ELECTRON MICROGRAPHS MADE FROM L FORMS OF PROTEUS AND TWO
HUMAN STRAINS OF PLEUROPNEUMONIA-LIKE ORGANISMS 1 2
L. DIENES
Department of Bacteriology, M1assachusetts General Hospital and the Robert W. Lovett Memorial
Foundation, Harvard Medical School, Boston, Massachusetts
Received for publication Februarv 27, 1953
The organisms in the cultures of the pleuro-
pneumonia group and of the L forms of bacteria
are visible with the light microscope. Their
morphology has remained controversial for a long
time because they are easily deformed in micro-
scopic preparations and give riise to bizarre forms.
It seems well established at present (Klieneberger
and Smiles, 1942; Dienes, 1945) that the cultures,
with a few exceptions, consist of round forms
which vary in size by continuous transition from
barely visible ones to those several micra in
diameter. The exceptions are the organisms of
bovine pleuropneumonia and of agalactia in the
cultures of which the small granules grow out
into fine branching filaments which later develop
swellings or break up into other granules. Accord-
ing to Freundt (1952), branching filaments also
are present occasionally in the cultures of other
pleuropneumonia-like strains. Reproduction oc-
curs either by binary fission as in bacteria or by
the production of small forms in varying numbers
inside the larger ones (Dienes and Weinberger,
1951). The size of the smallest viable element in
one bacterial L form, an old Li isolated from
Streptobacillus moniliformis (Klieneberger-Nobel,
1949), has been determined by filtration through
gradocol membranes. It was found to be at the
borderline of visibility between 0.175 and 0.25 I,u
about the same as in the pleuropneumonia group.
The electron micrographs of these organisms
which have been published have added little new
information on their morphology (Freundt, 1952;
Smith et al. 1948a, b). The fragility of the or-
ganisms made it difficult to make preparations
appropriate for electron micrography. We had
1 This is publication no. 145 of the Robert W.
Lovett Memorial Foundation for the Study of
Crippling Diseases, Harvard Medical School.
2 The expenses in connection with this investi-
gation were defrayed by grants from the Common-
wealth Fund and the United States Public Health
Service.
280
similar difficulties with agar cultures of L forms
of Salmonella and Proteus species and obtained
appropriate preparations only from the floating
cultures of the Proteus L described in the preced-
ing paper (Dienes, 1953). On the other hand,
repiesentative micrographs were obtained with-
out difficulty from the cultures of an oral and of a
genital strain of human pleuropneumonia-like
organisms (Dienes and Madoff, 1953). A selection
of these micrographs is presented in this paper.
To facilitate the interpretation of the electron
micrographs, photographs obtained with light
microscopy from stained preparations an(d with
phase contrast from the same cultures also are
presented.3
METHODS
The simplest method of preparation gave the
most useful screens. The Proteus L culture, lifted
from broth, was smeared lightly on horse serum
agar, and the screens were placed on the inocu-
lated surface and lifted immediately. They were
examined with the high dry lens of a light micro-
scope, and those in which the distribution of
organisms seemed appropriate were used. Im-
pressions were made directly from serum agar
cultures of pleuropneumonia-like organisms. Ex-
traction of the agar with distilled water, either
neutral or made slightly acid with phosphoric
acid before the screens were made, neither im-
proved nor harmed the preparations. The screens
weere surprisingly free of aitifacts, and the or-
ganisms were sufficiently opaque to permit the
making of pictures without shadowing.
RESULTS
Photographs made with phase contrast give the
best general impression of the various types of
forms present in the L cultures of Proteus. Photo-
' The electron micrographs were made by
Mrs.
Sokal in Dr. Jerome Gross' laboratory at the
Massachusetts General Hospital.
19531 ELECTRON MICROGRAPHS MADE FROM L FORMS
graph 1 shows the edge of a culture mass trans-
ferred to a thin layer of transparent agar. The
round, apparently empty, places are vacuolized
large bodies. The largest ones do not result from
the growth of a single organism but from the
coalescence of several smaller ones. They contain
liquid, and Brownian movement of granules is
visible in them. Germination of such large bodies
has never been observed, and they are probably
dead structures. In addition to these forms there
are dense granules in all transitions of size from
barely visible ones to large bodies of 3 to 5 an or
larger. The proportion of empty and full large
bodies varies in different cultures. Stained prep-
arations (photograph 3) show similar elements.
