Genetic studies on the viomycin resistance of Mycobacterium tuberculosis var. hominis, strain H37Rv, have been conducted and the following conclusions have been obtained:
1. Survivors appearing on media containing viomycin up to 10μg/ml can be produced by phenotypic variability within the wild genotype, and survivors appear-ing on media containing more than 20μg/ml of viomycin is produced by mutation.
2. Population structures of single clones obtained by the first-step selection of the parent strain, using more than 20μg/ml of viomycin, have been similar each other. Clones producing mutants resistant to higher levels of viomycin at higher ratios have been obtained by successive selections. However, increase of such mutants in ratio has remained to less extent even by multi-step selection.
3. The pattern of viomycin resistance is a multi-step type with a wide first-step like the pattern of furadroxyl resistance in Escherichia coli.
4. Viomycin resistance appears to be determined by multiple genotypes. It appears possible that these multiple genotypes are produced by mutations of a single gene (a main factor) and some conditional factors.
5. The emergence of viomycin resistance occurs within a narrow limitation. The upper limit of resistance remains within the tenfold increase of resistance from sensitive cells even after the multi-step selection.
6. It has been considered that the ability to survive on media containing 100μg/ml (the upper limit of survsval) is produced by phenotypic variability within any resistant genotype.

View full abstract

Cl. welchii Hobbs type has been isolated from the dishes served at the wedding party at the time of the outbreak of collective food poisoning. Cl. welchii has hitherto been frequently believed to be the cause of food poisoning in Europe and America. As far as we know, such a case has not yet been reported not only in Japan but also in the Far East. As pointed out repeatedly by Hobbs, Zeissler and other investigators, the most important medium of Cl. welchii food poisoning is believed to be meat, especially animal meat products. However, an outbreak of collective food poisoning due to the oyster contaminated with Cl. welchii was recently reported by Welti et al.⁽⁴⁾ Furthermore, the isolation of Cl. welchii from fish meat has already been recorded in this paper. It is, therefore, considered that not only animal meat products but also marine products can be the important medium of food poisoning, and so a great attention should be paid to the Cl. welchii food poisoning due to fish meat.
The room temperature, at the time of occurrence of the food poisoning, was 30°C and the moisture was 80%. The conditions are considered to be suitable for the germination and growth of the spores surviving the heating when cooked. Since the onset of the poisoning was seen in the persons who took the dishes which had been left under such conditions for more than 10 hours, it is presumed that the spores of Cl. welchii germinated and multiplied in the contaminated fish meat, and thus caused the illness.
Although epidemiological investigation was carried out, the process of contamination in the mackerel, from which the Cl. welchii was isolated, is still unknown.

View full abstract

Malachitegreen-fuchsin staining was demonstrated on Gram-negative bacteria, E. coli, a representative of this bacterial group, and quite similar results were obtained in the differential staining as well as in the correlation between the viability and the differential stainability just as in Mycobacteria. Accordingly, it was assumed that living cells stain green, while pink stained cells were the dead.
Pretreatment with tannic acid, in order to obtain differential staining of the cells, is very interesing in suggesting the differences of the cell wall structures among various bacterial groups.

View full abstract

Neocidin, a new, basic and water-soluble antibiotic extracted from bacterial cells of a number of the Bacillus subtilis-group has been demonstrated to exhibit in vitro and in vivo antituberculous action against Mycobacterium tuberculosis var, hominis (H37Rv) and Mycobacterium avium (Jucho). The antibiotic has been effective against experimental tuberculosis in mice infected with Mycobacterium tuberculosis var. hominis (H37Rv). The activity of this antibiotic was reduced by the presence of egg and agar in the medium. It was also demonstrated that there is no significant cross resistance of Mycobacterium avium (Jucho) to neocidin and other known antituber-culous agents, and that the appearance of neocidin resistance in Mycobacterium avium (Jucho) occurs in a step-wise manner.

View full abstract

Proteus vulgaris cultured in peptone solution showed growth rates which were different depending on the time it was inoculated. Culture inoculated in the morning achieved almost constantly better growth than that inoculated in the evening. In contrast to the growth, the production of NH₃-dissociable substance was lower in the group inoculated in the morning than that in the evening.
Both the rate of growth and the production of NH₃-dissociable substance of the organism underwent considerable change from day to day. The change was gradual towards one direction, probably a seasonal change, with definte fluctuations whose periods appeared mostly to be of a few days. Thus, the quantity or/and the quality of the agent concerned in this phenomenon seemed to change not only with night and day but also day after day, presumably changing with the season.

