A prospective stud)! was carried out to determine the epidemiology and etiology of acute gastroenteritis on the general infant ward oj The Montreal Children's Hospital in the late fall of /976. Diarrhea occurred in 4/ of /65 infants (25%), with probable nosocomial acquisition in 26 patients. Two infants each had two episodes of diarrhea. and one had three. A putative pathogen was found In 3/ of 45 case episodes (69%). Virus-like particles were present in 28 o] 45 patients, and in 24 of 74 asymptomatic room contacts. Particles belonging to six morphologic classes were identified: adenovirus. rota virus, minirotavlrus, calicivirus, plcorna-parvovirus, and coronavirus. More than one agent was identified in /2 infants with diarrhea and in five asymptomatic room contacts. No wardwide etiologic pal/ern was evident, but minirotavirus or caltcivirus or both were associated with diarrhea in 10 patients, accompanied by vomiting in /5 of these infants. Moreover, spread oj individual agents was almost entirely limited to minirotavirus and calicivirus, with diarrhea in six of ten. and four of seven, virus positive room contacts. respectively. These viruses were also identified in stools from 12 infants without diarrhea, seven oJ whom had repeated vomiting. Data support the etiologic role oj mlnirotavirus and calicivirus in diarrhea or vomiting or both in hospitalized infants.
Improved methods for studying the growth of Mycoplasma hominis (ATCC 14027) have been developed, involving modified growth conditions and preparation of the organisms under minimally distorting conditions. Cells so prepared from batch cultures show relatively uniform exponential growth and appear to be dividing by binary fission; but pleomorphic forms appear upon further incubation. Similar behavior was demonstrated by another laboratory-adapted strain and by three clinical isolates, and therefore seems characteristic of the species. The pleomorphic populations contain small forms having diameters within the 100to 250-nm size range reported for "elementary bodies." Such forms were isolated from this strain ofM. hominis by sequential filtration using gravity alone, after cell aggregates were dispersed by Pronase treatment. Of the small bodies which traversed membranes of 220-nm pore size, a negligible number grew in liquid or on solid media, suggesting that these were not essential reproductive units in a life cycle, but involution forms due to growth in an altered environment.The bewildering diversity of evidence and opinion on mycoplasma reproduction (1,9,21) partly reflects fundamental and clearly demonstrable differences between species. However, much of the confusion could have arisen from imperfect understanding of their growth requirements and from overlooking their liability to manipulative distortion. We attempted to determine the true morphological changes during growth of a readily cultivable species, Mycoplasma hominis, when cells were produced and prepared for microscopy under optimal conditions. Studies undertaken to determine a minimally distorting technique for microscopy are outlined elsewhere (23).Experiments are recorded in which the reproductive pattern of M. hominis was established.Filtration has been applied to the isolation of cells under pressure or negative pressure (5,15,20). For this study we adopted filtration through membranes by gravity alone. Special attention was given to the smallest cells, spheres of 100 to 250 nm in diameter, commonly known as "elementary bodies," which are regarded generally as the minimum reproductive units.(This work is based on a thesis submitted by J. Robertson to the faculty of graduate studies, McGill University, in partial fulfilment of the Ph.D. degree requirements.) ' Present address:
A method is presented whereby cells of Mycoplasma hominis can be prepared with minimal distortion for electron microscopy. After the addition of glutaraldehyde to broth cultures, incubation is continued for 1 h. The cells are then collected by centrifugation, washed in distilled water, and used for negativecontrast preparations.Disagreement on the gross morphology of organisms included in the family Mycoplasmataceae may partly reflect the heterogeneity of its members, but contributing factors are imperfect understanding of their growth requirements and their liability to deformity during preparation for microscopy. Various physical and chemical conditions, including gravitational force, osmotic change, and dehydration, have been implicated as distorting agents (1-5, 7, 11). Despite such wide recognition of the plasticity of these cells, no procedure was established for preparing these "soft-skinned" organisms with minimal distortion for electron microscopy. As an essential preliminary to a morphological study of the growth of Mycoplasma hominis, cells of strain ATCC 14027 were exposed to different conditions during their collection and preparation by the negative-contrast technique. (This work is based on a thesis submitted by J. Robertson to the faculty of graduate studies, McGill University, in partial fullfillment of the Ph.D. degree requirements.)To ensure that the proportion of cells in the stock culture which were irreparably damaged by freezing and thawing would be negligible in the final population, the cultures were prepared as follows. Stock (1 ml) was added to 9 ml of broth and incubated overnight at 37 C; 1 ml of this culture was added to 9 ml of fresh broth and, when nephelometry readings indicated that exponential growth was well advanced, 0.5 ml was used to inoculate 49.5 ml of broth. This test culture was incubated until midway through the logarithmic phase. The broth me-T6G2H7.dium used for this study (pH 7.3; 384 mosmol/kg) had been developed to furnish optimal growth ofM. hominis through short generation time, high cell yield, and a short rate of decline (9). Silicone-coated glassware was used throughout to prevent cell adhesion to containers used for cell culture or collection.To examine the effect of pre-fixation on morphology, a culture was divided into two parts. One part received 10% by volume of 5.0% (vol/vol) glutaraldehyde in Sorenson buffer (pH 7.4: 920 mosmollkg) and was incubated at 37 C, while the other part was placed at 4 C. After 1 h the cells of both samples were collected at 3,090 x g for 15 min at 4 C and negative-contrast preparations were made with 1% (wt/vol) phosphotungstic acid adjusted to pH 7.4 with NaOH (34 mosmol/kg). Many of the fixed cells were obscured by a layer of darkly stained material which was apparently removed by washing them twice in Dulbecco phosphate-buffered saline (PBS) (pH 7.2; 270 mosmol/kg). Clumping caused by PBS was circumvented by washing in deionized, glass-distilled water instead of PBS. This caused no morphological change. Figure 1 contains p...
Virus-like particles of 350 Å have been observed intracellularly in a strain of M. hominis. This appears to be the first example of such particles within cells of the order Mycoplasmatales.
A method is suggested for the negative staining of "difficult" bacteria like Arthrobacter globiformis for electron microscopy when the usual procedures do not give satisfactory results. It involves glutaraldehyde fixation, and thorough washing and drying of the cell suspension on a grid sitting on a bed of plain agar, followed by a rapid flushing with 2% phosphotungstic acid.
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