THE RESPONSE OF SELECTED MEMBERS OF THE ARCHAEA TO THE GRAM STAIN

Archaea possess a broader range of cell envelope structural formats th an eubacteria and their cell walls do not contain peptidoglycan. Some archaea have only a single S-layer as their cell wall (e.g. Methanococ cus jannaschii and Sulfolobus acidocaldarius), whereas others have mul tiple layers (e.g. Methanospirillum hungatei). Sometimes there can als o be a high proportion of tetraether lipids in membranes to make the e nvelope more resilient to environmental stress (e.g. Methanococcus jan naschii and Sulfolobus acidocaldarius grown at 70 degrees C). Since th e Gram reaction depends on both the structural format and the chemical composition of the cell envelope of eubacteria, it was important to d etermine if the same is true for archaea. Methanospirillum hungatei, M ethanosarcina mazei, Methanobacterium formicicum, Methanococcus jannas chii and Sulfolobus acidocaldarius, chosen because of their different envelope formats and chemistries, were subjected to a Gram stain that can be used for transmission electron microscopy. In this staining reg imen, the iodine is replaced by potassium trichloro(eta(2)-ethylene)pl atinate(II) as the mordant, and the platinum of the new compound is th e electron-scattering agent for electron microscopy. Of all these arch aea, only Methanobacterium formicicum stained Gram-positive since its pseudomurein wall remained intact; the platinum compound formed large electron-dense aggregates with the crystal violet that were located in the vicinity of the cell wall and the plasma membrane. All but the te rminal filament cells of Methanospirillum hungatei stained Gram-negati ve because the limiting porosity of its external sheath was so small t hat the Gram reagents could not enter the cells. The terminal cells of filaments stained Cram-positive because the staining reagents gained entry through the terminal plugs. All other archaea stained Gram-negat ive because their cell walls were so disrupted during staining that th e crystal violet-platinum complex could not be retained by the cells. Methanococcus jannaschii was grown at both 50 degrees C and 70 degrees C so that the tetraether lipids in its plasma membrane could be incre ased from 20% (50 degrees C) to 45% (70 degrees C) of the total lipids ; in both cases the cells stained Gram-negative.