Abstract
Thin sections of an anaerobic filamentous oral organism possibly related to Leptotrichia buccalis have been examined in the electron microscope. The organism exhibited a cell wall profile characteristic of Gram-negative bacteria, viz. a solid membrane and an outer double-layered membrane. The outer membrane could be removed by treating intact cells with trypsin or phenolwater. The organism contained numerous intracytoplasmic vacuoles.
Abstract
15-2
INTRODUCTION
Morphological and physiological properties of an anaerobic filamentous microorganism isolated from the human oral cavity have been reported (Hofstad, 1967a) . This micro-organism, which proved to be identical to a filamentous bacterium described by Theilade & Gilmour (1961) , appeared to be related to Leptotrichia buccalis (Gilmour, Howell & Bibby, 1961) , but differed from that species serologically and in showing a heterofermentative type of sugar metabolism. Like L. buccalis, very young cells of the atypical organism were Gram-positive, while most cells in cultures in the stationary phase of growth were Gram-negative.
INTRODUCTION
In the electron microscope, Leptotrichia buccalis exhibits a cell wall profile characteristic of Gram-negative bacteria (Hofstad & Selvig, 1969) . The fine structure of the related atypical filamentous micro-organism has been reported by Theilade, Theilade & Scott (1962) . These authors did not, however, study the cell wall structure of this organism.
INTRODUCTION
The present report concerns the ultrastructure of the filamentous micro-organism as revealed by electron microscopy of intact cells and cells treated with trypsin or phenol-water, with particular attention to the cell wall profile.
METHODS
The isolation of the filamentous organism, strains ~4 4 and ~4 9 , has been described earlier (Hofstad 1967a) . Cultivation was performed in enriched nutrient broth (Hofstad 1967a) , and the bacteria were harvested by centrifugation when in exponential phase of growth ( I or 2 days at 37').
METHODS
In preliminary experiments the harvested bacteria were washed with saline or phosphate buffer at neutral pH. However, the washings caused many organisms to break, and the remainder appeared fuzzy and with poorly resolved cell wall layers IP: 83.161.120.155
METHODS
On: Thu, 06 Dec 2018 06:27:58 222 T. HOFSTAD AND K. A. S E L V I G when examined in the electron microscope. Therefore, the washing was omitted and subsequent preparations were carried directly to fixative.
METHODS
Trypsin digestion was done on washed bacteria with crystalline trypsin (Trypure Novo, Novo Industri A/S, Copenhagen), freshly prepared in 0.02 M-tris buffer of pH 7.8, using 10 mg. trypsinlg. packed wet bacteria. The digestion was performed at 37" for 20 hr. The digested bacteria were centrifuged and washed twice in saline.
METHODS
Treatment with phenol-water was carried out by homogenizing equal volumes of an aqueous suspension of 200 mg. wet packed bacteria and 90 % phenol for 15 min. at 4'.
METHODS
Following centrifugation the water and phenol phases were pipetted off and the treated bacteria washed once in distilled water.
METHODS
Pellets of intact or treated bacteria were resuspended in acetate + veronal buffered osmium tetroxide fixative, transferred to agar and embedded in polyester resin according to the procedure suggested by Kellenberger, Ryter & SCchaud (1958) . Thin sections for electron microscopy were cut with glass knives, collected on carboncoated formvar membranes, and stained with 2 % aqueous phosphotungstic acid for 30 min., or with lead citrate (Reynolds, 1963) for 5 to 15 min. Some bacteria were resuspended in distilled water. Drops of the suspension were placed on specimen support grids coated with carbon and formvar membranes, dried and shadowed with palladium from an angle of 20 degrees. Specimens were examined in the electron microscope and photographed at plate magnifications of 5,000 to 20,000 diameters.
RESULTS

Intact organisms
The anaerobic filamentous organism, strains ~4 4 and ~4 9 , appeared in thin sections as elongated structures whose shorter diameter ranged from 0.4 to 0.6 p (Pl. I , fig. I ).
Intact organisms
The bacteria had rounded ends and were often adhering end to end. A moderate amount of floccular substance was seen adhering to the surface and in the surrounding medium.
Intact organisms
The concentric membranous structures surrounding the cytoplasm will be referred to as the plasma membrane, solid membrane and the outer membrane. The plasma membrane appeared as a double structure, 70 to 8 0 A in width, which followed a straight or slightly wavy course (Pl. I , fig. 2 ).
Intact organisms
The solid membrane appeared as a distinct single electron-dense line, less than 50 A in width (Pl. I, fig. 2 ). It followed the course of the plasma membrane, separated from it by a space of 50 to 150A. This space contained varying amounts of granular electron-dense material.
