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"9- . 1-71.21 :15“ I" 11‘ éJqfl‘qu ' '“' ‘20" L2 bu‘flg""“v ‘3‘... 4|. :31" f" mjjujj jigrmi‘ 31 9 5 2 'I- ‘ .' M JHEU’; '5 m _| w i \_ ,. g- . ‘ . 5 I STUDIES ON BAJTDRIUM BULLO&UM Submitted to the Faculty of the Kichigan Agricultural 3011336 in Partial Fulfillment of the Requirerents for the Degree of Master of Science. warm- major mmmm June, 1984. JHEmb‘ 1 fi‘f 1 NF“ 11:1 «b1: .Ll‘ 1.“) Introduction. Historical Resume. History of the cultures studied. Preparation of Media. MicroscOpic Studies. \ Effect of media on Korphology. Effect of Media on Staining Preperties. Jultural Studies. Biochemical Ghanges in Dunhem's Solution. Biochemical Jhenges 1n Nitrate-Eeptone Solution. Biochemical Changes in Litmus Kilk. Spec al Biochemical Tests. Eermentation Studies. 1'"periments to Determine the rectors Responsible for the Irre; ular Gas firodzcing Preperties of Bacterium Pullorum. Agglutinatil.g PrOperties of the Various Strains Studied. summary and Jonclusion. Acknowledgments. References. 1341”,, 10M 1'11““) STIDIES ON 331TJRIUH PULZOdJK Ill'i‘ iCDJ-J'i‘ICIJ Althoug h cons idereble work has been carried out on Bacterium Bullorum, both culturally and serolo: cally, still it was believed that work carried out on a large number of cultures obtained from different sources would either bring out any variations that had not been observed or it would be an excellent check and confirmation of previous work. Dr. Stafseth of this Station has had considerable difficulty in diagnostic serlegical work and it was desired to determine definitely ether the or5anisms used for antigen were giving satisfactory results. It is quite generally known that strains of some organisms vary considerably in their agglutinating power. Very little work concr. r1 in? this prOperty of Bacterium pullorum has been carried out. It was further honed that cultural and fermentation studies might shed some light on the causes of variation which are narticularly frequent Titmi 'P ot11iun :ullorum. TT ' Tqv‘ 1 “T‘~ ”—‘D'. a La ;‘..J. ‘J... J11 LL14...) 1L.'J. The study of chicken lusecs es dates back many years, 0'] althourh, a in other diseases, the work was largely observational. It is of interest, however, to know of these early investigators and their work. Hadley (1) gives a'very interesting historical resume of these early WorkerS. is I do not have these early articles at hen", I take the liberty of ducting Hadley. "Baroneo, a celebrated Lombardy (Italy) physician, appears to have been among the first to seriously consider the nature and the culse of serious typhoid-like epidemics which had oeen noted by poultry raisers even before this time. Toyather With Miocchi and drugna he examine With considerable Care the extensive epidemics which occurred among poultry in Lombardy between the years 1789- 179 8 and.Which were 3 mmonly referred to by t}1e ooultryuaa 01 the Turin district under the name 'cclcinsccio' and by the Eieontese as 'ccusiners'. Somefihut later similar epidemics in Eienonte were studied by “rugnone and T05gie. In vie J of the lees of bccteriolocicul knowledge and tec‘nique in these earl? hat there m g- H m C H 't is, of course, clear are fe.1 data, save tee description of the clinical features of the malady to serve as a oasis of connection with cholera-like p; iseases of poultr; as we know them at tee lresent day; and it is therefore impossible definitely to say whet 2cr they represented true fowl cholera or sonic other disess e. But in view of the circumstances that when advances in bacteriolo;ical knowledge and technique finally did permit a certain degree of differentiation (about 1875) the non-cholera type of infection appeared to be prevailing poultry malady of the Italian provinces, it can perhaps justifiably be assumed that, even in earlier years, it was a well established poultry scourge in these sections of southern Europe whence much of our poultry has been derived. "In the early days of the science, bacteriologists were laboring with the problem of anthrax and it is not sur- prising that Ercolani, who in 1861 made a study of many epi- demics, described some of the then prevailing types of poultry infection under the name of epidemic anthrax (carbon- ohis). Others who contributed to the early knowledge of these diseases were Lemaistre (1869) Delefond, Reynal and from 1877, considerable work on chicken diseases was carried on. All of this work centered around fowl typhoid and fowl cholera. No worker, apparently, isolated or studied the organism causing white diarrhea of chicks. It was not until 1899 that the organism causing this disease was discovered. At this time, Rettger (2) reported a case of fatal septicemia in young chicks. It is interesting to know that many bacteriolOgists were working on this problem at this time and were unable to make any pregress. Rettger -4... examined a number of chicks suffering from what he describes as a peculiar ailment. A bacteriological examination was made and an organism was isolated from the liver and the Spleen. This organism was injected into normal young chicks with fatal results. The organism was reisolated. In 1900, another epidemic was studied by Rettger (3) and again he was able to isolate the same organism. Inocula- tion experiments were again successful. In the same year, Pernot (4) at the Oregon Agricultural College published a report on chicken diseases in which under the heading indigestion, he mentions a heavy loss of incubator chicks through lack of understanding of their requirements when young. He states "Some of the chicks dissected were found to have their craws filled with food and a fair quantity of unabsorbed yolk in their abdomens. All the conditions gave evidence that they had died of an acute indigestion. Particles of food were found in all