The bacterial agglutination test has been conventionally employed for the titration of humoral antibodies for Shigella in human sera. In the field trials and also in the small scale trials for evaluating the prophylactic powers of Shigella vaccines, the determination of the degree of increase in bacterial agglutinins following vaccination has been considered as one of the important criteria as well as the survey of the morbidity rates among vaccinated population (Cooper and Keller, 1948; Ando et al., 1958) . By means of bacterial agglutination test, it is, however, generally impossible to detect the antibodies for Shigella sonnei in sera from the human subjects immunized with Shigella vaccines or from bacillary dysentery patients if any, whereas it is possible for Shigella flexneri to a considerable extent (Cooper and Keller, 1948; Cooper et al., 1948, 1949a, 1949b; Takigami and Tadokoro, 1953; Ando et al., 1958) . It has, therefore, been difficult to evaluate the vaccine prepared from Shigella sonnei by the conventional bacterial agglutination test.
On the other hand, it was established by many workers that bacterial hemagglutination test is generally more sensitive than the bacterial agglutination test (Neter, 1956) . In the case of Shigella, Neter and Gorzynski (1954) and Neter and Walker (1954) established the method to determine the agglutinin titers for Shigella by the bacterial hemagglutination test. They carried out with success the surveys of the distribution of hemagglutinin titers for Shigella among some population, using as antigens the erythrocytes modified with soluble antigenic components liberated in the supernatant from bacterial suspensions heated at 100°C for 1 hour. Chun and Park (1956) and Chun et al. (1957) reported the studies on the hemagglutination reactions of the rabbit antisera immunized with Shigella flexneri, by employing the tannic acid-treated erythrocytes sensitized with acidextracted antigens as well as the erythrocytes sensitized according to Neter and Gorzynski (1954) or Neter and Walker (1954) . Nakaya (1957) studied the relationships among the bacterial agglutinins, the hemagglutinins, and the passive mouse-protective powers of human and rabbit antisera for Shigella, resulting in that the highest sensitivity could be obtained by the hemagglutination test for determination of antibody titers.
In the present experiments, titrations of agglutinins were performed on sera from volunteers immunized with Shigella vaccines and from dysentery patients, by employing the method of hemagglutination test described by the Gastroenteritis Studies Group of the Bacteriology Department at Walter Reed Army Institute of Research (Young, 1959) . The present study aimed at the evaluation of the prophylactic powers of Shigella vaccines prepared by chrome alum treatment,
with respect to their immunogenicity to human subjects.
This has been accomplished by comparing the hemagglutinin titers of sera from vaccinated individuals with those from individuals recently convalescent from bacillary dysentery. In addition to this, sensitivity and specificity of hemagglutination have been compared with those of bacterial agglutination by using sera from vaccinated human subjects and hyper-immune rabbit sera. It will be presented that the hemagglutination test was very satisfactory at any respect in determination of agglutinin titers for Shigella, particularly in the case for Shigella sonnei.

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It seems to be generally believed that the ratio of Dlm to DRM or Drm of diphtheria toxin is constant, though only a few reports on the ratio have appeared thus far. The conclusion of Jensen (1933) based on results from the most rigid and precise experiments, in which various kinds of diphtheria toxins, fresh or matured, were tested, was that the ratio of Dlm/ (DRM) m was constant, the value being 3, 000. Utilizing this relation, he proposed a new method, by which one can indirectly compute Dim of a toxin by actural measurement of its (DRM) m. In Japan, Nakai and Ogawa (1941) agreed with Jensen in the respect that the ratio is constant, though the value obtained by them was somewhat higher than that of Jensen.
When Norlin (1943) made discussions on the production of diphtheria toxin, he employed Dlm computed from actually measured DRM according to the procedure and value proposed by Jensen. In his review, Pope (1954) offered an opinion that the intracutaneous method was an alternative for the classical Ehrlich's subcutaneous method for Dlm titration of diphtheria toxin.
However, a good number of diphtheria toxin were tested for their Dlm and DRM in our laboratory, showing that Jensen's conclusion was not always true. Though the ratio of Dlm/DRM of many matured toxins showed the figure close to that of Jensen, in most of fresh toxins the ratios were found to vary very widely, ranging from close to that given by Jensen to several tens thousand or more.
The present report describes the facts showing that the ratio is not constant.

