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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*  THE  CULTIVATION  OF  CHORIOALLANTOIC  BACTERIA  MEMBRANE  OF  UPON HEN'S  by  EDWARD  A  THESIS THE  i  H  THE EGGS  i  MILTON  DONALD  CLEVELAND,  SUBMITTED  IN  FULFILMENT  REQUIREMENTS  MASTER  PARTIAL FOR  THE  DEGREE  OF  OF  OF ARTS  .  IN  THE  .  O  DEPARTMENT F  BACTERIOLOGY and PREVENTIVE MEDICINE  WE  ACCEPT  STANDARD DEGREE  OF  THIS  THESIS  REQUIRED  AS  FROM  MASTER  CONFORMING  CANDIDATES  OF  TO  FOR  THE THE  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  September 1951  COLUMBIA  Abstract of Thesis The Cultivation of Bacteria upon the Chorioallantoic Membrane of Hen s Eggs 1  The literature pertaining to the cultivation of bacterial microorganisms i n f e r t i l e 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 accomplished by this method. Descriptions of other methods of inoculating the chick embryo have been added; Smooth and rough strains of S. typhimurium 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 immune serum or changes i n their type as evidenced by colonial morphology on nutrient agar or growth in nutrient broth; No change was noted; The minimal number of smooth and of rough type of organisms respectively necessary to infect fatally a ten day old chick embryo was approximately determined. In the course of the investigation a reduced susceptibility to infection with smooth type S. typhimurium organisms was noticed with increase i n 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 further pursuit and investigation of these advantages has been made; The thesis has been illustrated with diagrams and color photographs of the instruments used i n the course of the research and of the technique of chorioallantoic inoculation.  TABLE I II III IV  HISTORICAL  OUTLINE.  ANATOMICAL  DESCRIPTION  ARTIFICIAL  INCUBATION  INOCULATION  OF  CONTENTS 1  OF OF  THE  CHICK  EMBRYO.  16  EGGS  22  TECHNIQUES:  A  - CANDLING  B  - METHODS  C  -  TECHNIQUE OF  IN EGGS  CHORIOALLANTOIC  BEVERIOGES'  MODIFICATION  FOR  INOCULATION  TECHNIQUE OF  28  CHORIOALLANTOIC  TECHNIQUE. D  -  BEVERIDGE  E  -  BEVERIOGES'  31  AND  AMNIOTIC  BURNETS '  TECHNIQUE  OF  INOCULATION.  32  MODIFICATION  OF  AMNIOTIC  INOCULATION/TECHNIQUE  V  F  - HIRSTS'  MODIFICATION  G  - YOLK  H  -  I  -t I N T R A C E R E B R A L  J  - EICHORN'S  SAC  METHOD  ALLANTOIC  34  OF  AMNIOTIC  OF. I N O C U L A T I O N  C A V I T Y .METHOD, OF METHOD  OF  INTRAVENOUS  EXPERIMENTAL METHODS  TECHNIQUE  INOCULATION  •INSTRUMENTS  VII  VIII  A  - SE.RRATIA  B  - STAPHYLOCOCCUS  C  — MYCOBACTERIUM  D  - SALMONELLA  OF  AND  36  INOCULATION APPARATUS  MARCESCENS AL BUS  37 38  49 49 50  PHLEl  50  TYPHI MURIUM  51  60*63  DISCUSSION SUMMARY  64  -BIBLIOGRAPHY  DIAGRAMS  36  INOCULATION  METHOD  35 35  EXPERIMENTAL INFECTIONS "' '  VI  27  I II III  IV  65  Schematic section of four day chick embryo  20  Diagram of the arrangement of membranes in.the nine day embryo.  20  Diagrammatic 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 embryos.  21  The relationship of the a l l a n t o i c vein to the embryo.  21  V vi  VII VIII  PLATES (color plates)  I II  Diagram of egg candler  40  Diagram of Dunham egg inoculator  40  Diagram of adjustable camera stand  41  Diagram of Beverldges' egg inoculating stand.  42  I n s t r u m e n t s used i n the a l l a n t o i c technique  39  & III  STEPS  1 AND  TECHNIQUE  I I I & IV  STEPS  2  AND  TECHNIQUE  IV & V  STEPS  4  STEPS  OF  3  OF  43  CHORIOALLANTOIC  44  INOCULATION  S» IF  6. AND  CHORIOALLANTOIC  INOCULATION  OF  AND  TECHNIQUE  V & VI  1A OF  chorio-  »F  CHORIOALLANTOIC  INOCULATION  7  OF  45  CHIRIOALLANTOIC  TECHNIQUE  VII  46  & v m S T E P S 8 AND  9  OF  CHORIOALLANTOIC  TECHNIQUE.  I X 8s X  STEPS  10  47  AND  11  0F  C H 0 R j 0 A L L A N TO 1C  TECHNIQUE  XI & m  APPEARANCE ANTOIC  .48 OF  CULTIVATING UPON  HARVEST  SALMONELLA  STAIN OF  A  XIV  SIMPLE USED  OF  24  OF  SAFETY  TYPHIMURIUM  MADE  FROM  CULTURE UP«N  OF THE  CHOR-  MEMBRANE  WHEN  SMOOTH  SMEAR  HOUR  GLASS  .5.8 SH 1 E L D  WHICH  MAY  INOCULATING.  T W E N T Y ? F O U R - HOUR AND  HOURS  TYPHIMURIDM  TYPHIMURIUM  IOALLANTOIC  BE  CI 0 R 1 0 A L L •  24  58  SALMONELLA  A  OF  AFTER  IT.  WRIGHT'S  XIII  SURFACE  MEMBRANE  STRAINS  OLO  CULTURES  OF  IN N U T R I E N T  59 OF  ROUGH  SALMONELLA BROTH..  59  1 The use of the embryonated avian egg as a culture medium for the propagation and maintenance of cultures of l i v i n g microorganisms i s not new, but i t s history, l i k e that of the whole science of bacteriology, Is a short one* The intention of this outline i s to set forth the history of the use of the f e r t i l e egg as a dulture medium* together with those discoveries i n technique which were of importance to the development of the present methods. The cultivation of bacteria i s dealt with chiefly and not the use of the egg as a l i v i n g medium for the propagation of viruses and rickettslae*  It has been considered advantageous  though, to include the occasional reference to these other pathogenic agents i n 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 i s not u n t i l relatively redent times that any record i s found of the actual construction of a window i n the shell of a f e r t i l e egg through which the embryonic growth and development might be observed without either destruction of the embryo or i t s contamination from without. The f i r s t historical aeoount of the construction of such a window i s 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 t o view the gradual growth o f the embryo«  The  method which i s commonly used t o the present day o f s e a l i n g a hole cut  through the s h e l l of an embryonated egg with a c o v e r s l l p set on  a r i n g o f v a s e l i n e was f i r s t introduced by Soymclewicz (1815)* Apparently the f i r s t man ever t o 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 t o B r e l o c h , Ogston, as surgeon t o the Royal I n f i r m a r y at Aberdeen had an opportunity such as had not been afforded Koch, t o study the pathogenesis o f wound i n f e c t i o n .  I n a report i n the  B r i t i s h Medical Journal f o r 1881 upon Microorganisms i n S u r g i c a l Disease, Ogston r e l a t e d how he had conceived the i d e a o f c u l t i v a t i n g organisms found In abscessed wounds by i n o c u l a t i n g them i n t o hen's eggs, where he thought conditions would more c l o s e l y simulate those under which they grew I n animals than i n l i q u i d media*  He was  s u c c e s s f u l I n growing M i c r o c o c c i i n eggs, and i t was he who I n t r o * dueed the term Staphyloeoeel as a name f o r the cluster-forming c o c c i at the suggestion o f h i s f r i e n d Geddes, Ogston a l s o was s u c c e s s f u l i n o u l t u r i n g b a c i l l i and other b a c t e r i a i n eggs but g i v e r no d e t a i l s concerning these experiments. To understand the importance of Ogston's d i s c o v e r i e s i t i s necessary t o digress f o r a moment t o view h i s discovery i n the l i g h t of the times. I t was no simple and routine matter t o c u l t i v a t e any sort o f bacterium i n the year 1881.  