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Chicken tracheal organ cultures were made from embryos which were 19 to 20 days old. Transversely cut rings of trachea were placed in screw-capped tissue-culture tubes with Eagle's-N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) medium and incubated in roller drums. The method had advantages over other organ culture systems in that these cultures were prepared in numbers similar to conventional tissue cultures, ciliary activity was quickly and accurately evaluated, and contamination occurred less frequently than with organ cultures in petri dishes. Ciliary activity persisted for at least 1 month when the medium was changed at 5-to 7-day intervals and for 10 to 15 days without a change. Infectious bronchitis virus stopped ciliary movement, and this effect was used as a basis for titrating the virus and for determining the neutralizing capacity of immune mouse ascitic fluid. Twenty-four Mycoplasma strains were tested. Organisms of 17 strains, both avian and mammalian, multiplied in the organ cultures, and 7 strains, belonging to the species M. gallisepticum and M. mycoides var. capri, inhibited ciliary activity.

The growth and pathogenicity of Mycoplasma mycoides var. capri were studied in chicken embryo tracheal rings in rolled tubes. In these organ cultures, M. mycoides var. capri attained titers of 10(7) to 10(8) color-changing units/ml of Eagle's medium and there was inhibition of ciliary activity. The rapidity of inhibition was directly related to the number of organisms inoculated. Growth of organisms was closely associated with the tracheal tissue because multiplication was not demonstrated in "conditioned medium," that is Eagle's medium from which the rings had been removed. Mycoplasma growth occurred when the cultures were incubated at 37 C, 33 C, and room temperature, but the cilia-stopping effect (CSE) was most rapid at 37 C and was not demonstrable at room temperature. Furthermore, there was no CSE when cultures were maintained in medium in which M. mycoides var. capri had been grown but was either filtered to remove the organisms or treated with tetracycline to stop their multiplication. This indicated that the CSE of M. mycoides var. capri was dependent upon a close association of multiplying organisms with the tracheal rings and was not due to toxic products persisting in the medium. Chicken tracheal epithelial cells did not adsorb to colonies of M. mycoides var. capri but HeLa cells did. Although their adsorption was unaffected by prior neuraminidase treatment, the addition of neuraminidase to the tracheal organ culture system delayed the CSE of M. mycoides var. capri. Peroxide production was demonstrated in M. mycoides var. capri-infected tracheal rings but not in uninfected cultures. The addition of glucose to the organ cultures delayed the CSE of M. mycoides var. capri, possibly because of the stimulation of peroxidase activity. Similarily, catalase protected the cilia from the usual damaging effect of M. mycoides var. capri. This protective effect was partially reversed by the addition of 3-amino-1, 2, 4,-triazole and it did not occur if the catalase had been inactivated by boiling. The addition of hydrogen peroxide to tracheal cultures also resulted in loss of ciliary activity. The present results show that the liberation of peroxide is an important factor in the pathogenesis of M. mycoides var. capri infection of chicken tracheal organ cultures. It is speculated that this also may be so in the natural host.

A virus plaque method that consistently gives good visual contrast for macroscopic observation and also permits microscopic study is described. Cells are grown in plastic flasks and the gelled overlay medium can be of any desired agar concentration or volume. A fixing solution is used prior to removal of the agar overlay, and dye is added to the fixing solution or staining can follow fixation and agar removal. The bottom of the flask with the fixed monolayer is separated from the rest of the container and handled as a slide. A new mounting medium is described.

A simple parabiotic chamber which is easy to manipulate, does not leak, allows microscopic examination of organ cultures, and is easy to sterilize is described.