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The cultivation of bacteria upon the chorioallantoic membrane of hen's eggs Cleveland, Edward Milton Donald 1951

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(1* i H THE CULTIVATION OF BACTERIA UPON THE CHORIOALLANTOIC MEMBRANE OF HEN'S EGGS by i EDWARD MILTON DONALD C L E V E L A N D , A T H E S I S S U B M I T T E D I N P A R T I A L F U L F I L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F MASTER OF ARTS IN THE DEPARTMENT . . O F BACTERIOLOGY and PREVENTIVE MEDICINE WE A C C E P T T H I S T H E S I S AS CONFORMING TO THE STANDARD R E Q U I R E D FROM C A N D I D A T E S FOR THE DEGREE OF MASTER OF ARTS Members of the Department of B a c t e r i o l o g y and P r e v e n t i v e Medicine THE UNIVERSITY OF BRITISH COLUMBIA September 1951 Abstract of Thesis  The Cultivation of Bacteria upon the Chorioallantoic  Membrane of Hen1s Eggs The literature pertaining to the cultivation of bacterial micro-organisms in fertile eggs has been reviewed in an attempt to give the historical background of the subject. A technique of chorioallantoic inoculation of chick embryos has been described and by this means chick embryos were inoculated with Serratia marcescens, Staphylococcus albus. Mycobacterium phlei and Salmonella typhimurium; the cultivations of S . marcescens, M, phlei and S. typhimurium were successfully accom-plished by this method. Descriptions of other methods of inoculating the chick embryo have been added; Smooth and rough strains of S. typhi- murium were individually serially passaged through a number of chick embryos by means of the chorioallantoic inoculation technique in an attempt to discern any change in their agglutinability by specific im-mune serum or changes in their type as evidenced by colonial morphol-ogy on nutrient agar or growth in nutrient broth; No change was noted; The minimal number of smooth and of rough type of organisms respect-ively necessary to infect fatally a ten day old chick embryo was app-roximately determined. In the course of the investigation a reduced susceptibility to infection with smooth type S. typhimurium organisms was noticed with increase in the age of the embryo; Emphasis has been placed upon the advantages attaching to the use of the chick .embryo technique of cultivating bacterial organisms over the common practice of using laboratory animals and culture media, and a plea for the fur-ther pursuit and investigation of these advantages has been made; The thesis has been illustrated with diagrams and color photographs of the instruments used in the course of the research and of the technique of chorioallantoic inoculation. T A B L E OF C O N T E N T S I H I S T O R I C A L O U T L I N E . 1 II A N A T O M I C A L D E S C R I P T I O N O F T H E C H I C K E M B R Y O . 16 III A R T I F I C I A L I N C U B A T I O N O F E G G S 22 IV I N O C U L A T I O N T E C H N I Q U E S : A - C A N D L I N G T E C H N I Q U E IN EGGS FOR I N O C U L A T I O N 27 B - METHODS OF C H O R I O A L L A N T O I C T E C H N I Q U E 28 C - B E V E R I O G E S ' M O D I F I C A T I O N OF C H O R I O A L L A N T O I C T E C H N I Q U E . 31 D - B E V E R I D G E AND B U R N E T S ' T E C H N I Q U E OF A M N I O T I C I N O C U L A T I O N . 32 E - B E V E R I O G E S ' M O D I F I C A T I O N OF A M N I O T I C I N O C U L A T I O N / T E C H N I Q U E 34 F - H I R S T S ' M O D I F I C A T I O N OF A M N I O T I C T E C H N I Q U E 35 G - YOLK SAC METHOD OF. I N O C U L A T I O N 35 H - A L L A N T O I C C A V I T Y .METHOD, OF I N O C U L A T I O N 36 I -t I N T R A C E R E B R A L METHOD OF I N O C U L A T I O N 36 J - E I C H O R N ' S I N T R A V E N O U S METHOD OF I N O C U L A T I O N 37 V E X P E R I M E N T A L M E T H O D S • I N S T R U M E N T S AND A P P A R A T U S 38 EXPERIMENTAL INFECTIONS "' ' 49 A - SE.RRATIA M A R C E S C E N S 49 B - S T A P H Y L O C O C C U S AL BUS 50 C — M Y C O B A C T E R I U M P H L E l 50 D - S A L M O N E L L A T Y P H I MURIUM 51 VI D I S C U S S I O N 60*63 VII S U M M A R Y 64 VIII - B I B L I O G R A P H Y 65 DIAGRAMS I Schematic section of four day chick embryo 20 II Diagram of the arrangement of mem-branes in.the nine day embryo. 20 III Diagrammatic sketch showing f u l l dev-elopment of membranes and cavities in 12 to 15 day chick embryos. 21 IV The relationship of the allantoic vein to the embryo. 21 V Diagram of egg candler 40 v i Diagram of Dunham egg inoculator 40 VII Diagram of adjustable camera stand 41 VIII Diagram stand. of Beverldges' egg inoculating 42 PLATES I Instruments used i n the c h o r i o -a l l a n t o i c technique 3 9 (color plates) I I & I I I S T E P S 1 AND 1A OF C H O R I O A L L A N T O I C T E C H N I Q U E OF I N O C U L A T I O N 43 I I I & I V S T E P S 2 AND 3 OF C H O R I O A L L A N T O I C T E C H N I Q U E OF I N O C U L A T I O N 44 I V & V S T E P S 4 AND S» »F C H O R I O A L L A N T O I C T E C H N I Q U E I F I N O C U L A T I O N 4 5 V & V I S T E P S 6. AND 7 OF C H I R I O A L L A N T O I C T E C H N I Q U E 46 V I I & v m S T E P S 8 AND 9 OF C H O R I O A L L A N T O I C T E C H N I Q U E . 47 I X 8s X S T E P S 1 0 AND 11 0F CH0R j 0 A L L A N TO 1C T E C H N I Q U E .48 X I & m A P P E A R A N C E OF S U R F A C E OF CI 0 R 1 0 A L L• A N T O I C MEMBRANE A F T E R 2 4 HOURS OF C U L T I V A T I N G S A L M O N E L L A T Y P H I M U R I D M UPON I T . 58 WRIGHT'S S T A I N OF SMEAR MADE FROM H A R V E S T OF A 2 4 HOUR C U L T U R E OF S A L M O N E L L A T Y P H I M U R I U M UP«N THE CHOR-I O A L L A N T O I C MEMBRANE .5.8 X I I I A S I M P L E S A F E T Y G L A S S SH 1 ELD WHICH MAY BE USED WHEN I N O C U L A T I N G . 59 X I V T W E N T Y ? F O U R - HOUR OLO C U L T U R E S OF ROUGH AND SMOOTH S T R A I N S OF S A L M O N E L L A T Y P H I M U R I U M IN N U T R I E N T BROTH.. 59 1 The use of the embryonated avian egg as a culture medium for the propagation and maintenance of cultures of living microorganisms is not new, but its history, like that of the whole science of bacteriol-ogy, Is a short one* The intention of this outline i s to set forth the history of the use of the fertile egg as a dulture medium* together with those discoveries in technique which were of importance to the development of the present methods. The cultivation of bacteria is dealt with chiefly and not the use of the egg as a living medium for the prop-agation of viruses and rickettslae* It has been considered advantageous though, to include the occasional reference to these other pathogenic agents in order to convey some idea of the parallel development of their cultural methods with that of the culture of bacteria, as suoh, in eggs* Although numerous observers, from and ent times throughout the centuriesi had examined developing embryonated eggs and described their discoveries with more or less accuracy* i t is not until relatively redent times that any record is found of the actual construction of a window in the shell of a fertile egg through which the embryonic growth and development might be observed without either destruction of the embryo or its contamination from without. The first historical aeoount of the construction of such a window is by Beguelin 11749)j elted by Geriach (1886). Gerlaeh himself was responsible for the construction of a rather clumsy instrument, named by him the embryoscope. This piece of apparatus somewhat resembled the ocular of a microscope and oould be screwed into a threaded tube sealed permanently into a hole out through the 2 eggshell i n order to view the gradual growth of the embryo« The method which i s commonly used to the present day of sealing a hole cut through the s h e l l of an embryonated egg with a coversllp set on a r i n g of vaseline was f i r s t introduced by Soymclewicz (1815)* Apparently the f i r s t man ever to c u l t i v a t e microorganisms i n an egg, f e r t i l e or otherwise, was S i r Alexander Ogston (1844-1929), According to Breloch, Ogston, as surgeon to the Royal Infirmary at Aberdeen had an opportunity such as had not been afforded Koch, to study the pathogenesis of wound i n f e c t i o n . In a report i n the B r i t i s h Medical Journal for 1881 upon Microorganisms i n Surgical Disease, Ogston related how he had conceived the idea of c u l t i v a t i n g organisms found In abscessed wounds by inoculating them into hen's eggs, where he thought conditions would more closely simulate those under which they grew In animals than i n l i q u i d media* He was successful In growing Micrococci i n eggs, and i t was he who Intro* dueed the term Staphyloeoeel as a name f o r the cluster-forming cocci at the suggestion of his fri e n d Geddes, Ogston also was successful i n oulturing b a c i l l i and other bacteria i n eggs but giver no d e t a i l s concerning these experiments. To understand the importance of Ogston's discoveries i t i s necessary to digress f o r a moment to view his discovery i n the l i g h t of the times. I t was no simple and routine matter to c u l t i v a t e any sort of bacterium i n the year 1881. I t was only i n the year that Alexander Fleming was born that Koch described his method for the preparing of cultures on s o l i d mediae I t was "supposed" even by the most enlightened physicians of the day i n England, that bacteria 5 and micrococci were the cause not only of erysipelas, diphtheria and leprosy but also of v a r i o l a and malaria, and there was no certain method f o r either recognizing these organisms or of i s o l a t i n g them i n pure culture f o r study* Eberth of Zurich had only just published, i n Virchow's Arohiv, a series of observations which strongly pointed to the dependence of typhoid fever upon a certain rod«shaped organism, but Hans Gram (1884) did not describe his staining method, which would hare aided greatly i n the recognition of such an organism, u n t i l three years l a t e r * An e d i t o r i a l i n the B r i t i s h Medical Journal of the day Considered the great question of the times to be whether or not infectious diseases r e a l l y d i d depend upon the development of microorganisms i n the l i v i n g body, and considerable doubt was ex-pressed concerning the r e a l conference of immunity by protective inoculation, such as Pasteur had demonstrated with anthrax* -Even public sentiment interfered with the pursuit of s c i e n t i f i c medical knowledge* The ant i«*vivisect ion b i l l was i n parliament, and doubt-less i f much had been known about what' Ogston was doing to embryos i n eggs he would have found himself arraigned at Bow Street P o l i c e Court* But Ogston's method f o r c u l t i v a t i n g microorganisms was soon forgotten, and i t was not u n t i l the year 1905 that any record of an attempt to c u l t i v a t e anything but chicks i n f e r t i l e eggs was made* In that year, Oonstantin L e v a d i t l , a worker i n Bfetchnlkoff*s laboratory, reported a successful attempt to c u l t i v a t e the S p i r i l l u m gallinarum, an organism which had just been recently discovered i n B r a z i l by Marchoux and Salimbeni, i n f e r t i l e hen*a eggs* In his report Levaditi stated that he was merely repeating the e a r l i e r work of Borrelj but Borrel's reportj i f he ever made one* has been lost. Levaditl noted for the f i r s t time the