A parasitologic survey of human parasitic infections was made in Shizuoka Prefecture, Japan, during August and September, 1950 (Ritchie et al., 1951a) . The objectives included a determination of (i) the incidence of infections, (ii) helminth densities, (iii) factors predisposing infections, (iv) the status of schistosomiasis, (v) status of paragonimiasis and filariasis, and (vi) the efficiency of the intradermal test for the diagnosis of paragonimiasis.

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The fact that proteins having definite tuberculin activity are obtainable not only from the culture filtrate, but also from the bacterial cells of tubercle bacilli, has already been reported by a number of workers; among them, Heidelberger and Menzel (1934), Gronwall (1944), Heckly and Watson (1950), Baldwin et al. (1953), Seibert and Fabrizio (1952), and Ohtomo (1955) are included. Throughout these reports—in the opinion of the respective authors—the following points are the most conspicuous features of the bacillary protein: the high degree of purity and high potency (Seibert and Fabrizio, 1952) ; the remarkable ability in sensitizing animals (Baldwin et al., 1953) ; and the considerable amount of transfer of the protein from the bacterial cells into the culture filtrate when the entire culture was heated (Ohtomo, 1955) .
A discussion in the qualitative differences between the proteins of the culture filtrate and of the bacterial cells would pose the following problem: whether the protein present in the culture filtrate is merely the degradation product of the bacterial cells; or whether, in addition, it contains a secretory type of protein discharged from the bacterial cells themselves. If the former is the case, the protein of the culture filtrate and that of the bacterial cells should be the same in nature; and hence, on preparation of tuberculin protein, the extraction of bacterial cells would provide the best method as it would reserve the high potency and high purity (Seibert and Fabrizio, 1952) . But if the latter is the case, the protein obtained from the two sources should not be the same; and hence these sources would have to be differentiated, not only out of biological interest, but also from the practical standpoint of the preparation of the tuberculin protein—especially when the increasing demand for adopting the use of PPD in routine work in this country is taken into consideration.
Since there has appeared in a previous paper (Kasuya et al., 1956) a successful method for obtaining more detailed information on the chemical characteristics of tuberculin proteins obtained from culture filtrate, with respect to the nature of their constituent peptides, by the use of DNP-technique and countercurrent distribution, the task of the present author, in the light of this new method, has become one of elucidating the peptide constitution of the protein obtained from the bacterial cells of the human strains Aoyama-B and Frankfurt.

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The relationship between the intensity of the serological reaction and quantities of antigen and antibody concerned has been studied on the serological reactions in vitro. As for the precipitin reaction, for example, the two-dimentional pattern of precipitating zone was reported by Boyd (1941) and confirmed by many investigators. Similar findings were also observed in complement fixation test. As anaphylaxis is one of the antigen-antibody reaction, it is conceivable that there would be some quantitative relation among the intensity of anaphylactic reaction, and the amounts of antigen and antibody concerned. In this connection, attempts were made to clarify the quantitative relation among them on toxin-anaphylaxis of diphtheria.
The quantitative studies on anaphylaxis have been carried out by many investigators. For example, Kabat and Landow (1942) studied on the relation between the amount of antibody used for sensitization and the amount of antigen required to cause anaphylactic shock to death. As the result of this experiment, it was found that the severest anaphylactic response occured in the region of large antigen excess. However, it is clear that anaphylactic reaction is caused by antigen-antibody reaction in the tissues. In other words, the circulating antibody seems to take directly no part in anaphylaxis though sometimes it inhibits the occurrence of anaphylaxis (Ishizaka, 1953) . Moreover, according to Beserga et al. (1953), it was confirmed that the concentration of circulating antibody has not necessarily any parallelism with the degree of sensitization. Therefore, in order to clarify the quantitative relation between the intensity of anaphylactic reaction and the antigen and antibody concerned, the influence of the circulating antibody on anaphylactic reaction must be excluded and the concentration of tissue antibody should be discussed.
On the other hand, it seems that one of the difficulties in quantitative studies on anaphylaxis is to exclude the individual difference of experimental animals. In order to eliminate the individual difference as well as the influence of circulating antibody, anaphylaxis in vitro with the aid of Schultz-Dale's method was applied in the previous report (Ishizaka et al., 1954) . Namely, the intensity of anaphylactic reaction was determined in terms of acetylcholine concentration and the concentration of antigen required to cause specific contration comparable to the one caused by a certain concentration of acetylcholine was determined in each sensitized animal. As the result of this experiment, the correlation between the concentration of tissue antitoxin and the concentration of antigen required to cause a specific contraction in a certain degree was observed. However, the quantitative relation between them was not clear, because tissue antitoxin concentration was estimated from the disappearance rate of antitoxin from the circulation in the previous report.
In the present experiment, the concentration of tissue antitoxin was estimated exactly by using guinea pig's antitoxin labeled with I¹³¹ and the quantitative relation was studied in detail.

