It was reported previously that Pseudomonas aeruginosa multiplied, beyond doubt, in a simple solution which was composed of CaCl₂ and Na₂HPO₄. In this paper, further informations about the conditions necessary for the growth of bacilli in an inorganic solution are presented.
The results obtained are concluded as follows:
1) Unequivocal multiplications of Pseudomonas aeruginosa were observed in inorganic solutions which were composed of either CaCl₂-Na₂HPO₄, CaCl₂-NaH₂PO₄, MgCl₂-Na₂HPO₄, or MgCl₂-NaH₂PO₄ at the final concentration of 0.02%.
2) It was also noted that the ions essential for the growth of bacilli were i) Calcium ion (Ca⁺⁺) and either bivalent phosphate ion (HPO₄^--) or univalent phosphate ion. (H₂PO₄^-) ii) Magnesium ion (Mg⁺⁺) and either bivalent phosphate ion (HPO₄^--) or univalent phosphate ion (H₂PO₄^-). Thereby, it must be strongly mentioned, in both cases mentioned above, that sodium ion (Na⁺) and chloride ion (Cl^-) could not be omitted.
3) It was found that a limited final concentration of a mixture with CaC12Na2HPO4 which permited the growth of Preudomonas aeruginosa was 0.005%.

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Referring to the distribution of Group A streptococci in Japan, the data up to March 1969 have already been reported devidedly in separate papers, in which the annual details of the strains collected in each institution since April 1969 were described. Further a figure presenting the histograms of the main predominant types for the past 10 years was depicted to show retrospectively the transition of the types, at the time just a decade has past since we started T-typing.
During the whole survey period the situation of type 12 and type 4 has not essentially changed in the context that they represented two major types. This was to be seen in the tables as well as in the figure. The situation of other three types 6, 22 and 1 which had shown relative predominancy in incidence as compared with other types of minor incidence has also not changed throughout the period except in 1974 when a new type, type 3, joined for the first time in this group and, instead, type 6 dropped out of the group. Among these members types 22 and 1 were characteristic in the distribution,
i.e., their incidence was obviously higher in Niigata and Shimane the areas facing the Japan Sea. Similarly in Sapporo facing both the Japan Sea and the Pacific Ocean their incidence was moderately higher in 1969 to 1971. This was to be regarded as an exemplification of the areal aggregation of some specific types.
Through the period of 10 years the incidence of type 12 showed a marked continuous increase from the lowest incidence of 0.9% in 1964 to the highest level of 71.9% in 1974, a particularly impressive steep rise In contrast, type 4 took a different trend. In 1964 it took the premier rank and occupied 55.4%. However, the incidence declined relatively rapidly toward 1968 to 1969, and therefore the transition of the first ranking from type 4 to type 12 took place in 1967 to 1968 as we could see in the Fig. 1, a crossing of two “epidemic waves.”Thereafter the secondary ranking of type 4 being characterized with an undurate curve of incidence has been retained up to now.
The most recent peak in the total number of scarlet fever patient in Japan before 1971 was established in 1963 when the tetracycline resistance developed to a considerable degree after its initial appearance in 1962. In 1964, the following year, a sudden alteration of the premier rank took place and type 4, suppressing the type 6, took the absolute predominance as seen in the Fig. 1. The following mild peak of the number of patient was accomplished in 1971 when the macrolide highresistant strains appeared for the first time in Japan. The marked increase of resistant strains seemed to develope after 1972. The extraordinarily steep increase of the incidence of type 12 seemed to correspond to the maximum developement of the macrolide resistance. This might be clarified with our drug resistance survey which is now under proceeding.

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Studies were made on the antibiotic susceptibility of a total of 1, 542 strains, which were isolatedfrom the patients who were suffering from scarlet fever and hospitlized in Japan during the year 1973.
Results obtained are as follows:
1) All the strains tested were sensitive against 0.05mcg/ml of Penicillin-G (PC-G), and there was none of the PC-G resistant strain.
2) Remarkable increase of the resistant strain agianst Tetracycline (TC), Chloramphnicol (CP), Oleandmycin (OL) and Erythromycin (EM) was observed, and many of them showed a multiple drug resistance. Among them, there were many strains being i esistant to the concentration of greater than 1, 000mcg/ml of EM.
3) Most of the isolates obtained were serologically typed into the group A type 12. However, it was obscure whether there is any correlation between the serotype of the organism and their drug resistant characteristic or the development of a secondary disease.

