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Council Directive 93/85/EECShow full title
Council Directive 93/85/EEC of 4 October 1993 on the control of potato ring rot
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- Revised 07/07/20060.70 MB
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This is a legislation item that originated from the EU
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[F1Appendix 9 Gram stain procedure (Hucker's modification) (Doetsch, 1981) (1)
Textual Amendments
Crystal violet solution U.K.
Dissolve 2 g crystal violet in 20 ml 95 % ethanol.
Dissolve 0,8 g ammonium oxalate in 80 ml distilled water.
Mix the two solutions.
Lugol's iodine U.K.
| Iodine | 1 g |
| Potassium iodide | 2 g |
| Distilled water | 300 ml |
Grind the solids together in a pestle and mortar. Add to the water and stir to dissolve in a closed container.
Safranin counterstain solution U.K.
Stock solution:
| Safranin O | 2,5 g |
| 95 % ethanol | 100 ml |
Mix and store.
Dilute: 1:10 to obtain a working solution.
Staining procedure U.K.
1.
Prepare smears, air dry and heat fix.
2.
Flood slide with crystal violet solution for one minute.
3.
Wash briefly with tap water.
4.
Flood with Lugol's iodine for one minute.
5.
Wash with tap water and blot dry.
6.
Decolourise with 95 % ethanol, added dropwise, until no further colour is removed or immerse with gentle agitation for 30 seconds.
7.
Wash in tap water and blot dry.
8.
Flood with safranin solution for 10 seconds.
9.
Wash with tap water and blot dry.
Gram positive bacteria stain violet-blue; Gram negative bacteria stain pink-red.
REFERENCES U.K.
1. Anonymous, 1987. Scheme of the detection and diagnosis of the ring rot bacterium Corynebacterium sepedonicum in batches of potato tubers. Commission of the European Communities, Luxembourg. Publ EUR 11288 EN, 21 pp. U.K.
2. Bradbury, J. F., 1970. Isolation and preliminary study of bacteria from plants. Rev. Pl. Path., 49, 213-218. U.K.
3. Dinesen, I. G., 1984. The extraction and diagnosis of Corynebacterium sepedonicum from diseased potato tubers. EPPO Bull. 14 (2), 147-152. U.K.
4. Doetsch, R. N., 1981. Determinative methods of light microscopy. In: Manual of methods for general bacteriology, American Society for Microbiology, Washington, 21-23. U.K.
5. Hugh, R. and Leifson, F., 1953. The taxonomic significance of fermentative versus oxidative metabolism of carbohydrates by various gram-negative bacteria. J. Bact., 66, 24-26. U.K.
6. Janse, J. D., 1991. Infra- and intra-specific classification of Pseudomonas solanacearum strains using whole cell fatty-acid analysis. Systematic and Applied Microbiology 14; 335-345. U.K.
7. Janse, J. D. and J. Van Vaerenbergh. The interpretation of the EC method for the detection of latent ring rot infections ( Corynebacterium sepedonicum ) in potato. EPPO Bull., No 17, 1987, pp. 1-10. U.K.
8. Jansing, H. and K. Rudolph, 1998. Physiological capabilities of Clavibacter michiganensis ssp. sepedonicus and development of a semi-selective medium. Journal of Plant Diseases and Protection, 105, 590-601. U.K.
9. Kovacs, N., 1956. Identification of Pseudomonas pyocyanea by the oxidase reaction. Nature, Lond., 178, 703. U.K.
10. Klement Z.; Rudolph, K and D. C. Sands, 1990. Methods in Phytobacteriology. Akadémiai Kiadó, Budapest, 568 pp. U.K.
11. Lelliott, R. A., 1966. The plant pathogenic coryneform bacteria. J. appl. Bact., 29, 114-118. U.K.
12. Lelliott, R. A., E. Billing and A. C. Hayward, 1966. A determinative scheme for the fluorescent plant pathogenic pseudomonads J. appl. Bact., 29, 470-489. U.K.
13. Lelliott, R. A. and P. W., Sellar, 1976. The detection of latent ring rot ( Corynebacterium sepedonicum (Spiek. et Kotth.) Skapt. et Burkh.) in potato stocks. EPPO Bull., 6 (2), 101-106. U.K.
14. Li, X. and S.H. de Boer, 1995. Selection of Polymerase Chain Reaction primers from RNA intergenic spacer region for specific detection of Clavibacter michiganensis ssp. sepedonicus . Phytopathology, 85, 837-842. U.K.
15. Mills, D., Russell, B., W. and J., W. Hanus, 1997. Specific detection of Clavibacter michiganensis ssp. sepedonicus by amplification of three unique DNA sequences isolated by subtraction hybridization. Phytopathology, 87, 8, 853-861. U.K.
16. Pastrik, K.-H. and R.A. Rainey. 1999. Identification and differentiation of Clavibacter michiganensis subspecies by polymerase chain reaction-based techniques. J. Phytopathology 147; 687-693. U.K.
17. Pastrik, K.-H., 2000. Detection of Clavibacter michiganensis ssp. sepedonicus in potato tubers by multiplex PCR with coamplification of host DNA. European Journal of Plant Pathology, 106, 155-165. U.K.
18. Ramamurthi, C. S., 1959. Comparative studies on some Gram-positive phytopathogenic bacteria and their relationship to the Corynebacteria. Mem. Cornell agric. Exp. Sta., 366, 52 pp. U.K.
19. Schaad, W., Berthier-Schaad, Y., Sechler, A. and Knorr, D. (1999) Detection of Clavibacter michiganensis subsp. sepedonicus in potato tubers by BIO-PCR and an automated real-time fluorescence detection system. Plant Disease 83; 1095–1100. U.K.
20. Schaad, W. 2001. Laboratory guide for identification of plant pathogenic bacteria. Schaad [Hrsg.]. — 3. ed.; St. Paul, Minnesota:, 373 pp. U.K.
21. Skerman, V. B. D., 1967. A guide to the identification of the genera of bacteria. 2nd ed., William and Wilkins Company, Baltimore. U.K.
22. Smith, N. C.; Hennesy, J; Stead, D.E., 2001. Repetetive sequence-derived PCR profiling using the BOX-A1 Ralstonia solanacearum primer for rapid identification of plant pathogen Clavibacter michiganensis ssp. sepedonicus . European Journal of Plant Pathology, 107 (7), 739-748. U.K.
23. Sneath, P. H. A. and V. G. Collins, 1974. A study in test reproductibility between laboratories: report of Pseudomonas working party. Antonie van Leeuwenhoek, 40, 481-527. U.K.
24. Stead, D.E. 1992. Grouping of plant pathogenic and some other Pseudomonas spp. using cellular fatty-acid profiles. International Journal of Systematic Bacteriology 42; 281-295. U.K.
25. Wullings, B. A.; van Beuningen, A. R.; Janse, J. D. and A. D. L. Akkermans, 1998. Detection of Ralstonia solanacearum , which causes brown rot of potato, by fluorescent in situ hybridization with 23s rRNA-targeted probes. Appl. Environ. Microbiol. 64, 4546–4554.] U.K.
(1)
[F1Commercially available solutions and staining kits can also be used.]
Textual Amendments
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