important fact that the organisms were only propagable in the presence of a living embryo* In the course of investigating the Spirillum, which Levaditl consid-ered to resemble the spirochaete of syphilisj he found a second Important fact, namely that maternal antibody is present in the embryo, a new discovery, although i t had been previously demonstrated by Dziergowski (1901), that diphtheria antitoxin concentrates in the yolks of eggs from Immunized hens* However, again the usefulness of the embryonated egg for bact-erial research lapsed into obscurity and seems to have remained for-gotten until 1911 when Sous and Murphy found i t to be a useful medium for the transmission and cultivation of a tumor transplant, later to be immortalized In virological literature as Rous chicken sarcoma* These two workers rediscovered what Levaditl had already noted, namely that the ohlck, although capable of being passively Immunized by maternal antibody, had no antibody producing mechanism of its own* It is from 1911 onwards that the use of the chick embryo for the cultivation of tissue transplants becomes more frequent* The history of virus cultivation in the chick embryo probably begins with the discoveries of a pair of practically forgotten French research workers, Joiian and Staub, who in 1920 reported their pre-war investigations upon the virus of avian plague* As the result of a fortuitous discovery of the virus in an unlaid fertile hen*s egg, they had evolved a simple method for the propagation of the v i r a l agent, previously expensively cultivated by transmission from chicken to chicken. The present day techniques of inoculating chick embryos with various pathogenic agents are based upon the work of Clark* who in 1920 evolved a successful method for operating upon chick embryos. His methods were later developed and elaborated upon by several workers including Goodpasture, Beveridge, Galloway and Burnet* From 1905 to 1933* the only reference to be found in the literature of an experimental infection of the chick embryo with bacteria is by Askanazy (1933), who mentions the production of tuberculous chicks by the infection of fertile hen's eggs with Mycobacterium tuberculosis A A strange feature of the research of most of the early workers with organisms In eggs is their failure to recognize the essentiality of the l i f e of the chick embryo to the propagation of their pathogenic agents* It was only gradually and in a never definitely stated form, that i t came to be generally recognized that the vital ingredient of the egg, as a medium* was the living cell* Indeed, the cultivation of Organisms upon the living embryo was carried on for some time with-out any recognition of what exactly was occuring. Rous and Murphy were actually propagating a virus when they grew their fowl sarcoma in tissue culture on the chorioallantois,but they did not know It. In fact the credit for the first successful propagation of a virus really known to be such in a controlled experiment goes to Woodruff and Goodpasture* who in 1931 inoculated the chorioallantoic membrane of living chick embryos with the virus of fowl pox. They followed this work with the first successful propagation of vaccinia virus (1932), on the chorioallantoic membrane, a feat previously attempted 6 by Gay and Thompson (1929)• In 1932 Goulston and Mottram described their technique for the removal of the shell and the exposure of the shell membrane without the production of histological changes in the chorioallantois* They also experimented with the effects of incubation upon the exposed membrane surface; but it was not until 1934 that Burnet and Galloway discovered, when culturing the virus of vesicular stomatitis on the chorioallantoic membrane, that they eould create an ar t i f i c i a l air space between the shell membrane and the chorioallantois which would enable the introduction of larger amounts of material into the egg than was previously possible. The possibility of utilizing the chick embryo, by then widely accepted as the routine method of cultivating viruses, as a host for bacteria, with the end in view of noting pathogenic effects, was first suggested by Goodpasture in 1933. However, his remarks in this regard seem to have been largely overlooked* Zia (1934), gives the credit to Goodpasture for the suggestion of a method of cultivating those practically virus-like organisms, the rickettsiae, which he had been attempting to cultivate with ex-treme difficulty. His success with the cultivation of Mexican and European typhus rickettsiae on the chorioallantoic membrane was reported by him in 1934, but the growth by this method was not part-icularly abundant. It remained for Cox, in 1939, to find the present method for successfully propagating rickettsiae ln large numbers by inoculating them into t h e yolk sacs of embryonated eggs. There the rickettsiae proliferate within the lining of t h e cells of the yolk sac, being liberated as the cells disintegrate* The presence of maternal antibody in eggs was rediscovered by the Japanese Ozawa (1956) who noticed that embryos from the eggs of hens immunized with diphtheria toxoid were resistant to poisoning with diphtheria toxin placed upon the chorioallantoic membrane* This is one of the fi r s t suggestions of the possibility of the use of the chick embryo as an indicator in toxin-antitoxin titrations* In the same year, 1936, the first cultivation of an actinomycete, Streptobacillus moniliformis, upon the chick embryo, was accomplished. This was by van Hooyen, who succeeded in carrying the organisms through three successive transfers* The year 1937 was one of considerable advance in the use of the chick embryo for bacteriological research* Goodpasture and Anderson (1937), reported the successful cultivation of a number of bacteria upon the chorioallantoic membrane of fertile hen's eggs* They found that Streptococcus virldans. Aerobacter aerogenes, Bberthella typhosa. Brucella abortus and Mycobacterium tuberculosis avium were able to . i " " utilize the living intracellular environment of such a medium for growth. They also noted that Staphylococcus aureus and Streptococcus  haemolytlcus. although persisting in the host for a time, seem to be incapable of growing intracellularly within it.# From their research they concluded that cultivation upon the chick chorioallantoic mem-brane was an excellent practical method for studying the problems of Infection, especially the early stages about which so l i t t l e was known. In the same year, Gallavan (1939), cultivated Haemophilus 8 influenzae on the chorioallantois, and in cooperation with Good-pasture also demonstrated the infection of chick embryos with H,  pertussis, showing that the pulmonary lesions of human whooping cough were reproduced in detail in the embryo's lungs, thus in-criminating i t as the causative agent of human whooping cough. Meanwhile, Buddingh and Polk were cultivating the meningo-coccus for the first time upon the chorioallantois. In their discussion of this experiment they again brought up the possibility of using the chick embryo as a handy tool for the analysis of the effects of antisera and antitoxins on disease processes. Pandit,Rao and Short in India undertook an investigation in 1937 to determine the reaction of the chorioallantoic membrane to materials containing a hypothetical virus, not readily transmiss-able to animals. In order to be able to recognize suoh a reaction i t was necessary to know what responses would be induced by a variety of substances known to have no virus content. In the course of their experimental efforts, they found that not only Proteus X19 but also E. typhi, as previously noted by Goodpasture and Anderson, were capable of being cultivated upon the chorio-allantoic membrane; and that filtrates, presumably cell free, of the ground bacteria were capable of reproducing the lesions caused by the growth of these organisms upon the membrane. This they suspected was due to a filtrable form of the bacteria. In 1938 Evans carried further the work of Ogawa, conducting experiments upon the action of the toxin formed by Corynebacterium  diphtheriae upon chick embryos and devising a method of titrating the toxin of various strains upon them. In the same year (1938), Morrow, Syverton,Stiles and Berry succeeded for the first time in cultivating Leptospira ictero- haemorrhagica upon the chorioallantoic membrane* This is the first record of the successful cultivation of any member of the Spirochaetales upon the chick embryo, xliey were able to grow this organism, the agent of Weil*s disease, on ten-day-embryos and to effect its transfer through some twenty egg passages, finding no evidence of any alteration in its virulence as a result of this passage* The year 1939 saw the propagation for the first time of several species of Trypanosomas in the chick embryo by Longley, Clausen and Tatum. In the same year, Anderson and Snow isolated and cultivated H.-dmcc'eyi on the chorioallantois, by inoculating i t j after slight mechanical injury, with drops of pus from soft chancres* This was an Important and useful discovery, because no experimental animal, except possibly the monkey, is consistently infectible* However, the organisms proved to have a low patho-genicity for chick embryos, and could not even be induced to grow upon the chorioallantoic membrane without previous abrasion, which made serial passage very difficult. Swift and Brown made the first cultivation of pleuropneumonia-like organisms obtained from acute rheumatic exudate and the tissues of patients suffering from acute rheumatic fever. They found that characteristic lesions were produced, although not readily, since several transfers were necessary before the lesions were regularly reproduced, suggesting a possibility of seme adaptation to this environment* 10 Oag succeeded in growing Borellia duttoni in embryonic chicks, without the death of the embryos, although with the production of a general infection as in man, again emphasizing the similarity in reaction to infection between man and chick* Buddingh and Polk made a study of passive immunity to infection with the meningococcus in the chick embryo, immunizing the embryos intravenously with anti-toxin and antiserum; This was. a milestone in the development of the use of the chick as a test animal for antigen-antibody titration. They found that the antiserum inhibited the growth of the organism, but did not prevent infection, although apparently neutralizing the injurious products produced by the organisms* The Intravenous technique for the inoculation of chick embryos, a rather difficult method requiring considerable s k i l l and care in the performance, was described by Eichom in 1940* Again recording the similarity between the reaction of man and chick embryo, Oromartie (1940), experimented with the use of the chick as an indicator in toxin-antitoxin titration. Using Goryne- bacterium diphtheriae as the test organism, he noted that the essential characteristics of the human disease are shown by the chick embryo* In the same year* Jimlnez and Buddingh (1940) suc-ceeded in infecting the chick embryo with Bartonella baclllformls, and Peterson (1940), experimentally infected chick embryos with some nine strains of Listerella monocytogenes, by injecting both the allantois and the chorioallantoic membrane* Paterson noted that rough strains failed to set up an infection, whereas smooth strains gave rise to an infection affecting the heart, liver and central nervous system, which they apparently reached via the blood stream. 