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 pleuropneumonia 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 microscopic 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. According to Freundt (1952), branching filaments also
are present occasionally in the cultures of other
pleuropneumonia-like strains. Reproduction occurs 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 organisms 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 investigation were defrayed by grants from the Commonwealth 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 preceding paper (Dienes, 1953). On the other hand,
repiesentative micrographs were obtained without 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 inoculated surface and lifted immediately. They were
examined with the high dry lens of a light microscope, and those in which the distribution of
organisms seemed appropriate were used. Impressions were made directly from serum agar
cultures of pleuropneumonia-like organisms. Extraction of the agar with distilled water, either
neutral or made slightly acid with phosphoric
acid before the screens were made, neither improved nor harmed the preparations. The screens
weere surprisingly free of aitifacts, and the organisms 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 transferred 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 preparations (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 culture 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 preparations 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 granules of different size, is similar in the photographs
made by light and by electron microscopy (photographs 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 preparations (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 preparations, 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 sufficient 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 appearance of organisms in micrographs 16 and 17 suggests 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 micrographs 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

needs further study. The disintegration of the

organisms into several smaller ones is not their
only method of reproduction. The young colonies
extend on the surface and into the agar in irregular strands like bacterial colonies, and the organisms at the edge of these colonies probably
reproduce in the same way as bacteria.
DISCUSSION

The morphology of two human strains of

pleuropneumonia-like organisms and of the L cultures of a Proteus strain, as demonstrated by the
electron microscope, appeared similar to that
seen with the light microscope. The pleuropneumonia-like strains consisted of fairly uniform
granules, the smallest being about the same size
as is indicated by filtration (0.17 to 0.25 ,u).
In 72 hour old cultures, many organisms appeared
to be empty. In young cultures of the oral strains,
the granules presented dense areas and seemed to
be about to disintegrate into smaller forms. It was
indicated earlier that the interpretation of these
forms is uncertain.
The gradual transition from small granules into
large bodies several As in size, characteristic of
many cultures of pleuropneumonia-like organisms, was not apparent in the electron micrographs of the examined strains. This transition
was visible in the micrographs made from Proteus
L cultures. The electron micrographs confirm the
morphology of these cultures observed with the
light microscope. It adds the new information
that the size of the smallest granules, which seem
analogous to the larger ones according to density

[VOL. 66

disintegration of the latter. It is possible that the

smallest granules represent the intracellular phase
of their development and they are not able to
grow isolated on our media.
It is of interest that the only characteristic
structures visible in the electron micrographs were
those seen also with the light miicroscope. No
flagella, no tailed granules like the phage particles,
and no filaments were seen in them. It should be
mentioned especially that filaments have not been
seen either by phase contrast or in stained preparations in the two pleuropneumonia-like strains
in any stage of their development on agar or in
broth. Oerskov's (1942) and Freundt's (1952) proposal that the pleuropneumonia group should be
defined by the development of a branching undivided mycelium is certainly not applicable to
these strains. The fine filaments which Tulasne
(1950) described in Proteus L cultures were not
observed either with phase contrast or with the
electron microscope, nor were the distorted forms
connected with short filaments, which some authors regard as characteristic of the pleuropneumonia group (Sabin, 1952).
SUMMARY

Electron micrographs from genital and oial

strains of human pleuropneumonia-like organisms and from L cultures of Proteus strains are
published in this paper. They show elements essentially similar to those visible with the light
microscope. However, in the Proteus L cultures,
there are granules the size of which is below the
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
forms of bacteria. J. Bact., 66, 274-279.
served, new growth always starts from the large
bodies. The viability of the granules of somewhat DIENEs, L., AND MADOFF, S. 1953 Differences
between the oral and genital strains of pleurolarger size (0.3 to 0.5 ul) is supported by the oborganisms. Proc. Soc. Exptl.
pneumonia-like
that
similar
out
servation
of the
granules grow
Biol. Med., 82, 36-38.
large bodies and form the young colonies. As DIENES,
L., AND WEINBERGER, H. J. 1951 The
mentioned above, the size of the smallest viable
L forms of bacteria. Bact. Revs., 15, 245-288.
in
L1
granules the was estimated by filtration to FREUNDT, E. A. 1952 Morphological studies of
be between 0.175 and 0.25 ,P. In the floating Prothe pleuropneumonia organism (Micromyces
teus L cultures, most of the granules develop inperipneumoniae bovis). Acta Path. Microside 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 Streptobacillus 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 peripneumonia 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, Philadelphia.

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 pleuropneumonia-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 arrangement 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 Proteus. 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 indicating 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 organisms 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 organisms in nos. 16 and 17 seem to contain several much smaller granules.