Morphology, ultrastructure, and mode of division of Mycoplasma fermentans, Mycoplasma hominis, Mycoplasma orale, and Mycoplasma salivarium.

The morphology of viable Mycoplasma fermentans, Mycoplasma hominis, Mycoplasma orale types 1 and 2, and Mycoplasma salivarium was studied in broth cultures by interference microscopy and in thin sections by electron microscopy. Only spherical cells were seen by interference microscopy. M. hominis had a capsule-like outer layer. Except for M. orale type 1, mycoplasmas in thin sections were 0.3-1 mum in diameter, with a bounding trilaminar membrane 7.5-10 nm thick. The mycoplasmas contained DNA fibrils and randomly distributed ribosomes. No polyribosomes were seen. Dividing mycoplasmas elongated slightly; the membrane invaginated, forming one bud. Sometimes M. hominis and M. salivarium produced one bud by elongation, and the bud was attached by a tube. This method of division is not considered as characteristic but rather as due to centrifugal force separating unfixed cells during preparation for electron microscopy. Cross-septa were never observed. In thin sections M. orale type 1 was elongated and without buds, an observation which suggested that preparation for electron microscopy distorted the mycoplasmas.

A study of the amino acids and proteins of some human T-mycoplasma membranes.

Purified membranes were prepared from seven human T-mycoplasmas. Their amino acid composition was determined and was similar to that of other biological membranes. Their proteins were examined by isoelectric focusing and 7 to 10 protein bands were detected mostly in the pH 4 to 7 range of the gel. T-mycoplasma T-McA also had one distinctive band at pH 3 while T-mycoplasma T-213 had a prominent basic band at pH 9. The proteins were denatured by storage at -25 degrees. Five to seven Periodic Schiff-positive bands also were observed but were not identified.

The preparation of membranes of some human T-mycoplasmas and the analysis of their carbohydrate content.

Purified membranes were prepared from seven human T-mycoplasmas of which four are laboratory strains and three isolates from patients with nonspecific urethritis. The T-mycoplasmas were resistant to osmotic shock and sonication. Membranes were obtained only after lysing most of the T-mycoplasmas by four passes through a cell fractionator at 40,000 psi and separating the membranes from the unlysed cells by differential centrifugation. The membranes contained between 1 and 7% carbohydrates by dry weight. Mannose, galactose, and glucose were identified with glucose in the largest concentration.

Adaptation of Mycoplasma hominis to an obligate parasitic existence in monkey kidney cell culture (BSC-1).

During attempts to eliminate Mycoplasma hominis from a monkey kidney BSC-1 cell line with antibiotics, the mycoplasmas were isolated repeatedly. However, the organisms ultimately failed to grow on medium although electron microscopy confirmed that the cell culture still contained mycoplasmas. Thus, the mycoplasma had adapted to an environment in which viable cells were required for growth. Budding mycoplasmas which are indicative of replication were seen associated with viable cells extracellularly. Moreover, structures resembling cycoplasmas were observed budding from the cells which suggests that the mycoplasmas replicate within the cells and are similar to many viruses in their manner of release from the cells.

T-mycoplasmas: a study of the morphology, ultrastructure and mode of division of some human strains.

The morphology of 10 strains of T-mycoplasma was studied in wet preparations of viable cells by darkfield, phase-contrast and interference microscopy, and in fixed preparations by various techniques of electron microscopy. Mycoplasma-like artefacts in the horse-serum component of the medium were eliminated by filtration. All 10 strains were similar. Individual cells were spherical, 0-25-1-0 mum in size, with a bounding trilaminar membrane, 10 nm thick and containing 7-5-12-5-nm particles, and a layer of pilus-like projections, 5-8 nm long, on the outer surface. A possible capsular matrix was observed only by the pseudoreplica technique. The cells contained 12-15-nm ribosomes, nuclear fibroids 7-5-9 nm wide, and vacuoles. During replication, the cell elongated slightly and the ribsomes migrated to the ends of the cell leaving a ribosome-free area into which the bounding membrane invaginated to form a bud. The bud eventually separated by completion of the process of invagination; a cross-septum did not form. Usually only a single bud developed but sometimes two appeared simultaneously.

Morphological changes induced during fixation of Mycoplasma mycoides for electron microscopy.

Only spherical mycoplasmas 0.6-0.9 mum diam were observed by darkfield microscopy in single cell suspensions prepared from exponential broth cultures of Mycoplasma mycoides var mycoides and Mycoplasma mycoides var capri. Similar cells were seen in pseudoreplicas by electron microscopy and they are considered characteristic of the morphology of M. mycoides. When the mycoplasmas were fixed by the addition of 10% formalin to the suspensions or washed, centrifuged cells were fixed by glutaraldehyde, filamentous and ring forms were observed in electron micrographs. These are not considered typical of the morphology of M. mycoides but are artifacts produced during the preparation of the mycoplasmas for electron microscopy.

A cytochemical and radioautographic study of human tissue culture cell nucleoli.

Fine structural aspects of human tissue culture cell nucleoli were studied by cytochemical and radioautographic methods. Ribonuclease and pepsin digestions were carried out on glutaraldehyde-fixed cells that, in some instances, were labeled with thymidine-(3)H prior to digestion. Double digestion by ribonuclease and pepsin revealed a fine fibrillar reticulum that appears to be the supportive structure of nucleolonemal threads. The nature of the reticulum remains to be determined. The question of whether it may represent a dispersed form of chromatin was raised. Structural findings suggested such an hypothesis but the results of radioautographic studies do not support it. The reticulum showed a striking absence of radioactive labeling following a 3 hr incorporation of thymidine-(3)H. Only few silver grains were observed occasionally in the fibrillar nucleolonema that may or may not be significant. The radioautographic results are believed to be inconclusive for the various reasons discussed. The possibility that the reticulum is composed of proteins has to be considered. It appears that basic proteins can resist pepsin digestion in aldehyde-fixed cells. Individual chromatin fibrils were found to be associated with the nucleolar reticulum. It is possible that these alone represent the dispersed genetically active chromatin of nucleoli.