11 This seemed to indicate, as Goodpasture and Anderson (1937), had suspected previously, that i n the chick embryo phagocytosis does not act particularly well as a protective mechanism but rather serves to carry the introduced infection to other parts of the embryo. The year 1941 saw the rise of the chick embryo as an instru-ment for the in vivo testing of chemotherapeutic agents; i t also saw the discovery, so important to virology* of the Hirst phenomenon. Weil and Gall (1941) in a series of experiments, tested the efficacy Of sodium sulfathiazole upon infections produced i n the chick embryo with Salmonella typhi, Shigella flexneri and S. pullorum. they found i t to be a simple way of demonstrating the therapeutic effect in the presence of l i v i n g c e l l s ; Emmart and Smith also introduced a method for the use of the chick embryo in chemotherapy, cultivating Mycobacterium tuberculosis on the chorioallantoic membrane. Again in the year 1941 a number of experimental cultivations of various pathogenic fungi were carried out by Moore, on the chorio-allantois, as well as the cultivation of various strains of Mycobact- er i a . Buddingh and Womack observed the infection of chick embryos with Pasteurella tularense. Brucella suis, abortus and melitensis as well as Pasteurella pestis, while Alture-Werber managed to cultivate Trypanosoma equiperdium in the uolk sac of embryonated eggs, and Wile and Snow propagated Spirochaeta pallida i n the chick embryo. The following year, 1942, saw the introduction by Dunham of an apparatus for holding eggs while collapsing the chorioallantoic membrane with suction, which he termed an inoculator. This f a c i l i -tated the separation of the shell membrane and the chorioallantois, 12 yet at the same time avoided injury of the delicate chorioallantoic membrane^  always a hazard In this technique* In the same year Moore inoculated the chorioallantoic membrane with a number of varieties of Mycobacteria and found a rapid means of differentiating these types by the lesions produced; He also discovered that the time required to reproduce: recognizable lesions, which is such an obstacle to the use of laboratory animals in diag-nosis, was greatly reduced in the chorioallantois, thus introducing a hew use for the chick embryo. In 1944 Green and Birkeland decided to investigate the possibility of using the chick embryo as a test animal for ascertaining the anti-bacterial effectiveness of various wound disinfectants in vivo. Using Staphylococcus aureus as a test organism they made a comparison of the highest dilution of the distinfectant toxic for living cells with the highest dilution of i t lethal for the bacteria; this result they expressed as a "toxicity index". They found the chick embryo to be highly susceptible to infection with the test organism. The results of their experiment, as demonstrated by this method, indicated that eatlonie detergents are superior to the mercurials, halogens and phenol in their anti-staphylococcal action. The following year, 1945, Stauber and van Dyke were successful in infecting duck embryos with the malarial Plasmodium, and Haas and Ewihg were equally fortunate in cultivating Plasmodium gallinaoeum in chick embryos. Their method of infecting the chick embryo was rather a novel one* Mosquitos were placed in a small tube covered at the open end with a net; this end was then applied to the exposed 1 3 shell membrane of a l i v i n g chick embryo* The mosquitoes could be observed to feed by penetrating the shell membrane. However the di f f i c u l t y with this method was the time and effort; involved, since i t took half a day to persuade eight or ten insects to feed in this fashion. In the same year, Emmart used the chick embryo as an instrument for testing the value of antibiotics i n the treatment of tuberculosis. He treated tuberculous embryos with streptomycin and streptothricin finding that streptothricin was about four times as toxic to the embryo as streptomycin. Also in i945, Beveridge, working at the Institut Pasteur in Paris produced a simplified technique for the inoculation of chick embryosi This method solves to a considerable extent the problem of the research worker who does not possess a suitable d r i l l or dental engine for operating upon eggshell. Entrance into the egg i s effected by breaking away the shell with forceps at the blunt endj over the air sac. The next year, 1946, Silver and Kempe made further simplifications of the egg culture technique, but with the country physician rather than the research worker in mind. They solved the problem of penet-rating the eggshell by softening i t with concentrated n i t r i c acid solution confined to the desired area by a ring of petroleum j e l l y * In the same year, Meyer and Ordal used the chick embryo as a test animal for the action of antibiotics upon fungoid infection* Choosing the organism Blastomyces dermatitidis as the infective agent, they treated i t with streptomycin, gliotoxin, streptothricin and 14 p e n e c i l l i n . They f o u n d t h a t w i t h t h e e x c e p t i o n o f s t r e p t o m y c i n , t h e s e s u b s t a n c e s were e i t h e r e x t r e m e l y t o x i c t o t h e c h i c k embryo o r j u s t a g g r a v a t e d t h e l e s i o n s . T h e y a l s o d e m o n s t r a t e d t h e p a t h -o g e n i c i t y o f v a r i o u s C a n d i d a s p e c i e s f o r t h e c h i c k e m b r y o ; A l t h o u g h t h e c u l t i v a t i o n o f o r g a n i s m s , s u c h a s t h e r i c k e t t -s i a e i n t h e y o l k sac o f t h e egg has b e e n g r e a t l y u s e d a s a means o f o b t a i n i n g l a r g e numbers o f o r g a n i s m s f r o m w h i c h t o p r e p a r e v a c c i n e s , t h e r e ha s a l w a y s b e e n some q u e s t i o n a s t o t h e p o s s i b l e d a n g e r o f u n t o w a r d r e a c t i o n s f r o m s u c h v a c c i n e s i n egg s e n s i t i v e p e r s o n s , 4 r e p o r t o f s u c h a n o c c u r r e n c e , f a t a l a n a p h y l a x i s i n a n e g g s e n s i t i v e i n d i v i d u a l f o l l o w i n g a t y p h u s i m m u n i z a t i o n i n -j e c t i o n was made b y W a l k e r i n 1 9 4 8 , I n 1949 N o e l and M a r i e - S u z a n n e r e p o r t e d u p o n t h e p r o p a g a t i o n o f S t e f a n s k y ' s b a c t e r i u m u p o n t h e c h o r i o a l l a n t o i s , a n d M c N e l l y and P e d d e l l r e v i e w e d t h e u s e s o f t h e e m b r y o n a t e d egg i n t h e c u l t u r e o f M y c o b a c t e r i u m t u b e r c u l o s i s , B u r n e t , J o y c e and E d n e y r e p o r t e d i n 1950 t h a t t h e y had c o n -c l u s i v e l y d e m o n s t r a t e d t h e f a i l u r e o f a n t i b o d y f o r m a t i o n i n t h e c h i c k e m b r y o , a f a c t c o n c e r n i n g w h i c h t h e r e had been c o n s i d e r -a b l e q u e s t i o n f o r many y e a r s . They u s e d f o r e i g n r e d b l o o d c e l l s , b a c t e r i a l v i r u s and l i v i n g i n f l u e n z a v i r u s a s a n t i g e n s b u t c o u l d f i n d no a n t i b o d y r e s p o n s e t o t h e s e , a l l e x c e l l e n t a n t i g e n s . These r e s u l t s were i n agreement w i t h t h e e a r l i e r w o r k o f B u r n e t i n t h i s r e g a r d w i t h i n f l u e n z a v i r u s , and a l s o w i t h t h a t o f G r a s s e t , who h a d f o u n d no r e s p o n s e t o d i p h t h e r i a t o x o i d i n t h e c h i c k e m b r y o ; E a r l y i n 1 9 5 1 , E v a n s r e p o r t e d u p o n t h e r e s p o n s e o f e m b r y o n a t e d 15 eggs to cultures of Qorynebacterla. With the intention of develop-ing a p r a c t i c a l laboratory method for the t e s t i n g of the t o x i n -producing a b i l i t y of various strains of 6orynebacteria which might eliminate those uneconomic nuisances, the white mouse and the guinea-pi g , she tested t h i r t y - f o u r strains of Oorynebacteria i n f e r t i l e eggs, guinea pigs, rabbits and chicks. She concluded that the r e s u l t s i n both the usual test animals and i n chick embryos corresponded exactlyj thus perhaps forecasting the decline and f a l l of the guinea-Pig* 16 The chorioallantoic membrane of the embryonic chick presents an excellent medium for the propagation of many microorganisms since i t contains the tissues from three germinal layers at the same time which are also readily, and almost simultaneously, available upon opening the sufficiently developed egg* The outermost of these layers the ectoderm, eonsisting of epithelial cells, is interrupted at numerous points by many capillaries which underlie and protrude through i t * Thus the chorioallantoic membrane lends itself admirably to the cultivation of numerous bacteria, since they may invade the embryonic bloodstream and be carried throughout i t by the embryo*s own phagocytes or, at least, grow and produce characteristic effects upon the membrane itself* In view of the fact that the worker should be familiar with the various stages of growth of the embryonic chick, i t has been thought necessary at this point to describe the structure and formation of the developing chorioallantois in some detail, together with those structures most frequently inoculated* The calcium of the shell with its outer layer of mucous is deposited upon a fibrous shell membrane, which is a secretion product of the isthmus of the hen's uterus* To begin with, the chick embryo is only a sheet of cells lying over a pole of the yolk, but as incubation proceeds the three primary germinal layers, termed the entoderm, mesoderm and ectoderm(take form* Gradually the lateral ^ portion of the embryonic structure divides itself into the dorsal somatopleure, which is composed of ectoderm and mesoderm, and the ventral splanchnopleure, consisting of entoderm and mesoderm with 17 the incept ire coelomic cavity lying between* By a process of folding and overgrowth the ohorlon and the amnion arise from the somatoplenre until from the fi f t h day of incubation onwards the amnion is drawn entirely around the embryo,enfolding i t almost completely, except for the yolk stalk* The chorion develops rapidly, until by the tenth day i t has completely surrounded a l l the contents of the egg, but is pressed in close oontaot with the shell membrane at a l l points* The allantois originates within the body of the embryo itself^ being a sort of diverticulum of the ventral wall of the hindgut which appears on the third day of incubation* It enlarges quite rapidly, growing out into the extra-embryonic body cavity* The allantoic cavity is lined with entodermal epithelium, backed with mesodermal tissue, and as i t increases in size its mesodermal aspect fuses with the chorion to form the chorioallantoic membrane on the outside, and with the amnion internally* This fusion, which is practically complete by the tenth day, almost completely obliterates the extra-embryonic cavity. However, smooth muscle fibres, which are obviously designed to move the embryo from time to time and so prevent its adhesion, form between the epithelial layers of