Yanagimoto Manufacturing Company completed in 1926 the Polarograph No. 1 in Japan under the guidances of Prof. Shikata and Prof. Tachi, which was used thereafter in Yokohama Technical College. After the second War the applications of polarography was rapidly developed and we improved it so that the polarograms might be recorded photographically without the dark room. And since 1954 we have offered the pen-recording polarography, which are based upon the device of Prof. Takahashi, Prof. Niki and others at the Institute of Industrial Science, Univ. of Tokyo. We have followed the developments of polarography in these thirty-five years and so have paid attention to AC polarography, oscillographic polarography, chronopotentiometry and so on. Among them we have been able to present the bridge-type AC polarograph developed by Prof. Niki and the high-sensitive polarograph favourably.

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Ordinary polarography was introduced to our company in 1949 for the analysis of lead and cadmium in zinc, copper and zinc in ore and has been used for the analysis of metallurgical products and others.It has recently been ascertained too by our experiments that square wave polarographic method is superior to the ordinary polarographic method for the determination of minute quantity elements in high purity metal. In this paper, outline of square wave polarography was described with the reference of the reports of Barker, Miler and others and then practical use in our laboratory was described. For instance: the analytical method of zin in cadmium; Copper and lead in high purity tin or selenium; bismuth and lead in silver; bismuth, copper and lead in gold; tellurium in crude copper, selenium and silver; lead, Copper and cadmium in zinc. By the use of square wave polarographic method, time for the analysis of impurities in high pure metal were saved and reliabilty for the analytical results of minute quantities were increased in our daily work.

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In spite of many attempts to modify the filtrate reaction of Brdicka's cancer test, none of them gave better results than the original one. We already reported that in patients with acute leukemia the first maximum of Brdicka's filtrate wave was often higher than the second maximum. Such phenomenon presumably depends on the high contents of polysaccharides in molecules of these blood mucoproteins. In this paper further efforts were made to clarify the factors influencing on the shape of the protein double wave, and to find out the polarographic characteristics and clinical significance of electrophoretically separated mucoprotein with special reference to the wave form. Among many experimental conditions, the temperature in polarographic cells was found to be most sensitive factor to affect the protein wave shape, so that all experiments were carried out at the constant temperature (20°C±0.05). Serum mucoprotein was evidently separated into two fractions, M1 fraction an M2 fraction, by paper electroyhoresis in McIlvaine's buffer of pH 4, 4. These two fractions showed polarographically different shape each other. The M₁ fraction, the most rapidly anodic moving one, showed left side elevation in shape; in other words, the first maximum was higher than the second maximum. On the contraly, M₂ fraction showed right side elevation in shape; the second maximum was higher than the first maximum. Furthermore, it was very interesting from clinical point of view that the characteristic shape of M₁ fraction was to some extent variable depending on diseases. From the clinical observation on 259 cases including 119 cases of cancer and 11 cases of leukemia, it was revealed that in neoplastic patients the wave shape of M₁ fraction showed clearcut difference from those in normal; Δh (distance from the second maximum to the first maximum) of M₁ fraction in neoplastic patients was larger than that in normal. Thus, the cancer detection rate has markedly elevated as compared with the original filtrate test, namely from 77% to 90%o in our study.

