Primary cultures of all b u t one of the milky-disease organisms studied could be obtained routinely by inoculating deep tubes of the semisolid sulfite-treated brain-heart infusion m e d i u m with 0.1 percent starch added and incubating the tubes at 30°C. T h e m e d i u m should be freshly prepared, sterilized, and cooled in a tempering oven before inoculation.
Spore suspensions are prepared, counted, and diluted so that the n u m b e r of spores introduced into the m e d i u m is about 2000 spores per tube.
W i t h spore suspensions from clean dried-blood film containing large numbers of spores, if the usual aseptic bacteriological techniques are
used, all inoculated tubes should yield growth of the milky-disease organ
isms and be free from contaminants. Many lots of Bacto dehydrated brain-heart infusion m e d i u m have been tested and all have been suitable for this purpose. W i t h some lots, growth is established sooner and more consistently than with others, so several lots should be tested and the best of these selected a n d reserved for this purpose. If care is taken to see that the bottle is tightly capped after opening, the selected lots will give uniform results for several years.
As the milky-disease organisms make little growth in the isolation medium, and most organisms that might be contaminants grow well, tubes that show appreciable turbidity are suspect. Tests with B. popilliae show that using a million spores per tube the contamination rate is about one tube in fifty inoculated; with smaller inocula, the rate is negligible. Growth of B. popilliae can be expected in about 10 percent of tubes inoculated with 2 spores per tube, 50 percent of those with 20 spores, and nearly 100 percent of those inoculated with 200 spores or more. Growth and reduction of pigment of the m e d i u m is observed m u c h earlier at the higher levels of inoculation.
These cultures can be tested for purity by plating them out on agar media, and cultures obtained from single cells may then be reisolated.
T h i s m e t h o d could also be used directly with spore suspensions, b u t usually less than half of the spores inoculated produce colonies, and large numbers per plate are required to establish growth consistently.
Also, the organisms are short-lived in the media adequately nutritious to produce visible colonies. By using anaerobic techniques, or flooding plates with a layer of sterile agar m e d i u m , after plating to produce sub
surface colonies, a high proportion of colonies produced isolates with B. popilliae. M u c h poorer results, however, were obtained in tests with B. lentimorbus.
Selected strains can be maintained indefinitely by injecting larvae with cultures and preparing dried blood films from infected larvae.
Those strains whose lethality is so great that larvae die before sporula
tion cannot be preserved in this manner. Haynes et al. (1961) show that cultures of at least some of the milky-disease organisms can be preserved by lyophilization.
VI. CONCLUDING REMARKS
T h e milky-disease organisms, with their ability to grow and sporu-late within the living host for relatively long periods, afford a wonderful opportunity to study the effect of disease on the physiology of the host, including interference of disease with the normal processes of metab
olism and m a t u r a t i o n and the influence of these processes on the devel
o p m e n t of the pathogen.
T h e resistance of the spores to heat (Bonnefoi et al., 1959), to desic
cation, to radiation (White, 1946), and their remarkable longevity in dried-blood films and in soil, make it possible to utilize these organisms for basic research studies as well as for the control of a n u m b e r of de
structive insect pests.
Determination of the cultural requirements for vegetative growth and sporulation should yield information of value not only for a better understanding of these organisms, b u t also to microbiology as a whole.
T h e questions of their taxonomy and relationships between the several types of organisms in the g r o u p should be clarified when more information is available, particularly in respect to their artificial culture.
REFERENCES
Adams, J. Α., and Wheeler, Ε. H. 1946. Rate of development of milky disease in Japanese beetle populations. / . Econ. Entomol., 39, 248-254.
Beard, R. L. 1944. Susceptibility of Japanese beetle larvae to Bacillus popilliae.
J. Econ. Entomol., 37, 702-708.
Beard, R. L. 1945. Studies on the milky disease of Japanese beetle larvae. Conn.
Agr. Expt. Sta. Bull. 491, 505-581.
Beard, R. L. 1956. T w o milky diseases of Australian Scarabaeidae. Can. Entomol
ogist, 88, 640-647.
Boving, A. G. 1939. Descriptions of the three larval instars of the Japanese beetle, Popillia japonica N e w m . (Coleoptera-Scarabaeidae). Proc. Entomol. Soc. Wash., 41, 183-191.
Bonnefoi, Α., Toucas, M., and Chaumont, H. 1959. Essais de thermo-resistance de l'organisme responsable de la maladie laiteuse de la larve du h a n n e t o n (Melolontha melolontha). Entomophaga, 4, 227-231.
Carter, W . 1945. N o t e s o n milky disease of Japanese beetle larvae. Proc. Hawaii Acad. Set. 16th Ann. Meeting, p p . 8-9.
Cory, Ε. N., and Langford, G. S. 1950. T h e utilization of Bacillus popilliae Dutky against Popillia japonica N e w m . Intern. Congr. Entomol. Proc. 8th Congr. Stock
holm 1950, p p . 883-886.
D u m b l e t o n , L. J. 1945. Bacterial and nematode parasites of soil insects. New Zealand J. Sei. Technol., A27, 76-81.
Dutky, S. R. 1937. Investigation of the diseases of the immature stages of the Japanese beetle. Ph.D. Thesis, 113 p p . Rutgers Univ., N e w Brunswick, N e w Jersey.
