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THE USE OF THE CHORIOALLANTOIC MEMBRANE OF THE DEVELOPING

CHICK FOR CULTURE OF DERMATOPHYTES—A MODIFIED TECHNIC
A PRELIMINARY REPORT UPON ITS USE FOR SERIAL PASSAGE*
BETTY M. PARTRIDGE, M.A.

The chorioallantoic membrane of the develop- downy and granular strains in culture is T. men-
ing chick embryo provides a suitable living sub- tagrophytes. A conversion of these downy forms
strate for the cultivation of pathogenic fungi. into granular forms, during the course of serial
This was first demonstrated by Moore (1) in passage in guinea-pigs, was observed by Georg
a histo-pathological study using a variety of (4). As the growth and production of lesions on
fungi, including four dermatophytes: Micro- the chorioallantoic membrane by dermato-
sporum canis, Epidermophyton floccosum (E. phytes was known, from earlier workers, to be
inguinale), Trichophyton mentagrophytes (T. rapid compared with other laboratory animals,
gypseum) and T. schoenleini (Achorion Schoen- it was decided to investigate the use of this
leini). More recently, Showalter (2) studied technic as a method for passage of cultures. By
the morphological changes of the first three the serial passage of T. rubrum from one chick
dermatophytes, listed above, when in contact embryo membrane to another, it was hoped to
with the living chick membrane. determine whether there was correlation between
The object of the investigation, described strains and any alteration in their morphology
in the present paper, was primarily to develop and pathogenicity after passage.
an improved in vivo technic for cultivating
METHODS AND MATERIALS
dermatophytes in the chorioallantois, and to
compare the results so obtained with those of The cultivation technic employed by Moore
previous workers. The dermatophyte chosen was based on that described by Goodpasture (5)
for investigation was Trichophyton rubrum, an and his collaborators in their work on viruses.
organism which was not grown on the chori- Briefly this involved preparing 10 to 14 day old
oallantoic membrane by either Moore or Sho- fertile hens eggs by cutting a 'window' in the
waiter. shell, 1cm. square, with a carborundum disc.
Trichophyton rubrum is of particular interest The underlying shell membrane was also cut
because of the frequency with which it causes and the shell and its membrane carefully re-
long-standing ringworm in man. A study of the moved to expose the chorioallantoic membrane,
clinical manifestations of infections caused by which was then inoculated with a saline suspen-
this fungus, and the morphology of the strains sion of the fungous culture. The 'window' was
isolated, has shown little direct relationship. A closed by surrounding it with a layer of vase-
similar conclusion was reached by Silva, Kesten line-paraffin and applying a cover slip. The
and Benham (3) in studies of their T. rubrum eggs were incubated, 'window' uppermost, at
isolates. Cultures of T. rubrum vary consider- 34°C.
ably and range from white, downy forms to Burnet (6) described a modification of Good-
granular and occasionally dysgonic forms; the pasture's technic for chorioallantoic inoculation,
rate of pigment production also varies with the in which an artificial air-space was created above
strain. Both granular and downy forms of this the chorioallantois to give a larger area for in-
organism may occasionally produce hair infec- oculation. This tchnic has been widely utilized
tion, usually merely a follicular invasion; simi- in the study of viruses and rickettsiae (Bever-
larly for glabrous skin and nail infections. idge and Burnet, (7)), and was used by Sho-
Another dermatophyte which produces both walter in his dermatophyte studies. He em-
ployed Moore's technic of cutting a 'window' in
* From the Department of Medical Mycology, the shell and shell membrane to form a large
Institute of Dermatology, London, W.C.2., Eng- opening into the artificial air-space. The inocu-
land.
Received for publication September 8, 1958. lum was applied by means of an inoculating
605
606 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