parts of the intestinal tract." Jithout doubt, Pernot was describing white diarrhea of chicks, but he apparently knew nothing of its cause. In 1908, Milks (5) working in Louisiana isolated an organism from the liver of a chick dying from the so-called white diarrhea. He gives a description of the organism that checks very closely with the one isolated by Rettger in 1829 tum.1900. His inoculation expermments on young chicks were Successful. flettger and Stoneburn (6) in 1909 in further studies give a complete description of the organism, which Rettger at this time named Bacterium pullorum. In commenting on the properties of the organism he states "Some of the strains do not produce gas in any of the sugar media, though acid production is quite apparent." I quote this statement because of its bearing on work presented in this paper. Their conclusions show that the nother hen is the original source of the disease and that a certain nercet- age of the chicks oninfected farms have the disease when hatched. A little earlier, 1908, Horse (7) published a paper in which he stated that much of the so-called trouble in chickens was due to coccidiosis, identifying the parasite as Coccidium tenellum. In 1909, Hadley (8) claimed that the etiological factor in white diarrhea was Coccidium cuniculi which was found in the ceca. He believed the organism to be similar to that causing blackhead in turkeys. In 1909, Rettger (9) investigated the significance of coccidium tenellum. He states that he found Bacterium pullorum in cases where Coccidium tenellum was present and concludes that Bacterium pullorum is the etiolozical factor and not Coccidium tenellum. At this time, he again states that considerable variation was found in gas wroduction by the strains. He states "The behavior of the organism on agar is quite constant. The same thing is true with reference to the develonment in gelatin, milk and potato. The different strains vary, however, in their gas producing power in dextrose bouillon. some of the organisms which I have isolated have failed and still fail to produce gas in this medium. On the other hand, two particular strains feriented the sugar with 5 percent and 25 percent reapectively, in this medium. The more active ferments lost this prOperty within a period of seven to eight months so that for a while, it was strictly anerogenic. At the present time, the fermenting power is gradually being rednred. Work on this particular deportment is being carried on from this standpoint of variation, and some Very interesting results have already been obtained. These will be reserved for a later publication." 1all of the strains of organisms studied produced acid in dextrose bouillon. Lactose is apparently not affected. Mannite is fermented by some strains but not by others." Jones (10) in 1910 confirmed the work of Rettger and Stoneburn and further found that the disease might be induced through intravenous injection. In 1911, Gage (11) again donfirmed the work of Rettger and Hadley in their contention that the disease is transmitted to the chick from the hen through the egg. In 1913, Jones (18) reports an epidemic where adult birds were infected by feeding infected incubated eggs. In the same year, in another paper (18) he again suggests the use of the agglutination test for detecting birds infected with white diarrhea. This test was first suggested for use by Jones in 1911 (1‘). He recommends that in using the test the antigen should be made up of several strains of the organism and that dilutions of 1-50, 1—100, 1-200 for routine should be used. An efficiency of one hundred percent is claimed for the test. The -7- latter claim is rather a little premature as the number of tests is not sufficient for such conclusions. dettger, Kirkpatrick and Stoneburn (15) in 1912 state that the period of greatest danger from bacillary white diarrhea of young chicks lies within the first 24 hours after hatching. lhey further state that hens may become carriers of the disease after they have reached maturity. 1'hey believe that the ovaries may he become infected by contact of the hens with.infected hens and that the infection is likely acquired through the mouth. In 1914, a second report (16) considers the relationship between infection as chicks and the carrier state when mature. They state ”Of 138 chicks which grew to maturity and lited until the termina- tion of the eXperiment, 88 were-infected with bouillon cultures of Bacterium pgllorum when quite young, while the remaining 57 were not subjected to this treatment, but served throughout the investigation as checks. Of the 88 that were infected as chicks, 21 gave positive agglutination tests when about a year old and i at the name time,they were killed all of the positive agglutina- tion tested pullets showed unmistakable evidence of ovarian infection with Bacterium pullorum. In many instances, the ovaries were in advanced stages of infection. hora than 25 percent that were artidicially infected as chicks became permanent carriers." This also confirms Joncs' macroscOpic agglutination test and shows that all birds showing positive reactions had infected ovaries. Gage (17) in the same year also confirms the value of the aggluti- nation test as a means of detecting carriers. He also agrees with Jones in the use of polyvalent antigens. He confirmed Jones' method of infecting the ovaries through intravenous injection. In the following year (1915) dettger, Kirkpatrick and Jones (18) presented an extensive check on the use of the agglutination test. They state that they examined 14,617 birds. Of this number, 1440 were reactors, or 9.85 percent. The number of hens tested was 15,851, of which 1417 were reactors, or 10.24 percent. The number of males tested was 786, of which 25, or 2.9 percent,were reactors. The following year some