Miller-Schroth medium
From Bugwoodwiki
Purpose
This medium is diagnostic for Erwinia spp. Erwinia colonies turn the medium yellow or orange in 1-3 days. The colonies then change back to blue-green.
Ingredients
Part 1
| Ingredient | 1 L |
|---|---|
| Distilled water | 800 mL |
| Agar | 20 g |
Part 2
| Ingredient | 1 L |
|---|---|
| Tergitol 7 (sodium heptadecyl sulfate) | 0.1 mL |
| 2% nitrilotriacetic acid in 1.46% KOH (20 g + 14.6 g KOH/L) | 10 mL |
| 0.5% bromothymol blue (Na salt)2 | 9 mL |
| 0.5% neutral red | 2.5 mL |
| 5 N NaOH (do not use if a percipitate forms) | 1 mL |
| 1% thallium nitrate (TlNO3)3 | 1.7 mL |
| 14 mM CoCl2 (0.83g/250 ml H2O)4 | 50 mL |
Part 3
| Ingredient | 1 L |
|---|---|
| mannitol (or sorbitol1) | 10 g |
| nicotinic acid | 0.5 g |
| L-asparagine (anhydrous) | 3 g |
| K2HPO4 dibasic powder | 2 g |
| MgSO4 • 7 H2O | 0.2 g |
| sodium taurocholate (Difco) | 2.5 g |
Instructions
- Autoclave Part 1 and keep heated and stirring on heated stir plate.
- While autoclaving media, combine the Part 2 ingredients in a flask. Add in the order listed and gently mix by hand after each addition. Parafilm flask until time to add to media.
- Add the Part 3 ingredients to autoclaved media in the order listed. Be sure each ingredient is dissolved before adding the next.
- Add previously combined liquid ingredients to media.
- Bring total volume to 1000 ml with dH2O. (If isolating from soil: after autoclaving add 0.05 g cycloheximide, dissolved in a few ml of sterile distilled water.)
- The pH should be about 7.3. It should be blue-green in color. The medium may be poured into sterile prescription bottles for storage and melted and poured as need.
Notes
- MS is a time-consuming medium to make, but is worth the effort. Stock solutions may be made and stored to save time for the next batch. MS is the best isolation media for Erwinia spp. Many Pseudomonas species will grow on MS. However, they never turn yellow-orange, but always remaining blue-green. Plates should be observed every day for 3-4 days. After 3-5 days Erwinia and Pseudomonas spp. both appear blue-green.
- When selecting for soft rotters, use mannitol and look for rough colonies with convoluted margins. Erwnia herbicola and E. amylovora develop smooth colonies with translucent margins (seen by transmitted light with a dissecting microscope).
- Tergitol 7 can be omitted per the 2001 Ed. of Schaad et.al.
- Use sorbitol instead of mannitol for E. amylovora; sorbitol is not readily used by E. herbicola and may restrict some soft rot bacteria as well.
- The sulfone form is not water-soluble.
- Available from Aldrich Chemical.
- The cobaltous chloride is a reducing agent that allows for extended shelf life. If the cobaltous chloride is old or has not been stored in the refrigerator, the medium will not be the correct color.
References
- Miller, T. D., and M. N. Schroth. 1972. Monitoring the epiphytic populations of Erwinia amylovora on pear with a selective medium. Phytopathology 62: 1175 1185.
- Schaad, N.W. 1980. Laboratory Guide for the Identification of Plant Pathogenic Bacteria. The American Phytopathological Society, St. Paul, MN. 67 pp. (pp. 7, 26).
- Schaad, N.W. 2001. Laboratory Guide for the Identification of Plant Pathogenic Bacteria. The American Phytopathological Society, St. Paul, MN. 67 pp. (p. 43).
Contributed by
From the Virginia Polytechnic Institute and State University Mediabook; Orignially created by Robert Wick; contributed by Mary Ann Hansen.