Dutky, S. R. 1940. T w o new spore-forming bacteria causing milky diseases of Japanese beetle larvae. / . Agr. Research, 61, 57-68.
Dutky, S. R. 1941a. Method for the control of Japanese beetle. U.S. Patent 2,258,319.
Dutky, S. R. 1941b. Susceptibility of certain scarabaeid larvae to infection by type A milky disease. / . Econ. Entomol., 34, 215-216.
Dutky, S. R. 1941c. Testing the possible value of milky diseases for control of soil-inhabiting larvae. / . Econ. Entomol., 34, 217-218.
Dutky, S. R. 1942a. Process for propagating bacteria. U.S. Patent 2,293,890.
Dutky, S. R. 1942b. Method for the preparation of spore-dust mixtures of type A milky disease of Japanese beetle larvae for field inoculation. U.S. Dept. Agr. Bur.
Entomol. Plant Quarantine, ET192, 10 pp. (processed).
Dutky, S. R. 1947. Preliminary observations on the growth requirements of Bacillus popilliae Dutky and Bacillus lentimorbus Dutky. / . Bacteriol., 54, 257.
Dutky, S. R. 1957. Report on white grub control project in Chile. Agr. tec. Santiago, 17, 92-105.
Dutky, S. R. 1959. Insect microbiology. Advances in Appl. Microbiol. 1, 175-200.
Dutky, S. R., and Fest, W . C. 1942. Microinjector. U.S. Patent 2,270,804.
Easter, S. S. 1947. Control of Japanese beetles at Second Army Posts. / . Econ.
Entomol., 40, 632-634.
Harris, E. D. 1959. Observations on the occurrence of a milky disease among larvae of the northern masked chafer, Cyclocephala borealis Arrow. Florida Entomologist, 42, 181-183.
Haynes, W. C , St. Julian, G., Jr., Shekleton, M. C , Hall, Η. H., and Tashiro, H.
1961. Preservation of infectious milky disease bacteria by lyophilization. / . Insect Pathol, 3, 55-61.
Hurpin, B. 1955. Sur une maladie laiteuse des larves de Melolontha melolontha L.
(Coleopt. Scarabeidae). Compt. rend soc. biol, 149, 1966-1967.
Hurpin, B. 1959. Etude de diverses souches de maladie laiteuse sur les larves de Melolontha melolontha L. et sur Celles de quelques especes voisines. Entomophaga, 4, 233-248.
Kern, F. 1950. Untersuchungen an Amphimallus solstitialis L. mit Versuchen zur bakteriologischen Bekämpfung von Engerlingen. 75 pp. Dissertation, Zurich.
Langford, G. S., Vincent, R. H., and Cory, Ε. Ν . 1942. T h e adult Japanese beetle as host and disseminator of type A milky disease. / . Econ. Entomol, 35, 165-169.
Polivka, J. B. 1956. Effectiveness of milky disease in curtailing Japanese beetle in Ohio. / . Econ. Entomol, 49, 4-6.
Shotwell, O. L., Bennett, G. Α., Hall, Η. H., van Etten, C. H., and Jackson, R. W.
1962. A m i n o acids in the h e m o l y m p h of Popillia japonica N e w m a n . / . Insect Physiol [In press; accepted for publication August 8, 1962.]
Steinkraus, Κ. Η. 1957. Studies o n the milky disease organisms. II. Saprophytic growth of Bacillus popilliae. J. Bacteriol, 74, 625-632.
Steinkraus, Κ. Η., and Provvidenti, Μ. L. 1958. Studies on the milky disease organ
isms. III. Variability among strains of Bacillus popilliae sporulating o n artificial media. / . Bacteriol, 75, 38-42.
Tashiro, H., and W h i t e , R. T . 1954. iMilky diseases of European chafer larvae. / . Econ. Entomol, 47, 1087-1092.
Vago, C , and Delahaye, F. 1961. Etude au microscope electronique de la bacterie cristallophore Bacillus popilliae Dutky. Mikroskopie, 16, 198-206.
White, R. T . 1946. Effect of the sun's rays u p o n the viability of spores of Bacillus popilliae Dutky, the organism causing milky disease of Japanese beetle larvae.
U.S. Dept. Agr. Bur. Plant Quarantine E-703, 4 p p . (processed).
W h i t e , R. T . 1947. Milky disease infecting Cyclocephala larva in the field. / . Econ.
Entomol, 40, 912-914.
White, R. T., and Dutky, S. R. 1940. Effect of introduction of milky diseases on populations of Japanese beetle larvae. / . Econ. Entomol, 33, 306-309.
W h i t e , R. T., and Dutky, S. R. 1942. Cooperative distribution of organisms causing milky disease of Japanese beetle grubs. / . Econ. Entomol, 35, 679-682.
White, R. T., and McCabe, P. J. 1951. Colonization of the organism causing milky disease of the Japanese beetle larvae. 1939-1949. U.S. Dept. Agr. Bur. Entomol.
Plant Quarantine, E-816, 10 pp. (processed).
Wille, Η. 1956. Bacillus fribourgensis, n. sp., Erreger einer "milky disease" im Engerling von Melolontha melolontha L. Mitt. Schweiz, entomol. Ges., 29, 271-282.