I t was only i n the year that  Alexander Fleming was born that Koch described h i s method f o r the preparing of c u l t u r e s on s o l i d mediae  I t was "supposed" even by  the most enlightened p h y s i c i a n s of the day i n England, that b a c t e r i a  5 and micrococci were the cause not o n l y of e r y s i p e l a s , d i p h t h e r i a and l e p r o s y but a l s o o f v a r i o l a and m a l a r i a , and there was no c e r t a i n method f o r e i t h e r r e c o g n i z i n g these organisms or of i s o l a t i n g them i n pure c u l t u r e f o r study*  Eberth of Z u r i c h had only just published, i n  Virchow's Arohiv, a s e r i e s of observations which s t r o n g l y pointed t o the dependence o f typhoid fever upon a c e r t a i n rod«shaped organism, but Hans Gram (1884) d i d not describe h i s s t a i n i n g method, which would hare aided g r e a t l y i n the r e c o g n i t i o n 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 o f  the day Considered the great question of the times t o be whether or not i n f e c t i o u s 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 p r o t e c t i v e i n o c u l a t i o n , such as Pasteur had demonstrated w i t h anthrax*  -Even  p u b l i c sentiment i n t e r f e r e d w i t h the p u r s u i t o f s c i e n t i f i c medical knowledge*  The ant i«*vivisect i o n b i l l was i n parliament, and doubt-  l e s s i f much had been known about what' Ogston was doing t o embryos i n eggs he would have found h i m s e l f arraigned at Bow S t r e e t 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 f o r g o t t e n , and i t was not u n t i l the year 1905 that any record of an attempt t o 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  l a b o r a t o r y , reported a s u c c e s s f u l attempt t o 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 r e c e n t l y 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 h i s  report L e v a d i t i 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 i n the presence of a l i v i n g embryo* In the course of investigating the Spirillum, which Levaditl considered to resemble the spirochaete of syphilisj he found a second Important fact, namely that maternal antibody i s 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 bacterial research lapsed into obscurity and seems to have remained forgotten u n t i l 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 i t s own* It i s 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 i n 1920 reported their prewar investigations upon the virus of avian plague*  As the result of  a fortuitous discovery of the virus i n an unlaid f e r t i l e 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 i n 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 i n the literature of an experimental infection of the chick embryo with bacteria i s by Askanazy (1933), who mentions the production of tuberculous chicks by the infection of f e r t i l e hen's eggs with Mycobacterium tuberculosis A A strange feature of the research of most of the early workers with organisms In eggs i s 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 i n a never definitely stated form,  that i t came to be generally recognized that the v i t a l ingredient of the egg, as a medium* was the l i v i n g c e l l *  Indeed, the cultivation  of Organisms upon the living embryo was carried on for some time without 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 I t . In fact the credit f o r the first successful propagation of a virus really known to be such i n 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 f i r s t 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 i t was not u n t i l 1934 that Burnet and Galloway discovered, when culturing the virus of vesicular stomatitis on the chorioallantoic membrane, that they eould create an a r 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 u t i l i z i n g 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 f i r s t suggested by Goodpasture i n 1933.  However, his remarks i n  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 extreme d i f f i c u l t y .  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, i n 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 i n 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 i s one of the f i 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 f i r s t cultivation of an actinomycete, Streptobacillus moniliformis, upon the chick embryo, was accomplished. This was by van Hooyen, who succeeded i n carrying the organisms through three successive transfers* The year 1937 was one of considerable advance i n 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 f e r t i l e hen's eggs* They found that Streptococcus virldans. Aerobacter aerogenes, Bberthella typhosa. Brucella abortus and Mycobacterium tuberculosis avium were able to .i"  "  u t i l i z e the l i v i n g intracellular environment of such a medium for growth. They also noted that Staphylococcus aureus and Streptococcus haemolytlcus. although persisting i n 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 membrane 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 i n cooperation with Goodpasture 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 i n criminating i t as the causative agent of human whooping cough. Meanwhile, Buddingh and Polk were cultivating the meningococcus for the f i r s t 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 transmissable 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 chorioallantoic membrane; and that f i l t r a t e s , 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 f i r s t time i n cultivating Leptospira icterohaemorrhagica upon the chorioallantoic membrane* This i s the f i r s t 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 i t s transfer through some twenty egg passages, finding no evidence of any alteration i n i t s virulence as a result of this passage* The year 1939 saw the propagation for the f i r s t time of several species of Trypanosomas i n 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, i s 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 d i f f i c u l t .  Swift and Brown made the f i r s t  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 i n growing Borellia duttoni i n embryonic chicks, without the death of the embryos, although with the production of a general infection as i n man, again emphasizing the similarity i n 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 antitoxin and antiserum;  This was. a milestone i n 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 d i f f i c u l t method requiring considerable s k i l l and care i n the performance, was described by Eichom i n 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 i n 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 i n 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 p a r t i c u l a r l y well as a protective mechanism but rather serves to carry the introduced The year 1941  i n f e c t i o n to other parts of the embryo.  