needle to the exposed surface of the choriollan- lying chorioallantoic membrane. Using a standard
toic membrane, and the 'window' sealed with rounded steel burr, a circular 'window', 3 mm.
a cover slip and vaseline-paraffin as before. diameter, was carefully drilled through the shell
to expose the shell membrane. By this procedure,
I. General Technic accidental damage to the underlying membranes
was minimised, compared with previous tech-
The technic employed in the present study niques of cutting and picking away a larger area
also embodies the use of the artificial air-space of the shell. A smaller aperture into the air-space
above the chorioallantois, with modifications was made by drilling through the shell and shell
by Alexander (8). The principle feature of this membrane with a finer burr, or puncturing them
method is the small amount of manipulation with a needle. (Diagram I, A.)
of the chorioallantoic membrane required dur- Separation of membranes—A drop of sterile
ing inoculation. This decreases the development physiological saline was placed over the circular
of non-specific lesions due to trauma which area of exposed shell membrane which was then
may cause difficulty in the interpretation of carefully slit, using a sterile needle and pressing
down and backwards at right angles to the fibres.
any fungous lesions produced. Little trouble is Air was withdrawn from the air-space by gentle
caused by air-borne contaminants, probably suction with a rubber teat placed over the aper-
because of the natural resistance of the egg ture, and the chorioallantoic membrane separated
tissues. However, as this work had to be carried and dropped away from the shell membrane to
out in a routine bacteriology and virus labo- produce an artificial air-space with the chorio-
ratory, any risk of contamination and dissemi- allantoic membrane as its 'floor'. The saline was
nation of fungus spores, due to cover-slip dis- utilised as a fluid wedge during this stage and its
placement, for example, had to be minimized. disappearance acted as an indicator that the mem-
The following method has been used and proved branes had separated. Prior to inoculation, the
egg was again candled to determine the extent and
effective. position of the artificial air-space, and to ensure
Inoculation
that the original air-space had been obliterated.
The area of chorioallantoic membrane available
Pre-incubation—Fertilised eggs were incubated for inoculation was approximately 2 to 2.5 cm. in
in a standard electric, thermostat-controlled egg diameter, with the overlying shell membrane
incubator, with a temperature of 37°C, forced air- about 5 mm. above its centre. (Diagram I, B.)
circulation and relative humidity of 60 per cent. It was important to drop the chorioallantoic
The eggs were automatically turned twice daily. membrane and carry out inoculation as soon as
Ten days incubation was allowed—the minimum possible after drilling, otherwise it tended to ad-
period fnr develnpment nf the chorioallantois. here to the shell membrane, making it difficult to
Candling end drilling—The fertile eggs were avoid puncture of the chorioallantois and causing
selected by candling and the limits of the air- minor trauma in the area corresponding to the
space marked nn the shell. The pnsitinn of the opening in the shell.
embryo was noted by its spontaneous movements, Inoculum—The unexposed chorioallantoic mem-
and a point marked above it on the shell, care brane was inoculated by introducing a saline sus-
being taken to avoid blood vessels in the under- pension of the fungus into the artificial air-space.

.1

-AIR SPACE

HEM ERA NE S

DIAGRAM 1. Arrangement of membranes and cavities in a 10-day old chick embryo, (A) before, and
(B) after preparation for inoculation, as seen in longitudinal section. Points of drilling (arrowed).
 CHORTOALLANTOIC MEMBRANE FOR CULTURE OF DERMATOPHYTES 607