the chorion and allantois fusion* Primarily the chorloallantois serves as the embryonic lung and so is richly supplied with blood vessels* There is a dense capillary network in its mesoderm which is in close contact with, and at times i even protrudes through, the outer ectodermal layer which is pressed closely against the shell membrane* There are two main arteries which run from the yolk stalk to the chorioallantoic membrane* and the blood returns from i t by way of three main vessels * two accompanying the arteries while the third, which is the main allantoic vein* lies 18 separately. The chief allantoic vein is quite large and free to move, having its origin at the junction of the two large chorio-allantoic veins near the margin of the air-space* It is the point of origin of the ehorio-allantoic vein which is used as a landmark by the inoculator for determining the position of the embryo in the egg. The yolk sac, which has as i t s function the transport of nut-riment from the yolk contents to the embryoj consists of a steadily enlarging sheet of ectodermal cells and associated blood vessels, lying on the surface of the yolk. As the embryo grows* the surface of the yolk sac is Increased in area considerably by the development of v i l l i which increase in size and number as the embryo develops. Daring its final day or two in the egg* the embryo withdraws what is left of the yolk sae into its abdominal cavity to serve i t as a reserve food supply during its first week of hatched l i f e * It is in the white of the egg that most of the water is con-tained, but as development proceeds, moisture is transferred from the white to the yolk with its consequent enlargement* reaching a maximum by the eighth or ninth day. During the process of incubation* there is a constant loss of water through the shell by transpiration. The allantoic fluid increases in volume to about the eleventh day of incubation, attaining a maximum by the thirteenth day, but from that time on i t decreases until by the day of hatching no fluid at a l l remains in the cavities. The fluid in the allantoic and amniotic cavities consists in essence of a simple solution of the physiological salts, being a clear fluid with a low protein content. However, from the twelfth 19 day onwards, the protein contents and viscosity of the fluid is considerably increased due to the entry of albumen into the amniotic cavity from the albumen sac of the egg, situated near the pointed end. The fluid in the allantoic cavity shows a gradual increase in non-protein nitrogen throughout the whole incubation period,con-sisting mainly of urates, since i t receives the waste from the kidneys. In fact, from the twelfth or thirteenth day onwards the allantoic fluid i f chilled will show a precipitate of urates. According to Needham, the pH of the allantoic fluid varies with the stage of development, being slightly alkaline during the period from seven to twelve days, and falling to pH 6 towards the end of the incubation period. 20 Schematic section of 4-day chick embryo to show the early development of the extra-embryonic membranes. The three germinal layers are indicated in this figure ectoderm by a heavy black line entoderm by a red line mesoderm by green color C h r A U Diagram of the arrangement of membranes in a 9-day embryo. The three germinal layers are indicated in this figure as in the figure above. Amn. - amnion Ch.All. - chorioallantois A l l . - allantois A.S. - albumen sac Ch. - chorion Y . S . - yolk sao All.Cav. - allantoic cavity E.E.B.C. - extra-embryonic body cavity 21 „ C- A. Semidiagrammatio sketch showing f u l l development of membranes and c a v i t i e s i n 12-to 15-day chick embryo. The three germinal layers are indicated i n t h i s f i g u r e as i n the two preceding f i g u r e s . A.S. - albumen sac Am.O. - Amniotic ca v i t y A.C. - a i r c e l l V. * v i l l i C A . - c h o r i o a l l a n t o i s Y.S. - yolk sac E.E.B.C. - extra-embryonic body cavity yo\K s a c o \ t Diagram of the r e l a t i o n of the o r i g i n of the a l l a n t o i c vein to the d i s p o s i t i o n of the embryo and the yolk sac as seen from the a i r space end of the egg. A l l diagrams a f t e r Beveridge and Burnet 22 The A r t i f i c i a l Incubation of, %gs_. for Experimental Purposes ' There are many makes of incubators upon the market, a l l capable of giving satisfactory performance under the proper conditions of man-agement, but there are actually only two types of these machines in general use* The two types are the "cabinet" type and the " s t i l l air" type* The former relies for ventilation upon forced draught provided by electric fans, whereas the latter depends upon the movement of air by convection currents. The cabinet type is the type used by commercial egg hatcheries where large numbers of chicks must be hatched with a minimum of labor* It is thermostatically controlled as to temperature, and properly humidified and ventilated by mech-anical means, even the eggs being rotated automatically in the latest and most efficient models* The " s t i l l air" machine is usually a much smaller incubator than the "cabinet" type, being used in small poultry-raising operations. Some of the " s t i l l air" machines are small enough to be placed upon an ordinary table and will handle some five or six dozen eggs only. The heating, as In the "cabinet" type, is electrical and thermostatically controlled, but there is no provision for the controlling of humidity, or for the mechanical turning of the eggs* With the Intention of determining the practicality of the " s t i l l air" type of incubator for laboratory incubation of fertile aggs, prior to inoculation, one of that type was obtained* The machine was placed in a dark corner of the laboratory and away frofe 25 radiators in order to preserve i t as much as practically possible from the influence of exterior variations in temperature. In order to check the dependability of the machine's temperature controlj the incubator was set in operation and allowed to run steadily for several days, but without any eggs in i t . The incubator thermometer was checked for accuracy against a standard laboratory thermometer, and having been found to be sufficiently dependable, was used as a means of cheeking upon the temperature of the incubator. The thermo-static control of the incubator was adjusted until a steady temper-ature of between 102° and 103© F. was maintained, this being the temperature which* according to various authorities* is the correct one for the incubation of fertile eggs in this type of machine. The egg capacity of the machine was only sixty eggs* but no more than three dozen were ever maintained in i t at any one time. The eggs, reputedly fertile* were placed in the incubator blunt end up and standing supported in pressed paper cartons. The eggs were tilted from side to side once or twice a day* according to convenience. The results were far from satisfactory* many of the eggs appeared to be infertile to begin with, and others after developing to a small extent and showing some signs of possessing an embryo, suddenly ceased to progress farther and showed what is known as a blood ring* a dark band encircling the egg visible only when the egg is examined in the light of a candling device. Some eggs did develop to f u l l term* and a few chicks were even hatched out* but they proved to be weak and deformed. No definite number of eggs 24 could be depended upon to develop to a s u f f i c i e n t maturity for inoculation procedures, although frequently f i f t y percent did so. The uncertainty of having a s u f f i c i e n t number of we l l developed, viable embryos for experimental inoculations interfered seriously with the work attempted* The eventual solution was found to be to obtain embryonated eggs, developed to the required stage, from the commercial type incubators of the hatchery on the University's experimental poultry farm* The small incubator was used thereafter either to store unused eggs u n t i l required* or to incubate eggs which had been operated upon and inoculated, for both of which purposes i t was found to be completely s a t i s f a c t o r y . The reason f o r the f a i l u r e of the small machine to supply the required embryos was not due to either incorrect temperature control or lack of humidity, but simply to the i n a b i l i t y to arrange for the s u f f i c i e n t l y frequent number of turnings of the eggs* which accord-ing to various authorities should be at least every eight hours. The f i r s t ten days of incubation are the c r i t i c a l ones during which ro t a t i o n of the eggs must be practiced* since when the egg remains i n any one position for a length of time, the yolk tends to r i s e through the white and becomes stuck to the s h e l l membrane* When t h i s occurs the embryo i s unable to develop further and soon d i e s . In large machines equipped with mechanical devices f o r turning the eggs, i t i s not unusual to f i n d the eggs being rotated every three hours. The eggs are set i n cradles with t h e i r long axis almost v e r t i c a l and the blunt end uppermost, then the cradle i s rotated slowly from side to side through an arc of approximately ninety 2? degrees* the eggs being permitted to remain at each of three positions, vertical or lying on its right or left side for an equal period of time* Important also to the proper development of the embryo in an incubator is the proper degree of humidity* Dry air in an incubator chamber causes rapid evaporation of water from the egg and results in a toughening and drying out of the shell membranes which renders them somewhat more refractory to manipulation in the various techniques. The correct humidity for hen's eggs during the process of incubation stands between seventy and eighty per cent. It Is a waste of time obviously to incubate infertile eggs, but nevertheless this is frequently done since a hundred percent fe r t i l i t y rate is only possible theoretically. The usual fert-i l i t y rate at best is only eighty-five percent. Since there is no way of recognizing a fertile egg until the embryo has developed sufficiently to be recognizable in the candling operation, i t is necessary to incubate fertile or supposedly fertile eggs for sev-eral days before candling them* With this in mind, a candling device was constructed, as detailed elsewhere, for the routine candling of and detection of infertile and dead embryos. In practice i t was found most convenient to candle the eggs before bringing them away from the poultry farm. In this way infertile eggs and undeveloped embryos could be eliminated at the source* However it was usual to re-examine the fertile eggs after bringing them to the laboratory in order to mark the edge of the air-space and the position of the allantoic vein* Although i t is preferable to have 26 a c o m p l e t e l y d a r k e n e d r o o m , o r a l e a s t a camera c l o t h w h i c h c a n be drawn o v e r t h e h e a d and c a n d l i n g m a c h i n e f o r t h e o b s e r v a t i o n o f gggs b y t h i s m e t h o d , i t was f o u n d t h a t w i t h a l i t t l e p r a c t i c e , t h e m a r g i n o f t h e a i r - s p a c e c a n be s een i f t h e egg i s shaded f r o m d i r e c t l i g h t w i t h the hand w h i l e t h e