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The Use of the Chorioallantoic Membrane of the Developing Chick

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The Use of the Chorioallantoic Membrane of the Developing Chick View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector 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) inpassage 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) studiedtechnic as a method for passage of cultures. By the morphological changes of the first threethe serial passage of T. rubrum from one chick dermatophytes, listed above, when in contactembryo membrane to another, it was hoped to with the living chick membrane. determine whether there was correlation between The object of the investigation, describedstrains and any alteration in their morphology in the present paper, was primarily to developand pathogenicity after passage. an improved in vivo technic for cultivating dermatophytes in the chorioallantois, and to METHODS AND MATERIALS compare the results so obtained with those of The cultivation technic employed by Moore previous workers. The dermatophyte chosenwas based on that described by Goodpasture (5) for investigation was Trichophyton rubrum, anand 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 interestThe underlying shell membrane was also cut because of the frequency with which it causesand the shell and its membrane carefully re- long-standing ringworm in man. A study of themoved to expose the chorioallantoic membrane, clinical manifestations of infections caused bywhich was then inoculated with a saline suspen- this fungus, and the morphology of the strainssion of the fungous culture. The 'window' was isolated, has shown little direct relationship. Aclosed by surrounding it with a layer of vase- similar conclusion was reached by Silva, Kestenline-paraffin and applying a cover slip. The and Benham (3) in studies of their T. rubrumeggs 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; thepasture's technic for chorioallantoic inoculation, rate of pigment production also varies with thein which an artificial air-space was created above strain. Both granular and downy forms of thisthe 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 bothwalter in his dermatophyte studies. He em- ployed Moore's technic of cutting a 'window' in *Fromthe Department of Medical Mycology,the shell and shell membrane to form a large Institute of Dermatology, London, W.C.2., Eng- land. opening into the artificial air-space. The inocu- Received for publication September 8, 1958. lum was applied by means of an inoculating 605 606 THEJOURNAL OF INVESTIGATIVE DERMATOLOGY needle to the exposed surface of the choriollan-lying chorioallantoic membrane. Using a standard toic membrane, and the 'window' sealed withrounded 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 studyniques of cutting and picking away a larger area also embodies the use of the artificial air-spaceof the shell. A smaller aperture into the air-space above the chorioallantois, with modificationswas made by drilling through the shell and shell by Alexander (8). The principle feature of thismembrane with a finer burr, or puncturing them method is the small amount of manipulationwith a needle. (Diagram I, A.) of the chorioallantoic membrane required dur- Separation of membranes—A drop of sterile ing inoculation. This decreases the developmentphysiological saline was placed over the circular of non-specific lesions due to trauma whicharea of exposed shell membrane which was then carefully slit, using a sterile needle and pressing may cause difficulty in the interpretation ofdown and backwards at right angles to the fibres. any fungous lesions produced. Little trouble isAir was withdrawn from the air-space by gentle caused by air-borne contaminants, probablysuction with a rubber teat placed over the aper- because of the natural resistance of the eggture, and the chorioallantoic membrane separated tissues. However, as this work had to be carriedand 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 provedbranes 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 that the original air-space had been obliterated. Inoculation The area of chorioallantoic membrane available Pre-incubation—Fertilised eggs were incubatedfor inoculation was approximately 2 to 2.5 cm. in in a standard electric, thermostat-controlled eggdiameter, 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 minimumpossible 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 wereavoid 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 theopening in the shell. embryo was noted by its spontaneous movements, Inoculum—The unexposed chorioallantoic mem- and a point marked above it on the shell, carebrane 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 B 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 byThese were stained by the periodic-acid-schiff vigorous shaking of a loopful of mycelium andmethod in order to trace the invasiveness of the spores from a fresh Sabouraud's agar slope culturefungus, and with haematoxylin and eosin to de- in 2 to 3 ml. of sterile physiological saline. Largertect 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 sporessaline 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 chorioallantoicstrains were chosen. Single-spore cultures were membrane without touching either it or the shellobtained 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'sspores (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 damagecolony 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- tures in the shell were sealed with triangles ofinvolvement. "Sellotape". The eggs were placed on slatted wire Strain(5), No. 4497—semi-downy, well- trays, with the circular aperture uppermost, andpigmenting colony, isolatcd from a groin in- incubated at 34°C.
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