of these flocks were retested with the following results. Four flocks out of 13 failed to show any reactors and in the other 9 the percentage of infection varied from 0.6 to 25.7 percent, the number in each instance being decidedly less than before. smith and TenBroeck (19) in the same year published a paper showing differences between Bacterium pullorum and Bacterium sanquinarum. The following differences are given: Action on maltose Bacterium sanguinarmn produces acid Bacterium pullorum does not produce acid Gas production on dextrose and mannite Bacterium sanguinarum - ho was \) Bacterium pullorum - Gas produced Milk Bacterium sanguinarum - alkaline Bacterium pullorum - no alkaline Rettger and Koser (20) in 1916 working independently from Smith and TenBroeck made a similar study in which they found a few additional differences. morphOIOgically, they state that (O Bacterium sanquinarum is a shorter and thicker rod than Bacterium pullorum. They found that Bacterium sanquinarum produced appre- ciable acidity in maltose, dextrin and duloite, while Bacterium pullorum had a tendency to alkali production in these sugars. They also found, which is partially confirmatory, that gas production by Bacterium pullorum is quite variable in mannite and galactose. In immunizing eXperiments carried out at the same time they found that where birds were immunized with the fowl cholera organism, they were not protected against fatal doses of Bacterium pullorum, whereas when the birds were immunized with Bacterium pullorum they were immune to the fowl cholera organism. These eXperiments were carried out on a small scale and so do not justify any conclusions. ward and Gallagher (21) in 1817 suggest the use of an intradermal test for Bacterium pullorum in place of the agglutina- tion test which they consider macumbersome and impractical as compared with the above test. Although they do not claim one hundred percent efficiency for the test, they do urge its use in the field in comparison with the agglutination test. One of the most complete studies of the fermenting prOper- ties of Bacterium pullorum was carried out by Goldberg (22) in 1917. He studied 5 strains of Bacterium pullorum and determined the fermentative prOperties on 20 sugars and alcohols, as shown in table I. -10.. TABLE I Gas Production by farious strains of hacterium pullorum on Various Garbohydrates. dtra in No. Dextrose Lactose Sucrose hannite Dextrin Inulin Galactose 1 B - - 13 - - + 3 + - - B - - + 5 B - ~ B - - + 4 + - - + - - + 5 - .. .. .. .. .. .. Einfiinl levulose daffinose Amy:dalin arabinose adonite Dulcitc ‘1 L10. 1 B - ~ - + ~ - 3 B - - + - - 5 B - - + - - 4 B — - + - - 5 - - - + - - 1‘ ‘r b strand Xylose starch Isodulcite Salacin hannose Glycerin Erythol he. 1 - a. «- + - I- + u— u— 3 4 - - + - + - - 5 Mulsow (23) in 1919 reported a study of various strains of Bacterium pullorum. fable 11 gives the results that he obtained. An examination of fables 1 and II shows a marked irregularity in gas-forming characteristics. Both Goldberg ( Table I) and mulsow (Table 11) show a strain that is a non—gas-producer. Several organisms in fable 11 show a decided variation on arabinose, rhamnose, sorbite and xylose. Mulsow states that two strains of Bacterium pullorum produced acid and gas in maltose and a few strains produced slight acidity after several days. Berry (24) found three.strains of Bacterium pullorum that fermented lactose with gas. rhese three strains were other- :nse normal. Hadley 25) in 1917 confirmed Goldberg's work on fermentation. He also confirmed Jettger‘s fermentative studies and susscsts that the gas-producing type of Bacterium pullorum _)!) be called Bacterium pullorum Alpha,and the non-gas-producing type be called Bacterium pullorum Beta. Hadley states that the Alpha type is pathOgenic to young chicks only, while the Beta type is able to produce a generalized infection in adult fowls, but ohly to a slight degree, if at all, in young cni In 1919, Hadley, daldwell and Heath (36) again observed the two types of Bacterium pullorum and state that the anaerogenic (Beta) type is uncommon. fhey believe the difference between the two types depends upon onvrionmental factors and is independent of the S“801f10 fermentahle sugars. ) u- ,4 I- TABLE 11 Acid and Gas roduction of Various Strains of Bacterium pullorum on Various Sugars. Strain Dextrose 1.1a1tose Arabinose Rhamnose b‘orbite Xylose Dulci te A G A G A G A G A G A G A G 4 + + + - + + + + + + + + - - 5 + + + - + + + + + + + + - - 6 + + - - + - + + + - + - - - 7 + + - — + + + + + ~ + — - ~ 8 + - - - + - + - + - + ~ - - 9 + + - - + + + + + - + + - - 10 + + + - + - + + + - + - - - 16 + + - - + + + - + - + - - - 1'7 + + + - + + + + + ~ + + - - 19 + + + - + + + - + + + + - - 30 + + + + + + + + + + + + - - 21 + + - - + + + + + - + - — ~ 22 + + + ~ + - + + + + + + - - 25 + + - - + - + + + - + - - - 84 + + - - + + + + + + + + - - 35 + + ~ ~ + - + + + - + - - - 36 + + - - + - + + + - + + - - 27 + + - - + + + + + - + + - - £8 + + - - + + + + + + + - - - 39 + + + - + + + + + - + - - - 50 + + - - + + + + + - + - ~ - 31 + + - - + + + + + + + + - - . . C . . . . . , , . ., ._ _ - , . _ - . - ,_ , . - . . . . - .. .. ... . c . .. . , -r ... .. -' _ t ’- H . '— 7 5“ '4 H .4‘ I ' ‘ t .. . V . v ... .c _ . .. ‘ ; .. ‘ .. - ‘ , A ' - . . , . , . .. .. . . _. ._ . - ‘ . 4 . V , .. . . ,. _ ,. - . , r . l . v , .— . . v-o . - _. . . . . i ' , w - v n 1 G. A y. . —.. .— ., l . . ‘ i t 4 . . ,. — v t . r - F. F .— .. .- 1 - — — ._. . I -I .— H H . ,—-. N 4 . F 1 § . _. .. w . A ' -_ ( _’ . . p-q — I ‘ I . . i — , .— , - I . I 1 4 ~ . ,1, c .- - . . . H 3- _' 7‘ u . . _ ‘ .