saw the r i s e of the chick embryo as an i n s t r u -  ment for the i n vivo t e s t i n g of chemotherapeutic agents; i t also saw the discovery, so important to virology* of the Hirst phenomenon. Weil and G a l l (1941) i n a series of experiments, tested the e f f i c a c y Of sodium sulfathiazole upon infections produced i n the chick embryo with Salmonella typhi, S h i g e l l a f l e x n e r i and S. pullorum. they found i t to be a simple way  of demonstrating the therapeutic e f f e c t  i n the presence of l i v i n g c e l l s ; Emmart and Smith also  introduced  a method f o r the use of the chick embryo i n chemotherapy, c u l t i v a t i n g Mycobacterium tuberculosis on the chorioallantoic membrane. Again i n the year 1941  a number of experimental c u l t i v a t i o n s of  various pathogenic fungi were carried out by Moore, on the  chorio-  a l l a n t o i s , as well as the c u l t i v a t i o n of various strains of Mycobacte r i a . Buddingh and Womack observed the i n f e c t i o n of chick embryos with Pasteurella tularense. Brucella s u i s , abortus and melitensis as well as Pasteurella p e s t i s , while Alture-Werber managed to cultivate Trypanosoma equiperdium i n the uolk sac of embryonated eggs, and Wile and Snow propagated Spirochaeta The following year, 1942,  saw  p a l l i d a i n the chick embryo.  the introduction by Dunham of  an apparatus f o r 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 s h e l l membrane and the c h o r i o a l l a n t o i s ,  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 i s such an obstacle to the use of laboratory animals i n diagnosis, was greatly reduced i n 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 antibacterial 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 i n 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 i n cultivating Plasmodium gallinaoeum in chick embryos. Their method of infecting the chick embryo was rather a novel one* Mosquitos were placed i n a small tube covered at the open end with a net; this end was then applied to the exposed  13 s h e l l membrane of a l i v i n g chick embryo* The mosquitoes could be observed to feed by penetrating the s h e l l membrane. However the d i f f i c u l t y with t h i s method was the time and effort; involved, since i t took half a day to persuade eight or ten insects to feed in t h i s fashion. In the same year, Emmart used the chick embryo as an instrument for testing the value of a n t i b i o t i c s i n the treatment of tuberculosis. He treated tuberculous embryos with streptomycin and s t r e p t o t h r i c i n finding that s t r e p t o t h r i c i n was about four times as toxic to the embryo as streptomycin. Also i n i945, Beveridge, working at the I n s t i t u t Pasteur i n Paris produced a s i m p l i f i e d technique f o r 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 s h e l l with forceps at the blunt endj over the a i r sac. The next year, 1946, S i l v e r and Kempe made further s i m p l i f i c a t i o n s of the egg culture technique, but with the country physician rather than the research worker i n mind. They solved the problem of penetrating the eggshell by softening i t with concentrated n i t r i c acid solution confined to the desired area by a r i n g of petroleum  jelly*  In the same year, Meyer and Ordal used the chick embryo as a test animal f o r the action of a n t i b i o t i c s upon fungoid infection* Choosing the organism Blastomyces dermatitidis as the infective agent, they treated i t with streptomycin, g l i o t o x i n , s t r e p t o t h r i c i n and  14  penecillin.  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  streptomycin,  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 or  j u s t aggravated the  o g e n i c i t y of v a r i o u s Although the siae  lesions.  o f t h e egg  has  always  an egg  4 report  sensitive  of  has  been g r e a t l y  individual  from such vaccines  u s e d as  o f S t e f a n s k y ' s b a c t e r i u m upon the  Burnet,Joyce  c h i c k embryo, a f a c t  anaphylaxis  the f a i l u r e  immunization i n -  chorioallantois,  regard  the  con-  a n t i g e n s but  and a l s o w i t h t h a t  the  consider-  They u s e d f o r e i g n r e d b l o o d  i n f l u e n z a v i r u s as  with influenza virus,  t h e y had  had been  i n agreement w i t h the e a r l i e r work o f  cells, could  antigens. Burnet i n  These this  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 E a r l y i n 1951,  in  of antibody formation i n  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 r e s u l t s were  propagation  and M c N e l l y  egg  i n 1950 t h a t  concerning which there  v i r u s and l i v i n g  in  tuberculosis,  a b l e q u e s t i o n f o r many y e a r s . bacterial  possible sensitive  r e p o r t e d upon the  and Edney r e p o r t e d  c l u s i v e l y demonstrated  prepare  to the  and P e d d e l l r e v i e w e d the uses o f the embryonated of Mycobacterium  a means  1948,  I n 1949 N o e l and M a r i e - S u z a n n e  culture  path-  rickett-  i n egg  fatal  f o l l o w i n g a typhus  j e c t i o n was made b y W a l k e r i n  the  organisms from which to  such an occurrence,  the  c h i c k embryo;  b e e n some q u e s t i o n a s  danger of untoward r e a c t i o n s persons,  the  c u l t i v a t i o n o f o r g a n i s m s , such as  o f o b t a i n i n g l a r g e numbers o f there  They a l s o demonstrated  Candida species f o r  i n t h e y o l k sac  vaccines,  c h i c k embryo  c h i c k embryo;  Evans r e p o r t e d upon the response o f  embryonated  15 eggs t o cultures of Qorynebacterla.  With the i n t e n t i o n of develop-  i n g a p r a c t i c a l l a b o r a t o r y method f o r 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 s t r a i n s of 6orynebacteria which might eliminate those uneconomic nuisances, the white mouse and the guineapig,  she tested t h i r t y - f o u r s t r a i n s of Oorynebacteria i n f e r t i l e eggs,  guinea p i g s , r a b b i t s and c h i c k s .  She concluded that the r e s u l t s i n  both the u s u a l t e s t animals and i n chick embryos corresponded e x a c t l y j thus perhaps f o r e c a s t i n g the decline and f a l l of the guineaPig*  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 c e l l s , is interrupted at numerous points by many capillaries which underlie and protrude through i t * Thus the chorioallantoic membrane lends i t s e l f 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 i t s e l f * 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 i t s outer layer of mucous i s deposited upon a fibrous shell membrane, which i s a secretion product of the isthmus of the hen's uterus* To begin with, the chick embryo i s 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 i t s e l f into the dorsal somatopleure, which i s 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 u n t i l from the f i f t h day of incubation onwards the amnion i s drawn entirely around the embryo,enfolding i t almost completely, except for the yolk stalk*  The chorion develops rapidly, u n t i l by the tenth day  i t has completely surrounded a l l the contents of the egg, but i s pressed in close oontaot with the shell membrane at a l l points* The allantois originates within the body of the embryo i t s e l f ^ 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 i s lined with  entodermal epithelium, backed with mesodermal tissue, and as i t increases i n size i t s mesodermal aspect fuses with the chorion to form the chorioallantoic membrane on the outside, and with the amnion internally* This fusion, which i s 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 i t s adhesion, form between the epithelial layers of the chorion and allantois fusion* Primarily the chorloallantois serves as the embryonic lung and so i s richly supplied with blood vessels* There i s a dense capillary network i n i t s mesoderm which i s i n close contact with, and at times  i even protrudes through, the outer ectodermal layer which i s 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 i s the main allantoic vein* l i e s  18  separately.  