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We have already reported that, by gel diffusion and immunoelectrophoresis using antisera prepared with heat-killed bacteria, 3 types of heat-stable antigens were present in a strain of group C or G hemolytic streptococci of human origin. They are the protein antigenically common to the group C and G, and the trypsin-resistant antigens specific for the strain of the group C or G, in addition to the group specific polysaccharide antigens.
The present study describes further characterization of these antigens by the Sephadex gel filtration, and their distribution among various standard and wild strains belonging to the group C or G by the micro-slide gel diffusion technique. The results are summarized as follows.
1. The protein antigen, immunologically common to the group C and G strains and physicochemically resembling to the M protein of the group A type 12 strain, showed an estimated molecular weight between 70, 000 and 90, 000 by the Sephadex gel filtration using G-75, G-100 and G-200. This antigen was widely distributed among various wild strains belonging to the group C or G of human origin; 40 out of 43 strains of group C, and 37 out of 48 strains of group G possessed this antigen. Among standard strains of group C or G, however, the rate of strains lacking this antigen was significantly high; 4 out of 7 group C standard strains, and only 1 out of 4 group G standard strains possessed this antigen.
2. The trypsin-resistant antigens, specific for the group C and G strains used for the antiserum preparation, respectively, showed a little higher molecular weight than that of the protein antigen that is estimated at 100, 000 or more in the Sephadex gel filtration. The distribution of these antigens among other strains was scarce; 4 out of 50 strains (3 in 43 wild strains and 1 in 7 standard strains) in case of the group C, and only 1 out of 52 strains (1 in 48 wild strains and 0 in 4 standard strains) in the group G contained these antigens. The trypsin-resistant antigens, being specific for certain types of group C and G, respectively, may play an important role in establishing further classification of the group C and G hemolytic streptococci.

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Studies were carried out to develop and evaluate a electrosyneresis for the rapid, sensitive, and specific grouping of beta hemolytic streptococci and typing of group A hemolytic streptococci. In grouping and typing of strep ococci, well defined and centrally loca ed precipitin bands were produced by using a gel composed of Special Agar Noble: Agarose A-45=1: 2. Electrophoresis was carried out for 20 min. by using 9 mA/cm at room temperature with group specific or type specific antiserum. The precipitin bands were detectable during the electrophoresis. In such conditions, 1.7 μg/ml of purified group A polysaccharide could be detected.
When a antiserum well was placed opposite to two antigen wells, group and types of group A streptococci were identified at a same time with non-absorbed respective type of group A antiserum.

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Two hundred eighty strains of beta hemolytic streptococci isolated from patients were examined in parallel by precipitin, bacitracin. disc, Kobayashi's Methods and electrosyneresis. With both of the latter two methods, correct identification of Group A streptococci was obtained when compared with the results using the Lancefield precipitin method, and no false reaction occurred. With some variation on the different lots of same manufacturer of the bacitracin disc, 98.2% of the group A streptococci were sensitive, while 34.0% of strains of groups B, C, F, and G were sensitive.
In addition, 213 strains of group A hemolytic streptococci were also typing with electrosyn.eresis as compared with Lancefield precipitin method. This identification was accurately correlated with the results obtained with the precipitin test, and 53.7% cf non-typable strains with precipitin test were typable with this method.

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A total of 278 laboratory stock strains of streptococci, consisting of 169 of A, 32 of B, 47 of C and 30 of G, which originally isolated from human patients, was used in this investigation. Different sensitivity of the organisms against Bacitracin was observed according to their groups and groups A and C were easilly discriminated from other two types, when organisms were tested by plate dilution method of Bacitracin. However, when applicability of the 3 different commercial Bacitracin disks was tested by the use of the same organisms, it was found that the results obtained varied depending on the soft of pulp disk or different experimental conditions such as the adoption of pre-diffusion or direct application of disks.

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An epidemic of scarlet fever caused by type T-12 group A streptococcus which showed no sensitivity to erythromycine and tetracycline occured in Kakunodate, Akita Prefecture, from October to December in 1973. The morbidity of scarlet fever was 279.5 per100, 000 of population and 85 5 of patients were children 3 to 6 years old. Sera obtained from 331 children in two kidergartens, of which 38 children developed scarlet fever, in the middle of and after the epidemic and sera from 100 healthy inhabitants in this district in 1971 were examined for a seroepidemiological survey on type T-12 group A streptococcus.The obtained results were as follows.
1. Before the epidemic, the geometric mean ASLO titer of sera collected from children 4 to 6 years old was 1: 33.8 and the highest ASLO titer was observed in age group 10 to 12 years of old. ASLO titers of sera from children 3 to 6 years of age in the middle of the epidemic apparently increased higher than those of sera from children 4 to 6 years old before the epidemic. Particularly, ASLO titers of 1:>320 were observed in 47.1% of children whose sera were collected either in the middle of or after the epidemic, as compared with 6 % in sera obtained before the epidemic.
2. Eleven (57.9%) out of 19 children 4 to 6 years old before the epidemic revealed T agglutinin titers of 1: 20 against type T-12 group A streptococcus and the geometric mean titer was 1: 28.2, whereas 80% of sera from children in two kindergartens after the epidemic showed the T agglutinin titers 1:>20 and the geometric mean titer increased remarkably to 1: 365.
3. Significant rises (1: 8) of T agglutinin titer to the type T-12 after the epidemic were detected in 124 (64.9%) out of 191 children whose sera were collected both in the middle of and after the epidemic.
4. The rate at which children in two kindergartens were infected with the type 1-12 streptococcus was estimated as 66.0-84.6 5 on the basis of three indexes of significant rise of the T agglutinin titer, patient with scarlet fever and carrier of the type T-12 streptococcus. About 78.7 to 86.7 5 out of the rate were estimated by the index of the T agglutinin titers. Thus, it may be useful for seroepidemiological surveys on group A streptococci to measure T agglutinin titers against each type T streptococcus in human sera.
5. The rate of inapparent infection with the type T-12 group A streptococcus during this epidemic was estimated as 79.8-84.2%.

