MICROMORPHOLOGY OF DERMIATOPHILUS CONGOLENSIS

MORRIS A. GORDON AND MERCEDES R. EDWARDS

Division of Laboratories and Research, New York State Department of
Health, Albany, New York
Received for publication 8 July 1963

ABSTRACT coccoid elements, which are embedded in a

GORDON, MORRIS A. (Division of Laboratories gelatinous matrix (Fig. 1), then escape, leaving an
and Research, New York State Department of empty sheath, and assume motility, thus com-
Health, Albany), AND MERCEDES R. EDWARDS. pleting the cycle. Alternatively, the cocci remain
Micromorphology of Dermatophilus congolensis. attached and germinate in situ. An excellent
J. Bacteriol. 86:1101-1115. 1963.-As seen in account of the life cycle was given by Roberts
electron micrographs of thin sections, Dermato- (1961).
philus congolensis is a holocarpic actinomycete The Sarcina-like clusters and size of the in-
that fragments, after formation of septa in several dividual elements (about 0.5 It for the cocci and
planes, into Sarcina-like packets and then into 0.5 to 1.0 ,u for motile spores) suggest that
individual cocci. Release of coccal forms from Dermatophilus is a bacterium; its early fila-
the filaments and packets is by dissolution of a mentous stage (Fig. 2) and the texture and
capsular matrix, which is a product of degrada- morphology of its colonies would place it among
tion of the cell wall. The plasmalemma is a "unit the actinomycetes. Its characteristic pleomor-
membrane." Regularly occurring plasmalem- phism and the resemblance of various stages to
mosomes ("onion bodies") of uniform structure actinomycetes and other bacteria are reflected in
are apparently related to septum formation. A some of its synonyms, which include Actinomyces,
typical bacterial nucleoid is seen in most sections, Tetragenus, Nocardia, Rhizobium, and Strepto-
and ribosomes are scattered throughout the thrix. As with other diseases attributable to
cytoplasm. Specimens for electron microscopy actinomycetes, the study of those caused by
were prepared by a modification of Kellenber- Dermatophilus has been assumed by mycologists
ger's method. (see Ainsworth and Austwick, 1959, for a review
of streptotrichosis and the genus Dermatophilus),
and efforts have been made to integrate this
Dermatophilus congolensis (Van Saceghem, genus into the order Actinomycetales (Austwick,
1915) is the etiological agent of cutaneous strepto- 1958; Hesseltine, 1960). We have undertaken by
trichosis of cattle ("dermatose contagieuse" of means of electron microscopy to determine more
Van Saceghem, 1915), mycotic dermatitis precisely the ontogeny and affinities of this
("lumpy wool") of sheep (Actinomyces dermato- unusual organism and to compare it with Actino-
nomus; Bull, 1929), and strawberry foot-rot of myces bovis (Edwards and Gordon, 1962),
sheep (Polysepta pedis; Thompson and Bisset, Listeria monocytogenes (Edwards and Stevens,
1957). Recently, it has been found to be trans- 1963), Streptomyces coelicolor (Glauert and Hop-
missible from deer to man (Dean et al., 1961). wood, 1959), Streptomyces noursei (Stuart, 1959),
It displays a peculiar and possibly unique de. Actinoplanes philippinensis, and Streptospor-
velopmental morphology. Light microscopy angium roseum (currently being studied in this
reveals motile spores, reportedly flagellate laboratory).
(Thompson, 1954), that germinate to form fine,
septate hyphae. These proceed by means of MATERIALS AND MIETHODS
perpendicular branching, continued transverse Four strains of D. congolensis were examined:
septation, and longitudinal segmentation in two O (orange) and G (gray) are variants of the deer
or more planes, ultimately to form branched, isolate from New York state (Dean et al., 1961);
distally tapering filaments comprising a series of A-19, from a bovine skin infection, was received
coccal packets resembling those of Sarcina. The from D. A. Barnum, Ontario Veterinary College;
1101
1102 GORDON AND EDWARDS J. BACTERIOL .

IL

A.
wk.
OMF

M.U.

FIG. 1. Segmenting filament and coccoid clusters of Dermatophilus congolensis from agar slant, showing
capsules. India ink preparation. X 430.
FIG. 2. Young colony of Dermatophilus congolensis grown on agar plug under cover slip. Giemsa stain.
X 960.

