Effect of preparatory techniques on the gross morphology of Mycoplasma hominis

Abstract

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...

Mycoplasma hominis: growth, reproduction, and isolation of small viable cells

“…Cells of M. hominis, cultured and prepared for electron microscopy as described, showed no changes in gross morphology during the relatively short lag phase. Cells in actively growing cultures were typically round to rod shaped, as previously reported (23). Some of the rods were dumbbell shaped ( Fig.…”

“…Inoculum preparation and incubation conditions. Preparation of inocula has been described (23). Fifty-milliliter cultures in 250-ml Erlenmeyer flasks were incubated in a gyratory shaker (New Brunswick Scientific Co., New Brunswick, N.J.) at 125 rpm, and always at 37 C. Growth measurement.…”

“…Electron microscopy. Cells were pre-fixed in situ by a method reported elsewhere (23). Negative-contrast preparations were made with 1% (wt/vol) phosphotungstic acid (PTA) (pH 7.4; 34 mosmol/kg), isoosmolal ammonium molybdate (pH 7.4; 384 mosmollkg) or 1% (wt/vol) sodium zirconium glycolate (SZG) (pH 7.4; 115 mosmol/kg).…”

“…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).…”

Cytoplasmic helical structure associated with Acholeplasma laidlawii

“…(iv) Fixation and embedding. Cells of A. laidlawii were prefixed in situ by the method of Robertson et al (16), fixed in 5% glutaraldehyde in sodium-phosphate buffer (0.1 M, pH 7.4) for 1 h at 4°C followed by washing, and maintained overnight in the same buffer. Cells were postfixed in 2% osmium tetroxide in the same buffer for 1 h at room temperature and washed with double distilled water.…”