View full abstract

UV-treated temperate phage β⁺ can be reactivated by the host cell KA. The mechanism of reactivation was divided into two classes in which one is caused by the undamaged cell, while the other only by UV-treated cell.
Among the progeny of reactivated phage appearance of plaque type mutants was demonstrated.
On the basis of some reasons already discussed, it was considered that these reactivations were the results of recombination due to partial genetic homology to be found in the phage and its host bacterium.
In the temperate phage and its virulent mutant employed in the present study no difference in their host dependency was evident.
In addition, the evidence that the prophage and vegetative phage have a common genome was far more strengthened.
The author wishes to express his appreciation to Prof. Y. Kawakita for his kind advice and encouragement.

View full abstract

Test tubes containing peptone solution in which Proteus vulgaris was inoculated were covered with various materials, such as water, lead, and glass, and were left in an incubator for one or two days. It was found that the growth of the bacteria was strikingly affected by the materials. The effect was observed not only when the bacterial cultures were covered by a material but also when they were located in its close proximity.
The effect appeared to be characteristic to the material, but it was found that there were factors which determined the effect more than the material. The first of such factors was the location of the material, and the second was the time at which the observation was made.
It is believed that the effect of materials upon the growth of bacteria is produced by the X-agent. The agent may be altered in some way after coming across a material, thereby a secondary agent being yielded. This secondary agent may mostly affect the bacteria placed near a material.
The effect was demonstrated in all directions around a material, but it differed with the direction.

View full abstract

Coagulase-positive staphylococci obtained from 358 consecutive patients with staphylococcal lesions during 2 years ended April 1959 were phage-typed. Also, staphylococci were isolated from carriers among 55 hospital staffs, 116 trainee nurses, 92 babies and their mothers, and 40 children in an orphanage where an outbreak of boils was in progress.
(1) There was a striking difference between staphylococcal lesions acquired in and outside the hospital. In the majority of instances, group I or type 80 strains were responsible for the hospital infections and group II or type 55/71 and others for the non-hospital cases. In the second year of survey, group I, particularly type 80 strains, have increased markedly and disseminated widely into the general public, while group II strains have decreased, showing a tendency of gradual elimination.
(2) A definite difference of phage-group distribution found among lesion-strains was also found among carrier-strains. Discrepancies in phage-group distribution between lesion-strains and carrier-strains in both hospital and non-hospital groups might suggest differences in pathogenic ability of strains, showing groups I and II strains to be more virulent and group III and the non-typable to be feebly pathogenic.
(3) The highest rate of antibiotic resistance was found in group III, nearly in group I, and the lowest in group II. The rate was higher among hospital strains than among the non-hospital. The rate of isolation of penicillin-resistant strains was fairly high in both lesions and carriers, but the streptomycin-resistants were of rare occurrence among non-hospital carriers-strains at large although they comprised 89% of type 80 strains from hospital lesions. During the second year of survey, antibioticresistant strains have remarkably increased in general, but rather decreased among hospital lesion-strains. Considering that hospital infections have recently increased in both frequency and severity in spite of this finding, resistance to antibiotics may not be taken as a prerequisite for pathogenicity of the strain.
(4) A serial survey of carriers in 2 groups of trainee nurses has shown that during the first 3 months upon entering the wards, group I strains gained an ascendancy over group II, leading to the phage-group distribution typical of hospitalstrains. This changeover of predominant types did not take place during the 2 months of preliminary training, showing that cross infection did not take place in the dormitory. There was some evidence that new flora colonized a sterile site and the carriage of one type protected against the acquisition of another. The nasal and throat mucosa of infants seem to be more susceptible to colonization by staphylococci, which are prevalent in the maternity unit, soon after birth than their mothers.
(5) Although the potentialities of Staphylococcus species to produce any kind of pyogenic conditions are indisputable, phage-typing results appear to show that some differences exist between strains in their ability to produce a particular lesion or lesions. Type 55/71, 52/52A/80, and 80/54/73 showed a higher incidence of skin lesions than the average. A larger number of skin lesions due to type 80, however, may be attributable to the prevalence of strains and not to its unusual propensity, because the incidence of skin lesions due to type 80 strains was found to be below the average. On the other hand, type 80, 80/73, and 55/71 were shown to be more liable to produce breast abscess. It seems that strains from osteomyelitis belong mostly to group II, whereas those from myositis mostly to group I.

