g | 0.5 g | 0.25 g |
| MgSO4 • 7H2O | 0.5 g | 0.25 g | 0.125 g |
| 5% ferric ammonium citrate sol. (5g/100 ml) | 1 ml | 0.5 ml | 0.25 ml |
| 0.5% CaCl2 sol. dihydrate (0.66g/100 ml) | 1 ml | 0.5 ml | 0.25 ml |
| Noble agar | 10 g | 5 g | 2.5 g |
| Part 2 | |||
| 1% carbon source (1g/100ml) | 100 ml | 50 ml | 25 ml |
Instructions
- Mix and autoclave ingredients from part 1.
- Add the ingredient from part 2, filter-steralized.
- Make up the basal solution, heat to melt agar, and divide into as many aliquots as there are carbohydrates to test, plus one aliquot as a control without any carbon source.
- After autoclaving, cool to 50 55°C and add the filter sterilized carbohydrate. Mix well, but avoid bubbles, and pour into Petri dishes. Standard mineral base may be stored at 40°C.
- To inoculate, use a 24 hr culture suspended in sterile water (barely cloudy) and spot with a loop onto the agar surface. Incubate at 27°C for one week. Xanthomonas campestris can be used as a control on SMB without added carbohydrate to test for purity and freedom from contaminating carbohydrates.
Notes
- Care should be taken to make sure no other carbon source besides the one being tested contaminates the medium. Glassware should be clean and rinsed in distilled water 5 times before use. Use only ion agar or Noble agar in a 1% concentration, as softer agar is sufficient for spotting inoculum and this grade of agar is very expensive. At least 10 bacterial cultures can be spotted onto one Petri dish, so only a few plates need be poured for each carbohydrate.
- Palleroni and Douderoff report that starch can be safely autoclaved in the basal medium. They also recommend various concentrations of carbohydrate, depending on the substrate. Geraniol is added as 1 drop to the lid of an inverted Petri dish and the test strains utilize the vapor. Fahy also suggests transferring test isolates 3 times on the above basal medium containing only glucose before proceeding with the tests.
References
- Fahy, P. C., and Persley, G. J. 1983. Plant Bacterial Diseases, A Diagnostic Guide. Academic Press, NY, NY. 393 pp. (p. 351).
- Palleroni, N. J., and Doudoroff, M. 1972. Some properties and taxonomic subdivisions of the genus Pseudomonas. Annual Review of Phytopathology 10: 73-100.
Contributed by
From the Culture Media for Plant Pathogenic Fungi and Bacteria, University of Massachusetts: Contributed by Robert L. Wick
