Crystal violet pectate medium (modified)
From Bugwoodwiki
Purpose
For isolation of pectolytic bacteria.
Ingredients
Part1
| Ingredient | ~500mL |
|---|---|
| aqueous crystal violet solution 0.075% (w/v)(final concentration=1.5 ug/ml crystal violet) | 1 mL |
| 1 N NaOH (8 g NaOH/200 ml distilled water) | 4.5 mL |
| 10% CaCl2•2H2O1 | 6.8 mL |
| Davis agar | 2 g |
| NaNO3 | 1g |
| trisodium citrate dihydrate2 | 2.5 g |
| tryptone (Oxoid, L42)3 | 0.5 g |
Part 2
| Ingredient | Amount needed |
|---|---|
| Bulmer's sodium polypectate4 | 9 g |
Part 3
| Ingredient | Amount needed |
|---|---|
| 1% aqueous solution of novobiocin (sodium salt, BDH)3 | 1 ml |
Instructions
- Preheat a Waring blender by rinsing with boiling water.
- Place 500 ml boiling distilled water in the blender.
- Using a rheostat to control the speed, start the blender at a low speed and add Part 1 Ingredients
- Blend at high speed for 15 seconds
- Slowly add Part 2 while blending so that the sodium polypectate will not clump
- Blend at high speed for 15 seconds
- Divide medium into 2 lots of ~250 mL in 800 mL bottles and make sure the caps are loosely fitted on bottles.
- Autoclave for 25 min at 120°C (15 lb/in2). Do not permit medium to solidify before autoclaving. Allow pressure to drop slowly to avoid bubbles.
- While medium is still molten, add Part 3 and mix well while avoiding excessive frothing.
- Check the pH5 (6.9-7.4) and pour plates as soon as possible.
- It is essential to dry the plates thoroughly before use. No surface water should be present at the time plates are used. This can be achieved by holding the plates with lids ajar in a ventilated hot air oven at 60°C for 40 minutes.
Notes
- 1Use fresh solution. Do not store stock solution for more than 2 weeks.
- 2In the presence of sodium citrate, strains of Pseudomonas marginalis and pectolytic strains of P. fluorescens either do not form pits or pits are greatly reduced in depth. Thus, it is much easier to detect the colonies of pectolytic strains of Erwinia in background populations when isolations are made from decaying plant material or from soil (Di, Y.B. and Kelman, A., unpublished data).
- 3Addition of tryptone is essential to allow growth of E.c. ssp atroseptica and novobiocin is needed to restore selectivity.
- 4The protocol applies only to the commercially available batch of Na polypectate from H.P. Bulmer, Ltd., Hereford LR4 OLE, England
- 5The pH should be within the stated limits. If not, adjust the volume of NaOH added initially; it is easier to raise than lower the pH.
References
- Cuppels, D. and Kelman, A. 1974. Evaluation of selective media for isolation of soft-rot bacteria from soil and plant tissue. Phytopathology 64:468 475.
- Schaad, N. W. 1988. Laboratory Guide for the Identification of Plant Pathogenic Bacteria, 2nd ed., APS Press, St. Paul, Minnesota. 158 pp.
Contributed by
From the Virginia Polytechnic Institute and State University Mediabook; Contributed by Mary Ann Hansen
