1) In the previous part of this series of studies, a new genus, Halococcus, was discussed from the view point of the taxonomical system of the family Coccaceae and on the basis of Adanson's concept, Sneath's similarity value, and the senior author's concept of center species.
The specific characteristics of the genus Halococcus, induced theoretically are as follows: Gramnegative, spherical in shape, present in pair, aerobic, oxidase-positive, halophilic or halotolerant, and negative for acid production from glucose.
Three new species were isolated from salted fish and tested for 120 features by the authors.
2) In this experiment, the new genus was examined for about 70-80 new features, in addition to those (about 40-50 features) described in the previous part, in relation to the classical Micrococcus denitricans ATCC 13543, M. halodenitrificans ATCC 13511, and M. cryophilus ATCC 15174. These species were reoriented from the genus Micrococcus to Halococcus by the authors.
It was proved that the key features induced theoretically and the findings of the present experiment were perfectly correlated. Thus, it was considered that the taxonomy based on the concepts and methods described above might be reasonable.
3) The genus Halococcus was defined as one which has the following characteristics: Gram-negative, spherical in shape, occurring singly, in pair, in chain, or in clusters, but never in packet, aerobic, containing metachromatic bodies, halophilic or halotolerant, catalase-positive, oxidase-positive, and negative for acid production from glucose or any other carbohydrate.
4) The following tests were used to clarify the differential characteristics of the six species of the genus Halococcus: PPA test, KNO₃ reduction, urease and Voges-Proskauer reaction, gelatin liquefaction, spontaneous agglutination, and red pigmentation.
5) The nomenclature of the three species of the genus Halococcus isolated by the authors was as follows: Halococcus acetoinfaciens nov. sp. (strain H7-1), Halococcus agglomegatus nov. sp. (strain H8-1), and Halococcus nondenitrificans nov. sp. (strain H11-4).
6) The species reoriented from the genus Micrococcus to Halococcus should be renamed in the following manner: Micrococcus denitrificans to Halococcus denitrificans, Microc. halodenitrificans to Haloc. halodenitrificans, and Microc. cryophilus to Haloc. cryophilus.

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Studies were perfomed on the unique E 46 and E 97 strains of staphylococci, which had been described in the preceding part of this series, to find any evidence for high virulence for mice by intraperitoneal inoculation. The results were compared with those obtained from the typical Staphylococcus aureus and S. epidermidis represented by the E 111 and B 217 strains, respectively.
When inoculated into mice by the intraperitoneal route, organisms of both unique strains were hardly ingested by the intrapeitoneal cells within about 2 hours. They began to multiply in abundance in the peritoneal cavity several hours later and invaded into the phagocytes almost simultaneously. Their multiplication finally led to destruction of the host cells. Then the organisms seemed to move into the blood stream and spread into almost all the tissues and organs to cause septicemia and death of the host, judging from the results of infection experiments mentioned in the preceding report. On the contrary, organisms of the B 217 strain were easily ingested and digested by the phagocytes. Those of the E 111 strain behaved in an intermediate manner between the above two.
The unique strains were able to grow well in human and rabbit sera like the E 111 strain. The B 217 strain, however, decreased in number in these sera. In mouse serum the growth of any tested strain was not affected at all.
The unique strains were proved to be negative for coagulase and α-hemolysin activities in routine quantitative assay. Every tested strain was positive for these activities, but very low in hemolysin titer for mouse erythrocytes.
Thus, it may be concluded that the high virulence of both unique staphylococcal strains for mice by intraperitoneal inoculation is derived mainly from their strong resistance against phagocytosis by the intraperitoneal cells at the early stage of infection and also from their vigorous ability to multiply inside and outside the phagocytes. The resistance may be attributed to the surface structure and function of those unique strains of whose cells were demonstrated to have a capsule-like structure and contain cell-wall ribitol, as described in the preceding paper.

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The lipase test was examined for usefulness as an aid in the identification of pathogenic staphylococci, with special reference to the sort of substrates used. It was compared with the egg-yolk test with regard to interrelationships among coagulase, deoxyribonuclease DNase and other biological attributes.
The test strains employed consisted of 577 coagulase-positive and 150 coagulase-negative staphylococcal strains freshly isolated from human and swine clinical materials. The basic media used were Gillespie's agar, Starr's agar, heart infusion agar and, as control, egg-yolk agar. Several oils and Tween 20 and 80 were employed as substrates. The results obtained are as follows.
All the test strains of human origin showed the closest correlation between the coagulase and DNase activity production like those in the preceding investigation. The results of the egg-yolk test were inferior to those of the DNase test with regard to coagulase production when both tests were performed with media containing various oils. The correlation between the coagulase and lipase activities was very high as to the coagulase-positive strains, but considerably low in identity as to the coagulase-negative ones. The heart infusion agar containing Tween 80, however, gave a relatively good result in this point. Especially, the oil or Tween media supplemented with the egg-yolk protein fraction, which had beed devised by the authors, behaved in almost the same fashion as the egg-yolk medium.
Therefore, it may be conculuded that the medium containing Tween 80 and egg-yolk protein is the most appropriate for testing the lipase activity of staphylococci, considering the enzyme specificity.
It should be noted that the strains of swine origin, both coagulase-negative and coagulase-positive, showed a better interrelationship among the lipase, egg-yolk, and coagulase tests than those of human origin.

