Staining Rickettsiae in Yolk-Sac Cultures

Rickettsiae in yolk sacs are not stained well by the Macchiavello technique, and experiments were undertaken to understand the mechanisms involved. It was found that the citric acid destaining step was not effective and that most of the basic fuchsin was lost from the rickettsiae during the application of methylene blue, another basic dye. A staining technique was then evolved with carbol basic fuchsin in pH 7.45 phosphate buffer (0.4% dye, 0.4% phenol, 0.07 M buffer), followed directly by 0.8% aqueous malachite green oxalate. This technique worked well for R. mooseri, R. prowazeki, R. rickettsii, R. akari, and R. buretii, but for R. tsutsugumushi a modification was needed, whereby 4% aqueous Fe(NO₃)₃·9H₂O was used as destaining solution, and 0.5% aqueous fast green as the counter-stain.     

Evolutionary relationship of Rickettsiae and mitochondria.

Phylogenetic data support an origin of mitochondria from the alpha-proteobacterial order Rickettsiales. This high-rank taxon comprises exceptionally obligate intracellular endosymbionts of eukaryotic cells, and includes family Rickettsiaceae and a group of microorganisms termed Rickettsia-like endosymbionts (RLEs). Most detailed phylogenetic analyses of small subunit rRNA and chaperonin 60 sequences consistently show the RLEs to have emerged before Rickettsiaceae and mitochondria sister clades. These data suggest that the origin of mitochondria and Rickettsiae has been preceded by the long-term mutualistic relationship of an intracellular bacterium with a pro-eukaryote, in which an invader has lost many dispensable genes, yet evolved carrier proteins to exchange respiration-derived ATP for host metabolites as envisaged in classic endosymbiont theory.     

Taxonomy of coagulase-negative staphylococci: a comparison of two widely used classification schemes

In a comparative study 351 strains of Micrococcus subgroups 1–3 (Baird-Parker, 1963, 1965, 1974) were classified according to the Kloos and Schleifer scheme (1975b). The results showed that Micrococcus subgroups 1, 2 and 3 are heterogeneous groups in the Kloos and Schleifer scheme. Strains belonging to Micrococcus subgroups 1 and 2 were mostly classified by the Kloos and Schleifer criteria as Staphylococcus hominis, Micrococcus subgroup 3 strains from the skin as S. cohnii, while Micrococcus subgroup 3 strains from urinary infections were classified mainly as S. saprophyticus. The correlation of novobiocin resistant S. saprophyticus biotype III (Baird-Parker, 1974) with S. saprophyticus (Kloos and Schleifer, 1975a, 1975b) when isolated from urine, was 80%. Although the Kloos and Schleifer scheme provides more information about biochemical characters, doubts are expressed about the validity of some of the species so delineated.     

Phylogenetic diversity of the Rickettsiae.

Small subunit rRNA sequences have been determined for representative strains of six species of the family Rickettsiaceae: Rickettsia rickettsii, Rickettsia prowazekii, Rickettsia typhi, Coxiella burnetii, Ehrlichia risticii, and Wolbachia persica. The relationships among these sequences and those of other eubacteria show that all members of the family Rickettsiaceae belong to the so-called purple bacterial phylum. The three representatives of the genus Rickettsia form a tight monophyletic cluster within the alpha subdivision of the purple bacteria. E. risticii also belongs to the alpha subdivision and shows a distant yet specific relationship to the genus Rickettsia. However, the family as a whole is not monophyletic, in that C. burnetii and W. persica are members of the gamma subdivision. The former appears to show a specific, but rather distant, relationship to the genus Legionella.     

Taxonomic position of the Rickettsiae: Current knowledge

Abstract: The term rickettsiae initially encompassed all intracellular bacteria. Early rickettsial taxonomy was based on a comparison of a few phenotypic characteristics and recently, molecular studies brought new bases for rickettsial taxonomy. All rickettsial species studied so far belong to the alpha and gamma groups of the Proteobacteria. Ehrlichiae complex groups Cowdria ruminantium, Anaplasma marginate and Wolbachia pipientis and the related parthenogenesis and cytoplasmic incompatibility bacteria, whereas Rochalimaea species group with Bartonella bacilliformis. Rickettsia tsutsugainushi may form an independent lineage, whereas molecular data allow to regroup serologically defined typhus and spotted fever group rickettsiae. The true scale of Rickettsia and Coxiella genera remain to be determined.     

Taxonomy of Selaginella: a study of characters, techniques, and classification in the Hong Kong species

This study of 11 Hung Kong species of Selaginella indicates that sufficient characters exist by which they may be conveniently, consistently, and conclusively distinguished, and implies that the same is true for the rest of the genus, which suffers from chronic taxonomic confusion. Characters assessed are: leaf size, shape, epidermal cell patterns and mesophyll structure; ligule shape; patterns seen in stem transections; sporophyll shape; microsporangium shape; sporangial distribution; and spore surface ornamentation. Of these, the shape of median leaves, the maximum size of lateral leaves and epidermal cell patterns are the most significant and distinctive foliar features. These, together with spore surface ornamentation, are the most useful for distinguishing species. Correlations between the shapes of ligules, sporophylls, microsporangia, and vascular bundles indicate natural subgeneric groups.
These studies suggest that there are two ways by which taxonomic investigations of Selaginella can be improved. First leaves and spores should be prepared in permanent mountant (e.g. Hoyer's solution) for accurate observation of size, shape, and epidermal patterns. Secondly, data should be collected on a wide range of features.     

Plaque Assay for Q Fever and Scrub Typhus Rickettsiae

The plaque assay procedure developed for spotted fever and typhus group rickettsiae is also appropriate for scrub typhus and Q fever rickettsiae. The plaque titers of suspensions of Rickettsia tsutsugamushi and Coxiella burnetii compared favorably with end points obtained by titrations in mice.     

Plaque Assay of Rickettsiae in a Mammalian Cell Line

Clear-cut and repeatable plaque assays were obtained for three rickettsiae of the spotted fever group (Rickettsia rickettsi, R. conori, and R. montana) in Vero cells used in a manner similar to that for arboviruses. In addition, three typhus group agents (R. typhi, R. canada, R. prowazeki) induced plaques in these cells. In preliminary tests Coxiella burneti (Nine Mile strain) failed to produce plaques. Comparable results were obtained in plastic flasks and plastic culture trays incubated in ambient air with or without addition of N-2-hydroxyethyl-piperazine-N'-2-ethanesulfinic acid buffer. Larger and more well defined R. rickettsi plaques were produced when cultures were overlaid with Leibovitz (L15) medium than with either medium 199 or Eagle medium. Phosphate-buffered saline containing bovine plasma albumin (fraction V), in contrast to brain heart infusion broth, as a diluent for preparing inocula consistently permitted development of larger and more numerous plaques with three agents: R. rickettsi, R. conori, and R. montana. When R. rickettsi and R. typhi were assayed in parallel in primary chicken embryo cultures and Vero cells, comparable results were obtained, but with R. canada results in Vero cells were superior. In contrast, R. prowazeki produced inconsistent results in Vero cells.