Poly-B hydroxyrate accumulation
From Bugwoodwiki
Purpose
For detection of "poly-beta" granules.
Ingredients
| Ingredient | 1 L | 500 ml |
|---|---|---|
| Distilled water | 1000 ml | 500 ml |
| (NH4)2SO4 | 0.2 g | 0.1 g |
| KCl | 0.2 g | 0.1 g |
| MgSO4 • 7H2O | 0.2 g | 0.1 g |
| DL-β-Hydroxybutyrate | 5 g | 2.5 g |
| Agar | 15 g | 7.5 g |
Instructions
- Adjust to pH 7.2, and autoclave.
- Culture the bacteria for 12-24 hrs.
- Prepare Sudan black B solution (0.3 g in 100 ml of 70% ethanol). After most of the dye has
dissolved, shake the solution at intervals, then allow to stand overnight before use. The solution remains stable for several months at room temperature if kept in a tightly closed container.
- Make a bacterial smear from a 12-24hr-old agar culture on a glass slide; air-dry and heat fix.
- Flood the entire slide with Sudan black B solution and leave undisturbed for 10-15 min.
- Drain off excess dye, blot dry, and clean slide with xylene in a Coplin jar or by adding from a drop bottle.
- Blot the cleared slide to dryness and counter-stain with safranine (0.5% aqueous solution) for 5-10 seconds. Avoid over-staining with counter-stain. Wash in water, blot and dry the slide, then examine under oil immersion with a light microscope. The poly-beta granules are dark blueblack.
Notes
- Detection of "poly-beta" granules is done by phase contrast microscopy. The accumulation of cellular organic reserve materials is favored with some bacteria under conditions of nitrogen starvation.
References
- Schaad, N. W., Jones, J. B. and Chun, W. 2001. Laboratory guide for the identification of plant pathogenic bacteria, 3rd edition. The American Phytopathological Society, St. Paul, MN. 373pp.
Contributed by
From the Culture Media for Plant Pathogenic Fungi and Bacteria, University of Massachusetts: Contributed by Robert L. Wick