View full abstract

1) Throughly homogenized leprous nodules were treated with trypsin. The digested material was dialyzed and then centrifuged at 10, 000 r.p.m.. The precipi-tate obtained consisted of leprosy bacilli in almost a pure state after being separate from the tissue components.
2) The leprosy bacilli (3 mg) thus obtained and incorporated with Freund's adjuvant were injected into guinea pigs.
Sensitivity to lepromin turned positive as early as 3 weeks after immunization. This sensitivity lasted for at least 24 hours. In addition, these animals responded more strongly to lepromin of 20 mcg per 0.1 ml than to a 1: 100 dilution of OT.
3) There was a slight difference in lepromin sensitivity between guinea pigs inoculated with 3 mg of leprosy bacilli than those which received 15 mg.
4) Guinea pig injected with leprosy bacilli (3 mg) in combination with 2 mg of the pure wax of tubercle bacillus responded to both tuberculin and lepromin with larger size of skin reaction than those immunized with leprosy bacilli alone. This was particularly true with the former antigen. Consequently, no difference was observed in the reaction size between these two kinds of skin reaction.
5) Skin reactions to both lepromin and tuberculin at 18 weeks after immuniza-tion reached their maximum intensity in 10 to 24 hours after the injection of the antigen. The affected area then gradually decreased until the reaction lesion faded away completely after 7 days.

View full abstract

1. O(H) substance of bacteria. Anti-O(H) antibody in anti-S. Poona immune chicken serum was inhibited in its reaction by L-fucose. This indicates that blood group specific activity of O(H) substance in S. Poona, would be determined by L-fucose. However, anti-O(H) antibody in anti-Sh. dysenteriae immune chicken serum was principally inhibited by D-galactose and raffinose. Therefore, the O(H) substance in S. Poona and that in Sh. dysenteriae would be somewhat different in structure, and the former would be in higher stage of development.
2. FA and A substances in bacteria. The reaction of anti-A antibody, which was obtained by immunizing rabbits with S. paratyphi B 8006, containing FA sub-stance, or with S. riogrande or E. freundii B90, both of which contain A substance, was inhibited strongly by N-acetyl-D-galactosamine, and weakly by N-acetyl-D-glucosamine. This indicates that FA and A subsances in bacteria and human beings similarly would have specific activities of their blood groups determined by N-acetylhexosamine, especially by N-acetylgalactosamine.
3. B substance in bacteria. Anti-B antibody in anti-E. coli O86 immune chicken serum was principally inhibited in its reaction by D-galactose and raffinose. This indicates that B substance, both in bacteria and human beings, similarly would have its blood group specific activity determined by D-galactose.
4. C(A+B) substance in bacteria. Specific activity of the C(A+B) substance found in A substance is determined by N-acetylgalactosamine. Specific activity of the C substance found in B substance can be determined by D-galactose. However, anti-B-containing anti-C agglutinin in anti-Sh. dysenteriae R form immune chicken serum was inhibited by both sugars. Therefore the C substance in this case would be found in a stage preceding the development into A and B substances.
5. Somatic antigens 5 and 1 in bacteria. The specific activity of somatic antigen 5, which is related to FA substance, is determined by N-acetylgalactosamine. The specific activity of somatic antigen 1, which was reported by Iseki and Kashiwagi(17) to induce phage conversion, is determined by sugars containing glucose.