The dense granules and full large bodies are
darkly stained. The empty large bodies are visible
only in wet preparations. In dry preparations,
they appear as unstained spots.
The electron micrographs were made from
areas of the preparations where few organisms
were present and from the edges of thicker cul-
ture masses. The largest empty bodies are absent
from these places, probably because they lose
their identity during drying. The largest forms in
the photographs are about 2 u in diameter. Some
of these are dark and correspond to the full, well
stained bodies, visible with a light microscope.
The faint shadows probably correspond to empty
bodies. The smallest forms observed in the prep-
arations made from the floating cultures were
about 0.15 u. Continuous transition in size is
present between the smallest and the largest. The
smallest granules visible with light microscope
are considerably larger (0.3 to 0.5 ,u) than those
which can be seen in the electron micrographs.
The stained preparations and phase contrast
evidently do not show the smallest elements of
the culture. The arrangement of the organisms
in pairs or short chains, often consisting of gran-
ules of different size, is similar in the photographs
made by light and by electron microscopy (photo-
graphs 4 and 5), suggesting that the smallest
granules seen in the electron micrographs are of
the same nature as the larger ones. In some
preparations made from the floating cultures,
there are many round granules 0.15 to 0.25 u in
size (photograph 5). In others, somewhat larger
granules 0.25 to 0.5 p are more numerous. The
large forms do not always have uniform density.
They present dark areas wnich in some cases are
entirely similar to the small granules. Two such
large bodies are marked with arrows in micro-
2 81t
graphs 6 and 7. Similar structures, large bodies
containing granules, can be seen in stained prep-
arations (Dienes and Weinberger, 1951) and with
phase contrast (photograph 2). They indicate
the production of granules inside the large bodies.
The organisms were visible less clearly in
screens made from 3A L type cultures grown on
agar. In micrograph 8, made from a slightly
shadowed preparation, the granules are somewhat
larger than in the floating cultures. In some prep-
arations, granules of considerably smaller size,
less than 0.1 u, were present (micrographs 9 and
10). Their arrangement in a group or in a short
chain with the larger ones suggests in this case
also that all these granules are of similar nature.
We were unable to obtain micrographs with suffi-
cient contrast from these structures. They were
visible clearly in the original micrograph of no.
9 and 10.
The organisms in both oral and genital strains
of pleuropneumonia-like organisms appeared as
small granules in stained preparations and with
phase contrast. The appearance of 48 to 72 hour
old cultures in electron micrographs was similar.
The whole culture seemed to consist of small
granules. The size of the granules varies 0.2 to 1
, in nmicrographs 12 and 13 made from the genital
strain. No structure is visible in the granules,
but there is a marked variation in density. Some
appear dark; some are transparent and only their
contours are visible. The average size of the
granules varied in different cultures of the same
strain and, for example, it was markedly smaller
in micrograph 13 than in 12.
The organisms in the preparations made from
72 hour cultures of oral strains were similar but
somewhat smaller than in the genital strains. The
average size is around 0.3 u. In the preparations
made from 24 hour cultures, the organisms are
even smaller, many being only 0.2 u. In contrast
to older cultures, their shapes were not regular
and their density was not uniform. The appear-
ance of organisms in micrographs 16 and 17 sug-
gests that transplanted organisms disintegrate to
smaller ones less than 0.1 p in size. The dense
areas in the larger granules probably correspond
to the formation of smaller granules within them.
Disintegration of the organisms into small forms
can be observed in the large bodies with the light
microscope. Whether the forms seen in the micro-
graphs show a similar process and indicate that
growing elements less than 0.1 p in diameter may
be present in the cultures is only a conjecture and
282 L. DIENES [VOL. 66

needs further study. The disintegration of the disintegration of the latter. It is possible that the
organisms into several smaller ones is not their smallest granules represent the intracellular phase
only method of reproduction. The young colonies of their development and they are not able to
extend on the surface and into the agar in irregu- grow isolated on our media.