For this study, fungous suspensions were made by These were stained by the periodic-acid-schiff
vigorous shaking of a loopful of mycelium and method in order to trace the invasiveness of the
spores from a fresh Sabouraud's agar slope culture fungus, and with haematoxylin and eosin to de-
in 2 to 3 ml. of sterile physiological saline. Larger tect any cellular reaction. The other half was
fragments of mycelium were allowed to settle out, ground up with a few drops of sterile physiological
and the cloudy supernatant fluid containing spores saline in a Griffith's tube, and the emulsion used
and hyphal fragments was used for inoculation. for culture.
For large-scale work a uniform inoculum is recom-
mended. Using a 1 ml. sterile syringe, the fungous II. Technic for Serial Passage
suspension was introduced by inscrting the tip of Materials Used—For this prelimiuary survey,
the needle through the slit in the shell membrane, 3 forms of recently isolated Trichophyton rubrurn
and by holding the syringe vertically, slowly
ejecting the inoculum on to the chorioallantoic strains were chosen. Single-spore cultures were
membrane without touching either it or the shell obtained by growing spore suspensions on corn-
membrane. Extensive trauma of the chorioal- meal agar and isolating single germinating
lantois must have been caused by Showalter's spores (Georg, (9). A typical single-spore cul-
technic of inoculating the exposed membrane di- ture of each strain was selected. The type of
rectly with an inoculating needle, hut such damage colony it produced on a Sabouraud's agar plate,
was practically eliminated by the above pro- and its origin were as follows:
cedure. In this pilot study, 0.1 and 0.2 ml. fungous Strain (a), No. 4244—granular, well-pigment-
suspension was used as an inoculum. ing colony, isolated from a scalp kerion with hair
Incubation—After inoculation, the two aper- involvement.
tures in the shell were sealed with triangles of
"Sellotape". The eggs were placed on slatted wire Strain (5), No. 4497—semi-downy, well-
trays, with the circular aperture uppermost, and pigmenting colony, isolatcd from a groin in-
incubated at 34°C. fection.
Throughout this technic, care was taken to Strain (c), No. 4974—white, downy colony,
avoid shaking the live embryo or allowing the egg with slow pigment production, also isolated
to cool down. Once the circular aperture was cut, from a groin and axilla infection.
the egg was kept with this uppermost to avoid All 3 strains produced abundant, typical micro-
disturbing the position of the artificial air-space. conidia, and red pigment on cornmeal dextrose
agar. A few macroconidia were present in
Harvesting
strain (a).
The eggs were usually harvested after 4 and 7 Inoculation and Harvesting—Using the tech-
days incubation, when the chick embryos were 14 nic described, two or more eggs (depending on
and 17 days old respectively. A longer incubation availability) were inoculated with each strain,
period was found to be unwise, as the period of in-
cubation before the chick hatches is approxi- and the membranes harvested after 4 and 7 days
mately 21 days. incubation at 34°C.
On harvesting, the egg shell was cut in half Culture and Passage—Two sets of cultures
horizontally, using fine scissors, and the upper were made from the emulsion prepared from
half, containing the chorioallantoic membrane, re- each infected membrane. Each set consisted of
moved and upturned in a petri dish for examina- one plate and two slants of Sabouraud's agar,
tion of the artificial air-space. With a fine scalpel, and two malt tellurite agar slants (for any bac-
a cut was made, leaving a centimetre margin terial inhibition). These were then incubated,
around the periphery of its 'floor', and the chorio- one set at 25°C. (our standard temperature for
allantoic membrane gently stripped away and dermatophyte growth) and one at 30°C. to
placed in a petri dish in sterile physiological
saline. Stained smears were made of any fluid boost the growth rate. The cultures were ex-
present within the artificial air-space to detect the amined weekly and subcultures made when
presence of germinating or non-germinating necessary. As the dermatophyte used in this
fungus spores, mycelial fragments and any con- survey was a relatively slow grower, no cultures
taminating organisms. The chorioallantoic mem- were discarded under a month. Of the cultures
brane was examined against a dark background recovered from the infected membranes, a
and the presence of opacities and foci of infection single clean isolate from a 7 day infection of
noted. It was then divided into two. One half was each strain was selected and used for inoculating
placed in formol-saline and used for sections. a new batch of 10 day old fertile eggs. By this
608 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

method, 6 serial passages, referred to as pas- haps the inoculum was most concentrated, al-
sage I, II, etc., were made throughout the course though there was always the possibility that
of approximately one year. part of the tissue reaction in this area was non-
Of the isolates recovered after each serial pas- specific, resulting from slight trauma associ-
sage, a subculture was kept and a comparison ated with inoculation. As a result of separation,
of the morphological changes was made after the area of membrane forming the 'floor' of the
each passage, and at the end of the series. artificial air-space was always slightly thicker
Plates of Sabouraud's agar (25 ml. per plate) compared with the surrounding chorioallantoic
were inoculated singly with each series of iso- membrane in contact with the shell membrane.
lates, and the plates incubated at 30°C. Macro- Slight hemorrhage always occurred with sepa-
scopic and microscopic examination of each ration of the two membranes, giving rise to
colony was made. slight opacity or granulation around the pe-
riphery of the 'floor'. Hemorrhage was clearly
RESULTS visible as pigmentation on occasions where this
I. General Observations operation had been too hasty or clumsy.
The general appearance of the infection was
Macroscopic appearance of focal lesions the same for all 3 strains of T. rubrum. Four
By using the technic described for inocula- days after inoculation, the shortest period be-
tion of the chorioallantoic membrane, the foci fore harvesting, the lesions were manifest as
of infection were well distributed, with a slight opaque, almost white nodules, varying in size
tendency to accumulate around the periphery from 1 to 3 mm. in diameter. With a longer
and also to develop in the centre, where per- incubation period, i.e. 7 or 8 days after inocu-

