A number of viruses of different kinds have been newly discovered as causative agents of respiratory tract infections since techniques for virus isolation especially by tissue culture method improved recently. Etiological and clinical significances of these agents are gradually being elucidated, and many publications have been devoted to the adenoviruses especially in this direction. Recently Chanock (1956) and Chanock et al. (1958) have reported the isolations of CA virus (croup associated virus), HA1 and HA2 viruses from respiratory diseases and suggested the importance of these viruses in such diseases.
In February, 1959, the health authority of Tokyo Prefecture received from an elementary school a report that an influenza-like disease was prevailing there especially in classes of younger ages. In Japan, Asian type influenza ceased almost in March 1958, and thereafter no case or outbreak had been reported until the end of 1958, but it was an important problem to know whether Asian influenza would recur in the winter of 1958-1959. Therefore, detailed examinations were performed about the nature of the outbreak mentioned above, and it was not proved either serologically or virologically to be due to any type of influenza virus. Further studies, however, revealed that it had been caused by a virus belonging to HA2 virus. The present paper will describe our findings in the outbreak in question in detail.

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In the study of diphtheria, no one has made doubt of the curative effect of antitoxin and of the preventive effect of toxoid. A number of evidences have been accumulated suggesting that the toxin elaborated by the causative organism plays an important role in the development of the disease (Pappenheimer, 1936; Mueller, 1941a, 1941b; Amies, 1954; Tasman and Lansberg, 1957) . It is easily conceivable that a certain amounts of toxin might have already been taken up by various tissues of the body of the patient when the clinical diagnosis of diphtheria can be confirmed. The absorbed toxin is said to be scarcely neutralized by the antitoxin administered passively even in large amount. Therefore, the disorder such as late paralysis or heart disease may frequently follow the appearent recovery from the acute symptoms of the disease (Amies, 1954; Tasman and Lansberg, 1957) . For this reason the prevention of the disease is particularly important. Despite of the adoption of the active immunization by means of toxoid, the morbidity of diphtheria has recently risen again in Japan. The lowest rate in the last decade was 9.8 per 100, 000 population in 1952, but the rate rose gradually and grew to 20 in 1956. Besides, change in the type of the causative organism has been often reported; i.e., most of the strains isolated recently belong to the gravis type while the mitis type was predominant in former epidemics (Rata, 1951; Fujimoto, 1958; Kurokawa et al., 1959) . Some researchers began to think that the immunization by toxoid alone was not satisfactory to prevent the infection due to the gravis type organism and inclined to reconsider the role of antibacterial immunity (Etris, 1934; Frobisher et al., 1947; 1950) .
In many experiments hitherto reported, the tests of virulence have been done by injecting the causative organism in the susceptive animal. It is doubtful whether this conventional technic may furnish the fruitful information regarding the virulence of diphtheria bacilli in the natural situation and the development of the immunity against the virulent organism contacted to the natural portals of entry. Therefore, the authors decided to reinvestigate the problem of virulence and immunity in diphtheria by the different approach which stimulates the natural process of the disease involving the conjunctiva as the site of infection. In the first paper of this series, the authors wish to state the technical details of the methods and the general process of the experimental disease.