The chief allantoic vein i s quite large and free to  move, having i t s origin at the junction of the two large chorioallantoic veins near the margin of the air-space*  It i s 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 i n the egg. The yolk sac, which has as i t s function the transport of nutriment 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 i s Increased i n area considerably by the development of v i l l i which increase i n size and number as the embryo develops. Daring i t s final day or two i n the egg* the embryo withdraws what i s left of the yolk sae into i t s abdominal cavity to serve i t as a reserve food supply during i t s f i r s t week of hatched l i f e * It i s i n the white of the egg that most of the water i s contained, but as development proceeds, moisture i s transferred from the white to the yolk with i t s consequent enlargement* reaching a maximum by the eighth or ninth day. During the process of incubation* there i s a constant loss of water through the shell by transpiration. The allantoic fluid increases i n 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 i n the cavities. The f l u i d i n the allantoic and amniotic cavities consists i n essence of a simple solution of the physiological salts, being a clear f l u i d with a low protein content. However, from the twelfth  19 day onwards, the protein contents and viscosity of the fluid i s 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 i n the allantoic cavity shows a gradual increase in non-protein nitrogen throughout the whole incubation period,consisting 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 w i l l 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 i n this figure ectoderm by a heavy black line entoderm by a red line mesoderm by green color  ChrAU  Diagram of the arrangement of membranes i n a 9-day embryo. The three germinal layers are indicated i n 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 s k e t c h showing f u l l development o f membranes and c a v i t i e s i n 12-to 15-day c h i c k embryo. The t h r e e g e r m i n a l l a y e r s a r e i n d i c a t e d i n t h i s f i g u r e as i n the two p r e c e d i n g f i g u r e s . A.S. - albumen sac Am.O. - A m n i o t i c c a v i t y A.C. - a i r c e l l V. * v i l l i C A . - chorioallantois Y.S. - y o l k sac E.E.B.C. - extra-embryonic body c a v i t y  o\t  yo\K  sac  Diagram o f t h e r e l a t i o n o f t h e o r i g i n o f t h e a l l a n t o i c t o t h e d i s p o s i t i o n o f t h e embryo and t h e y o l k sac as seen f r o m t h e a i r space end o f the egg. All  diagrams a f t e r B e v e r i d g e and Burnet  vein  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 management, but there are actually only two types of these machines i n general use* The two types are the "cabinet" type and the " s t i l l a i r " 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 i s the type used by commercial egg hatcheries where large numbers of chicks must be hatched with a minimum of labor*  It i s thermostatically controlled  as to temperature, and properly humidified and ventilated by mechanical means, even the eggs being rotated automatically i n the latest and most efficient models*  The " s t i l l a i r " machine is  usually a much smaller incubator than the "cabinet" type, being used i n small poultry-raising operations.  Some of the " s t i l l a i r "  machines are small enough to be placed upon an ordinary table and w i l l handle some five or six dozen eggs only. The heating, as In the "cabinet" type, i s electrical and thermostatically controlled, but there i s 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 a i r " type of incubator for laboratory incubation of f e r t i l e 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 i n 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 i n 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 u n t i l a steady temperature of between 102° and 103© F. was maintained, this being the temperature which* according to various authorities* i s the correct one for the incubation of f e r t i l e eggs i n 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 f e r t i l e * were placed in the incubator blunt end up and standing supported i n 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 i s known as a blood ring* a dark band encircling the egg visible only when the egg i s examined i n 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 t o a s u f f i c i e n t maturity f o r i n o c u l a t i o n procedures, although f r e q u e n t l y f i f t y percent d i d so. The u n c e r t a i n t y of having a s u f f i c i e n t number of w e l l developed, v i a b l e embryos f o r experimental i n o c u l a t i o n s i n t e r f e r e d s e r i o u s l y with the work attempted*  The eventual s o l u t i o n was found t o be t o o b t a i n  embryonated eggs, developed to the required stage, from the commercial type incubators of the hatchery on the U n i v e r s i t y ' s experimental p o u l t r y farm*  The s m a l l i n c u b a t o r was used t h e r e a f t e r e i t h e r to  store unused eggs u n t i l required* o r to incubate eggs which had been operated upon and inoculated, f o r 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 o f the s m a l l machine t o supply the required embryos was not due to e i t h e r i n c o r r e c t temperature c o n t r o l or l a c k of humidity, but simply t o the i n a b i l i t y to arrange f o r the s u f f i c i e n t l y frequent number o f turnings o f the eggs* which according to various a u t h o r i t i e s should be at l e a s t every eight hours. The f i r s t t e n days of incubation are the c r i t i c a l ones d u r i n g which r o t a t i o n of the eggs must be p r a c t i c e d * since when the egg remains i n any one p o s i t i o n f o r a l e n g t h o f time, the yolk tends t o 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 f u r t h e r and soon d i e s . In l a r g e machines equipped w i t h mechanical devices f o r t u r n i n g the eggs, i t i s not unusual t o 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 a x i s almost  v e r t i c a l and the blunt end uppermost, then the cradle i s rotated s l o w l y from side to side through an arc o f approximately n i n e t y  2?  degrees* the eggs being permitted to remain at each of three positions, vertical or lying on i t s right or l e f t side for an equal period of time* Important also to the proper development of the embryo i n an incubator i s the proper degree of humidity*  Dry a i r 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 i n 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 f e r t i l i t y rate i s only possible theoretically.  The usual fert-  i l i t y rate at best i s only eighty-five percent.  