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Chlamydial inclusions were demonstrated by indirect immunofluorescence (IF) with antiserum to the chlamydial group antigen when McCoy cell monolayers infected with either Chlamydia trachomatis or Chlamydia psittaci were fixed in formaldehyde or paraformaldehyde, provided the monolayer was not allowed to dry. If these monolayers were then air dried and restained by IF with the same antiserum but with a different fluorescence conjugate, group antigen associated with inclusion-containing McCoy cells but independent of the inclusions was revealed. This antigen was not restricted to infected cells but appeared to radiate out from them, suggesting that group antigen was released from infected cells. Similar host cell-associated antigen could be shown by IF of glutaraldehyde-fixed, air-dried monolayers, but inclusions could not be stained by IF before these preparations were dried, presumably because antibody could not penetrate glutaraldehyde-fixed cells. Electron microscopic immunoperoxidase studies of paraformaldehyde-fixed, wet monolayers located group antigen within inclusions on the outer membrane of chlamydial organisms and on single-membrane vesicles. However, when dried monolayers were labeled with the same immunoperoxidase technique, no intracellular labeling occurred, but dense staining was seen at the surface of infected cells and on adjacent membranous material. These observations are compatible with the postulate that replicating chlamydiae produce outer membrane blebs containing group antigen, which are excreted by the host cell during the chlamydial developmental cycle. Chlamydiae are obligate intracellular procaryotic parasites. They possess a double-unit membrane cell envelope similar to that of gramnegative bacteria (4), and they multiply within eucaryotic cytoplasm in phagosomes known as inclusions. These inclusions can be demonstrated in infected eucaryotic cells fixed in methanol or acetone by immunofluorescence (IF) with antiserum to the group antigen (10). This is a lipopolysaccharide antigen common to all members of the genus (7, 8) and is probably located in the outer membrane (OM) of both infectious chlamydial particles (elementary bodies) and replicating forms (reticulate bodies) (6). Recent IF studies of glutaraldehyde (GTA)fixed, air-dried McCoy cells revealed group antigen independent of inclusions that is associated with infected host cell cytoplasm and appears to be released from infected cells (9). It has been suggested that OM blebs which contain group antigen are produced during chlamydial repli

Activity patterns of limpets were studied at two adjacent sites in an Irish sea lough, Lough Hyne, in order to relate timing of activity to physical and biological influences. Activity was suppressed during heavy rainfall, and osmotic stress from dilution appears to have led to increased mortality in transplanted limpets. Activity increased as tides progressed from neaps to springs, and for limpets low on the shore it was enhanced by wave action. It is suggested that lack of activity in calm water may reduce predation pressure from crabs, which caused high mortality in transplanted limpets. Either low relative humidity or dryness of the rock diminished activity of low-water limpets. Differences in feeding activity between low-water limpets and high-water limpets may relate to food supply, since more food was available low on the shore, and the guts of low-water limpets contained more diatoms than those of high-water individuals. Differences between the two sites are at present unexplained but may relate to differences in micro habitats.

The growth of a genital trachoma-inclusion conjunctivitis agent strain of Chlamydia trachomatis in McCoy cells treated with cytochalasin B was studied by quantitative infectivity estimations and by light and electron microscopy. Provided that infection of the monolayer was initiated by centrifuging the infectious particles on to the cells before incubation, this chlamydial strain grew as fast and to as high a titre [approximately 107 inclusion-forming units (i.f.u.) per culture] as those chlamydiae which infect cell cultures ipl vitro without centrifugation. Each i.f.u. inoculated yielded approximately 600 i.f.u., and extracellular infectivity was detected soon after intracellular infectivity appeared. Inclusions were recognized by fluorescent antibody staining techniques early in the developmental cycle when cultures were not infectious and when only reticulate bodies were seen by electron microscopy. Inclusions were recognized in Giemsa-stained preparations examined by dark ground microscopy only when elementary bodies appeared in the inclusions. Iodine staining was not a reliable indicator either of the number of inclusions present or of their infectivity.