egg i s h e l d o v e r t h e c a n d l i n g l i g h t . A h e a v y shadow, e x t e n d i n g a l m o s t t o t h e p o i n t e d end o f t h e e g g , i n d i c a t e s t h e p o s i t i o n o f t h e most d e v e l o p e d s i d e o f t h e c h o r i o a l l a n t o i s , and may be s i m i l a r l y s een b y r o l l i m g t h e egg w h i l e p r e s s e d o v e r t h e c a n d l i n g l i g h t ' s o r i f i c e and s h a d i n g f r o m r o o m l i g h t w i t h t h e f r e e h a n d . How-e v e r , f o r t e c h n i q u e s i n w h i c h t h e p o s i t i o n o f t h e embryo must be p r e -c i s e l y k n o w n , t h e r e i s no s u b s t i t u t e f o r a d a r k - r o o m . Once f o u n d , t h e p o s i t i o n o f t h e embryo can be e a s i l y o u t l i n e d upon t h e s h e l l w i t h a s o f t l e a d p e n c i l . I f a number o f eggs o f a b o u t t h e same age a r e b e i n g c a n d l e d a t t h e same t i m e , i t i s r e l a t i v e l y e a s y t o r e c o g n i z e d e a d e m b r y o s , j-hese show l e s s d e v e l o p m e n t , t h a t i s , c a s t l e s s shadow t h a n t h e l i v i n g o n e s i n t h e g r o u p ; t h i s i s , o f c o u r s e , o n l y i f i t h a s b e e n dead f o r t w e n t y - f o u r h o u r s o r m o r e . W i t h v e r y r e c e n t l y dead embryos w h i c h a r e o f the same s i z e as t h e o t h e r s i n t h e g r o u p , t h e r e i s no s u r e method o f r e c o g n i z i n g t h e f a c t , a n d i t may n o t be r e a l i z e d u n t i l t h e e g g h a s a c t u a l l y b e e n o p e r a t e d u p o n and t h e c h o r i o a l l a n t o i s l a i d b a r e , t h a t t h e embryo i s d e a d . E g g s t h a t a r e n o n - f e r t i l e o r embryos w h i c h have d i e d i n t h e e a r l y s t a g e s o f d e v e l o p m e n t a r e u s u a l l y r e a d i l y r e c o g n i z e d , e i t h e r because t h e e g g i s q u i t e c l e a r e x c e p t f o r t h e l i g h t shadow o f t h e y o l k , o r b e c a u s e t h e d a r k band o f t h e b l o o d r i n t may be s een e n c i r c l i n g 27 the egg at its equator* The most satisfactory system of candling eggs was found to be as follows* First, the eggs ready for Inoculation are examined rapidly and those showing tip as "clears", or possessing blood rings, are dis-carded* Second* the eggs chosen for inoculation by the chorioallantoic technique are examined carefully in order to determine the position of the margin of the air-space, which is almost always to be found at the blunt end of the egg* The edge of the air space is then marked on the egg shell with a soft lead pencil on the side where its shadow indicates that the chorioallantois is best developed* Third, the position of important veins, such as the main allantoic vein is carefully marked* Finally, a triangular outline is drawn in the area to be operated upon^ care being taken to avoid placing this directly over any Important blood-vessels, the rupture of which by clumsy manipulation might lead not only to the possible death of the embryo at an inopportune time, but to lesions which could not be accounted for after inoculation* It was found that eggs from ten to fifteen days of age could be used successfully for culturing bacteria upon by means of the chorio-allantoic technique. The age preferred for cultivation by this technique was from ten to twelve days, the reason being that at that age the chorioallantoic membrane is well developed, and that with oider embryos difficulty was frequently experienced in effecting a ready separation of the shell and chorioallantoic membranes* In the following descriptions of technique It should be fully realized that aseptic procedure is adhered to as far as possible* Sterile syringes* needles and pipettes are used both for making 28 inoculations and for the harvesting of eggs, and a l l instruments coming in contact with the egg membranes are sterilized by flaming, which although destructive to the temper of instruments, i s effectively bactericidal. The following chorioallantoic technique i s a slight modification of that described by Beveridge and Burnet ( 1946 ). The eggs selected for inoculation are esamined, as previously described, with the aid of a candling device. The side of the egg opposite that where the growing edges of the chorioallantois have united, should f i r s t be found, then the edge of the air-space i n the blunt end of the egg should be noted and i t s limit marked with a pencil on the 8 h e l l . An equilateral triangle may then be outlined with sides from 10 to 15mm. in length, and approximately a third the distance from the previously outlined edge of the air space to the pointed end of the egg. The surface of the eggshell immediately surrounding the marked area and the blunt tip of the egg should then be swabbed with a suitable disinfectant. An aqueous solution of sephiran ( alkyldimethyl-benzylammonium chloride, 1/1000), was found effective for this purpose, and preferable to iodine, since i t exerts no destructive action upon the d r i l l mandrel or other instruments; A small e l e c t r i c a l hand d r i l l ( Hand-ee Grinder ) f i t t e d with a dental mandrel for the mounting of carborundum separating discs ( Baker, 5/8 i n . ) was found to be a satisfactory tool for cutting eggshell. Flat double sided carborundum dental separating discs w i l l withstand autoclaving i f placed on a cheese-cloth pad i n a petri dish. However, they may quite satisfactorily and more 29 conveniently be sterilized by soaking in aqueous Zephiran ( l/lOOO ) solution when mounted on the mandrel just previous to use« The egg is manipulated as illustrated in the process of chorio-allantoic inoculation technique* A triangle of shell is cut out with the dental disc, and a cut is also made through shell and shell membrane into the air sac of the egg* The egg is then placed in the Dunham type inoculating stand and suction applied to the air sac end while the triangle of shell is prized gently out, the saline wedge inserted, and the separation of the chorioallantoic and shell membranes effected* The adhesive cellophane tape* which has been found quite satisfactory for sealing the egg with, is attached at one side of the triangular hole before removing the egg from the Dunham inoculator* The inoculum should not be added while the egg is in the Dunham inoculator because of the inconvenienoe of ster-ilizing this instrument should i t become accidently contaminated. Instead, the egg is placed upon a small inoculating stand such as the type illustrated, which was designed by Beveridge* If such a stand is not available, a pressed paper egg-box may be substituted, since i f this is contaminated i t may be either sterilized by autoclaving or easily destroyed in an incinerator* Egg-boxes of this type proved to be very useful for holding eggs which had been inoculated* during their subsequent incubation* The hole in the shell may be sealed in a variety of ways, the classical method being to ring the area with petroleum jelly* sterile of course, and to place a sterile cover slip over this* thus exclud-ing extraneous organisms from the egg. This method was found to be 50 rather inconvenient, and a seal of ordinary adhesive cellulose tape ( Scotoh tape ) was found to serve the purpose better* Although this tape was not sterilized, its use did not appear to predjudice the results of the experiments in any way by contributing to con-tamination of the egg contents, and trials with i t on uninoculated, but opened eggs, gave negative results when tests were conducted for bacterial contamination, even after several days incubation. Eggs were harvested after incubation of organisms in them by means of sterile physiological saline introduced with a sterile capillary pipette* The saline was added after cutting out the sealing tape over the triangular area and allowed to remain in the egg, which was tilted gently from side to side in order to wash any growth off the chorioallantoic membrane* This harvest was then withdrawn with the pipette. Several cc. of saline may be added to the egg when washing out the harvest. It was found that i f the egg were tilted over to one side the saline harvest could be easily withdrawn, but care must be exercised to avoid using heavy suction when so doing* or else the membrane itself may be sucked up, effect-ively blocking the pipette. For convenience in harvesting^ narrow glass test tubes containing sterile saline were used as receptacles for the harvest, and these instead of being plugged with non-absorbent cotton were covered with inverted gas vials* Tubes capped in this manner may be covered and uncovered a number of times* whereas tubes with the usual cotton plugs do not lend them-selves to too frequent removal of their cotton plugs, which lose their shape rather easily. Another advantage of the phial-topped tube is that i t can be placed in the centrifuge and the harvest 51 thrown down without the necessity of fastening a cotton plug in place with an elastic band; as is the usual practice* A modification of the chorioallantoic inoculation technique is that of Beveridge (1947)* The egg Is candled and the margin of the air-space marked as usual* The shell is disinfected and then a hole is made by jabbing with a sterile forceps a few mm* from • the margin of the air-space* over the air-space. The shell membrane is pierced and the shell-hole enlarged until 1 em* long and 3/4 mm; wide^is developed^running parallel with the margin of the air-space* Next the shell* without the attached shell membrane* is broken away for three to four mm* beyond the margin of the air-space* The exposed outer shell membrane is then torn away and the inner layer of shell membrane is found to have a fold about 1 mm* wide marking the margin of the air-space* A fine pointed pair of forceps i s used to tear the shell membrane along this fold* The egg is held in the hand up to this stage and in an approximately horizontal position, but is not tilted with the air-space downwards* In the case of eleven-day embryos the chorioallantois usually falls readily, giving an a r t i f i c i a l air-space in the same position as with the standard technique* More difficulty may be experienced in separating the shell membrane from the chorioallantois in the case of older embryos, but tapping the egg with the fingers usually serves to effeot the separation* The egg is returned to the horizontal position and the inoculation effected* the shell being sealed with adhesive cellulose tape. This method was tried since i t appeared to be a simple way 52 of opening a fertile egg and one not requiring the use of a d r i l l . However, i t was found to be somewhat difficult to control the breaking of the egg-shell and to avoid damaging the egg membranes* and far this reason was not continued. Perhaps with practice this might be found to be a reasonably convenient method. The method of chorioallantoic inoculation was the one used in the cultivation of bacteria* but for the sake of completeness the following methods of inoculation were attempted though not with 7 the use of any bacterial inoculum. For the Beveridge and Burnet (1946) technique of amniotic inoc-ulation, which is widely used in the cultivation of influenza virus* embryos aged anywhere from eight to fourteen days may be used. The