Since there i s  no way of recognizing a f e r t i l e egg u n t i l the embryo has developed sufficiently to be recognizable i n the candling operation, i t i s necessary to incubate f e r t i l e or supposedly f e r t i l e eggs for several days before candling them* With this i n 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 i n f e r t i l e eggs and undeveloped embryos could be eliminated at the source*  However  i t was usual to re-examine the f e r t i l e eggs after bringing them to the laboratory i n order to mark the edge of the air-space and the position of the allantoic vein*  Although i t i s 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 head and c a n d l i n g machine f o r t h e o b s e r v a t i o n o f 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 o f the a i r - s p a c e  c a n be s e e n i f  w i t h the hand w h i l e  the egg  shadow, e x t e n d i n g almost  is  the egg i s  shaded  h e l d over the  side of the  the margin  from d i r e c t  the egg,  indicates  ever,  o r i f i c e and s h a d i n g f r o m room l i g h t w i t h t h e f r e e  for techniques  c i s e l y known, there  i n which the p o s i t i o n o f is  no s u b s t i t u t e  p o s i t i o n o f t h e embryo c a n be e a s i l y soft  lead If  the  candling  h a n d . How-  t h e e m b r y o m u s t be  a number o f e g g s o f a b o u t  f o r a d a r k - r o o m . Once f o u n d ,  the  o u t l i n e d upon the  a  the  same age  are  i s r e l a t i v e l y easy to recognize  j-hese show l e s s d e v e l o p m e n t , l i v i n g ones i n the group;  shell with  that  this is,  is,  cast l e s s  of  course,  being candled dead  shadow t h a n  only i f  which are o f  the  same s i z e  as  the o t h e r s  sure method o f r e c o g n i z i n g the f a c t , the egg has  l a i d bare,that  i n the g r o u p ,  a n d i t may n o t be  a c t u a l l y been o p e r a t e d upon and the  t h e embryo i s  dead. Eggs t h a t  are  the  i t has  there  f o r t h e l i g h t shadow o f t h e y o l k ,  t h e b l o o d r i n t may be s e e n e n c i r c l i n g  is  no  realized chorioallantois  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 development is  been embryos  non-fertile  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 the egg  at  embryos,  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  except  pre-  pencil.  same t i m e , i t  until  the  c h o r i o a l l a n t o i s , a n d may  be s i m i l a r l y s e e n b y r o l l i m g t h e e g g w h i l e p r e s s e d o v e r t h e light's  light  c a n d l i n g l i g h t . A heavy  t o t h e p o i n t e d end o f  p o s i t i o n o f the most d e v e l o p e d  practice,  gggs  quite  or  are clear  o r because the d a r k band  of  27  the egg at i t s equator* The most satisfactory system of candling eggs was found to be as follows*  F i r s t , the eggs ready for Inoculation are examined rapidly  and those showing tip as "clears", or possessing blood rings, are discarded*  Second* the eggs chosen for inoculation by the chorioallantoic  technique are examined carefully i n order to determine the position of the margin of the air-space, which i s almost always to be found at the blunt end of the egg* The edge of the air space i s then marked on the egg shell with a soft lead pencil on the side where i t s shadow indicates that the chorioallantois i s best developed*  Third, the position of  important veins, such as the main allantoic vein i s carefully marked* Finally, a triangular outline i s drawn i n 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 chorioallantoic 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 i s well developed, and that with oider embryos d i f f i c u l t y was frequently experienced i n effecting a ready separation of the shell and chorioallantoic membranes* In the following descriptions of technique It should be f u l l y realized that aseptic procedure i s adhered to as far as possible* Sterile syringes* needles and pipettes are used both for making  28  inoculations and f o r the harvesting of eggs, and a l l instruments  coming  in contact with the egg membranes are s t e r i l i z e d by flaming, which although destructive to the temper of instruments, i s e f f e c t i v e l y b a c t e r i c i d a l . The following chorioallantoic technique  i s a slight  modification of that described by Beveridge and Burnet ( 1946  ).  The eggs selected f o r inoculation are esamined, as previously described, with the a i d 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 l i m i t marked with a p e n c i l on the 8 h e l l . An e q u i l a t e r a l triangle may sides from 10 to 15mm.  then be outlined with  i n length, and approximately  a t h i r d the distance  from the previously outlined edge of the a i r space to the pointed end of the  egg.  The surface of the eggshell immediately surrounding the marked area and the blunt t i p of the egg should then be swabbed with a suitable d i s i n f e c t a n t . An aqueous solution of sephiran ( alkyldimethylbenzylammonium chloride, 1/1000), was  found e f f e c t i v e f o r t h i s  purpose, and preferable to iodine, since i t exerts no destructive action upon the d r i l l mandrel or other A small e l e c t r i c a l hand d r i l l  instruments;  ( Hand-ee Grinder ) f i t t e d with a  dental mandrel f o r the mounting of carborundum separating discs ( Baker, 5/8 i n . ) was found to be a s a t i s f a c t o r y t o o l f o r 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 p e t r i d i s h . However, they may  quite s a t i s f a c t o r i l y and more  29  conveniently be sterilized by soaking i n aqueous Zephiran ( l/lOOO ) solution when mounted on the mandrel just previous to use« The egg i s manipulated as illustrated i n the process of chorioallantoic inoculation technique*  A triangle of shell is cut out  with the dental disc, and a cut i s also made through shell and shell membrane into the a i r sac of the egg*  The egg i s then placed i n  the Dunham type inoculating stand and suction applied to the air sac end while the triangle of shell i s 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, i s 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 steri l i z i n g this instrument should i t become accidently contaminated. Instead, the egg i s placed upon a small inoculating stand such as the type illustrated, which was designed by Beveridge*  I f such a  stand i s not available, a pressed paper egg-box may be substituted, since i f this i s contaminated i t may be either sterilized by autoclaving or easily destroyed i n 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 i n the shell may be sealed i n a variety of ways, the classical method being to ring the area with petroleum jelly* sterile of course, and to place a sterile cover s l i p over this* thus excluding 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, i t s use did not appear to predjudice the results of the experiments in any way by contributing to contamination of the egg contents, and t r i a l s 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 i n 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 i n the egg, which was t i l t e d gently from side to side i n 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 t i l t e d 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 i t s e l f may be sucked up, effectively blocking the pipette. For convenience i n harvesting^ narrow glass test tubes containing sterile saline were used as receptacles for the harvest, and these instead of being plugged with nonabsorbent 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 themselves to too frequent removal of their cotton plugs, which lose their shape rather easily.  