FIG. 1. Chorioallantoic membrane showing foci of infection (arrowed) by Trichophyton rubrum. (7

day infection, strain (a). Stained Periodic-acid-schiff.) X2
 CHORIOALLANTOIC MEMBRANE FOR CULTURE OF DERMATOPHYTES 609

FIG. 2. Development of parasitic phase (arthrospores) on contact with ectoderm. (8 day infection,
strain (a), passage I. Stained P.A.S.) X600

lation, the general response was more pro- itself is a thin sheet of tissue composed of an
nounced (Fig. 1.) The membrane had become outer cctoderm and inner entoderm, each con-
even more thickened and nodular, the lesions sisting of a single layer of flattened cells. Be-
tending to become confluent and deepening to tween these lies the mcsodcrm, which acts as a
cream in color. Inoculation of one batch of supporting tissue to the network of blood ves-
eggs (passage II) was tried after only 7 days sels and capillaries.
incubation instead of the standard period of Trichophyton rubrum grew readily on these
10 days, and the membranes harvested after a tissues, and their response to fungous invasion
further 7 days incubation. Although the chori- followed the same pattern for all 3 strains. The
oallantois was not fully developed at this early development of this organism from the sapro-
stage, a mild infection was visible, comparable phytic phase, i.e. spores and hyphac present in
with a 4 day infection in the previous batch the inoculum, into a parasitic phase and its
(passage I.). adaptation to a parasitic mode of life was rapid.
Not infrequently, in later passages, a white Sections of the membrane harvested 4 days
aerial fungous mycelium was visible, growing after inoculation showed that this had already
up from the nodules. On occasions, and with taken place. The spores and hyphac of the inocu-
strain (c) in particular, fungous invasion ap- lum had germinated to form mycelium which
peared to have spread into the original air-space was frequently seen as a loose aerial web of
where it produced a white aerial growth on the hyphac over parts of the membrane. Penetra-
shell membrane. This spread may have been tion occurred where the hyphac were in con-
caused by the close position of the artificial and tact with the cctoderm. The initial reaction of
original air-spaces or to clumsy handling of the the membranes was hypcrplasia with prolifer-
egg after inoculation, but it was possibly due to ation of the cctoderm where fungal hyphac had
the increase in parasitism of the fungus, es- infiltrated. Little or no reaction was shown by
pecially as this phenomenon tended to occur the underlying mcsodcrm in these areas. Chains
in the later passages. That this fungous growth of arthrosporcs, the parasitic spore form, were
was not a contaminant but T. rubrum was usually produced, either on the surface where
proved by culture. Growth of dcrmatophytes they appeared large and rounded, or within the
on the shell membrane has also been demon- proliferating ectodcrm, where they germinated
strated by Neuhauser (10). She utilized the (Figs. 2 and 6). This picture of the germinating
shell membrane 'floor' of the air-space in non- hyphac producing chains of arthrospores cor-
fertile eggs as a means of isolating the 'common' responds with Showalter's observations on other
dermatophytcs from pathological material. dcrmatophytcs.
Increased parasitic activity of the fungus was
Histopatholoqy of the lesions shown by the growth of invasive branching
The chorioallantois is primarily the respira- hyphae into the rapidly increasing ectodcrm.
tory organ of the embryo and the membrane Beneath this hyperplastic cctodcrm, the mcso-
610 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

FIG. 3. Section through infected membrane, showing hyperplasia and proliferation of ectoderm
(7 day infection, strain (a), passage II. Stained P.A.S.) X26