eggs are candled as for the chorioallantoic technique, but particular note is taken of the location of the allantoic vein, a pencil mark being made upon the shell to indicate its point of emergence, which is usually close to the edge of the air-space. It is particularly important that the eggs chosen for this technique should have light eolored shells* since otherwise i t is difficult to see the veins well when candling. If the eggs are derived from white leghorns,,*^* (whose eggs are uniformly white), which is the usual practice* no difficulty with dark shelled eggs should be experienced* If* as sometimes occurs* the allantoic vein itself cannot be seen* its position may be determined by noting the point at whioh two large veins converge towards the air-sac end of the egg. The point for inoculation may then be determined by the following rule. If the egg is held with the air space end towards the operator, the 55 point for inoculation is approximately half an inch clockwise from the point marked and nearer the air-space end of the egg* The embryo should l i e immediately below this point* A hole is drilled through into the air-spaee as in the chorio-allantoic technique* Next a rough oval, approximately 2 cm* by l^cm. and with the long axis parallel with the long axis of the egg, is cut about this point with the separating disc* Two cuts are made in the oval at the blunt end of the egg to form a sector which will facilitate the removal of the area enclosed in the oval. Great care must be taken, as in the chorioallantoic technique, not to damage the shell membrane during the cutting process* The egg may be mounted in a Dunham type inoculator and suction applied to the hole in the air sao end to facilitate the collapse of the chorioallantoic membrane, which usually occurs without the addition of a drop of physiological saline* A l l operations upon embryonated eggs should be carrled out under the illumination of a powerful light* It has been found that a goose*heok desk lamp fitted with a 60 watt daylight type bulb is quite suitable for the purpose* The chorioallantois is grasped with a pair of curved sharp toothed forceps at a point where no large blood vessels are in evidence* and a cut is made with a sharp pointed scalpel about half a centimeter in length* oare being taken not to damage the amnion, which lies immediately beneath* In this regard also* one should be cautious when grasping the chorioallantois not to include the amnion in the pinch of the forceps or i t may be aeo-idently cut* The injectioh may be readily made with a bevel 54 edged capillary pipette, the amnion being heid taut with the curved toothed forceps* to enable its being pierced. It is of practical value to have 0.02 « 0.05 ml* of air below the inoculum in the pipette, since this may then be injected into the amnion firsthand the position of the air bubble will inform the operator whether or not he i s in the amniotic cavity. The egg may be sealed after inoculation with a cover slip set on a ring of sterile petroleum jelly made about the opening* but a wide piece of adhesive cellulose tape is more convenient, Beveridge has modified this amniotic inoculation technique for the use of researchers not possessing dril l s for cutting the egg-shell. The method is as follows: The preliminary incubation of the egg is carred out with the air sac end uppermost which is the usual position in commercial incubators. This position is essential in order to assure the embryo's being close to the margin of the air**space» A hole is breached into the air sac end of the egg in the same manner as in Beveridge*s modification of the chorioallantoic inoc-ulation technique* but the hole is not extended beyond the margin of the air-spaeei A sharp pointed forceps is thrust through the shell membrane and the underlying chorioallantoic membrane close to the margin of the air-space, and the hole is then extended for about 1 .cm, by blunt dissection. As air enters, the embryo* enclosed in the amnion, is presented at the hole 0 It may be necessary to remove some of the shell membrane adhering to the chorioallantois. Should an air bubble biook the hole, as sometimes occurs* i t may be dispersed with a touch of a heated needle. 55 The amnion is grasped with toothed forceps and held taut while the injection is made either with a beveled capillary pipette or a hypo* dermic syringe and needle* The hole in the shell may then be sealed with adhesive cellulose tape and the egg incubated in an upright position* Another method, that of Hirst (1942), is a simple one for the inoculation of the amniotic cavity in embryos of seven days or older* In this technique the egg shell Is merely pierced, with a small hole, and the injection made Into the amnion through It with a hypodermic needle of suitable size* The only disadvantage of this method is, that i t requires transillumination of the egg during the process of injection in order to observe the position of the needle* The yolk sac method of inoculation Is a technique usually performed upon very young embryos* that is, five to six days of age* The eggs are candled as usual and a cut made with a carborundum separating disc at the blunt end Of tfee egg immediately over the air-space, large enough to admit an inoculating needle, but taking care, of course, not to pieroe the shell membrane when cutting* The needle is passed ttrough the shell membrane, the egg being held perpendicularly In an inoculating stand, such as Beveridge's until the point is judged to have reached the oentre of the egg or a l i t t l e beyond i t * The inoculation being completed, the hole in the shell may be sealed with either a tiny piece of adhesive cellulose tape or a drop of paraffin wax* A carborundum cutting disc is not essential to the performance of this technique, as the shell may be simply pierced with a straight surgical needle mounted in a rubber 56 ^ung* The allantoic cavity method of inoculation is usually performed upon embryos of from ten to eleven days of age* Tne eggs are candled as usuali and an area noted where the ohorioallantois is well devel-oped but not showing any large blood vessels* A out is made in the shell at this point parallel with the long axis of the egg and about three mm, in length whioh is wide enough to admit a hypodermic needle easily, but without damaging the shell membrane* The inoculation may then be made by piercing the shell and chorioallantoic membranes for a few millimeters with a suitable hypo-dermic needle* The intracerebral method is applicable to eggs of from eight to thirteen days of incubation. They are candled as usual and the position of the embryo determined as in the amniotic inoculation method, an oval being marked upon the shell and a sector cut out from i t as ln that technique* The shell membrane is pierced and torn away within the oval area and then the chorioallantoic membrane cut with a scalpel* The head of the embryo is frequently presented at the opening, but i f i t is not* i t ean usually be manipulated into position with a needle* It has been found that a sharp half-inch 27-gauge needle mounted on a tuberculin syringe is most suit-able for inoculating with* The head, In the case of more fully developed embryos, is grasped with a forceps and *he needle 57 inserted into the skull, but with young embryos usually a sharp quick thrust at the skull will be sufficient to pierce i t . The intravenous method of inoculation is usually, practised with embryos ten days or older$ and is a delicate technique* The eggs are candled to determine the position of the larger veins in the chorioallantois* One of them is chosen, and its position together with the direction of the flow of blood marked with a peneil on the shell* A parallelogram, one by one and a half centimeters is described on the egg shell at this point with the vein centred in i t and running parallel to i t s long axis* This marked piece of shell 1 B then removed by cutting about i t with the separating disc and then prising out, great care being taken to in no way damage the shell membrane nor the underlying chorioallantois* The shell membrane may be rendered transparent by placing a drop of sterile mineral o i l upon i t and the point in the selected vein for inoculation be seen. A sharp 27-gauge needle mounted on a tuberculin syringe is most satisfactory for the inoculation. The needle is then rested against the edge of the cut shell in order to steady i t and then it is most carefully inserted, bevel upper-most Into the vein in the direction of the blood flow* Either a Dunham or a Beveridge type inoculating stand will hold the egg satisfactorily during the perfonaanee of this technique* After inoculation the needle is withdrawn slowly and carefully in order to avoid any risk of hemorrhage* The hole in the egg-shell need not be sealed over* 58 EXPERIMENTAL METHODS The fundamental Tools for Chorioallantoic ^ Inoculation* It was found necessary to obtain, and in some cases to construct certain pieces of equipment essential in the manip-ulation and inoculation of fertile eggs. These were of the simplest type possible, the intention being to achieve a maximum of successful work with a minimum outlay for equipment* The essential equipment was as follows: A small capacity " s t i l l air" type incubator. An electric hand grinder with accessories. An electric egg candler* A-Dunham egg inoculator* Beveridge type simple egg stands* Various needlesj forceps* scalpel and inooulating instruments* In addition to these* since i t was desired to record as fully as possible the method of performing the chorioallantoic inoculation technique, together with some of the results of this method of cultivation, i t was found necessary to obtain a camera with various supplementary lenses and floodlights for illuminating the work, together with an adjustable stand for use in recording the appear-ance of the embryonic chorioallantoic membrane* Illustrations of this apparatus will be found on the following pages together with a brief outline of their construction. Instruments used i n the chorioallantoic inoculation technique Background.left to right: Hooked needle used i n separating chorioallantoic and shell membranes Straight needle used i n prising up cut triangle of shell Forceps used for removing pieces of shell Scalpel used i n removing remains of shell membrane i n triang-ular area Forceps used in tearing out remains of shell membrane in t r i -angular area Foreground: Handee Grinder fi t t e d with dental mandrel upon whioh i s mounted a carborundum separating disc. 40 < Sponge vnbb a.