A-7, labeled Polysepta pedis, was supplied by R.
E. M. Thompson, Bland-Sutton Institute of
Pathology, London, England. A taxonomic paper
now in preparation will set forth evidence for
synonymy of P. pedis with D. congolensis.
The organisms were cultured at 35 C either in
Tryptone broth (Tryptone, 1 %; sodium chloride,
0.5%; pH 7.0 to 7.2) or on the same medium
solidified with 2 % Difco agar, and harvested
at periods from 2 to 48 hr and 1 week. They were
fixed with 1% osmium tetroxide in Veronal-
acetate buffer at pH 6.1 to 6.2 for 4 to 6 hr at
VO IJ. 86, 1963 MICROMORPHOLOGY OF D. CONGOLENSIS
room temperature and then overnight at 4 to 6
C, treated with uranyl acetate solution (Kellen-
berger, Ryter, and Sechaud, 1958) at pH 5.0 in
the same buffer for 1 hr, dehydrated rapidly
through a series of ethanol solutions, and left in
100% ethanol for 0.5 hr. The material was in-
filtrated with a mixture of nine parts n-butyl
and one part methyl methaerylate monomers for
2 to 3 hr, placed in the same mixture plus 1 %
benzoyl peroxide overnight in a refrigerator, and
embedded in another portion of the latter mixture
which had been prepolymerized. The blocks were
hardened at 45 C for 24 hr and cured at room
temperature for several days. Ultrathin sections
were cut with a Caracas diamond knife in a
Porter-Blum microtome and examined in a
Siemens Elmiskop I at magnifications from 5000
to 40,000 X, further enlargement being obtained
photographically.
RESULTS
Thin sections of Dermatophilus exhibited an
unusual variety of cell configurations, but the
fine structure of all was essentially the same.
Before describing the latter in detail, we shall
review the ontogeny of the organism as revealed
by the electron microscope. Filaments (Fig. 3)
arise as tubular protrusions resembling germ tubes
from coccal forms such as those in Fig. 4 and 19.
They attain a length of several microns preceding
septation, and give rise to true branches (Fig. 3),
revealed in a light microscope to be more or less
perpendicular to the main axis. Very early in their
development, the hyphae begin to form trans-
verse septa. According to Roberts (1961), septa-
tion proceeds centrifugally with always a region
of new undivided protoplasm for 5 to 30 , behind
the growing tip; segments resulting from forma-
tion of primary septa, which are 3 to 6 , apart,
are redivided by secondary and successive centrif-
ugal waves of septation until septa ultimately
are 0.3 to 0.5 ,u apart. Figures 5 and 6 display
early transverse septation; subsequently (Fig. 7
and 10), longitudinal septa are apparent in many
of the segments. Radial partition is sometimes
seen (Fig. 9). As longitudinal septation progresses,
the filament increases in diameter (Fig. 11 and
12), each segment giving rise to a packet of many
coccoid cells. Cleavage of the cytoplasm appears
always to be accompanied by cell-wall formation.
The gelatinous matrix in which the more mature
cells are embedded is revealed by electron micros-
copy to be formed by thickening of the cell wall
1103
with subsequent dissolution of both peripheral
and central intercellular laminae, this process
being particularly evident in older hyphae (Fig.
12, 16, 17, and 18). It results ultimately in the
liberation of the cocci, either singly or in small
packets (Fig. 4, 19, and 20). Individual free
cocci apparently become transformed into motile
spores (seen by light microscopy), which may
reinitiate the cycle by the process of germination
and filament formation. Often germ tubes arise
from coccoid cells still enmeshed in capsular
material along with other such cells, either in
packets or in the old filaments.
Peripheral layers. The cell wall is clearly defined
in coccal forms and young filaments (Fig. 3 to 7),
but its limits are not discernible in older stages
because of progressive dissolution of the outer
layers, with resultant formation of a mass of
gelatinous capsular material of unknown com-
position. The latter sloughs off at the extreme
periphery as a dense amorphous material (Fig. 15,
16, 17, 18, 21, and 22) while at the same time
the more central zones display a fine network of
fibrils (Fig. 19, 20) enmeshing the emerging coccal
forms, whose sharply delimited cell walls appear
to have been carved out of the old filament wall
(Fig. 12, 15).
The inner border of the cell wall is closely ad-
herent to the plasma membrane (Fig. 23), which
is differentiated with difficulty from the adjacent
cytoplasm. Where there is incipient plasmolysis,
with separation of the cytoplasm from the wall,
the plasma membrane remains attached to the
former (Fig. 21 and 22), and is seen to be a typical
"unit" membrane (Robertson, 1959, 1960) with a
characteristic pattern of two dark lines, each ca.
20 A thick, separated by a lighter core of ca. 30 A.
The plasma membrane is shown also in the proto-
plast of Dermatophilus (Fig. 24) and presumably
is responsible for the osmotic properties of the cell
surface (Robinow and Murray, 1953).
Internal membrane system (plasmalemmosomes).
Membranous organelles of various configurations
have been found in bacteria (reviewed by Robi-
now, 1960; Murray, 1960, 1962; Glauert, 1962).
In Dermatophilus, lamellated organelles resem-
bling the cross section of an onion are found in
most sections (Fig. 4, 6, 7, 10 to 15, 21, and 22).
These "onion bodies" may be quite large and
occupy a major portion of the area of the cell at
the plane of section (Fig. 13 and 14). They are
often associated with the development of septa
(Fig. 4). Some micrographs (Fig. 14 and 22)
1104 GORDON AND EDWARDS J. BACTERIOL.