derm showed a corresponding reaction, becom- ment and foci of invasion by the fungus, which
ing thickened by oedema and cellular prolifer- acted as a stimulus (Fig. 8).
ation, and by the migration of ectodermal cells
II. Results of Serial Passage
in the form of islets or whorls which became sub-
merged in the inflammatory tissue arising from Variation of Pathogenicity
it (Fig. 3). Following response of these tissues, Evidence that serial passage had brought
fungous elements were often found deep in the about an increase in the pathogenicity of the
mesoderm, always within whorls of proliferat- fungous strains used was insufficient, patho-
ing ectodermal cells (Fig. 4). genicity being assessed by the speed with which
Advance of the infection was shown by the the organism was able to invade the ehori-
loss of architecture and degeneration of the oallantoic tissues and bring about death of the
proliferating ectoderm in the area below the embryo. However, even in this series, eonsider
invading fungus (Figs. 5 and 6), together with ing the comparatively small number of eggs
a development of fibrous tissue in the form of inoculated and allowing for variation between
fibroblasts and chronic inflammatory infiltra- the batches of eggs used, a difference in the rate
tion in the superficial mesoderm lying below of invasion by the 3 strains of T. rubrurn was oh.
the region of infection (Fig. 7). The underlying served. There was no evidence that this was
entoderm in these areas showed a corresponding due to the size of the inoculum.
tendency to proliferation with the formation
Rate of invasion
of elongated papillae.
The overall reaction of the membrane tissues Strain (a) showed a more pronounced patho.
was a response towards the parasitic develop- genicity during serial passage compared with
 CHORIOALLANTOIC MEMBRANE FOR CULTURE OF DERMATOPHYTES 611

FIG. 4. A. Hyperplastie eetoderm and fungous elements within whorl of ectoderm proliferating ioto
mesoderm. X420

the other 2 strains. In passage I, white nodular
lesions were clearly visible on the membrane
4 days after inoculation. Sections of membrane
from an S day infection showed fungal infiltra-
tion and hyperplastic response of the ectoderm,
with a slight proliferation of cells into the meso-
derm. The fungus, recovered by culture, was
passaged through 7 and 10 day old eggs (pas-
sage II) and showed a marked increase in para-
sitic activity. Sections through a nodule from a
7 day infected membrane (inoculated after only
7 days incubation) showed a high degree of
response to the invading fungus, with hyper-
plasia and proliferation of the ectoderm, whorls
of cells containing fungous elements being
found in the actively increasing mesoderm
(Fig. 3). The chick embryos did not survive a
third passage with this strain. However, the
612 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

C, 0

FIG. 5. B. Mycelial invasion and degeneration of ectoderm. X610

membranes recovered from these eggs, 6 days first 3 passages, appeared to be less rapid than
after inoculation on this occasion (passage III), with the previous strain, and there was a tend-
showed evidence of fungous infection, and sec- ency of the fungus to develop a covering aerial
tions through one of them showed an intensive web of mycelium over the ectoderm.
reaction of all the tissues to the invading fungus, With strain(b), nodules on the membrane
with proliferation and degeneration of both were visible from passage I onwards. Sections
eetoderm and mesoderm. of an S day infection (passage I) showed ecto-
The reaction of the chorioallantoic tissues to dermal proliferation and hyperplasia where the
invasion by strains (b) and (c), as seen in the fungus penetrated, with little reaction in the
 CHORTOALLANTOIC MEMBRANE FOR CULTURE OF DERMATOPHYTES 613

FIG. 6. C. Germinating arthrospore of invading mycelium within degenerating ectoderm. X1200

mesoderm, compared with a 7 day infection branes from passages V and VI were generally
from passage VI, where sections showed a very thick and nodular. Sections through a
marked increase in parasitic activity of the 7 day infection from passage VI showed a thick
fungus, with a corresponding reaction of the cover of aerial myeelium with an acute reac-
tissues including the entoderm. tion of the underlying membrane tissues, compa-
With strain(c), there was no obvious para- rable with that seen with strain(a), (Fig. 7).
sitic development of the fungus until after the The fungus was found deep in the mesoderm
third passage, when nodules were visible on the within whorls of proliferating eetoderm. The
membrane 6 days after inoculation. The mem- mesoderm exhibited a marked inflammatory
614 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

II. - • P0 — — a.a — a—

si

S.