-r VAl..V\o]a. Diagram of egg candler 1 Diagram of Dunham egg inoculator 41 Adjustable camera stand used In photographing chorioallantoic membrane 42 Beveridge simple egg Inoculating stand This may be constructed with a 1/16 in. thick rubber base or rubber headed tacks may be used to serve as legs to prevent slipping when in use STEP ONE The method of cutting a hole through the shell and shell membrane at the blunt end of t h e egg. The hands are steadied by resting the forearms and wrists against the chest* STEP ONE Side T i e n of step one but with e g g held i n alternative position. Note the method of holding the d r i l l . STEP TWO Method of holding egg and d r i l l while cutting first side of triangle of shell which is to be removed. The shell is not cut completely through since that would entail damage to the underlying shell membrane. STEP THREE Method of holding egg while cutting second side of triangle. Note the position of the fingers. STEP FOUR Method of holding egg while cutting t h i r d side of t r i a n g l e . STEP FIVE Method of i n s e r t i n g needle,bevel up, under edge of corner of tr i a n g l e of s h e l l which i s to be prised up. Egg i s mounted i n Dunham inoculator. S h e l l membrane must not be pierced. -STEP s i r Method of p r i s i n g out t r i a n g l e of detached shell.Care must be taken to avoid tearing s h e l l membrane. Suction i s now being applied to a i r sac of egg. STEP SEVEN Placing drop of s t e r i l e p h y s i o l o g i c a l saline upon i n t a c t s h e l l membrane. The drop runs between the c h o r i o a l l a n t o i c and s h e l l membranes when the s h e l l membrane i s pierced beneath i t with a sterile,hooked needle thus acting as a f l u i d wedge and e f f e c t i n g t h e i r separation. STEP EIGHT Method of tearing s l i t i n s h e l l membrane with s t e r i l e hooked needle thus allowing f l u i d wedge of saline to enter and with the aid of suction dropping the chorio-a l l a n t o i c membrane away from the s h e l l membrane• STEP NINE Method of tearing out remains of s h e l l membrane i n triangular area with pair of s t e r i l e forceps after the dropping of the chorioallantoic membrane. STEP TEN Method of adding inoculum to egg. Egg is now mounted in a simple stand with its adhesive cellophane tape seal partly attached. STEP T^WVKN The egg is ready for incubation with its cellophane closure sealed. Egg must be incubated in this position. 49 With the intention in mind of testing the suitability of the chorioallantoic inoculation technique for the cultivation of bacterial microorganisms^experimental infections were attempted with Serratla marcescens, Staphylococcus albus. Mycobacterium  •phlei and Salmonella typhimurium* Ten to twelve day embryonated eggs were inoculated with Serratia marcescens by dropping onto the exposed chorioallantois membrane three to five drops from a capillary pipette of a twenty-four hour nutrient broth culture© The eggs were sealed with adhesive cellulose tape and incubated for up to five days with daily examination of the appearance of the membrane* A l l eggs inoculated were dead at the end of three days of incubation* Examination revealed a gradual growth of the inoc-ulated organism, at first about the edges of the membrane where the chorioallantois and the shell membrane come together* This growth gradually spread, becoming a bright pink in color with the passage of time* A peculiar feature of the infection whioh was noted, was the apparent jelling or partial solidification of the contents of the egg* A number of transfers were made from the cultured eggs to other similarly developed embryonated eggs with the same result* Nutrient agar spread plates made from harvests of the cultured eggs showed profuse typically pink growth. No colonies of organisms other than Serratla marcescens were seen on any of the spread plates, and It was eoncluded that the technique had been sufficiently aseptic In its performance to avoid any large amount of contamination* 50 • Ten to twelve day old embryonated eggs were inoculated with Staphylococcus albus. a laboratory strain used for B t u d e n t class work* and a white pigment producer* The inoculation was effected by dropping onto the exposed chorioallantoic membrane three to five drops from a capillary pipette of a twenty*fbur hour nutrient broth culture of the organism• Contrary to expectation, the eggs did not die* nor did the membrane surfaoe show any evidence of infection in so far as could be ascertained by ordinary inspection. The ^ anbryos continued to live and develop, and would undoubtedly have hatched had they not been purposely destroyed by refrigeration* Smears taken from the surface of the chorioallantoic membrane some eighteen to twenty-four hours after the introduction of the inoculum* and stained by Wright's method, showed numerous polymorphonuclear cells, some containing a few cocci but no further evidence of the presence of the inooulum* It was assumed that the strain used* which was one which had undergone numerous transfers upon ar t i f i c i a l media* had probably become avirulent and incapable of setting up an infection* The surface of the chorioallantoic membrane of a ten day old egg was inoculated with a loopful of Mycobacterium phlei taken from the surface of a three day old agar slant of the organism* The inoculated egg was then incubated for three days. At the end of this time a smooth, raised, pale yellow gelatinous growth of the Mycobacterium was to be seen at the point of inoculation* The embryo was dead, b u t whether this was due to the growth of the 5 1 Mycobacterium was not determined. In an attempt to find whether prolonged cultivation upon the chorioallantoic membrane of fertile hen*s eggs would in any way affect the organism, a stook culture of Salmonella typhimurium was treated In the following manner* The stook culture was inoculated into nutrient broth and incubated at 37° 0* for twenty-four hours, at the end of which time a loopful of the broth culture was inoculated into a motility tube* At the end of twenty-four hours of incubation motile organ-isms were picked from the motility tube into nutrient broth* At the end of twenty-four hours of incubation this culture was Centrifuged at 3000 r*p*m* for twenty minutes, the supernatant liquid discarded, physiological saline added to the precipitate to resuspend i t , and this suspension than centrifuged as before* The precipitate was then resuspended in sterile physiological saline and approximately 0*2 co. used to inoculate the surface of the chorioallantoic membrane of a ten day old fertile hen's egg* At the end of twenty-four hears of incubation the egg was a harvested as previously described by washing off the surface of the chorioallantoic membrane with sterile physiological saline* The harvest was then centrifuged, the precipitate resuspended in sterile physiological saline, recentrifuged and again resuspended as previously described* and 0*2 cc. of this suspension used to Inoculate the surface of the chorioallantoic membrane of another ten day old fertile hen*s egg* This system of procedure was carried on for approximately a month, during which time some 52 twelve transfers were made at twenty-four to fourty~eight hour intervals. At each harvest, samples of the inoculum were introduced into nutrient broth and spread upon the surface of nutrient agar plates which were subsequently incubated at 37° 0. for twenty-four hours and then checked for contamination with other than the Salmonella  typhimurium type organisms. The embryos were found regularly to succumb to the pathogen within twenty-four hours or less. Smears made from surface of the chorioallantoic membrane showed masses of Gram-negative rods and a few polymorphonuclear cells, some containing ingested rods. Hanging drops made of the harvests showed many highly motile rods, many occuring in pairs, end to end and frequently elongated to three or four times the normal length of these cells. The colonies produced upon the nutrient agar plates remained uniformly smooth in appearance, and this was confirmed by numerous picked colonies which, when inoculated into broth, gave uniform turbidity with only slight sedimentation in twenty-four hottrs. The organisms apparently grew well upon the chorioallantoic membrane or rather in the moisture upon its surface. If this surface was examined closely with a hand lens, floating plaques of turbid growth oould be seen at the end of twenty-four hours of incubation. The blood vessels seem to be engorged and thrombosed, and there also appeared to be extravasation into the membrane immediately surrounding the blood vessels^ giving it an erythe-matous appearance, as may be seen in the accompanying photograph* 55 ... No gross growth or colonial development was noted.. The grey plaques of growing organisms were noted to be in close proximity to the thrombosed blood vessels. Next, an attempt was made to see whether there was any dif- . ference in the agglutitiability with specific antiserum of the passaged strain of Salmonella typhimnrium as opposed to the un-passaged. The original unpassaged motile culture of the organism was inoculated onto the surfaoe of several large nutrient agar slants and incubated overnight, at the end of which time the slants were harvested by washing the; growth of their surfaces with sterile physiological saline. This harvest was then centrifuged and re«* suspended in further sterile physiological saline in order that its treatment might not differ from that of the egg-passaged strain, which was treated as follows: Ah egg in which was a twenty-four hour growth of the egg-passaged strain was harvested as usual and the harvest centrifuged. The precipitate was then resuspended in physiological saline to give a turbidity equivalent to that of a McFarland's standard, number two. This strain of the organism had by now been passaged through some dozen eggs over a period of a month. The unpassaged strain wat diluted with saline to give a turbidity equivalent to a McFarland's standard number two. Then both the passaged and the unpassaged strains were titrated against Salmonella typhlmurium specific antiserum by the method of doubling dilutions. After eighteen hours of incubation in a water bath at 37° e., 54 the titres of both the passaged and unpassaged organisms were recorded. In both oases the reading was the same, four plus agg-lutination with a l / 5 1 2 0 dilution of the antiserum and a traoe agglutination at a dilution of 1/40960 of the antiserum. In the process of Carrying out the egg passaging of the test organism i t had been noted that occasionally harvests would contain considerable numbers of chick blood cells* In order to determine at what age eggs should be inoculated in order to avoid these cells appearing in the harvests a number of eggs aged ten, twelve and fourteen days were inoculated with similar amounts of the organ-isms suspended in sterile physiological saline,and incubated for twenty-four hours* It was noted that the harvest from the twelve day oid eggs were heavier than those from the fourteen day eggs* but they contained considerable numbers of blood oells* sufficient to give the harvest a decidedly pink coloration. The ten day old egg harvests did not contain .very many blood cells and were heavier In turbidity than the fourteen day old egg harvests. It was ooncluded from this that ten day old eggs are the best for inoc-ulating with this test organism, i f a good harvest free from blood Cells is desired. In an attempt to determine whether S or B type organisms of S. typhimurium would show any obvious change during egg passage, a series of some twenty*egg passages were performed at two to three day intervals* The organisms for passage were selected by plating out the original stock culture of S. typhimurium onto nutrient agar plates and selecting S and B type colonies. The 55 colonies were inoculated into nutrient broth, and incubated for twenty-four hours. At the end of the incubation period a broth showing the least amount of precipitate and