The structure of the outer layers (cell wall and membrane) of Staphylococcus aureus was studied by electron microscope using a bacteriolytic enzyme from Flavobacterium sp. called the L-11 enzyme. Comparative studies on the morphology of bacteria before and after treatment with this enzyme and cell wall and membrane fractions obtained from bacteria after the enzyme treatment led to the following conclusions. (1) The cell wall of S. aureus is composed of morphologically distinct two layers which are both susceptible to the L-11 enzyme. (2) Between the cell wall and membrane, there is an electron opaque region which could not be stained using any of the methods tested. (3) Before treatment of bacteria with the enzyme the cell membrane could not be seen clcarly. However, after enzyme treatment the membrane was clearly seen. (4) The infolding of the inner layer of the cell wall, forming a structure like a mesosome, was liberated by extensive enzyme treatment.

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Characteristics of phosphatases of Vibrio parahaemolyticus A55 K⁺ 105 were investigated. Phosphatases wete extracted from intact cells in a 3% NaCl solution by gentle treatment on a reciprocal shaker for 15min. The pH dependence of phosphatase of the extracts showed two optima; one optimum was found at pH5.1-5.7, the other at pH10.5-11.1, and the results were identical with that of resting cells. The activities of both acid and alkaline phosphatases were maximum in the presence of 0.1M salt (KCl or NaCl). The production of alkaline phosphatase of resting cells in the induction metlium containing p-nitrophenylphosphate was largely supplessed by the addition of 0.5% phosphate into the medium. The alkaline phosphatase of the extracts was stimulated about 2.5-fold by the addition of 10-3M Mg⁺⁺ or Zn⁺⁺, whereas it was inhibited completely by 10-3M ethylenediaminetetaacetic acid. On the other hand, Co++ activated acid phosphatase to 98-fold. Both acid and alkaline phosphatases were found in the culture fiftrated after cultivation of the organism in a 3% NaCl-nutrient broth medium for 16hr.

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Spheroplasts of Candida albicans could be obtained by treating cells with a snail enzyme in the presence of 0.75M MgSO₄ as a stabilizer and cells could subsequently be regenerated by the thin-layer-agar plating method. The initial regeneration process was affected by stabilizers such as MgSO₈ MgCl₂, MnSO₄, CaCl₂, KCl, NaCl and sucrose. It was found that 0.68M MgSO₄ was the best stabilizer. Good stabilization was also achieved in any medium with 0.5-0.75M sucrose, although stabilizing agents were in general most effecive at concentrations giving about 22 atmospheres of osmolarity. This osmolarity was lower than that used to prepare spheroplasts. Using this thin-layer plating method at least three factors were essential for resynthesis of a wall layer: (1) a carbon source, (2) a stabilizer and (3) several minerals. Nitrogen in the form of an amino acid stimulated formation of the wall layer. Relationship between these factors and osmolarity of the medium during regeneration of the wall layer is discussed.

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A large epizootic of an actite respiratory disease of cattle occurred in Japan during the months from October 1968 to May 1969. A virus was recovered in primary cultures of calf kidney and testicle cells from nasal swabs of affected cattle. Neutralization tests revealed the virus to be closely related to the Long strain of human respirator) syncytial virus. The virus induced cytopathic changes including the formation of syncytia and acidophilic-cytoplasmic inclusions in calf kidney and testicle cell cultures. A calf inoculated with the virus by the respiratory route developed an illness resembling the natural disease. Most cattle clinically diagnosed as having the disease showed significant rises of neutralizing antibody titer for the isolated virus, whereas none or only small fractions of those animals showed serological evidence for recent infection with bovine ephemeral fever virus, infectious bovine rhinotracheitis virus, Ibaraki virus, bovine diarrhea virus, bovine adenovirus Type 7 and parainfluenza virus Type 3. Neutralization tests on paired sera revealed a wide dissemination of the isolated virus among cattle in many areas of the country during the epizootic All these findings leave no doubt that the epizootic was caused by bovine respiratory syncytial virus. This is the first study that ever shows the presence of infection of cattle with this virus in Japan.