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The mode of action of enduracidin was studied from the growth curves of Staphylococcus aureus and Bacillus cereus exposed to the antibiotic.
Enduracidin was added to cultures at the same time as, and 3 hours after, the inoculation of bacteria. In both cases, there was a significant decrease in value of viable bacterial count and optical density of these cultures, as compared with the control culture to which the antibiotic had not been added. Enduracidin exerted, however, neither bactericidal nor bacteriolytic action upon the stationary phase of growth.
The ability of enduracidin to inhibit the growth of Staphylococcus aureus was not affected by the addition of several amino acids, sugars, fatty acids, vitamins, or cations to the medium.

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E. coli strains isolated from 108 domestic fowls were examined for drug resistance and distribution of R factors.
Of them, 54 strains were resistant to at least one of 4 drugs; that is, tetracycline (TC), chloramphenicol (CM), streptomycin (SM), and sulfanilamide (SA). Of these strains, 24% were resistant to one drug alone and 76% to two drugs or more.
It was also found that 12 strains (22%) of these 54 carried R factors. Of the 12 strains, 4 were resistant to one drug alone and 8 to two drugs or more.
Animal food containing tetracycline and other drugs may be responsible for the high frequency at which resistant E. coli strains and R factors were isolated.

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The phosphorus jar method (PJM) was compared with the steel wool method (SWM), a new type of anaerobic culture method induced from Parker's iron wool method (1955).
The results obtained are as follows.
1) Almost the same results were obtained from the cultivation of Veillonella and many species of Clostridium by both methods.
2) Lactobacillus, Bifidobacterium, and Bacteroids could grow predominantly at any time when SWM was used. Especially, Bacteroides cultured by SWM was numerically much superior to that by PJM.
3) In the direct cultivation of rectal feces, SWM was predominant over PJM with regard to not only the number of colonies but also the number of strains obtained.
4) Some of the colonies grown by PJM were small and opaque.
5) However, when PJM with addition of 10% CO₂ was used, good results near those obtained by SWM were brought about.
6) PJM was very inconvenient because of the danger from poisonous gas and inflammability.

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In the previous report, it was made clear that inosine, the antibody-promoting factor, accelerated the biosynthesis of bovine serum gamma globulin (BSG) when applied to the quantitative precipitation test. In the present investigation, 13 substances were selected from among those related to inosine metabolic degradation, and examined in vitro to clarify the interrelations between their action and inosine in the biosynthesis of BSG.
I. Of these substances tested, cysteine increased the formation of BSG, whereas its derivatives, such as thiazolidon-4-carboxylate, cysteine-s-sulfonate, and taurin, were ineffective or inhibitory. When cysteine was used together with adenosine, it displayed a potential effect on the biosynthesis of BSG. Moreover, it displayed an additional effect when used together with either inosine or ADP.
2. XMP, CMP, uracil and orotic acid did not act effectively upon the biosynthesis of BSG.
3. SH-inhibiting substances, such as thiourea, potassium iodate, atoxyl, p-aminobenzoic acid, p-chlorobenzoic acid, and cobaltous ion, had an ineffective or inhibitory action upon the biosynthesis of BSG. UMP alone displayed such an accelerating effect as that of cysteine.
4. Pyridoxal, biotin and pantothenic acid had hardly any effect upon the biosynthesis of BSG.
5. When used alone, adrenalin exerted an inhibitory action on the biosynthesis of BSG and a stimulating effect upon the adenosine action.

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Recently, many isolates of the tsutsugamushi disease rickettsia from different sources have been classified antigenically into three types, Gilliam, Karp, and Kato, by means of the immunofluorescent and the complement fixation tests. Therefore, such type-specificity among the isolates would be worth while being evaluated from an epidemiological point of view. For instance, studies are required on the relations of any different pathogenicity for man, any particular type of strains causing a different seasonal incidence of disease in endemic areas, and any host-specificity in the vector trombiculid mites.
In the complement fixation and immunofluorescet tests, however, the serological patterns of sera from human patients or rodents are hardly so much type-specific as those of the antisera obtained by immunizing laboratory animals with the prototype strains. No reasons for this have been elucidated yet. Since a relatively high type-specific reactivity can be observed with a serum sample collected at an early stage of the illness and a group-specific one at a later stage, a species difference may be considered in the human case. On the other hand, there probably occurs a mixed or double infection among field rodents. So that, a cross-reactive pattern may be expected from rodent sera. Befor proving a possibility of occurrence of such kind of infection among field rodents in nature, it seems rather important to pursue any antigenic change in those prototype strains during serial passages after the mixed infection in laboratory animals. It is from these ideas that the present investigation was performed.
A mixed infection was made by 3 combinations of the prototype strains: that is, Gilliam-Karp, Kato-Gilliam and Kato-Karp. For every serial passage, 3 groups of mice were inoculated intraperitoneally with 10% emulsion of liver and spleen derived from mice which had been infected with a mixed inoculum for 25 passages in one experiment and 50 passages in the other at weekly intervals. In antigenic smears from the peritoneum of infected mice, rickettsial organisms were examined for antigenicity against labeled antibodies to Gilliam, Karp, and Kato, by means of the direct method of immunofluorescence at every passage.
The results obtained are summarized as follows.
1. In the Gilliam-Karp series, the population of rickettsial organisms became type-specific to the Kato strain at about 10th passage, with no change in antigenicity by further passages.
2. In the Kato-Gilliam series, Karp-type organisms began to appear at about the 4th passage.
3. In the Kato-Karp series, Gilliam-type organisms were never observed.
4. In the control series of separate passage of the prototype strains, no change was observed at all in the antigenicity as prototype throughout the course of passage.

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