'I

Fin. 7. Section through infected membrane, showing development of fibroblasts and chronic cellular
infiltration of mesoderm around fungus-containing ectoderm whorls, below degenerating invaded ecto-
derm. Note aerial web of mycelium over ectoderm. (7 day infection, strain (c), passage VI. Stained
P.A.S.) X350

response, with development of fibroblasts and vested on the fourth and seventh day after in-
chronic cellular infiltration below the degener- oculation, the approximate time of deaths
ating invaded ectoderm. occurring during the incubation periods was
estimated by the development and condition
Survival of Embryos of the embryo.
With strain (a), the chick embryos did not
The viability of the embryos during the course
of serial passage varied considerably. As the survive a third passage, even 4 days after
eggs were examined and the membranes har- inoculation. That the fungus had produced an
 CHORIOALLANTOIC MEMBRANE FOR CULTURE OF DERMATOPHYTES 615

—
FIG. S. Section through nodule showing response of eetoderm, mesoderm and entoderm to stimulus
of fungous invasion. (7 day infection, passage V. Stained P.A.S.) X13

infection, in this and subsequent passages, was Examination revealed a very slight increase in
shown by the presence of small nodules and the granular appearance of the colonies obtained.
thickening of the ehorioallantoic membrane, There was no apparent difference between the
even though autolysis of the egg contents had isolates of the same strain and passage recovered
frequently commenced. The fungus was re- after 4 or 7 days growth on the membranes. It
covered from these membranes, usually on the was found during the course of this work that
malt/tellurite agar. Death of the embryo in the higher temperature (30°C) was more favor-
these later passages may have been associated able for the growth of T. rubrum isolates, and
with a particularly virulent fungous strain. In was adopted for all subeulturing. This temper-
the eggs inoculated with the other two strains ature was also used by Silva, Kesten and Ben-
of T. rubrum, 50 per cent of the embryos sur- ham (3).
vived with strain (b) and a greater percentage On completion of the series, sets of Sabou-
with strain (c). Further study on a large-scale raud's agar plates (25 ml. per plate) were
investigation is required before any definite inoculated singly with each series of isolates
conclusions can be drawn. obtained from each passage, and incubated for
4 weeks. Morphological examination of each
Variation of Culture Strains series revealed the following features:
The 3 strains of T. rubrum were recovered in Strain (a)—Changes from the original granular
culture from the ehorioallantoie membranes type (Fig. 10) took place in this series, some
after each serial passage; subcultures were made colonies developing white, downy sectors. Micro-
on to Sabouraud's agar from the isolates ob- scopic examination revealed the presence of
tained. As the series progressed, there appeared macroeonidia in the original colony type and in
to be no radical change in the general macro- the granular sectors of the downy colonies: only
scopic appearance in tube cultures of the organ- mieroconidia were present in the downy por-
isms. Halfway through the series, sets of Sabou- tions of these colonies. The development of
raud's agar plates were made from the isolates. downy sectors in some colonies appeared spas-
616 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

modic, especially when compared with the tube

downy type of colony showed a tendency to
cultures, where the granular form was main- develop a slightly more granular form, notably
tained throughout the series. by the sixth passage. There was also an increase
Strain (b)—The isolates obtained by serial in the rate of pigment production. Again, macro-
passage showed an apparent morphological conidia not found in the original and early
transition from the original white semi-downy downy forms were observed in the later more
colony form with slight radial folds, to the granular colonies.
flatter, pink-tinted, finely granular form re-
JH5CU55JON
covered after the fifth and sixth passage (Fig.
10). Microscopically, macroconidia were not The results of this study showed that the
found in the original and early isolates, but chorioallantoic membrane of the developing
were plentiful in the granular sectors of the chick provided a suitable medium for the in
later forms. vim culture of Trichophyton rubrum. Growth
Strain (c)—IDuring serial passage, the original of this organism compared favorably with re-

•"W*
S41/

Fm. 9. Section through human skin, showing invasion of epidermis by T. rubrum. Stained by P.A.S.
X900 (by courtesy of Dr. C. Calnan, Royal Free Hospital & St. John's Hospital)
 CHORIOALLANTOIC MEMBRANE FOR CULTURE OF DERMATOPHYTES 617

FIG. 10. Cultures of T. rubrum on Sabouraud's agar, (4 weeks at 30°C.) A. Original granular type
colony, strain (a). B. Original semi-downy type colony, strain (b). C. Granular type colony, strain (b),
produced after sixth passage through chick membranes.