lar strands like bacterial colonies, and the or- It is of interest that the only characteristic
ganisms at the edge of these colonies probably structures visible in the electron micrographs were
reproduce in the same way as bacteria. those seen also with the light miicroscope. No
flagella, no tailed granules like the phage particles,
DISCUSSION and no filaments were seen in them. It should be
The morphology of two human strains of mentioned especially that filaments have not been
pleuropneumonia-like organisms and of the L cul- seen either by phase contrast or in stained prep-
tures of a Proteus strain, as demonstrated by the arations in the two pleuropneumonia-like strains
electron microscope, appeared similar to that in any stage of their development on agar or in
seen with the light microscope. The pleuropneu- broth. Oerskov's (1942) and Freundt's (1952) pro-
monia-like strains consisted of fairly uniform posal that the pleuropneumonia group should be
granules, the smallest being about the same size defined by the development of a branching un-
as is indicated by filtration (0.17 to 0.25 ,u). divided mycelium is certainly not applicable to
In 72 hour old cultures, many organisms appeared these strains. The fine filaments which Tulasne
to be empty. In young cultures of the oral strains, (1950) described in Proteus L cultures were not
the granules presented dense areas and seemed to observed either with phase contrast or with the
be about to disintegrate into smaller forms. It was electron microscope, nor were the distorted forms
indicated earlier that the interpretation of these connected with short filaments, which some au-
forms is uncertain. thors regard as characteristic of the pleuropneu-
The gradual transition from small granules into monia group (Sabin, 1952).
large bodies several As in size, characteristic of
many cultures of pleuropneumonia-like organ- SUMMARY
isms, was not apparent in the electron micro- Electron micrographs from genital and oial
graphs of the examined strains. This transition strains of human pleuropneumonia-like organ-
was visible in the micrographs made from Proteus isms and from L cultures of Proteus strains are
L cultures. The electron micrographs confirm the published in this paper. They show elements es-
morphology of these cultures observed with the sentially similar to those visible with the light
light microscope. It adds the new information microscope. However, in the Proteus L cultures,
that the size of the smallest granules, which seem there are granules the size of which is below the
analogous to the larger ones according to density resolving power of the light microscope; they are
and to their arrangement, is smaller than is ap- apparently similar in nature to the larger ones.
parent with visible light. It may be less than 0.1 No other characteristic forms were observed in
p. The viability of granules of this size cannot be the micrographs.
determined in our Proteus L cultures by filtration
because growth can be started only by heavy REFERENCES
inoculation. Filtrates even through coarse Mandel DIENEs, L. 1945 Morphology and nature of the
filters remain sterile. It is apparent that the small pleuropneumonia group of organisms. J.
forms develop into large ones in growing colonies, Bact., 50, 441-458.
but when the development of transplants is ob- DIENES, L. 1953 Some new observations on L
served, new growth always starts from the large forms of bacteria. J. Bact., 66, 274-279.
bodies. The viability of the granules of somewhat DIENEs, L., AND MADOFF, S. 1953 Differences
larger size (0.3 to 0.5 ul) is supported by the ob- between the oral and genital strains of pleuro-
servation that similar granules grow out of the pneumonia-like organisms. Proc. Soc. Exptl.
large bodies and form the young colonies. As DIENES, Biol. Med., 82, 36-38.
mentioned above, the size of the smallest viable L., AND WEINBERGER, H. J. 1951 The
L forms of bacteria. Bact. Revs., 15, 245-288.
L1
granules the was estimated by filtration to FREUNDT, E. A. 1952 Morphological studies of
in
be between 0.175 and 0.25 ,P. In the floating Pro- the pleuropneumonia organism (Micromyces
teus L cultures, most of the granules develop in- peripneumoniae bovis). Acta Path. Micro-
side the large bodies and become free by the biol. Scand., 31, 508-529.
19531 ELECTRON MICROGRAPHS MADE FROM L FORMS
KLIENEBERGER-NOBEL, E. 1949 On Streptobacil-
lus moniliformis and the filterability of its
L-form. J. Hyg., 47, 393-395.
KLIENEBERGER, E., AND SMILES, J. 1942 Some
observations on the developmental cycle of
the organisms of bovine pleuropneumonia and
related organisms. J. Hyg., 42, 110-123.
OERSKOV, J. 1942 On the morphology of peri-
pneumonia virus, agalactia virus and Seifiert's
microbes. Acta Path. Microbiol. Scand., 19,
586-590.