‘ — n . v They report that the kind of broth medium used has an effect upon gas production. They found that when the anaerogenic types from chicks were grown in dextrose extract broth no gas was produced while gas was produced in dextrose infusion bnoth. The anaerogenic strains isolated from adults gave negative gas produc- tion on bath kinds of media. In regard to occurrence of the two tynes of organisms (Alpha and Beta) they ask "(1) Are both types present in the intestinal tract of fowls and does the Beta type produce generalized infection because of a difference in aggressivity; or (2) may the Alpha type give rise to the Beta type during the progress of ovarian or intestinal infection as a result of adaptation or as a result of a selection of anaerOgenic mutants? Is Bacterium pullorum an instance of Bacterium gallinarum in the making?" Intavery extensive report (1918) Hadley, Blkins and daldwell draw the following conclusions: "The present methods for and results of the diagnosis of Bacterium pullorum infections by agglutination reactions are not satisfactory because they fail to differentiate (1) between ovarian and non-ovarian infections, (2) between culminated and current infections (3) between Bacterium pullorum and infections with the fowl cholera bacillus which is widely disseminated among adult stock; and (4) between infections caused by Bacterium pullorum Alpha and Bacterium pullorum Beta." A new view on the transmission of the disease in the adult fowl is given by Rettger, Kirkpatrick and Jard (28) in 19 9. They date that progressive infection in a flock is not confined to -14.. young chickens but may also be Spread through the adult fowls, particularly the females. The introduction of the organism into the cloaoa and oviduct of laying hens may readily lead to perma- nent ovarian infection. The r61e of the male has not been demonstrated but it undoubtedly plays a part as a passive carrier. Very few males, they state, show infection of the testicles although they did find a few cases. mason or his Jamaica STUDIES). cultures 4 and 5. These two cultures were isolated May 5, 1922, from a Rhode Island cockerel sent in for examination from Lawrence, hichigen. l’he gross examination showed epicarditis, heavy injec- tion of the intestinal vessels and also of the vessels of the testicles. The bird had shown signs of white diarrhea and leg weakness for a considerable period previous to death. The disease was a generalized septicemia as evidenced by the isolation of the organism from the liver, kidneys and the blood. The two cultures were picked from the same plate, two being fished because of a difference in the size of the colonies. Julture 4 was a small colony while Culture 5 produced a somewhat larger colony, culture 6. This culture was isolated April 22, 1922, from an adult bird (hen) from Bearborn, Hichigan. The bird was autOpsied in the field and was diagnosed without hesitation as white diarrhea. Only the liver was submitted for examination, from which the organism was isolated. 'Julture 7. This culture was isolated from a baby chick from Big Rapids, hichigan, April 14, 1922. The liver was studded with small necrotic foci. The culture was isolated from the liver. Jultures 8 and 9. These two cultures were isolated August 1, 1S22, from . chicks shipped to the laboratory from Vicksburg, Michigan. No gross patholOgical description was taken. The chicks all showed positive agglutination tests. The two cultures represent two colonies showing Slight cultural differences. Culture 10. This culture was obtained from Dr. Hadley of the University of hichigan in 1921. ¥he culture was carried to Michigan by Dr. Hadley when he became connected with the University. The culture was one he originally isolated while at the Rhode Island Experiment Station. The culture was originally an aerOgenic type, Bacterium pullorum Alpha. Culture 11. Ibis culture was isolated May 8, 1&22, from baby chicks shipped from st. Johns, hichiuan. The liversof these chicks were ochre color and showed typical white diarrhea lesions. Julture 12. Fwd This culture was isolated from baby chicks from Iillsdale, Michigan, May 9, 1922. Typical ochre colored livers were fohnd. Julture 15. This culture was isolated f“om baby chicks from Ann Arbor, Kichigan, May 19, 1922. Typical ochre colored livers were found. The culture was isolated from the liver. Culture 14. This culture was isolated from white Zeghorn baby chicks obtained from Plainville, Michigan, may 22, 922. he patholozical description was taken. - 17 _ Julture 15. This culture was obtained from baby chicks from Marion, inchigan, June 6, 1€22. Typical lesions were present on the liver. Julture 16. This culture was isolated June 20, 1922, from baby chicks shipped from Benzonia, Lichigan. Typical lesions were present on the liver. Julture 17. This organism, isolated June 11, 1922, was obtained from baby chicks shipped from Bately, hichigan. Typical lesions were present on the liver. culture 18. This culture was isolated June 21, 1:22, from baby chicks from Paw Paw, Hichigan. Ty-ical lesions were found on the liver. Oulture 19. This culture was isolated June 2 , 1922, from baby chicks from Grass Lake, Michigan. Typical lesions were present on the liver., Julture 20. this culture was isolated June 23, 1922, and was obtained from baby chicks from :Allegan, Michigan. Typical lesions were present on the liver. Julzure 21. This culture was isolated June 24, 1922, and was obtained from baby chicks from Emmet, Lichizan. Typical lesions were present on the liver. -18.. Julture 22. This culture was isolated June 28, 1922, from baby chicks sent in from Oakley, Kichigan. Typical lesions were present on the liver. Julture 23. This culture was isolated June 28, 1:22, from baby chicks obtained from Lawrence, hichigan. Typical lesions were present on the liver. Julture 24. This culture, isolated July 1, 1922, was obtained from baby chicks from Royal Oak, hichigan. The liver showed only one slightly colored Spot. Julture 