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P ath . Microbiol. 29: 459-169 (1966)
Military Institute of Hygiene, Epidemiology and Microbiology, Prague
S tru c tu ra l C hanges in A ging Serum
I.
By J. Cinátl and Z. Marhoul
The formation, under certain conditions, of particulate struc­
tures resembling microbial colonies lias been repeatedly observed
in culture media containing serum or plasma. Although the resemblence is only superficial, these structures have been the cause
of not infrequent mistakes and incorrect conclusions. This led us
to a thorough study of these phenomena.
Altogether 1166 individual and pooled samples of human serum
and sera of 13 animal species were investigated.
M aterials and Methods
1. P rep aration of scrum for incubation.
(a) Native and inactivated serum:
Calf and horse blood was collected at the slaughter house and the serum
was then filtered through an asbestos EKS-I Seitz filter. All other samples were
taken aseplically by venous or cardiac puncture.
The sera were inactivated at 56°C fo r (SO minutes and were then incubated
at 37CC, room temperature, and 4°C and examined at 25 days macro- and
microscopically.
(b) Ileal denalurated serum:
Prepared from sterile samples by autoclaving at 120 ('. for 30 to ISO minutes
in 100 ml bottles or Petri dishes. These were in turn incubated at 37 ('. and
examined after 10-14 days.
Received: 12 Nov., 1965.
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2. P reparation of sterile agar media with serum.
l’PI.O agar Difco was supplemented with 20% of the tested serum. The
ag ar plates were incubated at 37°C and examined after 14 and 25 days. Control
plates were incubated al room tem perature and I 'C. Simultaneously parallel
plates without the serum supplement were studied.
460
C.intitl and Marlioul, Structural Changes in Aging Serum
3. P rep aratio n of serum-free media.
Besides routine blood agar, Kudo agar and m eat peptone agar, we used
thioglycolale blood agar, Difco dried media: Brain Veal Agar. Tom ato Juice
Special Agar. Trvptose and Yeast Extract Agar.
4. P rep aratio n of cell suspensions for incubation on serum agar.
In the experiments ten stable cell lines and prim ary monkey kidney
cultures and trypsinized h u m an enibyro cells were used. After trypsinizalion,
the cells were either washed in a synthetic culture medium [Parker's M 1991
without serum or in PBS (pH 7.4) and transferred to serum agar where they
died within a few hours retaining their shape. The results were read after
14 flays.
5. Staining of the structures.
Staining is possible only after fixation. Flaming and comm on fixatives
such as Carnoy's solution, HgCL, with acetic acid, and formol were used. The
structures were then stained with hematoxylin, aceto-orcein, eosin and light
green. Polysaccharides were determined with periodic acid and Schiff's
reagent. Fats were demonstrated with Sudan III.
After 14 days of incubation structures 300// large were found
on till agar plates containing 20% calf serum. These structures were
either fan-shaped (Fig. 1) or were round and resembled colonies
of I--organisms (Fig. 2).
These were composed of structureless matter and refractile
granules of granular microstructure just barely discernible in the
optical microscope. Around the twentieth day il was observed that
globular formations of varying size, up to 60//, permeate the agar
(Fig. 3). These structures were also found on the agar surface and
among Ihem stages testifying for the possibility of the transition
from globular structures to structures with a structureless and gran­
ular periphery. All these structures, upon transfer in agar blocks to
fresh plates, induced further characteristic growth'. Direct studies
of sterile serum incubated at 37 C disclosed that it becomes opal­
escent and turbid around the twenty-second day. On its surface a
discontinuous greyish membrane is formed. These two phenom­
ena were repeatedly observed in every sample studied. Microscopic
examination revealed that the membrane and turbidity are com­
posed of a great amount of the above described structures; the tur­
bidity consisted almost entirely of globular structures and the mem­
brane of both, round and fan-shaped structures. If such a serum
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¡{exults
461
Fig. /. IM’I.O agar Difco with 20% calf scrum after 14-dav incubation al 37 C.