FIG. 3. Thin sections of Dermatophilus congolensis. Young filaments in longitudinal and cross section.
Branching is shown at B. The cell wall (cw) encloses dense cytoplasm (c) and less dense nuclear material (n).
Retraction of cytoplasm is attributed to improper fixation and embedding. X 20,000.
FIG. 4. Coccoid cells bounded by apparently rigid cell walls (cw), in various stages of division. "Onion
bodies," or plasmalemmosomes (white arrows) are found in all cells. In some (black arrow), these bodies are
located at the inner rim of the ingrowing septum. X 42,600.

clearly show the organelle to be continuous with (Fig. 8, 12, 15, 24), with an average diameter less
the plasmalemma and of identical structure. than that of the ribosomes of higher organisms.
Ribosomes. The cytoplasm contains numerous This difference may be an artifact of preparation.
dense particles, which we interpret as ribosomes The bodies are free in the ground cytoplasm and
V()I,. 8G,, 1963 MICROMORPHOLOGY OF D. CONGOLENSIS 1105

PMs
i.

R~~~~~~~~~~

FIG. 5 to 7. Thin sections of Dermatophilus congolensis. Younq filaments in progressive stages of cell
division. In Fig. 5, four segments are distinguished, each showinq nuiclear material (n) of low density as comn-
pared with the adjacent cytoplasm (c); X 30,000. In Fig. 6, transverse septation has occurred; plasmialemn-
mosomes (pns) are visible in most of the resultant segments, X 35,000. In Fig. 7, longitudinal septation is
seen; plasmalemmosomes are numerous. X 30 000.
are generally more concentrated in peripheral ments course throughout the length of the cell
areas (Fig. 12 and 15), but may appear anywhere (Fig. 8 and 24). At an early stage of cell division
inside the cell (Fig. 24). (Fig. 12, uppermost cell) the nuclear filaments are
Nuclear material (nucleoid, nuclear apparatus, stretched across the central area. Individual fila-
nucleoplasm). The nuclear apparatus consists of ments, whose appearance may be related to the
thin electron-dense filaments (about 25 to 50 , stage of nuclear division, are clearly seen in Fig.
in diameter) appearing as coiled or twisted long 15.
threads or, in cross section, as numerous dots
(Fig. 15 and 24). A ropelike appearance first de- DIscusSION
scribed by van Iterson and Robinow (1961) is Nlicrographic demonstration of filamentous
demonstrated in Fig. 24. Bundles of parallel fila- cells, and particularly of their branching, is criti-
1106 GORDON AND EDWARDS

:Agl'y.,Sj;_Mta:si-w3*f.,'§ .fr
:. aoW'
.,
;tS:,:-...ji_.tS.

-
1 _ - _K
_ji;
\./ *

s
J. BACTERIOL.

*~
u4<*# ~ 2__ 9.t>i

FIG. 8. Thin sections of Dermatophilus congolensis. Portion of an elongated cell (young filament) at
higher magnification (X 120,000), illustrating internal structure. The nuclear apparatus (n), composed of
long dense fibrils, and cytoplasm (c) with numerous dense ribosomes (R) are readily discerned.
FIG. 9. Cross section of a filament showing "pizza" type of division, i.e., longitudinal septation in several
planes. X 80,000.