Another advantage of the phial-topped  tube i s that i t can be placed in the centrifuge and the harvest  51 thrown down without the necessity of fastening a cotton plug i n place with an elastic band; as i s the usual practice* A modification of the chorioallantoic inoculation technique i s that of Beveridge (1947)* The egg Is candled and the margin of the air-space marked as usual*  The shell i s disinfected and then  a hole i s made by jabbing with a sterile forceps a few mm* from • the margin of the air-space* over the air-space.  The shell membrane  i s 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* i s broken away for three to four mm* beyond the margin of the air-space* The exposed outer shell membrane i s then torn away and the inner layer of shell membrane i s 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 i s held  i n the hand up to this stage and i n an approximately horizontal position, but i s not t i l t e d with the air-space downwards* In the case of eleven-day embryos the chorioallantois usually f a l l s readily, giving an a r t i f i c i a l air-space i n the same position as with the standard technique*  More difficulty may be experienced  i n separating the shell membrane from the chorioallantois i n the case of older embryos, but tapping the egg with the fingers usually serves to effeot the separation*  The egg i s 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 f e r t i l e egg and one not requiring the use of a d r i l l . However, i t was found to be somewhat d i f f i c u l t 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 inoculation, which is widely used i n 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 i s taken of the location of the allantoic vein, a pencil mark being made upon the shell to indicate i t s point of emergence, which i s 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 i s d i f f i c u l t to see the veins well when candling.  If the eggs are derived from white leghorns,,*^*  (whose eggs are uniformly white), which i s the usual practice* no d i f f i c u l t y with dark shelled eggs should be experienced* If* as sometimes occurs* the allantoic vein i t s e l f cannot be seen* i t s position may be determined by noting the point at whioh two large veins converge towards the air-sac end of the egg. point for inoculation may  The  then be determined by the following rule.  If the egg i s held with the a i r space end towards the operator, the  55 point for inoculation i s 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 i s drilled through into the air-spaee as i n the chorioallantoic technique*  Next a rough oval, approximately 2 cm* by  l^cm. and with the long axis parallel with the long axis of the egg, i s cut about this point with the separating disc*  Two cuts  are made i n the oval at the blunt end of the egg to form a sector which w i l l facilitate the removal of the area enclosed i n the oval. Great care must be taken, as i n the chorioallantoic technique, not to damage the shell membrane during the cutting process* The egg may be mounted i n a Dunham type inoculator and suction applied to the hole i n 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 i s quite suitable for the purpose* The chorioallantois i s grasped with a pair of curved sharp toothed forceps at a point where no large blood vessels are i n evidence* and a cut i s made with a sharp pointed scalpel about half a centimeter i n length* oare being taken not to damage the amnion, which l i e s immediately beneath*  In this regard also*  one should be cautious when grasping the chorioallantois not to include the amnion i n the pinch of the forceps or i t may be aeoidently 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 i t s being pierced. It i s of practical value to have 0.02 « 0.05 ml* of a i r below the inoculum i n the pipette, since this may then be injected into the amnion firsthand the position of the a i r bubble w i l l inform the operator whether or not he i s in the amniotic cavity. The egg may be sealed after inoculation with a cover s l i p set on a ring of sterile petroleum j e l l y made about the opening* but a wide piece of adhesive cellulose tape i s more convenient, Beveridge has modified this amniotic inoculation technique for the use of researchers not possessing d r i l l s for cutting the eggshell.  The method i s as follows:  The preliminary incubation of the egg i s carred out with the air sac end uppermost which i s the usual position i n commercial incubators. This position i s essential i n order to assure the embryo's being close to the margin of the air**space» A hole i s breached into the a i r sac end of the egg i n the same manner as i n Beveridge*s modification of the chorioallantoic inoculation technique* but the hole i s not extended beyond the margin of the air-spaeei  A sharp pointed forceps i s thrust through the  shell membrane and the underlying chorioallantoic membrane close to the margin of the air-space, and the hole i s then extended for about 1 .cm, by blunt dissection. As air enters, the embryo* enclosed in the amnion, i s presented at the hole  0  It may be necessary to  remove some of the shell membrane adhering to the chorioallantois. Should an a i r bubble biook the hole, as sometimes occurs* i t may be dispersed with a touch of a heated needle.  55 The amnion i s grasped with toothed forceps and held taut while the injection i s made either with a beveled capillary pipette or a hypo* dermic syringe and needle*  The hole i n the shell may then be sealed  with adhesive cellulose tape and the egg incubated i n an upright position* Another method, that of Hirst (1942), i s a simple one for the inoculation of the amniotic cavity i n 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 i n order to observe the position of the needle* The yolk sac method of inoculation Is a technique usually performed upon very young embryos* that i s , 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 i s passed ttrough the shell membrane, the egg being held perpendicularly In an inoculating stand, such as Beveridge's until the point i s 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 i n the shell  may be sealed with either a tiny piece of adhesive cellulose tape or a drop of paraffin wax* A carborundum cutting disc i s not essential to the performance of this technique, as the shell may be simply pierced with a straight surgical needle mounted i n a rubber  56 ^ung* The allantoic cavity method of inoculation i s 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 i s well developed but not showing any large blood vessels* A out i s made in the shell at this point parallel with the long axis of the egg and about three mm, i n length whioh i s 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 hypodermic needle* The intracerebral method i s applicable to eggs of from eight to thirteen days of incubation.  