Spontaneous fan-shaped structure, size 275 fi.
F ig .2. Another plate (as in Fig. 1) with structure resembling a colony
of 1.-organisms, size 330 u.
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Ciiuill and Mar haul. Structural Changes in Aging Serum
Cindtl and Murlwul. Structural Changes in Aging Serum
Fig. 3. 1’PLO agar Difco with calf serum after 2f> days of incubation at 37°G.
Spherical structures within layer of agar medium, size about 55/t.
Fig. !. I’I’I.O agar Difco with ‘20% calf serum. Sediment transferred from calf
serum incubated 12 days at 3 7 ' C. Structures which are surrounded with rays
of structureless matter lie in inoculum lines, size of spherical structures f>-.'i() u.
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462
4 «:$
FUj.it. I’PLO agar Difco with 20% rabbit serum, t o d a y s of incubation at 37°C.
A typical structure characterizing rabbit serum. Granular zone extends into
fine long strands of structureless matter, size 300 u.
Fit/.ii. Structures from a mem brane that formed on lop of a sample of rabbit
serum a lter 30 days of incubation at 37
The structures resemble aclinomyeclal colonies. An illustration of the presence of more than one type of
structure in a serum: size at lower rigid 200 u, upper left 0itu .
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t'.ináll and Murhoul, Structural Changes in Aging Serum
4 «4
('¡mill and Marhoul. Structural Changes in Aging Serum
Fig, 7. Sediment from horse serum incubated 14 days on tryptosc agar williout
serum. Signs of disintegration expressed by herniation of matter from Ilie
structures.
Fig. 8. A hu m an em bryo cell after 14 days of incubation on PPL.O agar with
'20% calf serum. Darkly marginated spherical formation cxcentrically located
in the cell nucleus is the center of the structure. From this there are o u t­
growths of fine ramified filaments of structureless matter. On the filaments
in the serum agar hordering the cell retractile granules are being formed. The
periphery of the structure then ends in abundantly ramified filaments of
structureless matter. Overall size of structure 90 «.
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is spun down and the resuspended sediment transl'ered onto serum
agar, most of these structures shall grow after 12 to 14 days of
incubation. Around these structures rays of structureless matter
are formed (Fig. 4) and they are the basis for the development of
fine refraclile granules. Upon transfer of this material to further
serum agar plates the formation of similar structures is again
induced, this time after an up to by two thirds shorter period of
incubation.
When serum denatured by high temperature is incubated ten
days, there appears material, within it and on its surface, which
does not resemble the above described structures but is able to
induce their formation upon transfer to a serum agar plate.
.Just like we have studied calf serum, we examined serum
samples of humans and animals. In each and every case under
appropriate conditions these structures appeared regularly. In
some sera, as for instance human, these structures were observable
on about day twenty, in other, e.g. equine sera they appeared ten
days earlier. There were also quantitative differences. In equine
serum several times as many structures appear than in calf serum.
In late spring call’ serum gives rise to more of them than in other
seasons.
The structures in the serum do not appear all at once. On
serum agar it is possible to observe how around such a structure
in five to ten days there appear many smaller globular and Ianshaped formations.
The structures which are formed in various normal sera and
serum agar of the same species are fundamentally identical,
although one serum may contain more than one morphological
type of structure (Fig. 6). On the other hand the structures in sera
of different species vary so that it is possible to recognize the
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Cimil l and Mitrhoul, Structural Changes in Aging Serum
465
8
4 (5(5
Cinatl and Mnrlioul, Structural Changes in Aging Serum
animal species by the morphologic characteristics of the structures
in the serum examined (Figs. 1 and 5).
When these structures are transferred to and incubated on me­
dia containing no serum, no changes occur or the structures dis­
integrate. Their disintegration is especially marked on tryptose
agar and on brain veal agar and consists in the formation of blebs
(Fig. 7) and the herniation of fluid matter from the structures. On
other occasions the structures became surrounded by an area
containing granules in Brown's molecular motion. These structures
underwent no changes on media containing blood.