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In the studies of the lipids of Mycobacterium tuberculosis, especially of BCG, each fraction separated by the method of Anderson (1929) and Lederer (1952) has been subjected to an extensive reinvestigation in this laboratory and some new findings in the fraction of Wax A, C and D were reported (Tsumita 1956 a, b ; Nojima 1959 a, b, Nojima et al., 1958) . The acetone soluble netural fats of BCG are the main object of this report.
Noll and Jackim (1958) and Noll (1958) demonstrated that the native lipid of acetone soluble fat from a virulent human strain, Brévannes, consisted of glyceride, phosphoglycolipids and 1, 4-naphthoquinone derivatives by infrared spectroscopy after chromatographic partitioning. In the present communication the separation of neutral fat was successively performed by Mg-silicate followed by silicic acid chromatographies. Each fraction eluted was tested on the homogeneity by infrared spectroscopy and analyzed for some chemical behaviours. As a result, neutral fat of BCG was fractionated into four constituents; ethyl or methyl ester of fatty acids, triglyceride, diglyceride and glyceryl monomycolate.

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“Purified wax” as isolated by Anderson (1929), is a complex lipid extracted with chloroform from cultures of acid fast bacilli. Extracting the purified wax with boiling acetone, Asselineau (1951) obtained a soluble portion termed “Wax C” and an insoluble residue, the “Wax D” fraction. Likewise, the alcohol-ether soluble part of acid fast bacilli was classified into several fractions. “Phosphatide” is a boiling acetone insoluble residue in the alcohol-ether soluble fraction, corresponding to Wax D of the alcohol-ether insoluble and chloroform soluble fraction. Therefore, the substances contained in Wax D can be migrated from “phosphatide” depending on the conditions of extraction and on the metabolic stage of bacterial cells.
Works in this laboratory elucidated the chemical components of Wax A, C (Tsumita, 1956 a, b) and Wax D (Nojima, 1959 a, b) of BCG in 4-6 weeks culture. In this experiment a special consideration was paid on the migration of substances in Wax D from “phosphatide” depending on the extracting condition and on the age of the culture. Therefore, the present author intended to compare the yield and some chemical natures of “phosphatide” with those of Wax D in one week as well as in 4 weeks culture of the bacterial cells. The data of the latter were already precisely reported though its chromatographic fractionation has not been described (Nojima et al., 1958) .
Substances containing phosphorus in “phosphatide” and Wax D will play an important role in the metabolic process of lipid in Mycobacterium. After chromatographic partitioning of these fractions phosphorus and inositol contents were analyzed and compared. On the other hand, special emphasis was placed on the yield of cord factor contained in Wax D depending on the age of the culture.

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In the field of chemical studies of the mycobacterial constituents the fractionation methed originally reported by Anderson and his collaborators (1939) has hitherto been followed by a number of workers interested not only in configurational studies of components but also in biological and pathological approaches, until Lederer and his school (1952) made an improvement in fractionating the purified wax of tubercle bacilli further into hot acetone-soluble and insoluble fractions, i. e. Wax C and Wax D.
These two methods were successfully used and works have come out from this laboratory, reporting on neutral fat (Aoyagi et al., 1960), phosphatide (Kondo, 1960 a, b), Wax A (Tsumita, 1956), Wax C (Tsumita, 1956 b) and Wax D (Nojima, 1958 a, b) . At the present, however, it should be emphasized that chemical studies on mycobacterial components with a more complex nature are needed than that on simple lipids and lipopolysaccharides which can be isolated by means of organic solvents.
It is our present purpose to extract complex materials such as lipopolysaccharides or lipoproteins from defatted Mycobacteyia and to survey some of their biological properties.