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In the titration of Dlm of a diphtheria toxin it is necessary to use a large number of guinea pigs, for example, several tens as many guinea pigs for an accurate estimation. Therefore, it is natural that attempts have been made to find an alternative and less expensive method which would be of value, at least for a preliminary work.
Jensen (1933) proposed a method by which a fairly precise estimation of Dlm was thought to be possible. His method was to compute Dlm from (DRM) m actually measured by the intracutaneous rabbit method, basing on the evidence obtained in his experiments that the Dlm/DRM ratio of diphtheria toxin of every kind was constant.
Lately, Pope (1954) offered an opinion that the intracutaneous method was an alternative for the subcutaneous method in the titration of diphtheria toxin and was advantageous in the point that many titrations might be made on one animal.
Gerwing (1957) recently proposed a more reasonable method of Drm titration, in which the parallel line assay method was introduced, saying that the minimal reacting dose was preferable to the Dlm to define a Schick test dose. However, they declared nothing as to the Dlm/DRM or Dlm/Drm ratio.
A good number of experiments carried out in our laboratory with various kinds of diphtheria toxins have shown that the assumption that the Dlm/DRM or Dlm/Drm ratio of diphtheria toxin of every kind was constant did not hold in the majority of cases; particularly with fresh toxins, the Dlm/DRM ratio frequently being proved to be far from constant (Kurokawa, Kondo and Kondo, to be published) . Therefore, it is clear that the method proposed by Jensen cannot be used as a method estimating Dlm.
As we were compelled to do a good number of fairly accurate titrations of Dlm in experiments pursuing the factor or factors concerning the Dlm/DRM ratio of diphtheria toxin (Kurokawa, Kondo and Kondo, to be published), an alternative method, less expensive and fairly precise, for the classical subcutaneous guinea pig method was looked for.
Two methods which may meet this demand are found in the literatures.
Frobisher and Parsons (Frobisher and Parsons, 1940; Frobisher, 1942) reported that mice inoculated intracerebrally with living culture of toxigenic diphtheria bacilli or toxic culture filtrates showed characteristic responses, which could be specifically neutralized with diphtheria antitoxin. According to their brief description, the minimal dose that killed almost all mice fairly corresponded with 1 guinea pig Dlm when the toxin was intracerebrally inoculated into mice. Shortly later, Suzuki (1944) confirmed Frobisher's report basing on his extensive experiments and showed that there was a certain relationship between Dlm and the minimal dose killing mice with certainty.
Meanwhile, Hosoya and his collaborators (Hosoya, Ozawa and Tanaka, 1934; Ozawa, 1935) described that domestic fowls and 3 to 4 weeks old chicks of White Leghorn having received intramuscularly diphtheria toxin developed a characteristic paralysis of wings and of legs, and died for the most parts. Inoue (1936) confirmed the report of Hosoya and his collaborators and reported that approximately one week old chicks were the most susceptible to diphtheria toxin and that the paralysis developed in chicks with the toxin of one-several tensth of Dlm. Frobisher and his collaborators (Frobisher, 1940; Frobisher, Parsons and Tung, 1942), probably independently from Japanese authors' works, reported briefly that White Leghorn or Plymouth Rock chicks could be used for toxigenicity test of diphtheria bacillus. Branham and Wormald (1954) used the chicks in titration of diphtheria antitoxin.
These methods may provide a new tool which enables the quantitative titration of diphtheria toxin, less expensively and fairly precisely.
However, few report directed to this purpose using mice or chicks has yet appeared.

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For the purpose of gaining massive cell population for chemical analysis, in order to obtain an ideal single cell-virus system, or from another cellular biological interest, many attempts have recently been made to grow mammalian cells in the state of fluid-suspension like bacterial cultures. According to investigators, variously devised equipments were introduced such as tumbling tubes by Owens et al. (1953, 1954) ; rapid roller-tubes by Earle et al. (1954), Graham et al. (1955), Siminovitch et al. (1957) and Takaoka (1958) ; a rotary shaker by Kuchler et al. (1956) ; a magnetic stirrer by McLimans et al. (1957) ; a glass stirrer by Danes (1957) ; and shaker flasks with aeration by Earle et al. (1956) and Bryant et al. (1958) . While the successful production of massive population and steady growth rate in suspension were made with a few special cell types, the comparatively low growth rate, which was suppcsedly due to mechanical injuries, and the inconstant growth mode were the general trend with other types.
The principle of the present experiment is to select or train cells to be gradually acquainted with the state of floaing and finally to be able to multiply freely without any mechanical treatment. In usual way of cultivating cells in vitro, cells which float away from the surface of a glass vessel into fluid medium are expelled when a culture is transferred or its medium is renewed. The remaining cells should naturally have a tendency to stick firmly to the glass surface. Even among cell lines established through many passages, a small numbers of cells are noticed floating in medium sometimes during cultivation. If these floating cells are viable and supported to propagate freely, it can be considered rather easy to obtain a fluid-suspension type of cultures. Such a supposition as the above has been corroborated to some extent, using HeLa and L strain cells.