The study of native preparations reveals that the structures
have two constituent parts, i.e. structureless matter and highly
retractile finely granulated granules. Staining makes Ibis even
more apparent. Upon fixation with methods commonly used for
live objects the structures can be stained with hematoxylin, eosin
or light green. They also lake up aceto-orcein dye. The reaction for
polysaccharides is positive. In line with observations of native
preparations fat-soluble dyes also reveal two constituent parts of
which the finely granulated granules take up the dyes.
The formation of the structures on serum agar can be induced
by nuclei of dead cells (Fig. 8). They never originate in the sole
presence of only mammalian erythrocytes or of cell fragments
without nuclei.
These structures which we observed in serum agar and sera
are also formed in incubated sterile plasma.
In these studies we are dealing with little known and from time
to lime rediscovered structures which are usually evaluated as
meaningless artefacts (Laidlaw . 1925). These structures often re­
semble microbial colonies, e.g. colonies of L-organisms (Fig. 2)
and. of course, of bacterial L-forms. Many structures resemble
colonies of bacteria and fungi. In some sera we found structures
imitating actinomycelal colonies in miniature from (Fig. 6). There­
fore we think that such structures may have been the cause of
various errors as they are described by Kelson (1958), or may have
been the basis for erroneous theories (Boshyan , 1949). The same
may have served as the source of the fantastic opinion of certain
Japanese researchers that filtrable forms of bacteria are present
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Discussion
C.inátl and Mitrlwtil. Structural Changes in Aging Serum
4(57
in the blood of every man and animal (Ushiama, 1958). This
similarity to PPLO colonies was also discussed by Laidlow (1925)
and Brown el al. (1940). In 1963 these structures were rediscovered
by Bonifas, judged to be special kinds of organisms, and named
‘Stegasnur (Bonifas . 1963). To the contrary, liny flick (1965) has
studied them in connection with tissue culture PPLO, evaluating
them again only as a potential source of error.
The examination of various sera revealed that the morphology
of these structures is species specific. Interesting results were also
obtained by the study of dead cells incubated on serum agar. The
fact that nuclei of dead cells are the centers of structure forma­
tion indicates that this problem is not only of theoretical but also
of practical importance. At the same lime we are of the opinion
that these findings could be the basis for a new approach to the
study of cellular constituents and cellular material as it has been
pursued by Lepeshinskaya (1950), discounting her erroneous con­
clusions and generalizations and as has been of late brought up
by Anderson (1959) and McClure and Roberts (1958).
Besides their superficial similarity to microbial colonies, it is
necessary to stress several peculiarities which may lead to grave
errors in the evaluation of these structures. For instance, the
induction of further structure formation upon their transfer lo
serum agar plates simulates the passaging of microbial entities.
After several such 'passages' from plate to plate an acceleration
of growth is observed and this in turn reminds us of the phenom­
enon of adaptation. On serum-free agar plates the structures dis­
integrate. The structures formed on the serum surface or on plates
differ from those found within the serum or agar, the source
material having access to the forming structures from all sides.
Notable is their polymorphy and that they are composed of two
substances with difieren! staining properties.
There is no doubt that the structures under study are the result
of abiogenic processes. Bui we are of the opinion that their connec­
tion with biological objects has been well demonstrated.
Structures, on the average .'500 u in size, resembling colonies of bacteria,
fungi and actinomycetes were found on sterile bacteriological agar plates co n ­
taining filtered serum, after long-term incubation at IS"
It was demonstrated
that these structures are not a chance agglomeration or precipitate and that
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Summitry
468
('.iiiiUI and Marhoitl. Structural Changes in Aging Serum
they originate in serum, in the presence of dead cells, and that their formation
is induced b y some material which is present in cell nuclei. Their growth can
he elicited on serum agar by material ‘precipitated' in serum incubated at 37°C
for several weeks. The form of the structures is specific for different anim al
species and may serve to identify the source of the serum sampled. Staining
disclosed that the structures are composed of two different substances of
which only one takes up fat-soluble dyes. The structures can be stained with
hematoxylin and cosin o r light green. The reaction for polysaccharides is
positive.
