Dcparozcni of .o\icrobiology, lfo(tYn Stotic ULnivcr.sitY School of' .1Icdicice, D)ctroit, Mlicligan 48207 Recci\ed clor publication 4 I-ebruar_\ 1967 Pyocin, a bacteriocin obtained from l1sates of ultraviolet-induced cultures of Pseuldcolionaces aceruiiginosa was clhLrLacterized in vitro and in vivo aLfter I,000-fold purification by chemical, column, and differential centrifugation procedures. Electron micrographs of negatively stained pyocin p)repalrations contaLined rod-shaped particles whichi resembled the contrcactile tcalil protein of the T-even phages of Escherichia coli. Although two septLrate anid distinct pyocin fractions were eluted from diethylamninoethyl cellulose (p1H 7.5) during the puriticatioll procedure, the pLar

The bacteriocin from Pseudomonas aeruginosa, pyocin, consists of a contractile sheath and inner core reminiscent of T-even coliphage tails. Contraction of the outer sheath was found to be promoted by 0.5 M magnesium chloride, 1 % Formalin, low pH, sonic treatment, and freezing or thawing or both. The contraction caused by 0.5 M magnesium chloride, however, was found to be reversible and occurred upon reduction of the salt concentration from 0.5 to 0.02 M. In addition, direct assay showed that pyocin activity was nearly proportional to the percentage of only uncontracted forms. Initial studies suggested that the adsorption of purified pyocin onto cell wall fragments from the sensitive indicator strain of P. aeruginosa occurs with the relaxed particle only and not with the contracted form. However, after adsorption, contraction occurred. Various morphological structures, such as tail fibers and base-platelike appendages, were also observed. Upon contraction, six tail fibers were observed on many particles, four of which appeared to originate from the sheath and two from the inner core. Polysheaths and polycores several hundred nanometers in length were also occasionally observed.

The mission of this study was to determine whether or not arteriovenous connections, indicative of a "closed" type of circulation, existed in the human spleen. Spleens from four patients requiring therapeutic splenectomy were the basis for this report. Scanning electron microscopy of plastic corrosion casts, prepared from these four spleens, revealed direct vascular conduits between splenic pulp arteries or arterial capillaries and the venous sinuses in the red pulp. Also demonstrated were a few arteriovenous shunts between pulp arteries or arterial capillaries and pulp or trabecular veins. Inclusion of sized microspheres in low-viscosity perfusion plastic illustrated that some diameters of the connecting shunts were 7-10 mum, with other shunts even smaller. Not only do arteriovenous connections exist in human spleens, but their frequency, as revealed by methods accentuating three-dimensional aspects of the splenic microcirculation, justify future reconsiderations of the functional significance of this closed type of circulation. Examination of samples of the same intact spleens, prepared by freeze-fracture and conventional critical-point drying, also revealed an "open" type circulation structure, namely, pore-patterned sinus walls that could facilitate blood cell movement from pulp cords into venous sinuses. Scanning electron microscopy thus has provided direct evidence that human spleens have both "open" and "closed" circulatory pathways in their microvasculature.

The ultrastructure of cell surface and tissue organization of reproductive tracts of female rabbits were observed by scanning electron microscopy. In the vagina, straight and shallow longitudinal folds were observed. Complex, very deep, narrow folds with small crypts were observed in the cervix uteri. Two types of cells were recognized : ciliated cells and secretory non-ciliated cells. The internal 0s area contained more ciliated cells than the external 0s area. In the uterus, two different patterns of fold formations were observed: shallow fold formation in a random direction or mosaic pattern in the lower part of the uterus, and wave-like folds in the middle or upper part of the uterus. The lower part of the uterus contained more ciliated cells than the mid and upper part of the uterus. At the uterotubal junction, four large folds and four small folds from the isthmus are projected into the uterine lumen forming a rosette-like structure.In the oviduct, longitudinal fold formations were observed through the isthmus to ampullae. The number of ciliated cells gradually increased from the isthmus to the ampullae. The fimbriae, made of several mucosal folds arranged like flower petals, were composed of a high percentage of ciliated cells.