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Ferguson and Henderson (1947) described the isolation of a motile coliform organism possessing the major somatic antigen of Shigella sonnei, form I, from the feces of a patient whose clinical history was not available. They called the organism strain C27. It was also found in man and animals by Wheeler (1947), van de Pitt (1953), and Schmid, Velaudapilli and Niles (1954) .
Recently, similar organisms have been repeatedly isolated from clinical materials in Japan and attracted much interest of the investigators. In this paper, the morphological, cultural, biochemical, and serological characteristics of C27 organism are described and its taxonomical position is discussed.

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In 1949 Coman reported that the loss of cellular adhesiveness of cancer cells might be correlated with calcium deficiency of the cell membrane, and studied the problem electronmicroscopically later (Coman, 1954) . Nishimura et al. (1955) demonstrated that the rigidity of cancer cells could be altered by changing the calcium content of cytoplasm by the treatment of disodium ethylenediamine tetraacetate. Little was known about the effect of calcium on the growth of tumors in vivo.
In this investigation, disodium ethylenediamine tetraacetate, a chelating agent for certain alkali earth elements, was used for the purpose of studying the effect of calcium deficiency on the growth behavior of cancer cells. It was found that disodium ethylenediamine tetraacetate enhanced the growth of a solid transplantable mammary cancer in dbr strain of mice. In order to analyze the above phenomenon, the effects of the agent on the intraperitoneal growth of Ehrlich ascites tumor cells, on one hand, and on the stromal reaction of the host against the Ehrlich ascites tumor cells subcutaneously implanted in a sponge matrix, on the other hand, were investigated.

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Since a method was established by Wisseman et al. (1951) for obtaining rickettsiae in a purified state almost free from contamination of yolk sac material, a considerable amount of information has been accumulated concerning the metabolic activities of this group of parasites.
The oxidation of various respiratory substrates was discovered in Rickettsia prowazeki and R. mooseri (Bovarnick and Snyder, 1949; Bovarnick and Miller, 1950; Wisseman et al., 1951, 1952) . The transamination and oxidative phosphorylation were demonstrated in the above mentioned typhus rickettsiae and in Rickettsia rickettsi (Bovarnick, 1956; Hopps et al., 1956; Price, 1953) . Recently, Hays et al. (1957) have demonstrated the oxido-reductive activity of the cytochrome system in R. mooseri by the aid of the sensitive spectrophotometric method originally developed by Chance.
Among the numerous difficulties encountered in this line of approach, the most serious are perhaps the extremely poor yield usually encountered in the purification of rikettsiae, and the fast unavoidable contamination of yolk sac components in the final rickettsial preparation. These difficulties are indeed doubled by the circumstance that the available methods of handling rikettsiae are very limited, because of their fragility towards some of the procedures ordinarily adopted in preparation of biological materials.
In the present study, an attempt was made to improve the purification method, using an ion-exchange resin instead of celite to remove yolk sac materials (Yamamoto and Kawamura, 1958) . A colorimetric method was also developed to determine the dehydrogenase activities with as small quantities as possible of purified rickettsiae thus obtained.
The presence of various dehydrogenases in R. mooseri were revealed by this method, with 2, 6-dichlorophenol indophenol (2, 6 DPIP) as hydrogen acceptor. The effects of various poisons and the immune sera on the dehydrogenase activity were also investigated. The experimental results obtained will be briefly described in the following.

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