Key words: WYO medium, New selective medium, Legionella species, Cooling tower water

Table 2. Results of isolation culture for the strains of Legionella species from the water specimens ot 15 cooling towers Figures in parenthesis indicate percent positive to the total number of specimens tested. Table 3. Histories and corresponding numbers of the 13 strains of 7 Legionella species tested ATCC American Type Culture Collection, Maryland. GIFU Department of Microbiology, Gifu University School of Medicine, Gifu. T Nomenclatural type for the species. Surgical Center, University of * Tokyo Hospital, Tokyo.

Table 6. Susceptibility of 13 strains of 7 Legionella species, Candida albicans, and Candida tropicalis against amphotericin B and cycloheximide

C. B., Cherry, W. B., Orrison, L. H. & Thacker, L.: Isolation of Legionella pneumo- phila from nonepidemic-related aquatic habitats. Appl. Environ. Microbiol., 37: 1239 2) Morris, G. K., Patton, C. M., Feeley, J. C., Johnson, S. E., Gorman, G., Martin, W. T., Skaliy, P., mallison, G. F., Politi, B. D. & Mackel, D. C.: Isolation of the Legionnaires' disease bacterium from environmental samples. Ann. Intern. Med., 90: 664-666, 1979. 3) Fliermans, C. B., Cherry, W. B., Orrison, L. H., Smith, S. J., Tison, D. L. & PoPe, D. H.: Ecological distribution of Legionella pneumophila. Appl. Environ. Microbiol., 41: 9-16, 1981. 4) Edelstein, P.H. & Finegold, S. M.: Use of a

semiselective medium to culture Legionella pneumophila from contaminated lung specimens. J. Clin. Microbiol., 10: 141-143, 1979. 5) Bopp, C. A., Summer, J. W., Morris, G. K. & Wells, J. G.: Isolation of Legionella spp. from environmental water samples by low ph treatment and use of a selective medium. J. Clin. Microbiol., 13: 714-719, 1981. 6) Pasculle, A. W., Feeley, J. C., Gibson, R. J., Cordes, L. G., Myerowitz, R. L., Patton, C. M., Gorman, G. W., Cormack, C. L., Ezzell, J. W. & Dowling, J. N.: Pittsburgh pneumonia agent: Direct isolation from human lung tissue. J. Infect. Dis., 141: 727-732, 1980. 7) Edelstein, P.H.: Improved semiselective medium for isolation of Legionella pneumophila from contaminated clinical and environmental specimens. J. Clin. Microbiol., 14: 298-303, 1981. 8) Wadowsky, R. M. & Yee, R. B.: A glycinecontaining selective medium for isolation of Legionellaceae from environmental specimens. Appl. Environ. Microbiol., 42: 768-772, 1981. 9) Edelstein, P.H.: Comparative study of selective media for isolation of Legionella pneumophila from potable water. J. Clin. Microbiol., 16: 697-699, 1982. 10) Greaves, P.W.: New methods for the isolation of Legionella pneumophila. J. Clin. Pathol., 33: 581-584, 1980. Dees, S. B., Mcdougal, L. K. & Dodd, D. J.: Characteristics of environmental isolates of Legionella pneumophila. Appl. Environ. Microbiol., 42: 109-115, 1981. 14) Vickers, R. M., Brown, A. & Garrity, G. M.: Dye-containing buffered charcol-yeast extract medium for differentiation of members of the family Legionellaceae. J. Clin. Microbiol., 13: 380-382, 1981. 15) Hebert, G. A.: Hippurate hydrolysis by Legionella pneumophila. J. Clin. Microbiol., 13 240-242, 1981. 16) Brenner, D. J., Steigerwalt, A. G. & McDade, J. E.: Classification of the Legionnaires' disease bacterium: Legionella pneumophia, genus novum, species nova, of the family Legionellaceae, familia nova. Ann. Int. Med., 90: 656 17) Edelstein, P.H., Meyer, R. D. & Finegold, S. M.: Isolation of a new serotype of Legionnaires' disease bacterium. Lancet, 2 (8101): 1172-1174, 1978. 18) England, A. C. III., Mckinney, R. M., Skaliy, P.& Gorman, G.W.: A fifth serogroup of Legionella pneumophila. Ann. Int. Med., 93: 58 19) McKinney, R. M., Wilkinson, H. W., Sommers, H. M., Fikes, B. J., Sasseville, K. R., Yungbluth, M. M. & Wolf, J. S.: Legionella pneumophila serogroup six : Isolation from cases of legionellosis, identification by immunofluorescens staining. and immunological response to infection. J. Clin. Micrbiol., 12: 395-401, 1980. 20) Brenner, D. J., Steigerwalt, A.G., Gorman, G. W., Weaver, R. E., Feeley, J. C., Cordes, L. G., Wilkinson, H. W., Patton, C., Thomason, B. M. & Sasseville, K. R. L.: Legionella bozemanii sp. nov. and Legionella dumoffii sp. nov.: Classification of two additional species of Legionella associated with human pneumonia. Curr. Microbiol., 4: 111-116, 1980. 21) Hebert, G. A., Steigerwalt, A. G. & Brenner, D. J.: Legionella micdadei species nova: Classification of a third species of Legionella associated with human pneumonia. Curr. Microbiol., 3: 255-257, 1980. 22) Morris, G.K., Steigerwalt, A. G., Feeley, J. C., Wong, E. S., Martin, W. T., Patton, C. M. & Brenner, D. J.: Legionella gormanii sp. nov. J. Clin. Microbiol., 12: 718-721, 1980. 23) McKinney, R.M., Porschen, R. K., Edelstein, P. 13) Orrison, L. H., Cherry, W. B., Fliermans, C. B., H., Bissett, M. L., Harris, P.A., Bondell, S. P., Steigerwalt, A. G., Weaver, R. E., Ein, M. E., Lindquist. D. S., Kops, R. S. & Brenner, D. J.: Legionella longbeachae species nova. another etiologic agent of human pneumonia. Annal. Internal Medicine. 94: 739-743, 1981. 24) Cherry, W. B., Gorman, G. W., Orrison, L. H., Moss, C. W., Steigerwalt, A. G., Wilkinson, H. W., Johnson, S. E., McKinney, R. M. & Brenner, D. J.: Legionella jordanis: A new species of Legionella isolated from water and sewage. J. Clin. Microbiol., 15: 290-297, 1982.

