GROWTH ON ARTIFICIAL MEDIUM OF AN AGENT ASSOCIATED WITH ATYPICAL PNEUMONIA AND ITS IDENTIFICATION AS A PPLO

Abstract

Recent volunteer and controlled epidemiologic field studies have provided evidence which firmly associates the agent first recovered by Eaton in 1944 with lower respiratory tract illness of man.'-' A serologic response to the Eaton agent occurs in approximately 90 per cent of pneumonia illnesses in which cold agglutinins develop during convalescence as well as in a significant but variable proportion of cold agglutinin-negative pneumonias. 2 4 The development of pneumonia and other forms of respiratory disease following the administration of tissue culture-grown Eaton agent to volunteers and the demonstration that naturally acquired antibody offered protection against such illness supports the contention that the agent is a respiratory tract pathogen.5 For many years, the agent was tentatively classified as a virus. The large size of the agent (180-250 mMA) and its sensitivity to streptomycin and various tetra-cycline derivatives, however, posed some difficulty with such a classification.6-8 Recently, Marmion and Goodburn were able to visualize small cocco-bacillary bodies on the mucous layer covering the bronchial epithelium of the Eaton agent infected chick embryo.9 The distribution of these bodies corresponded with the localization of Eaton agent as visualized by the fluorescent antibody technique. These workers also demonstrated that the Eaton agent was inhibited by an organic gold salt. Clyde visualized extracellular "colony-like" structures in stained preparations of infected tissue culture; these structures corresponded with the areas of specific immunofluorescence.'0 Both groups of workers suggested the possibility that the Eaton agent may be a "pleuropneumonia-like organism" (PPLO) rather than a virus. Cultivation of the organism in cell-free media, however, was not achieved. " Stimulated by the findings of Marmion and Goodburn, we attempted cultivation of the Eaton agent on an agar medium incorporating 2.5 per cent yeast extract and 20 per cent horse serum. The present report will describe the successful growth of the agent on agar and its identification as a PPLO. Materials and Methods.-Media: Agar plates, measuring 5 cm in diameter, were prepared with 7 parts Difco PPLO agar, 2 parts uninactivated horse serum and 1 part 25% yeast extract (prepared from active Baker's yeast and stored at-20'C). The agar was prepared in 70-ml quantities and sterilized by autoclaving. After cooling to about 450C, the agar was supplemented with the yeast extract and horse serum. In one passage series, antibiotics were not employed, while 500 units of penicillin/ml was added to the medium in a parallel series. The plates were generally used the same day they were prepared. Cultivation: Agar plates inoculated with Eaton agent were incubated at 360C. Initially, infected tissue culture fluid was streaked onto the agar. Subsequently, passages were initiated by rubbing a small block of agar, measuring 1-2 cm2 and representing approximately one twentieth to one tenth of the volume of the agar on a plate, over the surface of a fresh plate. Fluorescent antibody techniques: The method employed for the staining of Eaton agent antigen in infected chick embryo lung sections has been described previously.2 Briefly, chick embryos 41