They are candled as usual and the  position of the embryo determined as i n the amniotic inoculation method, an oval being marked upon the shell and a sector cut out from i t as l n 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 i s frequently presented  at the opening, but i f i t i s 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 i s most suitable for inoculating with*  The head, In the case of more fully  developed embryos, i s grasped with a forceps and *he needle  57  inserted into the skull, but with young embryos usually a sharp quick thrust at the skull w i l l be sufficient to pierce i t . The intravenous method of inoculation i s usually, practised with embryos ten days or older$ and i s a delicate technique* The eggs are candled to determine the position of the larger veins i n the chorioallantois*  One of them i s chosen, and i t s 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 i s described on the egg shell at this point with the vein centred i n 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 i n 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 i n the selected vein for inoculation be seen.  A sharp 27-gauge needle mounted on a  tuberculin syringe i s most satisfactory for the inoculation. The needle i s then rested against the edge of the cut shell i n order to steady i t and then i t i s most carefully inserted, bevel uppermost Into the vein i n the direction of the blood flow*  Either a  Dunham or a Beveridge type inoculating stand w i l l hold the egg satisfactorily during the perfonaanee of this technique*  After  inoculation the needle i s withdrawn slowly and carefully i n order to avoid any risk of hemorrhage* The hole i n 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 i n the manipulation and inoculation of f e r t i l e 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 a i r " 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 f u l l y 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 appearance of the embryonic chorioallantoic membrane* Illustrations of this apparatus w i l l 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 r i g h t : Hooked needle used i n separating chorioallantoic and s h e l l membranes Straight needle used i n prising up cut triangle of s h e l l Forceps used for removing pieces of s h e l l Scalpel used i n removing remains of s h e l l membrane i n triangular area Forceps used i n tearing out remains of s h e l l membrane i n t r i angular area Foreground: Handee Grinder f i 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 i n . 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 s h e l l and s h e l l 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 p o s i t i o n . Note the method o f holding the d r i l l .  STEP TWO Method of holding egg and d r i l l while cutting f i r s t side of triangle of shell which is to be removed. The shell i s 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 o f h o l d i n g egg w h i l e t h i r d side o f t r i a n g l e .  cutting  STEP FIVE Method o f i n s e r t i n g n e e d l e , b e v e l up, under edge o f corner of t r i a n g l e o f s h e l l which i s t o be p r i s e d up. Egg i s mounted i n Dunham i n o c u l a t o r . S h e l l membrane must n o t be p i e r c e d .  -  STEP s i r Method o f p r i s i n g out t r i a n g l e o f detached s h e l l . C a r e must be taken t o a v o i d t e a r i n g s h e l l membrane. S u c t i o n i s now b e i n g a p p l i e d t o a i r sac o f egg.  STEP SEVEN P l a c i n g drop o f s t e r i l e p h y s i o l o g i c a l s a l i n e upon i n t a c t s h e l l membrane. The drop r u n s 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 p i e r c e d beneath i t w i t h a s t e r i l e , h o o k e d needle thus a c t i n g a s 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 o f 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 s a l i n e to enter and with the a i d o f suction dropping the chorioa l l a n t o i c membrane away from the s h e l l membrane•  STEP NINE  Method o f tearing out remains of s h e l l membrane i n t r i a n g u l a r area with p a i r of s t e r i l e forceps a f t e r the dropping of the c h o r i o a l l a n t o i c membrane.  STEP TEN Method of adding inoculum to egg. Egg i s now mounted i n a simple stand with i t s adhesive cellophane tape seal partly attached.  STEP  T^WVKN  The egg i s ready for incubation with its cellophane closure sealed. Egg must be incubated in this position.  49 With the intention i n 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 twentyfour 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 i n 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 i t s 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 i n so far as could be ascertained by ordinary inspection.  The ^anbryos continued to  l i v e 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 twentyfour 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 a r 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 b e seen at the point of inoculation* The embryo was dead, b u t whether this was due to the growth of the  51  Mycobacterium was not determined. In an attempt to find whether prolonged cultivation upon the chorioallantoic membrane of f e r t i l e hen*s eggs would i n 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 i n sterile physiological saline and approximately 0*2 co. used to inoculate the surface of the chorioallantoic membrane of a ten day old f e r t i l e 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 f e r t i l e 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 c e l l s , 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 c e l l s .  The colonies  produced upon the nutrient agar plates remained uniformly smooth i n appearance, and this was confirmed by numerous picked colonies which, when inoculated into broth, gave uniform turbidity with only slight sedimentation i n twenty-four hottrs. The organisms apparently grew well upon the chorioallantoic membrane or rather in the moisture upon i t s surface.  I f 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 i t an erythematous appearance, as may be seen i n the accompanying photograph*  55  ...  No gross growth or colonial development was noted.. The grey plaques of growing organisms were noted to be i n close proximity to the thrombosed blood vessels. Next, an attempt was made to see whether there was any d i f - . ference i n the agglutitiability with specific antiserum of the passaged strain of Salmonella typhimnrium as opposed to the unpassaged. 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 i t s treatment might not differ from that of the egg-passaged strain, which was treated as follows: Ah egg i n which was a twenty-four hour growth of the eggpassaged strain was harvested as usual and the harvest centrifuged. The precipitate was then resuspended i n 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 i n 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 c e l l s *  In order to determine  at what age eggs should be inoculated i n order to avoid these cells appearing i n the harvests a number of eggs aged ten, twelve and fourteen days were inoculated with similar amounts of the organisms suspended i n 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 inoculating with this test organism, i f a good harvest free from blood Cells i s 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 A l 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 d i f f i c u l t i e s of supply were occasionally older or younger by a day. The two parallel series of transfers were continued for some twenty passages. 