25. This culture was isolated July 8, 1922, from baby chicks from Hilliamston, hishigan. A retained yolk the size of a pea was found. The adjoining tissue had a slight ochre color. ho out- smnuding lesions were present. Jultures 26 to 41. These cultures werz obtained from the Kansas Agricultural Jollege in 1922. No history of the organisms was given. The names of the organisms were: Gulture 26 was marked 2205-40 Julture 27 w " 3142-29 Julture 28 " " PP7 Julture 29 “ " 2248-50 culture 50 " " PPl culture 51 " " 3848-39 Gulture 32 " " PPS -19.. culture 33 was marked Jhick Julture 34 " " 2353-32 Julture 35 " " 2248-39 Julture 36 " ” 2648-37 Julture 37 " " 190-3 Julture 38 W " 2317-40 )ulture 39 " " 2533-Ad Julture 4O " " EPE culture 41 " " Bacterium pullorum Jultures 42 to 45 These cultures were obtained from Ohio State University in 1922. No history of these cultures is given. l'he cultures xmre labeled as follows: Julture 42 was marked Jacterium pullorum Julture 43 " " Rettger 216 Gulture 44 " ” jettger U17 Julture 45 " ” Jettger uhi meortunately strains 42, 43 and 45 were lost, in fact we were unable to grow strains 43 and 45 from the slants sent us. culture 46. JL’his culture was isolated July 20, £22, from a hen belczg'ing to Dr. t’liltneryliamiosedtyhim aslming; white diarrhea. Only the ovaries were brought in to the laboratory. The organism was isolated from these. culture 47.to 53 These cultures were obtained from Perdue University in 1922. No history of the cultures was given. They were labeled as follows: Julture Julture culture Culture Julture Julture culture 47 was marked 48 49 50 51 53 53 Y! W -20 W 01 Field Strain Field Strain 7 Field Strain Field Strain Field Strain n: r4 o. .s Field Strain (A Field Strain A map of the lower peninsula of Michigan is given in Figure I, showing the sources of the various cultures that were isolated in Michigan. This map shows that the cultures were obtained from a wide area representing practically the whole of the lower peninsula. Thus the cultures studied would represent many strains, as the disease would very likely be introduced from various sources, from outside states as well as the state of Hichigan. -2}- ' ‘- ""'".‘ ‘ ‘ ‘t..,‘~ r' '-- c 1.45-- .Li: 1.11.4...ba 4.1 .L 4.44.05.4- i . 1‘7} “""3 _j'.‘-_‘ U.‘ MAJ. J-Li‘lisii Amharil-J .".1‘1-~-1~r. LJIL'-.'.‘.L.UsI 1.1.2qu \- HI, "' r- ‘,)-1-; ',..,. \"' -," ~,‘-_‘ '. V1 .' 1 " ‘- \ JAuLJNULJil UAJ-u';-LUl.i ud--.iuu.4..) unaiub Culuki~.ud .Bsnaonim-le .MOVIOO ~ )5 :Bi E 'ids‘7 .Bite'ej m1; P “c.“ a... _ Pg3PAjATIUN 0E MEDIA USED considerable variation occurred in the preparation of the media used and this should be known as it may play some part in the reaction obtained as has been demonstrated by Aettger and others. Livarkgar. This medium was selected for growing Bacterium pullorum as Dr. Stafseth had highly successful results using it for carrying stock cultures. At first the medium was made according to the direction of Stafseth and Huddleson. Briefly, this method consisted in cooking the liver with tap water, added at the rate of 500 c.c. per pound of finely ground lean liver. The cooking was done in an Arnold steamer for about 1 hour. The liquid was pressed out and filtered through *1ass wool. The liver agar medium was now prepared, using the following ingredients: 500 c.c. liver infusion, 500 c.c. tap water, 15 gm. agar, 10 gm. peptone, 5 gm. salt. 1’he mixture was steamed until the agar melted, after which it was cooled to 60° c. and 10 gm. of egg albumin added. The mixture was now cooked another hour to coagulate the egg albumin and precipitate any other albuminous substances that would be preci- pitated by heat. After heating, the hot mixture was filtered until clear through glass wool. Due to the fact that the above method was quite difficult, modifications were introduced.‘ I might state here that the glass wool was used as it was believed (Torrey) that paper or cotton removed certain growth-producing substances. This has recently been proved by Hasley in a very striking manner with freshly isolated Bacterium abortus. However, the writer discarded the use of glass wool and substituted cotton and also dropped clarifying the medium with egg albumin. This made the preparation of the medium as simple as making ordinary agar. No change in the amount of growth of the Bacterium pullorum resulted, as was the case with bacterium abortus. Extract Broth. The extract broth was made according to the method recommended by the American Public Health Association for water analysis. The broth was fermented with Bacillus coli to remove any fermentable agar present and then adjusted to a titre of pH 6.8. The last run of fermentation tests (1924) was made on extract broth with a titre of pH 7. fihe sugars were used at the rate of 1 percent in the case of the cheaper sugars, as lactose, sucrose, mannite, dextrose, starch, inulin, maltose, glycerine and dextrin, and one tenth percent in the case of the more expensive sugars as adonite, dulcite, mannose, levulose, rhamnose, galactose, arabinose and .. f‘r‘lvv‘r 33.2-1.111 )SG. * Huddleson, Hasley and Stafseth now have a procedure whereby the above process of preparation is relatively simple. Infusion Broth. This broth was prepared from ground lean beef in the :mual manner. The resulting broth was fermented with Bacillus coli and the titre adjusted to a pH 6.8. Preparation of the Sugar hroths. All sugar broth used, with the exception of lactose, maltose and sucrose were prepared as usual and sterilized at 15 pounds pressure for 20 minutes. ”he three sugars mentioned, due to the fact that they are very sensitive to heat were prepared as follows: The required amount of broth needed was measured into a suitable flask and sterilized by heat. A 20 percent solution of the sugars to be used was prepared, using