suits obtained by Moore with other dermato- phyte infection, the difference being one of
phyte species. degree. Both tissues originate from the same
The mode of invasion and overall pattern of germinal layers. In man, the epidermis arises
tissue response to T. rubrum was the same for from the ectoderm, and the dermis with its
all 3 strains studied. The difference between vascular supply, is mesodermal in origin. In-
them lay in their rate of adaptation to a para- fection of the chorioallantoic membrane tended
sitic mode of life. Reversion to this phase was to cause chronic granuloma and development of
indicated by the germinating spores and hyphae fibrous tissue, resulting in nodular lesions.
of the saprophytie phase (the inoculum) pro- Acantholysis of the hyperplastic ectoderm in
ducing arthrospores and invasive hyphae on contact with the invading fungus was usual at
contact with animal tissue, i.e. ectoderm. this stage. The fungous mycelium, although
The reaction of the membrane to parasitic penetrating deep into the inflammatory meso-
invasion by T. rubrum was comparable with derm, was nearly always found within whorls
the response of the human skin to dermato- of proliferating ectodermal cells unless degener-
618 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

ation had taken place. The response to invasion would therefore appear from the previous tech-
of the human skin by T. rubrum produces a less nic employed that 'mechanical trauma' tender
marked cellular reaction, the most noticeable to mask the true picture of the type of fungous
feature in the epidermis being hyperkeratosis. infection produced.
Fungous growth is limited to the epidermis Although the 3 strains of T. rubum produced a
(Fig. 9). similar response in the chorioallantoic tissues
This procedure for in vivo culture proved both there appeared to be a difference in their inva-
simple and effective. Although it was not possi- siveness and the production of lesions. This was
ble to observe the daily growth of the fungus most obvious with strain (a) which quickly
through the 'glass window' as in the previous reverted to its parasitic phase and caused
technics employed, the condition of the em- infection. It was apparently a virulent strain
bryo and the artificial air-space created above producing a corresponding picture in human
the chorioallantois could always be checked tissues, where it caused a kerionic infection in a
daily by candling. child's scalp. The development of infections by
The advantage of this modified method lay the other two strains was less marked, though
not so much in the increased area of the unex- indications of an increase in parasitism became
posed chorioallantoic membrane available for more emphasised during serial passage. Strain
inoculation, but in the marked decrease in (c) showed a slower reversion to the parasitic
traumatic damage to the delicate membrane phase than strain (b). Even when this did occur
during its preparation and inoculation. The and an infection was well established, the sapro-
technique employed by Moore involved a cer- phytic phase was still evident as aerial growth
tain amount of mechanical trauma of the limited over the lesions.
region of underlying chorioallantois available The morphology of the isolates recovered after
for inoculation. The 'window' was first cut in membrane inoculation showed that during the
the shell, and the shell membrane penetrated course of 6 serial passages, there was a tendency
with a 'spear-point needle and cut along the for the semi-downy strain (b) and the downy
shell incision', both being removed to expose strain (c) to produce more granular colonies
the underlying chorioallantoic membrane which similar to those of strain (a). This observation
was inoculated direct with the fungus. would seem to correspond to a similar conversion
The results so obtained showed a violent from downy to granular forms, recorded by George
inflammatory reaction of the tissues. With M. in the case of T. mentagrophytes. Future world
cartis, E. floccosum and T. schoenleini, though toincludes a study of this organism.
a lesser extent with T. mentagrophytes, Moore With the development of granular type
recorded a loss of continuity in the ectoderm, colonies, a corresponding increase in the pro-
and its replacement with necrotic inflammatory duction of macroconidia by strains (b) and (c) was
tissue, arising from the mesoderm. The mesoderm also recorded. Benham (11) observed that are
itself showed marked oedema with perivascular artificial medium, blood agar base, stimulated
infiltration and an increased number of distended the production of macroconidia in culture, and
capillaries. The overall tissue response to invasion suggested that tryptose was the ingredient
by all 4 dermatophytes simulated the histological responsible. In this present study, the production
appearance of 'traumatic ulcers'. of macroconidia in cultures obtained on artificial
By use of a non-mechanical procedure for media (e.g. Sabouraud's agar) would appear to
separating the shell membrane and underlying have been stimulated by animal passage, i.e. by
chorioallantoic membrane, no breaks in the the chorioallantoic membrane. Macroconidia are
ectoderm were observed, and there was no notice- only occasionally seen in primary isolates froir
able change in the vascular supply, although the man. Conidia are a means of dispersing the fungus
mesoderm showed an inflammatory response. No in the saprophytic phase, and the thicker-walled
lesions resembling 'traumatic ulcers' were multicellular macrocondium constitutes a more
observed using this technic. Although a different resistant spore form than the single-celled micro-
organism was used in this study, it seems unlikely conidium. Their production in culture therefore
that it would produce less tissue reaction than the is a response of the fungus to environmental
other dermatophytes, especially strain (a). It conditions. The development of macroconidia in
 CHORTOALLANTOIC MFMBFANE FOR CULTURE OF DERMATOFHYTES 619

