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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- similar difficulties with agar cultures of L forms
pneumonia group and of the L forms of bacteria of Salmonella and Proteus species and obtained
are visible with the light microscope. Their appropriate preparations only from the floating
morphology has remained controversial for a long cultures of the Proteus L described in the preced-
time because they are easily deformed in micro- ing paper (Dienes, 1953). On the other hand,
scopic preparations and give riise to bizarre forms. repiesentative micrographs were obtained with-
It seems well established at present (Klieneberger out difficulty from the cultures of an oral and of a
and Smiles, 1942; Dienes, 1945) that the cultures, genital strain of human pleuropneumonia-like
with a few exceptions, consist of round forms organisms (Dienes and Madoff, 1953). A selection
which vary in size by continuous transition from of these micrographs is presented in this paper.
barely visible ones to those several micra in To facilitate the interpretation of the electron
diameter. The exceptions are the organisms of micrographs, photographs obtained with light
bovine pleuropneumonia and of agalactia in the microscopy from stained preparations an(d with
cultures of which the small granules grow out phase contrast from the same cultures also are
into fine branching filaments which later develop presented.3
swellings or break up into other granules. Accord-
ing to Freundt (1952), branching filaments also METHODS
are present occasionally in the cultures of other The simplest method of preparation gave the
pleuropneumonia-like strains. Reproduction oc- most useful screens. The Proteus L culture, lifted
curs either by binary fission as in bacteria or by from broth, was smeared lightly on horse serum
the production of small forms in varying numbers agar, and the screens were placed on the inocu-
inside the larger ones (Dienes and Weinberger, lated surface and lifted immediately. They were
1951). The size of the smallest viable element in examined with the high dry lens of a light micro-
one bacterial L form, an old Li isolated from scope, and those in which the distribution of
Streptobacillus moniliformis (Klieneberger-Nobel, organisms seemed appropriate were used. Im-
1949), has been determined by filtration through pressions were made directly from serum agar
gradocol membranes. It was found to be at the cultures of pleuropneumonia-like organisms. Ex-
borderline of visibility between 0.175 and 0.25 I,u traction of the agar with distilled water, either
about the same as in the pleuropneumonia group. neutral or made slightly acid with phosphoric
The electron micrographs of these organisms acid before the screens were made, neither im-
which have been published have added little new proved nor harmed the preparations. The screens
information on their morphology (Freundt, 1952; weere surprisingly free of aitifacts, and the or-
Smith et al. 1948a, b). The fragility of the or- ganisms were sufficiently opaque to permit the
ganisms made it difficult to make preparations making of pictures without shadowing.
appropriate for electron micrography. We had RESULTS
1 This is publication no. 145 of the Robert W.
Photographs made with phase contrast give the
Lovett Memorial Foundation for the Study of best general impression of the various types of
Crippling Diseases, Harvard Medical School. forms present in the L cultures of Proteus. Photo-
2 The expenses in connection with this investi-
gation were defrayed by grants from the Common- ' The electron micrographs were made by
Mrs.
wealth Fund and the United States Public Health Sokal in Dr. Jerome Gross' laboratory at the
Service. Massachusetts General Hospital.
280
19531 ELECTRON MICROGRAPHS MADE FROM L FORMS 2 81t

graph 1 shows the edge of a culture mass trans- graphs 6 and 7. Similar structures, large bodies
ferred to a thin layer of transparent agar. The containing granules, can be seen in stained prep-
round, apparently empty, places are vacuolized arations (Dienes and Weinberger, 1951) and with
large bodies. The largest ones do not result from phase contrast (photograph 2). They indicate
the growth of a single organism but from the the production of granules inside the large bodies.
coalescence of several smaller ones. They contain The organisms were visible less clearly in
liquid, and Brownian movement of granules is screens made from 3A L type cultures grown on
visible in them. Germination of such large bodies agar. In micrograph 8, made from a slightly
has never been observed, and they are probably shadowed preparation, the granules are somewhat
dead structures. In addition to these forms there larger than in the floating cultures. In some prep-
are dense granules in all transitions of size from arations, granules of considerably smaller size,
barely visible ones to large bodies of 3 to 5 an or less than 0.1 u, were present (micrographs 9 and
larger. The proportion of empty and full large 10). Their arrangement in a group or in a short
bodies varies in different cultures. Stained prep- chain with the larger ones suggests in this case
arations (photograph 3) show similar elements. also that all these granules are of similar nature.