sterile distilled water. This solution was now passed through a sterile Berkefeld filter to remove any organisms present. The sterile sugar filtrate was added, aseptically in the required amounts, to the sterile broth. Using the filling apparatus Figure II pictured in Figure 11, the broth was tubed aseptically. (This apparatus was not original with the writer.) The broth was incubated to detect contamination and any tubes showing growth or even a suspicion of growth were discarded. However, it was necessary to discard but few tubes. - 25 Indicator for Acidity. Ihe indicator used to determine acidity was Andrade's indicator, which was added at the rate of l c.c. per 100 of broth. KicroscOpic Studies of Bacterium pullorum. Transfers of all the cultures to be studied were made on liver agar slants. These were incubated at 37° 3. for 24 hours, and than ordinary stains were made to study the morphology and staining properties and to make sure that all strains were pure cultures. Jontrary to eXpectations, the microscOpe showed large heavy stained organisms inclined to and 03 asional apiculated forms, instead of fins long chains 1 observing typical bacterium gallorum cells. few cells appeared normal. The strains appeared to he contaminated with an entirely different type of organism. Save a1 strains showing pronounced differences, namely, cultures 10, 25, and 24 were plated out on liver agar, in order to determine whether they were contaminated or whether they were passing through some stage of pleomorphism. Solonies which were fished from these plates were planted on liver agar slants. stains were made and the following observations noted: Julture lO - btain showed fairly large rods which were mostly in singles, but still a few chains were observed. Julture 23 - Ihe stain showed smaller rods than Julture 10 and very few chains. The apiculated forms observed before were still in evidence. Julture 34 - This strain showed cells somewhat the same as strain 25 3:33 t that many more of the apiculated forms were nresent, which in this case were always in pairs. hone of the colonies fished ap eared normal (nicr0~ scooically) for Bacterium pullorum. Apparently the forms observed were involution forms or marked pleomorphic tynes. The colon- typhoid group is characterized by their decided pleomorphism and it would appear that Bacterium oullorum was no exception. In order to be able to make micrOSCOpical studies to determine whether all of the strains were the same it was neceSsary to find some medium upon which the organisms would apyear normal. As irevious observations were made from plain agar, this medium was selected. All of the strains were invigorated on liver agar and then liver agar and plain agar slants were made. These were inc bated at 57° for 24 hours when plain and Gram's stains were made. The results of these studies are tabulated in Table III. A close study of Table III shows that a distinct dif- ference exists in the apnearance of the cells on plain agar and on liver agar. It will be noticed that on plain agar, the size and shape of the cells is similar to earlier observations, the cell being slightly longer than wide with slightly rounded ends and with a size of about 1.25 by 0.75 microns. Ihe organisms appear generally as sinvies with an occasional pair. ho chains were observed in any of the strains. On liver agar, the cells are totally different, the cells being larger, staining more easily and more deeply and having a tendency to chain formation. dolls are seldom in singles as compared to the cells appearing on plain agar. v K Table III. 3 30mparison of the Korphologiccl Appearance of Bacterium Fullorum on 24 Hour flain igir and Liver Agar Slants. Strain C) 10 11 13 Plain Xgar Slants. Liver Agar Slants. Ho chains-orgon'sms Chains frequent-mostly singles- single-size i-2 x 0.5 size 2.4-5.5 x 0.75 microns Ho chains, organisms Long chains frequent, i 72 micron single- 1.2-1.8 X 0.5 chain observed-monerally single pairs freouent with ends slightly pointed-1.2-5 X 0.75 Ho chains-organisms Long chains-singles and pairs single-short rods Size 5.6-u X 0.75 n. nearly coccoid size 0.75 - 1.8 X 0.5 No chains-cells single 10 chains~cclls single, s i h short rods-inevcn stain- larger-even staining, 1-l.5 ing- 0.75-1.25 X 0025‘005 Io chains-cells gener- chains freouent- pairs common ally single, even even staining-1-l.5 X 0.5 u staining-1.25-2.5 X 0.5 u. ' Ho chains-cells single- Chain infreouent- pairs occa- even staining, short sional-even s dining-l-2.5 X coccoid rods- 1-1.5 X 0.5 u. 0.25 " 005 u. ho chains-coccoid to Chains very frequent- pairs slender rods- common- long rods- stain deeply 1-2.5 X 0.5 u. 1.5 -5 X 0.5 u. ho chains-very short Ho chains-heavy thick rod - rods-nearly round- short- stains deeply always single- 1.2 ~2 X 0.75 u. 9.75 -1025 X 005-0075 11. Ho chains-uneven stain- Chains present-2 types of cells ing-always single in regard to staining- one 1-2 X 0.5 u. deep and one liaht 105 “2.5 X 005 -0075 u. 30 chains-generally Shaina present-singles predom- single-vcry short to inate-stains evenly moderate rods- 1.5 -2.5 X 0.5 u. 0.75 “1.85 :{ 00511-0 - 28s - Table III continued. Strain Plain lger Elan 14 15 16 17 18 19 20 21 22 25 to 01 Fe ch1ins-rze11erzlly single short rods-nearly coccoid in some Cuces- stein evenly-0.75 X 0.5 170 cheins-sener lly single short rods-uniform size, even steining .75-1.5 0.5 So chuins-senerslly sinrly -very uneven steining 0078-105 X 0.5 U0 chcins-eencrsllv sinrle uneven steinin ~short rods 1.25 - 0.75 No cheins-singles-stein fuirly even- l-l.25 X 0.25 -007 I0 ch1ins-einrles- stein fairly even sltho vsris tion is notice1ble - l X 000 No chsi s-sinwles s-decided variation in 0‘11n1n 1-1.25 X 005 HO chuins—singles-decided variation in steining- 1-1.25 X .5 210 ch sim1 C‘inslP's-““Qeins 1vell & f irly even-l X 0.5 ._ __ _ 'I‘ noout the seas as 521- very li holy steined HO Chains-sinfles 8.0011 shaped-twice as long as wide l-l.25 X 0.5 10 chnins-s in; coli shuped- -l- 1.25 I“, e 1;:1 l s X COIN \J \ Esme us f25-not steined as well Liver Agar Slants. C sins pres ent-comnonly in short ch1ins of 2 and 5. Stein heavily and evenly 2.5 X 0.5- size uniform Chsins present-pairs freouent- stains deeply h evenly 1.2-5 X 0.5 Chains present-stains deeply & evenly-uniform size-pairs I" freeuent- 1.5-2.5 X 0.5 Puirs common-steins more even- ly-slightly larger Few ch1ins-peirs frequent- 1025-105 X 0.75 - Stained deeply & evenly. 