A permanently implantable in-series left ventricular assist device, the dynamic aortic patch (DAP), has been tested in chronic animal experiments. The DAP replaces a section of the intrathoracic aortic wall. Hemothorax and hematocele at the implantation site have been complications in recent experiments. Primary postoperative hemorrhage was ruled out, and the biocompatibility of all components was therefore examined. Dacron velour, Teflon felt, conductive polyurethane, segmented polyether polyurethane, and Teflon-coated polyester fiber sutures were implanted in the pleural cavities of dogs and tested in vitro by culturing canine saphenous vein explants on them. In vivo experiments demonstrated that all components elicited mild to moderate inflammatory reactions, but hematocele occurred only when the components were implanted in the aorta with direct blood contact and exposed to arterial blood pressures. In vitro, cells were cultured on all components with no signs of toxic reactions. These results indicated that the host tolerated all implant components without major inflammatory responses. However, histological data indicated that chronic slow bleeding into or through the Dacron velour in contact with the arterial blood serum could account for hemothorax or hematocele formation. Therefore, a configuration of the assist device using materials impermeable to blood may obviate these difficulties.

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Preparative techniques were developed for t,he purpose of visualizing, by negative contrast, some of the cytological characteristics of unsectioned cells of Pseudontonas ueruginosa.Concent,rated fimbriae free from cells had a length of approximately 1.5 pm and a diameter of 6 nm. Pyocin-like stroct'ures ranging in lengt,li from 32-64 11111 with a width of S.6 nm were "attached" to the terminal ends of a sniall percentage of t,hese finibriae. Microtubules and ringlets having a diameter of 10-20 nm were also visualized in the bacteria. Budding or evagination of cell wall material in multiple units up to 135 nm in length was frequently obscrved in older cultures grown on agar media.Pseudomonas aeruginosu is a graiii negative, motile, polar flagellated bacterium of increasing medical importance. Previous cytological studies have indicated that this organisin contains microtubule-like structures termed rhapidosorries ( YAMAMOTO 1907, BAECIILER et al. 1972) and a number of different types of nieinbranous inclusions, some of which are suggestive of iiiesosoiiies (CARRICK and BERK 1971). I n addition, several reports of one or more types of finihriae have been described for this bacteria (FUERST and HAYWARD , WEISS 1971, BRADLEY 1972.Preparative technicpes developed for the purpose of visualizing internal structures such as rhapidosonies inside unsectioned cells by negative contrast (BAECHLER and BERK 1972) have fortuitously exposed other cytological features such as discrete ringlets or spherical units, evaginations, and specific extrusions associated with autolysis. Consequently, the purpose of this manuscript is t o present sonic cytological features of P. aeruginosa which have not been previously described. Material and methodsOiganism: The strain of Pseudomonas aeruginosa employed for this study was originally a hospit,al isolate maintained in the culture collect.ion of the Department of Microbiology, Wayne State university, School of Medicine. This strain was auto-plaque positive and was designated C,AP+ by BERK (1966).Cnlture medium: Agar slants and plates contained 1.50,.6 (w/v) Bacto agar (DIFCO). The broth media consisted of 2% tryptone, 1% glucose, and 0.504 sodium chloride. The cultures were incubated a t 26 "C or 37 "C for various time periods. Prior to electron microscopy studies, agar slant cultures, incubated more than 72 h., were lightly washed with 0.02 M tris (hydroxymethyl) aminomethane (Tris-HC!) buffer, p H 7.8, containing 0.02 M MgCI, t'o selectively remove the older, autolyzed surface cells.Electron microscopy: Agar blocks (1.5%) of 1.5 cm2x0.5 cm were placed on the end of glass slides. A droplet of the specimen was placed on the surface of the agar and the majority of t'he water and salts was absorbed into i t leaving the specimen in a thin film of moisture 19*