and nonhospital plumbing fixtures. Appl. Environ. Microbiol., 43: 1104-1110, 1982. 30) Levy, J. S., Wong, E. W. & Schaffner, W.: An outbreak of Legionnaires' disease associated with a contaminated airconditioning cooling tower. N. Eng. J. Med., 302: 365-370, 1980. 31) Cordes, L. G., Fraser, D. W., Skaliy, Perlino, C. A., Elsea, W. R., Mallison, G. F. & Hayes, P.S.: Legionnaires' disease outbreak at an Atranta, Georgia, cuntry club : Evidence for spread from an evaporative condencer. Am. J. Epidemiol., 111: 425-431, 1980. 32) Morris, G. K., Patton, C. M., Feeley, J. C., Telle, R. D. & Bennett, J. V.: A major focus of Legionnaires' disease in Bloomington, Indiana. Ann. Intern. Med., 90: 587-591, 1979. Wadowsky-Yee-Okuda (WYO) Medium: A New Selective Medium for the Isolation of Legionellae Strains from Environmental Water Specimens Keiichi OKUDA1), Masanari IKEDO2) & Eiko YABUUCHI2) College of Medical Technology, Kitakyushu, Fukuoka 802, Japan of Microbiology, Gifu University School of Medicine, Tsukasamachi 40, Gifu 500, Japan In order to obtain more effective medium for the selective isolation of strains of Legionella species from environmental water specimens, 18 test media were prepared by adding each one of the 5 antifungal agents, amphotericin B, anisomycin, cycloheximide, nystatin, or miconazole, in various concentrations to GVP (glycine-vancomycin-polymyxin B) medium proposed by Wadowsky and Yee. In a comparative study to isolate legionellae from the water specimens of 15 cooling towers in Fukuoka Prefecture, 15 (100%) specimens were positive on the GVP medium added with either amphotericin B or anisomycin in a concentration of 80ƒÊg/ml, 14 (93%) on GVP added with 80ƒÊg/m Œ cycloheximide, whereas 11 (73%) on MWY (modification of Wadowsky-Yee) by Edelstein was the highest and only 1 (7%) on BMPA (buffered cefamandole-polymyxin B-anisomycin) medium was the lowest among the 6 previously reported selective media. All these 24 media were used in combination with acid-buffer treatment of the water specimens. Of these 3 effective antifungal agents, amphotericin B is of cheaper cost and of easier availability than anisomycin in Japan, and is less inhibitory to the growth of legionellae and more inhibitory to candidae than cycloheximide. Thus amphotericin B was chosen as antifungal agent to be incorporated to GVP medium in a concentration of 80pg/ml. This medium in herein named as WYO (Wadowsky-Yee- Okuda) medium and recommended for the selective isolation of legionellae in combination with acidbuffer treatment of the specimen. Growth rates of the 13 test strains of 7 Legionella species on WYO medium were almost similar to those on the original GVP medium. Although the inability of the type strain of L. gormanii to grow on WYO medium would suggests a limitation of such selective isolation device, WYO medium would be helpful for both ecological and epidemiological researches on legionellae and human legionellosis.