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Study on the distribution of group A hemolytic streptococci was carried out in throat of children of a primary school stituated in rural area of Saitama prefecture in which 206 pupils were enrolled.
From these children the isolation of steptococci has been attempted 7 times and drawing of blood specimens 3 times for the purpose of ASO test for the period from October 1968 to March 1970.
Frequency of distribution of steptococci among children varied by the time of examination, ranging from 1.0 percent (the lowest) in July 1969 to 20.3 per cent (the highest) in March 1970, and mean of 7 surveys was 10.7 percent.
Of them 114 strains were isolated and typed as follows: type 1 (0.7%), 3 (9.0%), 4 (43.8%), 6 (14.6%), 12 (10.4%) and 22 (0.7%).
As there was positive correlation between the ASO titer and isolation rate of streptococci, we could confirm the epidemic of group A streptococci from the variation of ASO titers, i. e., in the case of non-manifested epidemic due to type 4 streptococci which we found in November 1969.
By the examination of ASO test, carried out for 8 months, it has been revealed that there was maiked difference between the behevious of antibodies (ASO) of Strepococcus-positive and negative groups, namely the titei of 67 per cent of the former raised and of 60 per cent of the latter fall down.

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Through our daily practice and various informations concerned we had known that this Santama districts, the west part of Greater Tokyo, are relatively densely infiltrated areas by hemolytic streptococci (HS). Since the inauguration of this hospital, Tokyo Metropolitan Fuchu Hospital, in 1970, we have carried out isolations and group and type determinations of pharyngeal HS from child patients who visited the department of pediatrics of this hospital, the number of cases totaling 293. Additionally, 23 cases of tonsillectomized children were investigated for extra- and intra-tonsillar HS to contribute for not yet definitely secured evaluations of the operation. The period of the survey was from 1971 to 1973. The similar investigation had previously been done by us in Tokyo Metropolitan Komagome Hospital which located in almost central part of Tokyo during 1966-1968 and the both data were compared for areal difference.
The followings are the summarized results and conclusion.
The total positive rate of HS was 39.5% and A group rate in it was 63.9% around Fuchu hospital, and 20.2% and 78.0%, respectively, around Komagome hospital. In type distributions, the study in Komagome hospital revealed the prevalence of T1, T3, T6, T9, and T12, with some yearly fluctuations. In the case of Fuchu districts, T12 and T18 were the prevailing type with little number of T2, T3, T11 and T22. In the connection of this type study, we investigated 6 cases of scarlet fever in 1973 and got T12 from 4 cases, T18 from 1, and untypable from 1. This result seems to reinforce our viewpoint that T12 and T18 are main prevalent stocks around Fuchu Hospital.
It is interesting to see the prvailing inconsistency in type comparison of extra- and intra-tonsillar HS in tonsillectomized cases.

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It has been recongized that minocycline showes a potent antibacterial activity against tetracyclineresistantStaphylococcus. In order to examine the pattern of antibacterial activity of minocycline against tetracycline-resistant Streptococcus hemolyticus, a comparative study with regard to MIC (Minimal Inhibitory Concentration) of tetracycline and minocycline was conducted on 487 strains of A-Streptococcus hemolyticus isolated from the scarlet fever patients.
The study revealed that, under the MIC of more than 25 μg/ml, 76.4% of 487 strains tested were resistant to tetracycline and 16.1% to minocycline. Moreover, it would be worthy of notice that 67.1% of the tested strains were sensitive to minocycline with the MIC range of 3.12 to 12.5 μg/ml.
The clinical trials were made on 76 scarlet fever patients who were administrated minocycline, 4 mg/kg b.i.d., for a week. The summary of the trial results follows:
1. Within two days after the administration of minocycline, the causative A-Streptococcus hemolyticus at the pharynx became negative in 41.8% of the patients examined.
2. In less than three days after the treatment began, the body temperature came down below 37C in 57.7% of the cases.
3. In 73.3% of the cases, exanthema disappeared within three days after the treatment.
4. Clinical trials showed that minocycline was effective in 38.2% of the cases, good 28.9% and poor or unknown 32.9%.
5. In 26.7% of the cases examined, relapse of the causative A-Streptococcus hemolyticus at the pharynx was observed after the discontinuation of minocycline treatment.
6. No serious side effect was recognized and the result of the examination of liver function was normal.

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