The dense granules and full large bodies are We were unable to obtain micrographs with suffi-
darkly stained. The empty large bodies are visible cient contrast from these structures. They were
only in wet preparations. In dry preparations, visible clearly in the original micrograph of no.
they appear as unstained spots. 9 and 10.
The electron micrographs were made from The organisms in both oral and genital strains
areas of the preparations where few organisms of pleuropneumonia-like organisms appeared as
were present and from the edges of thicker cul- small granules in stained preparations and with
ture masses. The largest empty bodies are absent phase contrast. The appearance of 48 to 72 hour
from these places, probably because they lose old cultures in electron micrographs was similar.
their identity during drying. The largest forms in The whole culture seemed to consist of small
the photographs are about 2 u in diameter. Some granules. The size of the granules varies 0.2 to 1
of these are dark and correspond to the full, well , in nmicrographs 12 and 13 made from the genital
stained bodies, visible with a light microscope. strain. No structure is visible in the granules,
The faint shadows probably correspond to empty but there is a marked variation in density. Some
bodies. The smallest forms observed in the prep- appear dark; some are transparent and only their
arations made from the floating cultures were contours are visible. The average size of the
about 0.15 u. Continuous transition in size is granules varied in different cultures of the same
present between the smallest and the largest. The strain and, for example, it was markedly smaller
smallest granules visible with light microscope in micrograph 13 than in 12.
are considerably larger (0.3 to 0.5 ,u) than those The organisms in the preparations made from
which can be seen in the electron micrographs. 72 hour cultures of oral strains were similar but
The stained preparations and phase contrast somewhat smaller than in the genital strains. The
evidently do not show the smallest elements of average size is around 0.3 u. In the preparations
the culture. The arrangement of the organisms made from 24 hour cultures, the organisms are
in pairs or short chains, often consisting of gran- even smaller, many being only 0.2 u. In contrast
ules of different size, is similar in the photographs to older cultures, their shapes were not regular
made by light and by electron microscopy (photo- and their density was not uniform. The appear-
graphs 4 and 5), suggesting that the smallest ance of organisms in micrographs 16 and 17 sug-
granules seen in the electron micrographs are of gests that transplanted organisms disintegrate to
the same nature as the larger ones. In some smaller ones less than 0.1 p in size. The dense
preparations made from the floating cultures, areas in the larger granules probably correspond
there are many round granules 0.15 to 0.25 u in to the formation of smaller granules within them.
size (photograph 5). In others, somewhat larger Disintegration of the organisms into small forms
granules 0.25 to 0.5 p are more numerous. The can be observed in the large bodies with the light
large forms do not always have uniform density. microscope. Whether the forms seen in the micro-
They present dark areas wnich in some cases are graphs show a similar process and indicate that
entirely similar to the small granules. Two such growing elements less than 0.1 p in diameter may
large bodies are marked with arrows in micro- 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 283
KLIENEBERGER-NOBEL, E. 1949 On Streptobacil- SMITH, W. E., HILLIER, J., AND MUDD, S. 1948a
lus moniliformis and the filterability of its Electron micrograph studies of two strains of
L-form. J. Hyg., 47, 393-395. pleuropneumonia-like (L) organisms of human
KLIENEBERGER, E., AND SMILES, J. 1942 Some derivation. J. Bact., 56, 589-590.
observations on the developmental cycle of SMITH, W. E., MUDD, S., AND HILLIER, J. 1948b
the organisms of bovine pleuropneumonia and L-type variation and bacterial reproduction
related organisms. J. Hyg., 42, 110-123. by large bodies as seen in electron micrograph
OERSKOV, J. 1942 On the morphology of peri- studies of Bacteroides funduliformis. J.
pneumonia virus, agalactia virus and Seifiert's Bact., 56, 603-618.
microbes. Acta Path. Microbiol. Scand., 19, TULASNE, R. 1950 Quelques donn6es nouvelles
586-590. sur la formation et les caracteres de culture
SABIN, A. B. 1952 The pleuropneumonia group, des formes L du Proteus vulgaris. Compt.
pp. 621-633. Bacterial and Mycotic Infections rend. soc. biol., 144, 1200-1203.
of Man. Second Edition. Edited by R. J. WEISS, L. J. 1944 Electron micrographs of pleu-
Dubos. J. B. Lippincott Company, Phila- ropneumonia-like organisms. J. Bact., 47,
delphia. 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.