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As to the chemical nature of the antigen of the Middlebrook-Dubos hemagglutination reaction (Middlebrook and Dubos, 1948), it was preliminarily reported by the authors that the antigen was a macrbmolecular polysaccharide which had never been reported either chemically or biologically (Tsumita, Matsumoto and Mizuno, 1959) .
To elucidate the high adsorbability onto red blood cells and specificity against antituberculous sera which are essential features of the antigen, this paper covers details of the fact that the antigen could be obtained in a homogeneous state under ultracentrifugal forces and it contained lipid as a constituent, that is to say, it was a new lipopolysaccharide. The chemical and biological properties including stability of the antigen and specificity of the hemagglutination reaction are also reported in this paper.

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In the previous paper the author and her collaborators reported the isolation and purification of hemagglutination antigen of Mycobacterium tuberculosis Aoyama B (virulent human type) by zone electrophoresis and ultracentrifugation techniques (Tsumita, Matsumoto and Mizuno, 1959, 1960) . They isolated a macromolecular lipopolysaccharide which was electrophoretically and ultracentrifugally homogenous (26 S) . The component sugars were mannose and arabinose. It gave a high titer of hemagglutination reaction against anti-Aoyama B horse serum as well as against anti-BCG rabbit serum.
The present author intended to isolate and purify a hemagglutination antigen from BCG similar to that of Aoyama B strain and to elucidate as to whether it crosses with anti-Aoyama B serum or not.
A lipopolysaccharide composed of mannose, arabinose and glucose was isolated, giving a high titer in hemagglutination reaction both for anti-Aoyama B and anti-BCG sera.

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It was previously reported that thirty six compounds of 2-alkyl and 2-alkyl-5 (or 6) -nitro benzimidazol derivatives were synthesized and screened for antitumor effects using a solid type of Ehrlich ascites tumor cells (Komai et al., 1959) . Among these compounds, a slight antitumor effect was found with 2-propyl-benzimidazole, and its 5- (or 6) -nitro derivatives showed increased activities. It has been generally accepted that 9-β-D-ribofuranoside of purine antimetabolite is more active against tumor or more toxic to normal cells than its original free base (e.g. Biesele, 1956) . We intended to extend our previous findings in synthesizing 1-riboside of these benzimidazole derivatives.
The present report deals with the synthesis of 2-propyl- and 2-propyl-5 (or 6) -nitro benzimidazole-1-riboside and the test for antitumor activity of these substances.
On the other hand these substances now synthesized by us are related to antivirall activity. Tamm et al. (1951, 1953) examined extensively an anti-influenza virus activity in vitro of 2-substituted and halogenated ribofuranosyl benzimidazole derivatives andd reported a marked effect of isopropyl and n-butyl derivatives among 2-alkyl substitutedd benzimidazoles and a high inhibitory activity of certain halogenated ribofuranosyl benzimidazoles. These results led us to test the antiviral actions in ovo and in mice of 2-alkyl substituted benzimidazole derivatives previously synthesized and their riboside now synthesized by us.

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Paragonimus iloktsuenensis was first demonstrated by Chen (1940) in Canton Province, China. Afterwards Miyazaki (1944) found the same species in Japan. The cercaria and metacercaria of this worm was described by Chen (1940) in China, by Miyazaki (1944) and Yoshida (1959) in Japan. No one, however, elucidated the flame cell pattern of this worm.
The flame cell pattern of P. westermani was made clear by S. Yamaguti (1943), Komiya and Ito (1950) and that of P. ohirai by Yokogawa, Yoshimura and Komiya (1960) . The purpose of this work is to describe the flame cell pattern of P. iloktsuenensis and to compare it to the other Paragonimus species.

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