a maximum amount of generalized turbidity was chosen as the S type organism* and the broth with the most granular precipitate and flocculant growth chosen as the H type organism. For convenience, these types were denoted as Al for the B type and AS for the S type. Eggs were inoculated with centrifuged and resuspended cultures of these two types of organisms and transfers made to fresh ten to twelve day eggs at two to three day intervals. The eggs were usually ten days old when inoculated, but owing to difficulties of supply were occasionally older or younger by a day. The two parallel series of transfers were continued for some twenty pas-sages. A series of broth-to-broth passages of the test organisms were carried out at the same time as the e§gto egg passages of the S and B type organisms, in order to note any similarity in changes between those organisms egg-passaged and those not egg-passaged. At each harvesting, nutrient broths and agar spread plates were made of samples of the harvests in order to see i f any changes in the organisms had taken place. No 'change was noted. The Al strain, both in the broth and in the egg remained B in type and the AS strain similarly remained S in type. During the course of this investigation two facts were noted. First, harvests were best when taken at two day intervals; eggs left unharvested for longer than this time showing poorer and 56 poorer harvests until after five days of incubation very few organisms were present in the harvests* This was particularly the case with the S type organisms* Second, that from time to time some of the embryos infected with the H type organism Al failed to succumb* whereas those infected with the A8 or S type invariably died within eighteen to twenty-four hours* The quantity of inoculum used had been consistent throughout, one drop from a capillary pipette* so i t was concluded that R type strains were less pathogenic than R type strains for chick embryos* This of course raised the question of "How much dif-ference in pathogenicity"? An experiment was then undertaken to determine the approximate number of A8 and the approximate number of Al organisms required to k i l l a ten to fifteen day old ohick embryo in twenty-four hours. A number of eggs were inoculated with equal quantities of inocula but containing different concentrations of organisms* Different dilutions of S and R type organisms were made up* and two eggs inoculated with each dilution* After a number of rep-ititions of the experiment, i t was concluded that from 18 to 90 S type organisms were sufficient to k i l l a 10 day old chick embryo within twenty-four hours* The results also indicated that at least 18,000 R type organisms were required to achieve the same result* In order to dispose of embryos which had survived sub-lethal inocula of the R type organisms* they were in some instances inoculated with many times the determined lethal dose of S type organisms and in some instances, found to survive* Investigation of this phenomena, however, demonstrated that the effect arose from a reduced susceptibility, due not to the previous infection with the B type organisms, but to an increase in the age of the embryo• Eleven or twelve day old embryos which had survived sub-lethal doses of B type organisms for three days would be fourteen or fifteen days old when inoculated with the S type organisms in a dosage known to be fatal to ten to twelve day old embryos. Apparent-ly with this increase in age, a decreasing susceptibility to this organism is acquired, since doses of S organisms far in excess of those lethal for ten or twelve day old embryos failed to k i l l fifteen day old embryos not previously inoculated. Appearance of surface of chorioallantoic membrane a f t e r 24 hours of c u l t i v a t i n g Salmonella typhimnriiun upon I t * Wright's stain of smear made from harvest of a 24 hour culture of Salncnella typhimv.rlum upon the chorioallantoic membrane. A simple safety glass s h i e l d which may be used when in o c u l a t i n g . Twenty-four hour old cultures of rougkg and smooth s t r a i n s of Salmonella typhi-murium i n nutrient broth. Left:rough Right: smooth 60 In the process of the foregoing experiments, i t has been once again demonstrated that the chick embryo i s a satisfactory tool, or perhaps one should Bay a remarkable combination of test-tube and guinea pig, for the student of research in bacterial cultivation* It has also been shown that elaborate equipment is not essential to the performance of the various techniques of inoculating the embryon-ated egg with -very good results* The organisms, Serratia marcesoens and Salmonella typM™***™ »PW> successfully propagated by means of the chorioallantoic inoculation technique and also carried in the egg for a number of generations,0 without serious difficulty or trouble from contamination* The convenience of the chick embryo as a test animal for experimental infection has been adequately demon* strated, confirming the work of numerous other investigators in the field* It has been shown that i t is possible to demonstrate a dif-ference in susceptibility to the smooth and rough strains of Salmonella  typhimurium by inoculating a number of eggs with varying concentrations of organisms and noting the survivors, and i t has also been shown that there is an apparent difference in susceptibility to these strains of Salmonella typhimurium which alters with the age of the embryo* It i s hoped that the ground-work to future work in this departmeirt with the ehick embryo has been laid ln this thesis, especially as regards the techniques of inoculation* The use of the chick embryo in bacteriological research and probably even in virological research has not yet been either fully explored or exploited* 61 In view of Its wide application to the cultivation of viruses and rickettsiae It may perhaps be considered strange that relatively so.little use has been made of the embryonated egg for the propagation of bacteria. The reason why, however, should be obvious. The fact that viruses and rickettsiae do grow and grow well in such a readily available and relatively uniform a medium as the Ohiok embryo has indicated its use for their culture ever since t&e discov-ery of the method. It is the only completely satisfactory medium for the purpose known* Were the processes of Inoculation and propagation of infectious agents such as these twice as difficult as they are* i t would s t i l l be the medium of choice* On the other hand there are numerous media upon which, bacteria can be cultivated with comparative ease* many of them having selective and differential qualities which the chick embryo does not so obviously possess* These standard media are readily controllable as to conditions of manufacture and incub-ation* their formulae are thoroughly and completely known, and they lend themselves to rapid Inoculation by none too skilled technicians. The chick embryo is not strictly speaking a uniform medium, i t s manufacture Cannot be completely supervised, and its incubation Is not a simple matter, nor i s i t s formula completely and thoroughly known* Time and considerable s k i l l must be exercised in its inoc-ulation and, as with a l l living things, results cannot be completely foreseen* Time Is an important faetor in most laboratory routine work* and in few laboratories would It be economically feasible to spend as much of i t as is required to make the inoculation of bacteria into eggs, particularly by such a time-consuming process as the 62 chorioallantoic technique. Nevertheless, despite its fallings, the chick embryo method of cultivating bacterial microorganisms does have certain features which should recommend i t to the bacteriological research worker and perhaps even to the practical laboratory* These might be summarized as follows: (1) It is a very convenient type of laboratory experimental animal sinoe It requires neither feeding nor cleaning© (2} It has been found to be readily infectible by many pathogenic bacteria. (3) It is freer from latent bacterial or viral infections than laboratory experimental animals. (4) It is suitable for the propagation of organisms not readily propagated upon laboratory media. (5) It is an excellent in vivo test medium for toxins, antitoxins, antisera and antibiotics* (6 ) While the embryonic chick medium may not be strictly compar-able with the natural hosts for bacteria studied* the lesions produced are characteristic for the bacterial types* and closely resemble those found In the natural host. This indicates its useful-ness in the study of the pathogenesis of infection. Of course, the experimental egg has certain features which limit its use for experimental purposes. Cl) A certain amount of care must be exercised in the handling of eggs*, since although excellent, they are very fragile test-tubes. (2) They have definite requirements as to temperature and humidity whioh must be provided by a suitable incubator* 6 5 (3) 5hey do not keep, unlike the usual laboratory media which, when not required for immediate use, may be refrigerated until heeded* The experimenter must foresee the number of eggs that he will require for use in his experiments some ten days ahead of time, and they must be available and viable when required* Probably the most important limitation of the ehick embryo method of cultivating bacteria i s the supply of eggs. (4) The techniques of inoculation are somewhat difficult and time-consuming and the necessity for scrupulous attention to detail in the performance of the techniques demand patience and s k i l l , together with a certain delicacy of touch. Nevertheless i t may be concluded that the embryonated egg has a very definite future in a wide field of bacteriological investigation. 64 . STJ&MARY (1) The literature pertaining to the cultivation of bacterial microorganisms in the embryonated egg have been reviewed in en attempt to give the historical background of the subject* (2) Descriptions and diagrams of the fundamental tools for working with the chick embryo have been given* (3) A description and diagrams of embryonic development have been given* (4) The method of handling fertile eggs has been discussed* (5) The usual techniques of operating upon and inoculating the chick embryo have been described and the method of chorioallantoic cultiv-ation of bacteria has been described and illustrated* (6) Chick embryos have been inoculated with Serratla marcescens.  Staphyloccccus albus. Mycobacterium phlei and Salmonella typhimurium, and S* marcescens; M* -phlei and S*| typhimurium have been successfully propagated in this manner* (7) The effect upon its agglutlnability of embryonic cultivation of a laboratory strain of S, typhimurium has been investigated, and found to be unchanged following prolonged serial passage in fertile eggs* (8) Smooth and rough strains of S* typhimurium have been separately cultivated upon the chorioallantoic membrane of the developing chick embryo* and no change in their type of growth has been found attribut-able to such cultivation* (9} The minimal number of smooth, and the minimal number of rough type organisms of S; typhimurium required to infect fatally a ten day chick embryo have been determined approximately. 65 BIBLIOGBAFBY Alture-Werber,E. 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