3h11ns irecuent- 2 % 5's conrnon-even ste- ining- 1.25-2 X 0.5- 0.75 5 s and 4's common-singles fsirlJ so - 1.5-2 X 0.75 Chuins free ue -st11n he1vier out V1r11ble-s me r111y single- 1.25-1.5 X 0.5- 0. 75 1’5".'. 7’ " "F' H '_" .‘ Y. Cn11ns 1c1y frecdent-S1ngles '1? ' .- . nn 1 [x 1!. hr- 1ew-st11ns ne111ly - 1.25-2 X Chsins present - poorly stained Io chuins-p1irs occasional rods slenderer a slirhtly long- Pgirs freeuent-stuins well- la gcr 5 longer rods - 1025-2 K 005 Short chsins-peirs iomnon- variable but heevy st1ining- Q." 05w an I-SK U1 ue Updike we .. Ht." Ltr1in 27 28 29 ()1 O B 0] CO ()1 D] ()3 C31 (:1 CD 111in 1g1r L'o cn1ins-sine le evenlz-cells 911. more slender than 1-1.25 X 0.5 s-stdin 1" ‘lgt 1:," '50 '- 11”..“ L 30 ch11ns~sirgles~stdin well-reurly coccoid in some cases-1 X 0.5 No ch1ins-singles-s evenly-generally twice as lonchS wide- 0. 75 -l "0.5 C‘ C 11.1, lo cn1ins-s °nile tdin evenly- 1-l.25 X 0.5 no cells visible Io chains-sineles-stein 4.1.1. hauls C‘J'elflljf- 0.75-1925 }: 0075 Ho chains-singles-stain lirhtlv but evenly- 007 53-10215 X 0.0. No engine-singles-size sxne as fSS Ho ch1ins-sinjles-st1in well & evenly uniform size- l~l.25 X 0.5 No ch1ins-sinsles-st1in well & evenly-uniform size nearly coccoid- O.75-l.25 X 0.5 Io chdins-singles-st1in well-uniixmwn size,more slender than “7‘ 1 L1 No chdins-sinjlcs- -st in well out li‘ptlv-ur1rorm size-very sh01t rods 1 X 0.5 ”r: 9d. Liver lger 511nts. Che ins freouent- p1irs common- 1. *1 gen chains-stains very irregular- sice of cells irregular-s mewhat lerzer. Extremelv lens chains % very freouent-st1in heuvy- 1.25-1.75 X 0.75 30 chains-st;in deenlv-& evenly- consider1ole QéDqu-°O‘C“'at l1rger Io cells visible ?ew cteins consider! larger esent-steins deeply ‘pr Ml débris- somewhet Some chains-senerally pairs or 3's 1nd 4's-;t;inins extremely U”el,L-°t iT'S li t1?- 025 ‘01 5 X 0.75:: 3h1i ns fre De wt- in deenlv & evenly altho sove cells stein very poorly- ”-1.7 X 0.75 .C‘- Ch1in ireeuent-stdinin: even within the cells but decided variation in st1inine of different cells. Sells long & cylindiic11-1.5-2.5 X 0. 75 Chdins frecuent-some cells stain deeply-others no stlin at all- cells long and slender. 105-205 X 0075:: Jheins but short-sirnilar to 56 in su1ining and shape- 105-205 3': O. 5 Pronounced chdins-most cells poorly st1ined(degenereted :pwedrdnce)fst1ining and shape as in E56 SL418 Strnin 42 '53 O) 48 52 55 ch1e III continued. Plain leer Slcnts. Verv noorlv stained-appecrs Io choins-sinrles-sone extrerm :l" long-about twice as long as Wide,mny be pcirs altho no evidence shows- 1-2 X 0.5 10 cln.iz_s- tin les-sc;ir1 well-sli htlr longer the 4;?)8- 1-2 X 0.5 10 c sins-sin les-s din well-size uniform- O.75-l.25 X 0.5 Very poorly st: ined- -proowo- 1:7 1 1“- 1-9 fc/B Io chcins-very short rods- necrly coccoid-stein evenly 0.75-'1 X 0.5 Ho chcins-short rods- -necrly coc coid-stci n evenly- l-O.5 X 0.5 Too poorly steined Io croins-cells short- poorly stoined .1 Poor stein-cells coccoid no chains-cells evenly stained-some very short 0075-]. X 0.5 No chcins-cells evenly steined-verv short rods 0075‘]. X 0. 5 Liver Agar Slants. Very poorly stained-veneers its $58 Chains-much debris or degenerate anneerins cells-normal unpeerin: cells are 1.5-2 X 0.75 3hnins few-much debris-cells sli tlv l; r then on plain agar Io chwins observed-cells stein deenly-long and slender- 105- 3.5 X 0.75 Very poorly stained-seem to be long rods Ho chains-pairs co““on-°td1n1ne of cells irregular-i ended or polcr- 1.5-2 X 0.75 Io chains-pairs-stuining some- 'M: st 11 regular 1-2 X 0. 75 Engine-staining poor-appears to be long rods Slide dirty-cells clumped- appesr longer Chain ani long rods U0 chain-cells slirhtly lsrrer. Ho cLsin-cells longer and wider-slender appearing- 33 me T7511 T8 0 I‘ l. All strains do not behave the same on the same medium. dtrains IO and 11 and to a lesser extent strain 47 show a marked coccoid shape. Strainle and 11 were particularly prominent and stand out from the rest. However, the strains vary considerably in this characteristic and it is very doubtful whether the shape is constant and even if it was it is too unreliable to try to base a separation of strains on. On the liver agar, the production of chains varied considerably, but here again it is too inconstant to have any d'fferential value. This table shows that fiacterium pullorum is decidedly pleomorphic and hence too much stress should not be placed upon a micrOSCOpic examination in diagnosis. l‘he effect of the medium used on the }ram's stain was also studied. These stains were made at the same time that the plain stains were prepared (Table III) The results of the Gram's stains are shown in Table IV. The meiium does not apparently influence the Gram's stain to any extent although the results in Table IV show slightly better results where plain agar was used. The stains were made on the same slide in every case to avoid any variations in technic. The results show that occasioially (“trains 5 and 12) a positive stain will appear. This is not a constant characteristic and might occur on any stain. Its occurrence is not constant. However, in making Gram's stains, plain agar should be used where possible as the results obtained on liver agar are too variable. . a _ so _ TABlj If The Effect of the hedia on the Gram's Stain. Strain Plain Agar Liver Agar ,Strain I‘lain Agar Liver Agar Slant Slant , Slant Slant 4 - _? ' 27 - ho good I 5 - + ' d8 — - I 6 - - ' 25 ~ - I V No good No good ' 50 - - I 8 — — ' 51 — - I 9 " "' ' :52 a— .