SABIN, A. B. 1952 The pleuropneumonia group,
pp. 621-633. Bacterial and Mycotic Infections
of Man. Second Edition. Edited by R. J.
Dubos. J. B. Lippincott Company, Phila-
delphia.
283
SMITH, W. E., HILLIER, J., AND MUDD, S. 1948a
Electron micrograph studies of two strains of
pleuropneumonia-like (L) organisms of human
derivation. J. Bact., 56, 589-590.
SMITH, W. E., MUDD, S., AND HILLIER, J. 1948b
L-type variation and bacterial reproduction
by large bodies as seen in electron micrograph
studies of Bacteroides funduliformis. J.
Bact., 56, 603-618.
TULASNE, R. 1950 Quelques donn6es nouvelles
sur la formation et les caracteres de culture
des formes L du Proteus vulgaris. Compt.
rend. soc. biol., 144, 1200-1203.
WEISS, L. J. 1944 Electron micrographs of pleu-
ropneumonia-like organisms. J. Bact., 47,
523-527.
 PLATE I
The photographs in plate I were made from a floating Proteus L culture, no. 1 to 3 with the light
microscope and no. 4 to 7 with the electron microscope. The magnification in no. 1 and 3 is X 2,000; in
no. 2, X 3,000; in no. 4, 5, and 6, X 6,000; in no. 7, X 9,000.
Figure 1. Dark phase contrast. The mass of the culture appears as vacuolized large bodies. Only a
few of these large bodies appear dark and are full. The small granules are dark, and all transitional
forms between these and the large bodies are seen.
Figure 2. Dark phase contrast. The large body marked with an arrow contains 5 granules arranged at
the periphery.
Figure 8. Preparation stained with crystal violet. The granules and full large bodies are darkly
stained. The vacuolized empty large bodies are indicated only by the unstained round areas. The ar-
rangement of the granules in pairs and short chains and the variability of their size are characteristic.
Electron micrographs
Figure 4. The individual granules and their arrangement in short chains often consisting of granules
of different size are clearly shown. The granules in the short chains marked with an arrow correspond
in size to the smallest ones visible with the light microscope.
Figure 6. A thickly covered area was selected for this micrograph. Granules of 0.15 to 0.3 , in size
are present in large numbers connected to the large bodies with transitional forms.
Figure 6. The uneven density of several organisms is apparent. In one marked with an arrow, three
dark granules are visible.
Figure 7. The uneven density of the organisms is more apparent with higher magnification. Several
of the small granules seem to be surrounded by a less opaque fringe.

284
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PLATE II
Electron micrographs nos. 8, 9, and 10 were made from an agar culture of 3A L type colonies of Pro-
teus. The magnification is about X 14,000.
Figure 8. A lightly shadowed preparation showing round granules of 0.25 to 0.5 pA in size.
Figures 9 and 10. Nonshadowed preparations. At the upper center of no. 9, there are two organisms
about 1 p& in size partly vacuolized and surrounded with smaller granules. At the right lower corner small
granules varying in size from less than 0.1 to about 0.15 p& are arranged in a half circle probably indi-
cating their production inside of a larger form. In no. 10, many dense granules are less than 0.1 pU in
size.
Photograph no. 11 and micrographs 12 and 13 were made from a human pleuropneumonia-like organism
of genital origin.
Figure 11. Dark phase contrast. It shows small granules at the borderline of visibility. XC 2,000.
Figures 12 and 18. Electron micrographs. Impressions of two different agar cultures of the same strain,
magnification X 6,200 and X 5,900, respectively. The culture consists of round granules, the size of
which is noticeably smaller in no. 13 than in no. 12.
Micrographs 14 to 17 were made from agar cultures of an oral strain of pleuropneumonia-like organ-
isms with higher magnification. X 14,000 to X 14,500.
Figure 14. A 72 hour old culture. The organisms are of uniform size, about 0.3 p, and have an even
contour and density.
Figures 15, 16, and 17. A 24 hour old culture just starting to develop. The size of many organisms is
somewhat smaller than in no. 14, and they often have uneven contours and density. Some of the organ-
isms in nos. 16 and 17 seem to contain several much smaller granules.