Electron Microscopy of the 

 Legionnaires' Disease Bacterium 



F. W. Chandler. J. A. Blackmon, M.D. Hicklin, 

 R.M. Cole, and CS. Callaway 



We studied the Legionnaires' disease bacterium (LDB) by examining cultures, preparations 

 of iiuman lung tissue, and chicken egg yolk sacs with transmission electron microscopy (TEM). 

 Samples of consolidated iiuman lung were obtained at autopsy from twelve patients with con- 

 firmed Legionnaires" disease. Formalin-fixed pieces of these lung tissues were embedded in 

 paraffin, sectioned<5 /im, stained by the Dieterle silver impregnation method (2), and examined 

 by light microscopy. If numerous bacteria characteristic of the LDB were seen, the piece of tissue 

 from which the section was taken was diced into 1 mm cubes for TEM studies. Normal yolk sacs 

 and those infected with the LDB were fixed either in cold 4% (v/v) buffered paraformaldehyde 

 or cold 2% (v/v) glutaraldehyde in 0.2 M S-collidine buffer, pH 7.0, for 1 h. Both lung specimens 

 and yolk sacs were postfixed in 1% osmium tetroxide (v/v), buffered in 0.2 M S-collidine, pH 7.2 

 to 7.4 (7), for 1 h at 4°C. The tissue specimens were then dehydrated through graded alcohols, 

 embedded in Maraglas 732, and cut on a Reichert 0MU2 ultramicrotome fitted with a diamond 

 knife. Sections were picked up on uncoated copper grids and stained with saturated uranyl 

 acetate and lead citrate {13). Selected 4-day cultures of the LDB in Mueller-Hinton broth supple- 

 mented with IsoVitaleX were prepared by the method of Ryter and Kellenberger (6). For whole 

 cell preparations, broth cultures of the LDB were dried on Formvar-coated grids and stained with 

 2% aqueous uranyl acetate. All sections were examined with a Philips 200 transmission electron 

 microscope at 40 KV. 



The LDB is ultrastructurally identical in human lung tissue, experimental lesions of guinea 

 pigs, bacteriologic media, or yolk sac membranes of embryonated hens' eggs. In sections, the 

 LDB is a blunt or tapering rod which usually measures 0.3 to 0.9 ^tm in diameter and >2.0 /im 

 in length (Figs. 1 and 2). Its sides may not be parallel, and it varies considerably in length depend- 

 ing on its environment. After 4 to 7 days of growth, greatly elongated forms are commonly 

 found in cultures and less commonly found in yolk sac membranes; they are rarely found in 

 human lung or in experimentally induced lesions in guinea pigs. The LDB is clearly prokaryotic 

 (!S,9) in that it lacks eukaryotic features such as mitochondria, nuclear membranes, endoplasmic 

 reticulum, and mitotic division. Prominent features include electron-lucent, filamentous nucleoids 

 interspersed among areas of well-defined ribosomes; enclosure by a double envelope, each por- 

 tion of which consists of a triple-layered "unit" membrane (4.10): and division by a pinching, 

 nonseptate process (Figs. 2 and 3). This pinching type of division and double envelope enclosure 

 are characteristic of gram-negative bacteria (4. 5. 10. II). 



With the techniques used, we have not been able to see any definite structure that could 

 represent a peptidoglycan layer (11) in the periplasmic space. Cleanly circumscribed, round 

 electon-lucent vacuoles are frequently seen within the LDB (Fig. 1). These vacuoles stain readily 

 with Sudan black B in smears made from cultures, and their lipid content is probably removed by 

 the solvents used in preparing samples for electron microscopy. Their ultrastructural appearance 

 is suggestive of poly-jS-hydroxybutyrate granules in sections (-5,72). Rarely, membranous profiles 



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