| R. solanacearum / Culture Media |
Printer friendly  PDF file  |
| |
Find here a selection of recipes for Ralstonia solanacearum culture media
<Reference source> |
| More recipes from our Diagnostic and Sampling Protocols page |
| |
| |
Non-selective media
Non-selective media are generally used for growth of pure culture of R. solanacearum. (e.g. to retrieve cultures from frozen stocks or for successive plating of cells). These media can also be used for isolation of R. solanacearum from fresh, symptomatic plants, due to the high density of the pathogen in the tissues.
|
| |
| |
- Casamino acid-Peptone-Glucose (CPG) medium
<Reference>
| |
Per liter |
Casamino acid (casein hydrolysate)
Peptone
Glucose
For solid media (plates) add:
Agar |
1 g
10 g
5 g
17 g |
Adjust pH to 6.5-7.0 if necessary.
Autoclave at 121˚C for 20 minutes.
On solid medium, colonies of R. solanacearum usually are visible after 48-72 hours of incubation at 28ºC.
Colonies of the normal (or virulent type) are white or cream-colored, irregularly-round, fluidal, and opaque; and colonies of the mutant (or non-virulent) type are uniformly round, smaller, and butyrous (dry). This shift from virulent to non-virulent bacterial cells occurs during storage or under oxygen stress in liquid media.
|
| |
| |
- Tripheny tetrazolium chloride (TTC or TZC) medium
<Reference>
Prepare 1 liter of CPG medium:
| |
Per liter |
Casamino acid (casein hydrolysate)
Peptone
Glucose
For solid media (plates) add:
Agar |
1 g
10 g
5 g
17 g |
Adjust pH to 6.5-7.0 if necessary.
Autoclave at 121˚C for 20 minutes.
After autoclaving cool the medium to 55˚C and add 5 ml of a 1% stock solution of 2, 3, 5-tripheny tetrazolium chloride. The stock can be filter sterilized or autoclaved for 5 minutes at 121˚C, and stored at 4˚C or frozen.
On solid medium, colonies of R. solanacearum usually are visible after 48-72 hours of incubation at 28ºC.
This medium was developed to differentiate between the two colony types: virulent colonies appear white with pink centers and non-virulent colonies appear dark red.
|
| |
| |
| |
Semi-selective media
Semi-selective media can be used for isolation of the pathogen from nonsymptomatic plants or from water and soil samples. Although widely used, they do not support growth of all R. solanacearum strains and/or do not suppress growth of all related or unrelated Gram-negative bacteria.
|
| |
| |
- SM-1 medium
<Reference>
Prepare 1 liter of TTC (or TZC) medium:
| |
Per liter |
Casamino acid (casein hydrolysate)
Peptone
Glucose
For solid media (plates) add:
Agar |
1 g
10 g
5 g
17 g |
Adjust pH to 6.5-7.0 if necessary.
Autoclave at 121˚C for 20 minutes.
After autoclaving cool the medium to 55˚C and add 5 ml of a 1% stock solution of 2, 3, 5-tripheny tetrazolium chloride. The stock can be filter sterilized or autoclaved for 5 minutes at 121˚C, and stored at 4˚C or frozen.
| Add: |
Per liter |
Merthiolate tincture *
Crystal violet **
Polymyxin ß sulfate **
Tyrothricin **
Chloromycetin **
Cycloheximide **
|
5 to 50 µl
50 mg
100 mg
20 mg
5 mg
50 mg |
* Merthiolate tincture contains 1 part merthiolate per 1000 parts of 50% alcohol. Determine the best concentration to suppress local microflora, as suggested <Reference>.
** Dissolve in 5 ml of 70% ethanol 30 minutes prior to use.
|
| |
| |
- Modified SMSA medium
<Reference>
Prepare 1 liter of TTC (or TZC) medium, except substitute glycerol (5 ml per liter) for the glucose:
| |
Per liter |
Casamino acid (casein hydrolysate)
Peptone
Glycerol
For solid media (plates) add:
Agar |
1 g
10 g
5 ml
17 g |
Adjust pH to 6.5-7.0 if necessary.
Autoclave at 121˚C for 20 minutes.
After autoclaving cool the medium to 55˚C and add 5 ml of a 1% stock solution of 2, 3, 5-tripheny tetrazolium chloride. The stock can be filter sterilized or autoclaved for 5 minutes at 121˚C, and stored at 4˚C or frozen.
| Add: |
Per liter |
Crystal violet *
Polymyxin ß sulfate *
Bacitracin *
Chloromycetin *
Penicillin *
When inhibition of fungal contaminants id desirable, add:
Cycloheximide * |
5 mg
100 mg
25 mg
5 mg
0.5 mg
100 mg |
* Dissolve in 5 ml of 70% ethanol 30 minutes prior to use.
On solid medium, colonies of R. solanacearum usually are visible after 2-5 days of incubation at 28ºC.
Typical bacterial colonies appear fluidal, irregular in shape, and white with pink centers. |
| |
|
| |
Reference source
Denny, TP; Hayward, AC. 2001. Gram-negative bacteria: Ralstonia. Pages 151-174 in: Laboratory guide for identification of plant pathogenic bacteria, 3rd ed. Schaad, NW; Jones, JB; Chun, W, eds. APS Press, St. Paul, M. N.
|
| |
|
| |
Text references
- Casamino acid-Peptone-Glucose (CPG) medium
Kelman, A. 1954. The relationship of pathogenicity in Pseudomonas solanacearum to colony appearance on a tetrazolium medium. Phytopathology 44:693-695.
- Tripheny tetrazolium chloride (TTC or TZC) medium
Kelman, A. 1954. The relationship of pathogenicity in Pseudomonas solanacearum to colony appearance on a tetrazolium medium. Phytopathology 44:693-695.
- SM-1 medium
Granada, GA; Sequeira, L. 1983. A new selective medium for Pseudomonas solanacearum. Plant Disease 67:1084-1088.
- Modified SMSA medium
Elphinstone, JG; Hennessy, J; Wilson, JK; Stead, DE. 1996. Sensitivity of different methods for the detection of Pseudomonas solanacearum in potato tuber extracts. EPPO/OEPP Bulletin 26:663-678.
French, ER; Gutarra, L; Aley, P; Elphinstone, J. 1995. Culture media for Pseudomonas solanacearum isolation, identification and maintenance. Fitopatologia 30:126-130.
|