the cultures grown from emulsions of the infected ratories, Colindale, London, by courtesy of the
membranes harvested during serial passage, late director, Col. H. J. Bensted and Dr. C. H. P.
could thus be attributed to a reaction of the Bradstreet of the Standards Laboratory. My
fungus to previous parasitic stimulus within the thanks are due to Dr. J. J. O'Donnell, St. John's
egg. This hypothesis could equally well be applied Hospital for Diseases of the Skin, for assistance
to isolates from human infections, the type of with the histological interpretation, and to Mr.
colony produced being an indication of the R. Lunnon, Institute of Dermatology, for the
parasitic condition of the fungus itself within the photography.
tissues, not necessarily shown by the clinical
REFERENCES
appearance. Carrying this hypothesis further, it
might account for the slight variation in isolates 1. Moonx, M.: The chorioallantoie membrane of
of the same strain obtained from different areas the developing chick as a medium for the
cultivation and histopathologic study of the
of the body. pathogenic fungi. Science, 89: 514, 1939.
Am. J. Path., 17: 103, 1941.
SUMMARY 2. SHOWALTER, W. V.: Morphological studies of
dermatophytcs in chick embryo membranes.
The ehorioallantoic membrane of the develop- Tr. Kansas Acad. Sc., 57: 149, 1954.
ing chick was us9d for the in vivo culture of 3. Siiv, M., KESTEN, B. M. ANn BENHAM, R. W.:
Trichophyton rubrum infections: a clinical,
Trichophyton rubrum. The technic employed was mycologic and experimental study. J.
based on the creation of an artificial air-space Invest. Dermat., 25: 311, 1955.
4. CEORG, L. K.: The relationship between
above the membrane, which was inoculated with downy and granular forms of Trichophyton
a fungous suspension through a small aperture. mentagrophytes. J. Invest. Dermat., 23: 123
By this method, the chorioallantois remained 1954.
5. GOODPASTURE, E. W. AND BUDDINGH, G. J.:
unexposed and relatively undamaged during The preparation of antismallpox vaccine
preparation. by culture of the virus in the chorioallantoic
Results of histopathologie studies revealed membrane of chick embryos and its use in
human immunisation. Am. J. Hyg., 21: 319,
that the fungous lesions produced were not 1935.
masked by mechanical trauma compared with 6. BUENET, F. M.: A virus disease of the canary
of the fowl-pox group. J. Path. & Bact., 37:
previous technics. 107, 1933.
Three strains of T. rubrum were studied. The 7. BEYERJDGE, W. I. B. AND BURNET, F. M.: The
mode of invasion and overall pattern of tissue cultivation of viruses and Rickettsiae in the
response were the same for all 3, although the chick embryo. H.M.S.O. Special Report
Series, Med. Res. Council, London, No. 256,
rate of infection varied. 1946. (Reprinted 1955).
Preliminary observations on serial passage 8. ALExANnER, R. A.: Studies on the neurotropic
virus of horse sickness. VI. Propagation in
(6 were carried out), using the modified technic the developing chick embryo. Onderstepoort
described, indicated a slight increase in parasitic J. Vet. Sci., 11: 9, 1938.
adaptation by two strains, accompanied by a 9. GEORG, L. K.: A simple and rapid method for
gradual conversion from downy to granular type obtaining monospore cultures of fungi.
Mycologia, 39: 368, 1947.
colonies together with an increase in production 10. NEUHAUSER, I.: Avian egg-shell membrane as
of maeroconidia. a culture medium for dermatophytcs. Arch.
Dermat. & Syph., 75: 401, 1957.
ACKNOWLEDGEMENTS 11. BENHAM, R. W.: Effect of nutrition on growth
and morphology of the dermatophytes. I.
The experimental work in this study was Development of macroeonidia in Tricho-
carried out at the Central Public Health J4abo- phyton rubrum. Mycologia, 40: 232, 1948.