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From 1898, Shigellae shigae was discovered by Shiga, there are many excellent data, which are best reference on the bacteriology, epideminology, toxicology. But we know a few data that are on the reference of biochemistry, serology, and difference of the metabolic materials from pathogenic and non-pathogenic dysentery bacillus. I have studied on these points by polarography, and the results are as follows:1. The methods of exercise and thier materials. 1) By countercurrent of ethanol and water with saturated ammonium sulfate, produced crystals from the liquid medium of old cultured bacillus, detected from patients and germ-carriers. Paperchromatographed, measured by use of spectrophotometer and tested by use of experimental animals. Thus, we can observe the difference between the metabolic materials from pathogenic and those from non-pathogenic dysentery bacillus by the half wave potential of their polarogram. 2) Sh. flexneri bacillus was cultured in the suitable liquid medium for 10 days at 37°C. Then, the filtrate of the cultured medium is added 1/2 vol, of 99.5% ethanol, heated at 80°C and added ammonium sulfate at 55% and saturated. After further heating, extracted liquid is separated into two phase, upper being yellow and lower being orange. Flat six-angle crystals, six-surface columnar, four-surface columnar, conic columnar and octahedron crystals are produced from the lower phase. 3) Polarogram. E1/2 of flat six-surface columnar from cultured medium of pathogenic bacillus in the supporting electrolyte of 0.1 N-NH₄Cl +-ethanol abs. (1:4) is -1.12±0.02v, and E1/2 of flat six-surface columnar from cultured medium of non-pathogenic bacillus is -1.28±0.02.v. The former is β-monoacylglycerglphosphatildycholin and the latterr is a-monoacylglycerylphosphatidylcholin. Using 0.1M-Na₃PO₄(pH=10.0) and 0.1N-LiCI (pH=2.6) as electrolyte, E1/2 of the flat six-surface columnar from pathogenic bacillus 4a is -0.98v, and -1.44v., and E1/2 of the 1b is -0.96v. The former is D-mannoic acid and the latter is D-glucosamine. Using 0.1 N-N(CH₃)₄C1 and 0.1N-NH₃ + 0.1 N-NH₄C1 as electrolyte, E1/2 of the flat six-surface columnar from pathogenic bacillus is -0.90v, and -1.02v. The former is Fe³⁺ and the latter is Ni²⁺ Using 8% ethanol as electrolyte, E1/2 of the octahedron crystals from cultured medium of pathogenictivc bacillus is -1.02±0.02v. This is 2·4-dinitrophenyl-peptide. Thus we can identify the metabolic materials of pathogenic from those of non-pathogenic dysentery bacillus by the polarographie method. This method may be able to use for test on other toxins of bacteria and toxins from food poisoning.

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Contaminated by Cl. botulinum type E variant, lecithin within the internal organ of Pabylonia japonica living in bog was spalted by lecithinase A and D which is produced by that bacillus to give β-monoacylglycetylphosphatide as β-toxin from the internal organ. When man eat these Eabylonia japonica at hall-cooked, he will suffer from foodpoisoning and die in the worst case. This β-toxin contains organic phosphoric acid and combines with acetylcholinesterase owing to become active phosphoric acid in mammalial bodies. So β-toxin has very much faster poisoning action than that of Parathion which at first becomes Paraoxon by oxidation and then produces active phosphoric acid in mammalial bodies. α-and β-toxin in co-operation act mammals to food-poisoning. In the poisoned animal apparently acetylcholin is not produced in the end plates, and the action of the toxin is proximal to the point at which the toxin is produced. The toxin acts on the nerve filaments since acetylcholin is released following direct stimulation of the excised diaphragm of the animal, but not tetanization of the phrenic nerves. Neuromuscular paralysis observed in the poisoned animal is a result of interference with conduction in the terminal twigs of motor nerve. Venerupis philippinarum is contaminated by one of Pseudomonas and α-, β- and γ-toxins produced. These toxins to enclude, became so-called enteroxin and mammalian became food-poisoning by this toxin, α-Toxin is lecithinase A, β-toxin is β-monoacylglycerylphosphatidylcholin and γ-toxin is 2.4-dinitrophenyl-peptide. The poisoning action of α-and β-toxin is fatal and it dissolves blood corpuscle. 2·4-DNP-peptide is feverish and vomiting material. This β-toxin can be determined from its half-wave potential in polarography in the supporting electrolyte of 1N-NH₄C1 with pure etanol (1:4). Therefore we are able to know whether this material is β-type or α-type. β-Type is a hybrid of reasonance to α-type and has dipolemoment and is toxic, but α-type is not. The former E1/2=-1.12±0.02v. and the latter E1/2=-1.28±0.02v. γ-Toxin is able to determin by its half-wave potential in polarography using 8% ethanol. E1/2 of this is -1.02±0.02v. We can determin the poisoning grade by polarography using the solution of 1N-NH₄C1+99.5% ethanol and[Co(NH₃)₆]C1₃. The polarographic tests tell us about β-toxin produced, spalted cystine of patient's serum and thiol-radical produced, so we are able to know poisoning grade, measusing its distance of Co⁺⁺⁺ wave and Co wave. Thus we are able to determine food-poisoning assay. E1/2 of β-monoacylglycerylphosphatide is -1.20±0.02v., so we are able to know the existance of f3-toxin, produced by Cl. botulimum type E also.

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