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In the analyses of the lipid of Mycobacterium tuberculosis, Stodola, Lesuk and Anderson (1938) isolated a long chain hydroxyacid having a molecular formula of C₈₈H₁₇₆O₄ and showing acidfastness. The mycolic acid has been subjected to extensive studies by Lederer and his coworkers. In fact, various kinds of related fatty acids were isolated and purified by chromatography from lipids of human strains of tubercle bacilli, Test and Aeschbacher (Asselineau and Lederer, 1949; 1951), H37Ra and H37Rv (Asselineau, Demarteau and Lederer, 1950) and of bovine strains, Vallée (Demarteau, 1951), BCG (Ginsberg and Lederer, 1952), Marmorek (Demarteau and Lederer, 1952) and Mycobacterium phlei (Peck and Anderson, 1941; Barbier and Lederer, 1952) . In view of the fact that those acids isolated from various kinds of Mycobacteria have closely related chemical configulations, i.e. a long alkyl branch on α position and a hydroxy radical on β position, they were generally designated as mycolic acids.
So long as fatty acids in bacterial lipids are concerned, the presence of mycolic acids in Mycobacteria is certainly a distinguishable character from other bacteria. It has also been reported that the lipid of tubercle bacilli contained mycolic acids not only in the form of free acid but also of complexes with another components possessing biological activities.
Sabin, Doan and Forkner (1930) reported that the phosphatide and wax fractions were responsible for the formation of tubercle which was composed of epitheloid and giant cells, although other bacterial components, protein and polysaccharide, showed no bearing thereupon. The tubercle formation in the lesion of tuberculosis was, therefore, chiefly due to the lipid of tubercle bacilli. Later, mycolic acid (Gerstl, Tennant and Perzman, 1945), wax D (Delaunay, Asselineau and Lederer, 1951) and branched fatty acids (Sabin 1941; Husseini and Elberg, 1952) were proved to have the activity when injected into animals.As wax D is a lipopolysaccharide containing a lot of mycolic acids, it seems reasonable that mycolic acid is one of the important components for tubercle formation.
Since Anderson's pioneering studies, the free mycolic acid has been shown to have acid-fast property, which is one of the characteristic features of Mycobacteria. Furthermore, it should be emphasized that no other substance responsible for acid-fastness has ever been isolated from Mycobacteria than mycolic acid and related complexes. Accordingly, acid-fastness of tubercle bacilli should partly depend on the chemical nature of mycolic acids.
Choucroun (1947) reported the isolation of a lipopolysaccharide from tubercle bacilli by extraction with paraffin oil, which was capable of producing tubercle lesion in guinea pig's lung when injected intraperitoneally. As the toxic lipid gave mycolic acid and sugars on hydrolysis, it was an ester of mycolic acid with a polysaccharide and in all likelihood identical with wax D, a chloroform soluble and hot acetone insoluble wax derived from tubercle bacilli.
Bloch (1950) reported the presence of another toxic lipid, called cord factor, in the lipid of tubercle bacilli which inhibited strictly leucocyte migration and exerted delayed toxicity in injected mice which succumbed showing pulmonary hemorrhage and decrease in body weight. Extensive studies on the occurrence and chemical nature of the toxic lipid rendered evident that the toxic lipid could be found in the petroleum ether extract as well as the chloroform soluble wax, especially wax C. (Bloch, Sorkin and Erlenmeyer, 1953; Noll and Bloch 1953; Asselineau, Bloch and Lederer, 1953) .