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Twenty-three temperate phages of Lactobacillus salivarius isolated from human feces were studied as to their morphological, biological, and serological properties. (1) Among 30 strains of L. salivarius tested, 23 strains were lvscd by induction with mitomycin C (MC). In all these lysates, phage particles were detected by electron mioroscopic cxamination. (2) These phages were morphologically divided into three groups: particles with a regular hexagonal tead and a long flexible tail; particles having a regular hexagonal head with or without a short tail-like structure: particles with an elongated head and a long noncontractile tail. (3) Only two, phage 223 having an elongated head and phage 227 with a regular hexagonal head and a long noncontractile tail, produced tiny and very turbid plaques on several host bacteria_Six phages could produce only inhibition zones, rangnig from complete inhibition through partial inhibition to normal growth by a serial dilution spot test. (4) All these killer paiticles could also inhibit the growth of their producer cells. (5) .A serologieal relationship was observed between temperate phages and killer particles, and this was somewhat consistent with the morphological groupings.

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Rabbits pretreated by intrasenous injection with Newcastle diease virus (NDV) produced high-titered virus-inhibiting factor (IF) or interferon in the serum upon intravenous injection of Escherichia coli endotoxin 24hr later as compared with the IF production induced by endotoxin in rabbits without the NDV pretreatment. IF assays of different organs revealed that the NDV pretreatment rendered the spleen rather hyporeactive but other organs such as the liver, lungs and kidneys hyperreactive to the subsequent IF induction by endotoxin. The enhanced IF titer observed in the serum seems to be the summation of IF released from various organs rendered hypozeactive or hyperreactve by the NDV pretreatment. It is postulated that the late-appearing, heat-and acid-stable IF and the early-appearing, heat-and acid-labile IF are produced by different processes in different types of cells. The production of early IF consists of two steps, formation of "preinterferon" and conversion of pteintetferon to early IF. Pre interferon formation is induced by virus but not by endotoxin, while the conversion of preinterferon to early IF is effected by both endotoxin and virus. The formation of late IF is induced only by virus, taking a one-step process. This hypothesis seems to explain the linding; in the present shtdy.

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Electron-microscopic and biochemical studies on motphological changes in Staplrylococcus aureus following exposure to protein synthesis inhibitory agents such as lineomycin (LCM), clindamycin (CLM), eiythromycin (EM), and spiramycin (SP) are presented in this paper. It was demonstrated that bacterial cell walls beeanne extremly thickened usually with the formation of multilayers, when exposed to eaeh of the ahove-mentioned antibiotics. Furthermorc. electron density of the cytoplasm was higher in thosc cells exposed to drugs than in intact control cells. Incorporations of 41C-labeled L lysine into the cell-wall fraction and the protein fraction were measured for biochemical elucidation of these phenomena. Labeled lysine was selectively incorporated into the cell-wall fraetion when the test organism was exposed to the respective antibiotics. Uptake at 15min after exposure was about twice as large as that of intact control cells. SP and CLM inhibited protein synthesis while they stimulated cell-wall synthcsis. The evidences for thickening of, and formation of multilaycrs in the bacterial cell walls following exposure to drugs were elosely related to the slimulating action of these antibiotics on the cell-wall synthesizing system. Morphology of resistant clinical isolates following such antibiotic exposure was also investigated using two staphyloeoccal strains, one resistant to EM alone and the other completcly cross-resistant to all the macrolides.

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Delaved hypersensitivity against hamster crythrocyte antigen was examined after sensitization with hamster erythrocytes (HRBC) in Freund's complete adjuvant (FCA). Extent of the delayed hypersensitivity was determined by the migration inhibition test, the peritoneal macrophage disappearance test and the skin test in the car using solubilized HRBC as the test antigen. 1) Delayed hypersensitivity against HRBC deyeloped earlier in high-responder SL mice than in low-responder C57BL/6 mice after sensitization. The period required for development of the delayed hypersensitivity in AKR mice was intermediate between periods in high-responder SL mice and low-responder C57BL/6 mice. 2) After sensitization with HRBC in FCA, a delayed hypersensitive state without detectahle antibody production persisted until day 12 in highresponder SI. mice and until day 16 or later in low-responder C57BL/6 mice. 3) Delayed hypersensitivity against HRBC antigen persisted even after the appearance of circulating antibody which occurred late after sensitization with HRBC in FCA or after intravenous injection of HRBC into sensitized mice.

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Antibacterial activty of aminoglycosides toward Pseudomonas aernginosa was investigated by using 221 clinical isolates. One hundred and seventy-five strains were shown to be highly resistant against one or more of the antibiotics tested. Aminoglycoside resistance was found to be lost in some strains of pseudomonads, suggesting that the determinants would be located extrachromosomaliy. Loss of resistance in some strains was paralleled with the loss of an ability to inactivate the drug.

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