The mechanism of neutralization of T1 bacteriophage by the anti-T1 rabbit serum was investigated as to the polyvalency of the virus particle. T1 can develop nearly equal number of plaques on two Shigella, BIII and Shiga strains as well as on Escherichia coli, strain B, but on Shigella KA strain the efficiency of plating was about 0.2.
The rate and final survivors of T1 inactivation by the specific antiserum depend markedly upon the bacterium used for assay. However, the choice of host used in growing the phage stock has no effect. The T1 phage inactivation by the specific antiserum was partially reversible, i.e., a slight reactivation of the neutralized T1 phage occurred on dilution by broth media. The degree of the recovering was estimated to be about 10 to 20 per cent when it was assayed with B or BIII cells, but less than 1 per cent with Shiga cell.
From these findings we concluded that T1 particle has a rather complex antigenic structure in its infectious site, and that the pattern required for invasion to the different host cells may be different each other.

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Some new host-range mutants of T4 bacteriophage were isolated and their serological properties and mutation patterns were described.
Four new host-range mutant strains were isolated from an experiment in which a large amount of T4 wild phages had been inoculated with each of several Shigella strains that are all resistant to T4 wild type. These were designated as T4h_(BI), T4h₍₁₁₀₆₎, T4h_(BIII) and T4h_(KA) respectively, according to the name of Shigella strains on which they had been discovered. They were classified into two groups, A and B, from their host-range patterns.
In experiments of serological neutralization with these mutant phages, polyphasic neutralization such as reported in my previous papers with T1 and T2 phages was also observed, suggesting that these mutant phages have a rather complex antigenic structure in their infectious site too.
A discussion was attempted on the problem of the mechanism of phage infection based on these evidences.

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The kinetics of phage induction in artificial lysogenic strains of Shigella flexneri was investigated by means of X-ray irradiation.
The β phage induction in KA (βL) was found to be a kind of single-hit phenomenon, whose target size was estimated to be about 50mμ in diameter. The maximum induction rate was about 40 per cent.
Whereas, the target diameter responsible for colony inactivation was about 125mμ, indicating that this is different from the target responsible for phage induction.
The latent period of time and burst size of the induced KA (βL) bacteria by X-ray were about 60 minutes and about 100 independently of the X-ray doses from 5 to 90kilo roentgens.
The diameter of the sensitive area of the β phage particle (whose size in electron micrograph was about 40-50mμ in diameter) was estimated to be about 16mμ.

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In the past few years attention was focussed on heat resistant variant or mutant of polioviruses by several investigators (Melnick et al, Stanley et al, Dulbecco and Youngner). Along this line attempt was made to select heat resistant (t) mutant not only from a wild type such as MEF-1 t⁺ but from each parent type such as MEF-1 i t⁺ (a mutant resistant to inhibitors in normal bovine serum described by Takemori, Kitaoka et al) and MEF-1 i m t⁺ (a mutant giving small size plaque on HeLa cell-monolayer described by Takemori, Kitaoka et al). First the culture of a wild or a parental type especially type II poliovirus was heated at 50°C and variation in thermal resistance of individual infectious units was followed up by estimation of pfu/ml with exception of m-mutant stock which was titrated by cytopathic effect on HeLa cells in tubes, in 5, 10, 15, 20, 25, 30, 40, 50 and 60 minutes after exposure to the heat.
As a result surviving rate of wild type MEF-1 t⁺ fell down rapidly within 20 minutes after exposure to heat, that is, the heat sensitive virus was inactivated and approximately 0.3per cent of MEF-1 t⁺ was found to be heat resistant, phenotypically or genotypically, among a given virus population.
Herewith the selection of t-mutant was undertaken mainly according to Dulbecco's method, that is, by successive steps being propagation of survivors in subculture and heating at 50°C for 20 minutes one after another and if the surviving rates gave rise to 75per cent, two more subcultures were mabe through plaque passages or by the limiting dilution method to select possibly the thermostable clone.
The results so far obtained are as follows.
1) The t-mutant was selected at the 9th subculture of the wild type MEF-1 t⁺. Its surviving rate was more than 89per cent at 50°C for 20 minutes. It was found that progeny of different virus particles varied spontaneously in their rates of thermal inactivation.
2) The parental type MEF-1 i t⁺ was found to have 1.2 to 1.3 times much higher inactivation velosity constant than MEF-1 t⁺. The surviving rate of MEF-1 i t⁺ was 0.022per cent after heating at 50°C for 20 minutes. However, it increased and the incubation period became shorter giving cytopathic effect on HeLa cells at each generation one after another and the surviving rate of MEF-1 i t at the 4th and 5th generation was found to be more than 75per cent and the incubation period to give cytopathic effect became 3 days. It is worthy to note that progeny of MEF-1 i t retained its i-character in messing its plaque size on monolayer containing inhibitor in normal bovine serum and no differences was observed in i-character of the parent and mutant.
3) The difference between both TCID₅₀ of the parental type MEF-1 m t⁺ before and after heating at 50°C for 20 minutes was 10^3.0 TCID₅₀ by using HeLa cell tubes and the incubation period of the parental type giving cytopathic effect on HeLa was 6 days. However, the difference between both TCID₅₀ of 2nd subculture of servival was 10^1.0 before and after heating. The incubation period of cytopathic effect on HeLa cells was shortened by subcultures and it became 2 days at the 3rd subculture. Thus MEF-1 m t was obtained without difficulty.
4) Furthermore, the back mutant MEF-1 m^+t was selected from MEF-1 m t by serial passages in culture media containing bovine serum but its t-character was proven to be stable in spite of m-character back to m⁺.
5) The difference of TCID₅₀ of MEF-1 m i t⁺ before and after heating was 10^2.5 but no difference was found at the 9th subculture, that is, complete t-mutant was obtained. Such t-mutant was confirmed to retain m- and i-properties in messing plaque size on monolayer containing inhibitors in bovine serum.
6)

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Variola virus (Yamamoto strain) was shown to multiply in culture of HeLa cells, causing cytopathogenic effects. In succeeding passages, the effects appeared sooner and infectivity titer of intracellular virus increased. It was found that their occurred a drop in infectivity in the first few hours, followed by an increase in infectivity after 11 hours. The maximum yield of variola virus in a infected HeLa cell was about 20 infectious units per cell.
Intracytoplasmic inclusion bodies were observed. The inclusions were stained red with the Giemsa stain, blue or bluigh red with hematoxylin eosin stain. Electron microscopy of the infected cells revealed that they had so-called matrix type inclusions, which contained nothing associated with viral component in early stage of infection, but became contain several kinds of developmental form after 10-hour-infection. In the matrix a membrane-double membrane seemed progressively to enclose a pre-existing viroplasmic component.

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1. We have obtained various kinds of virulent mutant from temperate phage s₂ liberated from lysogenic strain, S. typhimurium 4066. Three strains of the virulent mutant of which virulence grade is variant and which can be clearly differentiated on the plaque were taken for study.
2. In order to examine the arrangement of four loci (tu, v₂, v_w, v_s) upon the chromosome of phage, a recombination test by single cell burst experiment among each phage has been carried out and the recombinant value has been calculated as below: tu×v_s: 8.95, tu×v_w: 8.32, V2×v_s: 1.35. Accordingly, linkage map of S. typhimurium 4066 phage which tu, v₂, v_w, v_s, were arranged upon one line at certain intervals was drawn.
3. From the result of above experiment, we studied a genetic observation on virulent mutation of temperate phage.

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