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In the previous study on mycolic acid complex in Mycobacterium tuberculosis it was established that glyceryl mono-mycolate was a component in wax C fraction of BCG (Tsumita, 1956) . However, considering the solubility of glyceryl monomycolate, it can be considered that a substance closely related with it might be present also in wax A fraction of BCG, which is extractable with alcoholether (1: 1) from tubercle bacilli and soluble in hot acetone.
Asselineau (1951) reported that wax A fraction of a human virulent strain of tubercle bacilli, Test, was composed of free mycolic acid, phthiocerylesters of fatty acids and a lipidic substance with a molecular formula of C₆₀H₁₂₀O.
On saponification of alcohol-ether soluble wax, Wieghard and Anderson (1938) obtained mycolic, hexacosanoic, stearic and palmitic acids from the fatty acid fraction and phthiocerol from the neutral fraction and glycerol and sugars from the water soluble fraction.
The author fractionated wax A of BCG into three portions by aluminum oxide chromatography. It is the purpose of this paper to describe the fact that one of them was glyceryl mono-mycolate, which gave two kinds of mycolic acid on hydrolysis.

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As regards the brackish water cercariae, several reports were found in Japan. Initial report was made by Fujita (1906, 1907) who described two species of cercariae, Cercaria pectinata Huet, 1891 (Trichocercous group) and C. tapidis Faust, 1924 (Long-tailed group) from mussel, Paphia philippinarum at Yawata in Tokyo Bay. Asada (1928) elucidated the life cycle of Heterophyes nocens and described its cercaria from brackish water snail, Tympanotonus microptera at Onoda in Yamaguchi Prefecture. From the same snail host at the same locality another parapleurolophocercous cercaria was reported by Ochi (1931) who decided it as the cercaria of Pygidiopsis summus. In the next year, Katsuta (1932) reported one species of cystophorous cercaria from a snail, Cerithium spp. in Formosa. Yamaguti (1934) described one of echinostome cercaria from the snail, Batillaria multiformis at Hyogo Prefecture and suggested to develop to genus Accanthoparyphium. In the same year Ozaki and Ishibashi (1934) described a new bucephaloid cercaria, Bucephalus margaritas from the pearl-oyster, Pinctada martensi. The latest report was made by Ogata (1943a, 1943b) who described a new echinostome cercaria, Cercaria granifera from a snail, Cerithidea spp. at Tokyo Bay, and a new gymnocephalous cercaria, Cercaria haplocoecum from a snail, Assiminea japonica at the same locality. Since then no report was found in Japan.
The locality, from where two cercariae were reported by Ogata (1943), is a muddy seashore at Urayasu-Cho near the mouth of Edo River in Chiba Prefecture. In this area several species of snails as well as bivalves and crustacean were found abundantly distributing on the wide tide-line. It was revealed that some species of snails, namely, Tympanotonus microptera (Kiener), Cerithidea (Cerithidea) largillierti (Philippi), Cerithidea (Cerithideopsilla) cingulata (Gmelin) and Assiminea japonica v. Martens, were infected with more than 20 species of cercariae by the present author's investigation in the year 1949, 1951 and 1952. While the fresh water cercariae were described more or less completely, most of brackish water ones remained undescribed up to the present day in Japan.
In this paper the description was made on two new furcocercous cercariae to which I proposed a name Cercaria ogatai and Cercaria tympanotoni respectively. The former name was designed to dedicate to the late Dr. Toji Ogata who carried out an initial investigation in this field, and the latter one to mean the genus name of its snail host.

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During the course of my investigation at Urayasu, two new species of peculiar long-tailed cercariae were encountered. One species was obtained from a brackish water snail, Tympanotonus microptera, and I proposed a new name, Cercaria komiyai n. sp. which was designed to dedicate to Dr. Yoshitaka Komiya, to whom I was much indebted for his generous encouragement. The other species emerging from a brackish water snail, Cerithidea largillierti was also long-tailed one but distinctly differed from the former species. While the only meagre observation could be made on the latter species, I tried to make a preliminary description with a new name, Cercaria nigrocaudata n. sp. The detailed description of the latter species will be performed in the future.
Materials and methods were briefly noted in this paper since they were given in details in the former paper of this series. The living larvae were mainly studied for details of the excretory system and the number of penetration glands. For these measurements they were killed with 10% hot formalin. All drawings were made to scale from such measurements.

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Cameron (1950) described that cells in tissue culture can withstand some degree of osmotic hypotonicity but proportionally a much smaller degree of hypertonicity, and this fact has been commonly accepted. Concerning mere maintenance or survival of the HeLa strain cells, the above-mentioned fact was confirmed by the present authors. However, when the influence of various grades of environmental osmotic pressure, around the isotonic one, was examined by estimating the cell population on the basis of nucleus enumeration, it was found that a slightly hypertonic environment was best for proliferation of the cells.

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