View full abstract

1. In Shigella dysenteriae, containing O(H) substance of lower structure, glucose, rhamnose, and hexosamine, besides galactose, which is considered to be related to heterophile human blood antigen in this strain, were demonstrated. Salmonella poo-na, containing O(H) substance, demonstrated the possession of galactose, glucose, and hexosamine besides fucose, which is related to O(H) substance. The presence of N-acetylhexosamine was presumed in both of them.
2. In Salmonella paratyphi B, containing FA substance, not only the presence of N-acetylhexosamine, which is related to blood group A specificity, was presumed, but also the presence of galactose, glucose, mannose, rhamnose, abequose, and hex-osamine was demostrated. And in Salmonella riogrande and Escherichia freundii B90, galactose, glucose, mannose, and hexosamine were demonstrated, and the presence of N-acetylhexosamine, which is related to blood group A specificity, was presumed.
3. In Escherichia coli O86, containing B substance, fucose, glucose, and hexosa-mine, in addition to galactose, which is related to blood group B specificity, were demonstrated. Also the presence of N-acetylhexosamine was presumed.
4. C (A+B) substance has been demonstrated especially in the rough variants of the above strains. N-acetylhexosamine, which is related to blood group C and A specificities, and galactose, which is related to blood group C and B specificities, could be demonstrated in all the strains. It is considered that a combination of these two with other sugars would be different from that in O(H) substance, and that this would play a part in the manifestation of group C (A+B) activity.
5. In bacteria, the presence of sugar, related to blood group specificity, did not always indicate the manifestation of blood group specificity. This suggests that the manner in which the sugars are arranged as determinant groups governs the blood group specificity.

View full abstract

Malachitegreen-fuchsin staining was used on Staphylococcus aureus, a representative of Gram-positive bacteria, and quite similar results were observed as in Mycabac-teria and Gram-negative bacteria. A very closed relationship was shown between the differential stainability and the viability of bacterial cells. The differential stainability by the authors' procedure is completely independent of the Gram's method, and the hydrolysis of RNA-compound with ribonuclease did not affect even the stainability to malachitegreen as well as to the viability of the bacterial cells. The decrease in the stainability using Gram's method in the course of preservation was obviously slower than that to malachitegreen.
From the results and the results obtained on Mycobacteria and E. coli, it seems most likely that the authors' malachitegreen-fuchsin staining method presents bio-logical significance from the stand point of differential staining the living from the dead bacterial cells. Because the staining procedures are slightly different, depending on the bacterial species used, to achieve similar staining effects, may suggest the differences in their cell wall structures to some extent.

View full abstract

The local pathological changes occurring in our experimental animals when Habu-venom was injected are classified as follows: (1) extensive hemorrhage by intracutaneous injection and (2) myolysis with extensive hemorrhage by intramus-cular injection. Both of these changes take place immediately after the injection of small quantity of venom.
It must be noted that there is a marked difference in the degree between changes caused by the intracutaneous injection and those by intramuscular. Even when the doses are similar in quality and quantity, and the animals used are the same, yet the latter produces far more violent changes. In an actual Habu-bite case, it is clear that the symptoms are more serious when the patient is bitten deep into the muscle.
People in Amami Oshima-island and other South-Western islands are afflicted by Habu-bite; several hundred cases are reported every year. Immediately after the bite, there is severe pain with the appearance of swollen area. Because the progress of these pathological changes is so quick and the additional first aid is so primitive, it is almost impossible the snake bite victims to receive adequate treat-ment. Ignorant to the character of the bite together with improper disinfection, the victims often invite inevitable reinfection, and this leave the patients maimed after the healing of the wounds.
A case in Nase City. A victim of a snake bite made a several hours trip across the mountain and sea in order to receive treatment at Nase-city, the only place where assistance from a surgical specialist could be obtained. His operation findings and histopathological examination showed serious myolysis and extensive hemorrhage (Fig. 7). His upper limb on the wounded side had already swollen. These findings agree quite well with the pathological changes caused by intramus-cular injection of animals in our experiment.
The quantity of the venom used in our experiment was within 10 mg dry weight. The maximum collectable amount of venom from living Habu-snake is 300 mg dry weight; one tenth of this amount equal to several times the maximum experimental dosage used.
Sawai and Makino(2, 3) reported on the neutralizing effect of anti-Habu serum against Habu venom. Neutralization test in vitro showed that in order to counteract myolysis effect of 300 mg Habu-venom, 160 ml of the serum was necessary. One or two 40 ml serum available in the Habu-prevailing districts is not sufficient. Moreover, by the time the patients can get to the village chief in whose house the serum is always kept, the symptoms have taken serious and quick progress. The counteracting power diminishes when the intramuscularly injected serum reaches the wound by the circulation of blood. Therefore, until now, the local effect of the serum therapy depended on what part of the body was bitten and how much of the venom has acted.
The parotid gland of the Habu is the organ which is toxic, and when its secre-tion is fully injected into small animals upon swallowing them, as we have seen above, the characteristic pathological change is myolysis in the muscle. These facts tell us that proteinase in the venom is the very cause of the pathological change.
As reported in other papers (4, 5, 6, 7), much proteinase is present in the Habu-venom. A dried and preserved material still possesses strong enzyme activity. A kind of proteinase, which is separeted and purified, also has strong myolysis action as well as venom's action (6, 8, 9) (Fig. 8).
In another report, we have stated that this enzyme consists of metallic Mn(5, 6, 7), which is chelated by EDTA (ethylenediamine tetraacetic acid). In chelating Mn, EDTA depresses enzyme action; it also depresses myolysis action of crude venom pathologically(6, 10, 11, 12).
We have suggested EDTA as a first aid to the Habu bite. As a stable and portable therapeutical medicine, we believe that its early application would be of great help.