Intact organisms
The outer membrane was separated from the solid membrane by a less electrondense layer, around 40 A in width. Where the multi-layered cell wall was best resolved, the outer membrane could be seen as a double-tracked structure. In other organisms the outer membrane appeared as a less distinct single line. The outer membrane was regularly coated by a more or less dense crust of granular or floccular material
Intact organisms
The cytoplasm consisted mainly of an evenly dispersed electron-dense granular material. Nuclear regions appeared as less electron-dense regions filled with fibrillar substance (Pl. I , fig. I ). Many cells contained oval or circular vacuoles (Pl. I , fig. I) .
Intact organisms
The contents of the vacuoles were less electron-dense than the surrounding cytoplasm.
Intact organisms
(Pl. I, fig. 2 Ultrastructure of a filamentous micro-organism 223 The filamentous shape of the micro-organism was confirmed in shadowed preparations. Some of the organisms were collapsed; these showed numerous rigid bodies which were interpreted as corresponding to the intracytoplasmic vacuoles (Pl. I , fig. 3) .
Intact organisms
Organisms treated with trypsin or phenol-water Following digestion with trypsin, most bacteria maintained their original shape, but the wall structure was considerably modified (Pl. I , fig. 4) . Generally, the cell wall consisted of a single electron-dense layer separated from the plasma membrane by a space of varying width. This space did not contain appreciable amounts of stainable material.
Intact organisms
After treatment with phenol-water the organisms were extensively altered (PI. I , fig. 5) . The cell content appeared as a homogeneous mass retracted from the wall. The cell wall appeared as a single line, frequently with attached outer cell wall material.
DISCUSSION
The cell wall profile of the filamentous oral organism conforms to the general description of the Gram-negative cell wall type (Murray, Steed & Elson, 1965; de Petris, 1965) , and is characteristically different from the cell wall profile of Grampositive bacteria (Glauert, 1962; Murray, 1962) . The various layers were not as clearly differentiated, however, as in the possibly related species Leptotrichia buccalis (Hofstad & Selvig, 1969) .
DISCUSSION
The finding of a Gram-negative cell wall type is in agreement with previous chemical studies of isolated walls (Hofstad, 1967b) . Cell walls of the filamentous organism contained a wide range of amino acids, and protein and lipid accounted for approximately 50 and 20 % of the dry weight, respectively. In contrast, the hexosamine content did not exceed 5 %. Furthermore, the cell wall preparations contained an aldoheptose, a component previously demonstrated in Gram-negative bacteria only.
DISCUSSION
The disappearance of the outer membrane from the trypsin-treated organisms indicates that this membrane of the filamentous organism is made up mainly of ptotein. This hypothesis is supported by chemical analysis of the material extracted from washed whole bacteria or cell wall preparations by digestion with trypsin. The extracted material contained 55 % by weight protein, 25 % lipid and 20 % neutral sugars (T . Hofstad, unpublished results) .
DISCUSSION
The removal of the outer membrane by trypsin digestion is somewhat at variance with the results of de Petris (1967), who reported on the cell wall structure of Escherichia coli. Digestion of intact or heat-treated organisms with proteolytic enzymes caused no removal of the outer membrane in this species. Rather, the double-layered outer membrane was sharply outlined and apparently unaffected by the treatment. On the other hand, this membrane could be removed by treatment with phenol-water, which in the present study was less effective than trypsin treatment in this respect.
DISCUSSION
Trypsin-digested cell walls of the filamentous organism contain approximately 25 % amino acids, 38 % neutral sugars and 4 % fatty acid esters (Hofstad, 1967b) . This indicates that the solid membrane comprises structures other than pure mucopeptide.
DISCUSSION
The removal of the stainable material between the plasma membrane and the solid membrane by digestion with trypsin shows that protein is interposed between these (Weidel, Frank & Martin, 1960; de Petris, 1967) .
DISCUSSION
The nature of the intracytoplasmic vacuoles is not clear. However, similar structures, interpreted as intracellular polysaccharide, have been found in cariogenic streptococci (Guggenheim & Schroeder, I 967) and in coccoidal, rodshaped and filamentous bacteria in human dental plaques overlying various and, to a lesser degree, normal enamel (Frank & Brendel, 1966) . The finding of rigid bodies within collapsed bacteria supports the assumption of a consolidated content of these vacuoles. (strain ~4 4 ) .
DISCUSSION
The cytoplasm is surrounded by three concentric structures: the double-tracked plasma membrane, the solid membrane and the double-tracked outer membrane. Numerous intracytoplasmic vacuoles are present, especially in the bacterium to the right. Small amounts of floccular material adhere to the cell surface. N-nuclear regions. Magnification x 40,000. The collapsed bacterium contains intracytoplasniic rigid structures. x 10,000. Fig. 4 . Result of trypsin digestion (strain LM). The bacteria have maintained their shape, but the outer membrane is missing. PM-plasma membrane, SM-solid membrane. x 40,000. Fig. 5 . Result of treatment with phenol-water (strain LM). The cell wall appears in general as a single electron dense line which corresponds to the solid membrane (SM). x 40,000.

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