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, i n order to note any similarity i n 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 i n the organisms had taken place. No 'change was noted. The A l strain, both i n the broth and in the egg remained B i n type and the AS strain similarly remained S i n type. During the course of this investigation two facts were noted. First, harvests were best when taken at two day intervals; eggs l e f t unharvested f o r longer than this time showing poorer and  56 poorer harvests until after five days of incubation very few organisms were present i n 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 A l 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 d i f ference i n pathogenicity"? An experiment was then undertaken to determine the approximate number of A8 and the approximate number of A l organisms required to k i l l a ten to fifteen day old  ohick embryo i n 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 i n some instances inoculated with many times the determined lethal dose of S type organisms and i n 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 i n the age of the embryo• Eleven or twelve day old embryos which had survived sublethal doses of B type organisms for three days would be fourteen or fifteen days old when inoculated with the S type organisms i n a dosage known to be fatal to ten to twelve day old embryos. Apparentl y with this increase i n age, a decreasing susceptibility to this organism i s acquired, since doses of S organisms far i n 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 o f s u r f a c e of c h o r i o a l l a n t o i c membrane a f t e r 24 hours of c u l t i v a t i n g S a l m o n e l l a typhimnriiun upon I t *  Wright's s t a i n of smear made from harvest o f a 24 hour culture o f S a l n c n e l l a typhimv.rlum upon the  c h o r i o a l l a n t o i c membrane.  A simple s a f e t y g l a s s s h i e l d which be used when i n o c u l a t i n g .  may  Twenty-four hour o l d c u l t u r e s o f rougkg and smooth s t r a i n s o f S a l m o n e l l a t y p h i murium i n n u t r i e n t b r o t h . Left:rough R i g h t : 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 i n bacterial cultivation* It has also been shown that elaborate equipment i s not essential to the performance of the various techniques of inoculating the embryonated egg with -very good results*  The organisms, Serratia marcesoens  and Salmonella t y p M ™ * * * ™ »PW> successfully propagated by means of the chorioallantoic inoculation technique and also carried i n the egg for a number of generations, without serious d i f f i c u l t y or 0  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 i n the field*  It has been shown that i t i s possible to demonstrate a d i f -  ference i n 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 i s an apparent difference i n 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 i n this departmeirt with the ehick embryo has been l a i d l n this thesis, especially as regards the techniques of inoculation*  The use of the chick embryo  i n bacteriological research and probably even i n virological research has not yet been either f u l l y 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 s o . l i t t l e 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 i n such a readily available and relatively uniform a medium as the Ohiok embryo has indicated i t s use for their culture ever since t&e discovery of the method. It i s the only completely satisfactory medium for the purpose known* Were the processes of Inoculation and propagation of infectious agents such as these twice as d i f f i c u l t 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 incubation* their formulae are thoroughly and completely known, and they lend themselves to rapid Inoculation by none too skilled technicians. The chick embryo i s not s t r i c t l y speaking a uniform medium, i t s manufacture Cannot be completely supervised, and i t s 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 i n i t s inoculation and, as with a l l l i v i n g things, results cannot be completely foreseen*  Time Is an important faetor i n most laboratory routine  work* and i n few laboratories would It be economically feasible to spend as much of i t as i s required to make the inoculation of bacteria into eggs, particularly by such a time-consuming process as the  62 chorioallantoic technique. Nevertheless, despite i t s 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 i s 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 i s freer from latent bacterial or v i r a l infections than  laboratory experimental animals. (4)  It i s suitable for the propagation of organisms not readily  propagated upon laboratory media. (5)  It i s an excellent i n vivo test medium for toxins, antitoxins,  antisera and antibiotics* (6)  While the embryonic chick medium may not be s t r i c t l y compar-  able with the natural hosts f o r bacteria studied* the lesions produced are characteristic for the bacterial types* and closely resemble those found In the natural host. This indicates i t s usefulness i n the study of the pathogenesis of infection. Of course, the experimental egg has certain features which limit i t s use for experimental purposes. Cl)  A certain amount of care must be exercised i n 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 (3)  5  5hey do not keep, unlike the usual laboratory media which,  when not required for immediate use, may be refrigerated u n t i l heeded* The experimenter must foresee the number of eggs that he w i l l require for use i n 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 d i f f i c u l t 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 i n a wide f i e l d of bacteriological investigation.  64 . STJ&MARY  (1)  The literature pertaining to the cultivation of bacterial  microorganisms i n the embryonated egg have been reviewed i n 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 f e r t i l e eggs has been discussed*  (5)  The usual techniques of operating upon and inoculating the chick  embryo have been described and the method of chorioallantoic cultivation 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 i n this manner* (7)  The effect upon i t s agglutlnability of embryonic cultivation  of a laboratory strain of S, typhimurium has been investigated, and found to be unchanged following prolonged serial passage i n f e r t i l e 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 i n their type of growth has been found attributable 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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J . , and Snow,J.S;: Chick embryo as a culture medium f o r Sp, p a l l i d a , J.Invest.Dermat. 4:103,1941, WoodruffjAJa.; and Goodpasture,E,W.: S u s c e p t i b i l i t y o f chorioa l l a n t o i c membrane o f chick embryos t o i n f e c t i o n with fowl-pox v i r u s , Am.J,Path. 7:209,1931. Zia,S.: C u l t i v a t i o n of Mexican and European typhus r i c k e t t s i a e i n the c h o r i o a l l a n t o i c membrane o f the developing c h i c k embryo, Am.J.Path. 10:211,1934*  i  


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