View full abstract

It has been confirmed by the reported experiment that 7H9 Tween- or Oleic Acid-Albumin medium permits early growth from small inocula of tubercle bacilli.It is shown that this type medium is the most preferable medium as evidenced by a 13-13.5 hour generation time (Table 8). When actively growing cultures of tubercle bacilli were transferred in dispersed fashion in this medium and incubated in a stationary manner the culture was characterized by arithmetic linear growth, independent of the age of the seed culture, or amount of inoculum. It was ob-served that if 7H9 cultures of mammalian tubercle bacilli were aerated by con-tinuous rotation aeration the growth is much greater than is observed in stationary incubated cultures. Yields of bacilli were increased many fold over those obtained in stationary cultures (two-fold greater at 4 days, three to four-fold at 7 days). It was found that with rotation aerated cultures, the average values of generation time calculated from experiments using four different inocula (Table 3) was con-siderably shorter than that of stationary cultures.
In the reported studies, the generation time of mammalian tubercle bacilli cal-culated by the formula of Monod(6), using logarithmic plotting with small inocula, is approximately 20.0 to 24.2 hours during exponential growth of early stage (Tables 4 and 5). Under these conditions of lag phase growth, at least, the stationary culture and rotation aerated culture are approximately identical. When the Values of growth over a long incubation period were taken (Tables 4 and 5), the genera-tion time calculated from rotation aerated cultures was quite short as compared with that obtained from stationary incubation over the same period.
It was shown that the period of logarithmic growth varies and is dependent upon the available supply of oxygen: a good oxygen supply permits continuousrapid growth of M. tuberculosis strains. A 7H-9 Tween Albumin culture of tubercle bacilli (H37Rv-strain) inoculated in a shallow layer of medium such as 30 ml volume medium contained in a 250ml Erlen-Meyer flask and agitated gently by hand once each day, gave a generation time of approximately 17.2 hours during early stage of growth (24-64 hours). This was somewhat shorter than that observed in station-ary tube cultures or in rotation aerated cultures: with stationary cultures it was 22.2 hours and with rotation aerated cultures it was 20.0 hours (Table 6). In the present experiments described the effect on the growth rate, when cultures of tubercle bacilli were rotation aerated, indicated that significant differences in growth rate enhancement was greatest with M. tuberculosis var. bovis (Ravenel-strain), decreasing in the order of M. tuberculosis virulent H37Rv-strain and then H37Ra-avirulent strain (Table 10). The difference in degree of stimulation by aeration on the growth rate of these strains appears to be explainable by a difference in the rate of meta-bolic utilization of oxygen in the Tween Albumin medium employed. In this study, investigations have been devoted to the determination of the optical concentrations of glucose and glycerol employed alone, and in various combinations in aerated cultures. The addition of glycerol, as low as 0.2 per cent final concentration, showed a marked initial stimulatory effect on the growth of the M. tuberculosis strains tested. Higher concentrations of glucose and glycerol (0.5 per cent) are inhibitory in stationary cultures, stimulatory for aerated cultures. It is apparent that considerable increases in oxygen uptake depend on the concentration of carbon sources.
Better growth in the initial stage of growth was obtained in cultures with glucose alone than with the same concentration glycerol alone. It was clearly shown that the oxidative breakdown of glucose in the initial stages of growth was a far more efficient means of obtaining energy than when glycerol was used.

View full abstract