cal to the taxonomic placement of Dermatophilus
among the actinomycetes, but preparation of this
material for electron microscopy was difficult, the
young hyphae being delicate and easily suscepti-
ble to lysis or mechanical breakage (Fig. 3). Ma-
terial fixed after only a few hours of incubation
(to obtain "germ tubes") appeared to be particu-
larly susceptible to damage during polymeriza-
tion. Best results (Fig. 8) appeared to follow pro-
longed fixation in osmium tetroxide (Chapman
and Hillier, 1953) and prolonged infiltration with
prepolymerized methacrylate at low temperatures
(4 to 6 C).
Electron microscopy confirms in almost every
VOL. 86, 1963 MICROMORPHOLOGY OF D. CONGOLENSIS 1107
d..-
..f
.I. ''
.:.,;8
.f-

pins

.A

FIG. 10 and 11. Thin sections of Dermatophilus congolensis. Portions of filaments of increased thickness
resulting fromi multiplication of cells within each segment. Symbols: n = nucleoid (nuclear apparatus); cw
= cell wall; R = ribosome; pms = plasmalemmosome (internal membrane); s = septum. X 60,000.

respect the concept of the Dermatophilus life cycle
derived from light microscopy. A question unre-
solved by the latter was the manner of liberation
of coccal forms from the mature holocarpic myce-
lium. Our studies reveal that this occurs by gelat-
inization and dissolution of the thickened cell-
wall material, both interlocular and peripheral,
with the layer immediately adjacent to each pro-
toplast remaining intact to constitute the spore
cell wall. The extracellular capsule or slime sheath
1108 GORDON AND EDWARDS J. BACTERIOL.

FIG. 12. Thin sections of Dermatophilus congolensis. Advanced stage of segmentation. In each cell, nuclear
material (n), ribosomes (R), and plasmalemmosomes (pms) are seen. The upper cell is in an early stage of
division. Note its ingrowing septum (s) and the dense nuclear fibrils across its center. The arrow indicates
sloughing cell-wall material, or capsule. The original cell wall (cw) is in process of dissolution, leaving a
thin wall around each developing spore. X 120,000.
voL. 86, 1963 MICROMORPHOLOGY OF D. CONGOLENSIS 1109

FIG. 13 to 15. Thin sections of Dermatophilus congolensis. Cells showing lamellate plasmalemmosonies
("onion bodies"). In Fig. 13, a large membranous body is located in the area of cross-wall formation (s);
X 80,000. In Fig. 14, the membranous structures (pms) occupy most of the area of the sections. The lower one
is continuous with the plasma membrane (pm); X 121,000. In Fig. 15, coiled nuclear fibrils (F) are seen;
arrows indicate capsular niaterial resulting from break-down of the cell wall. X 200,000.
1110 GORDON AND EDWARDS J. BACTER[OL.

FIG. 16 to 18. Thin sections of Dermatophilus congolensis. Portions of peripheral cells further demonstrat-
ing dissolution of wall material: (16) X 300,000; (17) X 120,000; (18) X 200,000.

may, therefore, in a sense be considered a primi-
tive multilocular sporangium. It does not always
undergo complete disintegration, "ghost forms"
composed of a branched, tubular, empty matrix
being observable by light microscopy. Sheaths
were recognized in Dermatophilus by Van Saceg-
hem (1934) and slime layers, or capsules, were
found by Pfennig (1958) to be common on vegeta-
tive hyphae of Streptomyces spp.
Examination of several strains of Dermatophilus
and a review of the literature reveal that there is
a great deal of variation in the time required for
completion of the life cycle. Formation of cocci
and their liberation as motile spores may take
place as early as 24 hr after initiation of hyphal
growth or may be delayed for several days. In
some strains, the spores germinate while still
within the sporangium, so that motile forms are
never found.
Our observations do not account for the motil-
VOL. 86, 1963 MICROMORPHOLOGY OF D. CONGOLENSIS 1111

FIG. 19. Thin sections of Dermatophilus congolensis. Group of mature coccoid (sporelike) cells enmeshed
in capsular material showing fine filamentous structure. Filaments are fewer and more widely spaced peri-
pherally (cm-2) than in center (cm-i). X 60,000.
FIG. 20. Capsular material under higher magnification (X 120,000) showing a network (arrows) of fine,
dense filaments.
ity of the freed cocci; thin sections have revealed vides valuable clues to its taxonomic status. The
no flagellar apparatus such as that demonstrated germination of coccoid forms to produce fine,
by Thompson (1954). This does not, of course, branching hyphae places this organism among the
deny the existence of flagella, since our failure to Actinomycetales, group B of Waksman and Hen-
find them may be attributable to inadequacy of rici (1943). Austwick (1958) proposed a new
the technique. family, Dermatophilaceae, to be related to BI
Ultrastructural analysis of Dermatophilus pro- Actinomycetaceae, the fragmenting actinomy-
1112 GORDON AND EDWARDS J. BACTERIOL.