Zusammenhang
Auf sterilen, filtriertes Serum enthaltenden Agnrplnttcn finden sich nach län­
gerer Bebrütung bei 37 0 ca. 300 » Durchmesser aufw eisende Strukturen, die
an Kolonien von Bakterien. Pilzen und Aktinomyceten erinnern. Die Gebilde
gehen nicht a u f zufällige Agglomerationen oder Präzipitationen zurück, so n ­
dern entstehen im Serum in Gegenwart toter Zellen, und zwar unter FinfluB
von im Zellkern enthaltenem Material. Präzipitale aus Serum, das während
m ehrerer W ochen bei 37 C gehalten wurde, können ihre Bildung auf SerumAgar auslösen. Die F orm der Strukturen ist für verschiedene Tierspecies
spezifisch und kann zur llerkunftsbestim m ung des Serums verwendet werden.
Die F ärbung zeigte, dal.! die Partikeln aus zwei verschiedenen Substanzen be­
stehen, von denen nur eine fett lösliche Farbstoffe aufnimml. Die Strukturen
lassen sieh mit IInomatnxylin-Fosin oder l.iehtgrün färben. Die Polvsaccharidrenktion ist positiv.
liésumé
Des plaques de gélose stérile contenant du sérum filtré présentent après
une longue incubation à 37 ('. des structures de 300 » de diamètre environ cpii
rappellent des colonies de bactéries, de champignons ou d'actinomycètes, (.es
formations ne sont pas la conséquence d'agglomérations ou de précipitations
mais apparaissent dans le sérum en présence de cellules mortes sous l'influence
spéciale de matériel nucléaire. Des précipités de sérum, conservés pendant plu­
sieurs semaines à 37 (., se défont sur la gélose au sérum. D’aspect des struc­
tures est spécifique p o ur chaque espèce anim ale el permet d'identifier l’origine
du sérum. L eur coloration met en évidence dans les particules deux substances
différentes dont une seule absorbe les colorants liposolubles. Files sont colorables p ar l'hématoxyline-éosine el p ar le vert brillant. I.a réaction des poly­
saccharides est positive.
A nderson. .V.
Studies on isolated cell components. X. Structure formation
by clear protein solutions. Fxp. Cell. Res. Hi: -11 (1951b.
lionifus. V. II.: Stegasma ‘Organisms' found in sterile serum of warm-blooded
animals. P ath . Microbiol. 26': 696 (1963).
Iloslujun, (1. M .: O prirode virusov i mikrobov. On the nature of viruses and
microbes. (Medgiz, Moscow 19+9).
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References
Cin/itl and Marhoul, Structural (Changes in Aging Semin
409
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B row n. T. M.; S w ift. II. .S', and W atson, II.
Pseudo-colonies simulating those
of pleuropncumonia-like microorganism. J. Bad. 40:857 |1940).
Ila y flic k . T i s s u e culture and M ycoplasm a. Texas Be|>. Biol. Med.. Snppl. 1,
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Laid law. I‘. I1.: On structures which develop in certain culture media and re­
semble colonies of micro-organism. Brit. J. exp. Path. (>: 30 (1925).
Lepeshinskaya, <>■ B.: Proiskhozhdenie kletok iz zhivovo veshchestva i rol' zhivovo veshchestva v organisme. The origin of cells from live matter and the
role of live matter in the organism (Medgiz, Moscow 1950).
McClure. I', and Roberts, B. B.: The formation of prolomorphs. in: First Sym­
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p. 151 i New York 1958).
X clson. E.
The development in vitro of particles from cytoplasm. 1. Obser­
vation on particle development in hacteriologic media. .1. exp. Med. It)7: 755
(1958).
I'shiyam a . .1.: A process for preparing antibiotic substance from special bac­
terium in h um an blood plasma an d earth. Patent specification 791,142 (The
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