FIG. 21 and 22. Thin sections of Dermatophilus congolensis. Cells in incipient plasmolysis promoted
by addition of sucrose (15%) to the fixative solution. The plasma membrane (pm) and membranes of the
plasmalemmosomes (pnis) are clearly visible; note the similarity between these "unit" membranes. Arrows
indicate capsular material. X 200,000.
FIG. 23. Portion of dividing cell, demonstrating relationships of cell wall (cw), ingrowing septum (s),
and plasma membrane (pm). X 200,000.

cetes. The Actinoplanaceae, with sporangia and
nonfragmenting mycelium, would be placed in
BII, together with the Streptomycetaceae. Elon-
gate cells similar to those of Dermatophilus have
been seen also in mycobacteria, among the Acti-
nomycetales. Hesseltine (1960) proposed a phylo-
genetic scheme for this order.
The internal structure of the cells is that of a
bacterium, with nucleoid and an endomembrane
system. The first clear demonstration of a bacte-
VOL. 86, 1963 MICROMORPHOLOGY OF D. CONGOLENSIS 1113

F'IG. 24. Thin section of Dermatophilus congolensis. An early stage of protoplast formation. The ropelike
appearance of the nuclear material (n) and the numerous ribosomes (R) are readily seen. Note that in this
condition the membranes of the plasmalemmosome (pms) are not well defined. X 200,000.
1114 GORDON AND EDWARDS
rial nuclear apparatus by electron microscopy
was that of Chapman and Hillier (1953). With
improved techniques, Kellenberger et al. (1958)
and Ryter and Kellenberger (1958) were able to
preserve the fine fibrils of deoxyribonucleic acid
in the nucleoplasm of Escherichia coli and to dem-
onstrate that certain coarse filamentous material
previously observed by others was in part arti-
fact. There have since been a number of reports
of a similar apparatus in various bacteria (for
reviews, see Robinow, 1962; Glauert, 1962). We
have obtained excellent preservation of the nu-
clear apparatus in Dermatophilus.
Plasmalemmosomes in the form of "onion bod-
ies" were found to be a consistent feature of the
cell structure. Similar but not identical lamellate
organelles have been reported in mycobacteria
(Takeya et al., 1959; Koike and Takeya, 1961;
Imaeda and Ogura, 1963), Streptomyces coelicolor
(Glauert and Hopwood, 1959), Bacillus spp.
(Fitz-James, 1960; Giesbrecht, 1960; Robinow,
1962), and Listeria monocytogenes (Edwards,
1963; Edwards and Stevens, 1963). However, in
none of these were the structures as uniform as
they appear in Dermatophilus. The continuity of
these organelles with the plasmalemma provides
additional justification for the proposed appella-
tion of plasmalemmosome (Edwards, 1962). The
inadequacy of other proposed names, such as
mesosome, was discussed by Edwards and Stevens
(1963). The term peripheral bodies (Chapman
and Hillier, 1953) is more appropriate to the or-
ganelles of Dermatophilus but lacks reference to
their association with the plasma membrane. The
frequency of occurrence of plasmalemmosomes in
dividing cells, and their location at the inner rim
of ingrowing cross walls, are highly suggestive of
a role related to septation and cell-wall synthesis.
Similar observations were reported by Chapman
and Hillier (1953) and Imaeda and Ogura (1963).
The exact function of the plasmalemmosomes in
Iermatophilus is as yet undetermined.
Bacterial plasma membranes have been de-
scribed by several authors (reviewed by Robinow,
1962) but there has been some difference in inter-
pretation of their structure (discussed by Ed-
wards and Stevens, 1963), possibly because of the
use of different techniques in the preparation of
material for electron microscopy. A classical
"unit" membrane has been demonstrated in sev-
eral organisms, such as Actinomyces bovis (Ed-
wards and Gordon, 1962) and Bacteroides (Bla-
den, 1963). In others, e.g., Bacillus spp.
J. BACTERIOL.
(Fitz-James, 1960) and Listeria (Edwards and
Stevens, 1963), the plasma membrane appears to
be more complex.
ACKNOWLEDGMENTS
The assistance of Robert J. Bendon and Robert
Alger is gratefully acknowledged. This work was
aided by grant A104589-02 from the National
Institute of Allergy and Infectious Diseases, U.S.
Public Health Service.
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