- l 10 - - ' 53 - - I ll - - ' 54 - - I 3 -? + ' "5 - - I 15 No good - ' 56 - - I 14 - - ' 37 - - I 15 - - ' 38 Do good - I 16 - - ' 39 I- - I 17 -? -? ' 4o - - I 18 - - ' 41 - - I 19 - - ' 42 _ - I 80 - - ' 46 + + I 21 " - I 47 .- I. I 32 — - ' 48 - -? I 33 - 110 good. ' 49 — -? ' long rods - 34 - No good ' 50 +? short coccoids + I 25 -? -? ' 51 — - I 26 - - ' 52 - - 8 ' 55 _ - — - indicates a negative Gram's stain, and + indicates a positive. GULTJiAL STUDIES Biochemical Jhanges Produced in Dunham's Solution. These tests were made in series of two years, using duplicate tubes in each year. Examinations, on the Dunham's Solution were made for hydrogen sulphide, ammonia, nitrites and indol. All tests were made after incubation of the broth at 57° for 7 days. The results obtained are given in the accompanying tables. Hydrogen Sulphide Production. Previous work by others (Hadley) has shown Bacterium pullorum quite a constant pro- ducer of hydroqen sulphide. The determinations(Table V) fail to show any confirmation of such work, in fact, it is just the Opposite. HydrOgen sulphide production appears to be extremely variable. It wiil be noticed that the last determinations showed very few positives (4 strains) while the 1922 determina- tions show 15 strains positive. The same strains in 1923 do not show necessarily the same as in 1922. Julture 10 showed negative in 1922 and slightly positive in 1925, and only cultures 7, 8 and 9 show nositive in both series of tests. considerable variation is shown but has no apparent significance. Ammonia éroduction. The production of ammonia by all strains is anearently constant. (Table VI) All strains with the exception of one tube showed approximately the same amount of ammonia. This one tube showed an increased amount. Ammonia pro— duction is then a constant prOperty of all Bacterium pullorum strains. Formation of a: V sulphide in Dunham's solution. by Bacterium pullorum. I strain Determinations Strain Determinations 923 725 ' 1922 1223 I 4 - - - - I 238 - - - .- I 7 +++ ++ + - ' 31 - - — - _ I 8 ++ - - + ' 52 - - - - '- I 9 +++ +++ + + ' 53 - - - - " " I 10 " " + 'I’ ' (54: - - I- I- — "‘ I ll ++ + — - ' 55 — - - - I 3 - - I. u- ' 136 - - - - I 13 +++ +++ - - ' 57 — - - - I 14 + ++ " - t ('8 - o- - - I 15 - ++ - A- ' 59 - - - - I 16 + ++ - - ' 40 - - - - I I 18 - n a. II- ' 44 no a - II. I I 20 + - _ _ ' 47 + + - - I 21 — ++ — I- ' {L8 II- - - - I I 25 + + - - ' 5O - - ~ ~ ‘ I I 25 - - - - ' 53 - - - - I I 37 .. .. .. .. ' - indicates negative + indicates questionable or very slight + indicates slight, ++ indicates fair, +++ indicates fairly strong. n,fl Ebrmation of Ammonia in Dunham's solution by Pact. pullorum. Strain Determinations . Strain Determinations 19238 C £3" , 1922). 19 25 4 + + + + I 28 + + + + 5 + + + + I 29 + + + + 5 + + + + I 50 + + + + '7 + + + + I 51 + + + + 8 - + + t I 153 + + + + 9 + + + + I 55 + + + J- 10 + + + + z 5" + + + + 11 + + + + z 55 + + + + 12 + + + + z 56 + + + + 15 + + + + I 5'7 4- + 4.. + 14 + + + + I 58 + + + + 15 + + +++ + z 59 + + + + 15 + + + + I 4!.) + + + + 1'7 + + + + : 41 + + + + 18 + + + + z 46 + + + + 1‘; + + + + : 47 + + + + 20 + + + + I 48 + + + + 21 + + + + I 115 + + + + 2.. + + + + I 50 + + + + 25 + + + + I 51 + + + + 24 + 1'. + + ' 52 + + + + I 25 + + + + ', 55 + O + + 26 + + + + ' - 54 - Nitrate Production. None of the strains studied were able to produce nitrites in Dunham's Solution. Ihis checks with previous work. ' Indol Production. Bacterium pullorum is not supposed to produce indol and the strains studied did not show any evidence of it in 1922, except strain 25 which showed positive in both tubes. In the 1923 determinations, strains 20 and 22 -showed positive in one tube only and strains 14, 15 and 19 showed very slight in one tube (Table VII). Tnblgfi VII Formation of Indol from Dunham's Solution by Bact. pullorum Strains Determinations 1522 1923 Strain Determinations lSZB 1925 0‘: 01 4h l l I I 23 - - - + 49 - - - - 23' - - - - 50 - - - - 31 + + ~ - 51 - — _ - 25 + - - - 52 — — - - Biochemical Shanzes Produced in Nitrate-Peptone Solution by Bacterium nullorum. our one set of determinations was made for the oxidation of nitrites in Hitrate-Peptone Solution. These determinations were made December 20, 1823. the results of these tests are given in Table VIII. Hadley (39) states that Bacterium Eullorum does not reduce nitrites, however, the results obtained show that this prOperty of lack of orOperty is variable. A number of tubes showed what might be designated as a slight nitrite test. This was confined, with one exception, to only one tube of the two tubes used of each organism. Ihe results obtained should be duplicated before any conclusions can be drawn. No ammonia was shown in any of the tubes tested. - 07 - 94313 TIII Biochemical Jhanges Produced in hitrate-Beptone solution by Bact. Eullorum. I Strain NHa Litrites ' strain Ammonia Iitrites I I "’ I I '7 C- - — I- ' 31 — — - u- I 8 - - - - ' 52 - - - I- I 10 - - - - ' "4 .. .. + . ll - - + - ' ”5 - - - - - I 13 n - + - ' 3!. c- - no I- I 13 - - + - ' b” - - - + [-J ,p I I I I (23 (:3 I I I I 2‘." - - - - {L9 - — - - I I .- I I 2.6 - - + - ' 55 - - + + N ‘3 I I I I -55... Jffect Produced on hilk by hacterium fiullorum flhe milk used was adjusted to a titre of 1.5 percent normal acid, using phenolphthalein as an.indicator. Tho percent azolitmin was added andthe medium was sterilized by autoclaving. Two series of tests were made, one in 1922 and the other in 1923. Duplicate tubes were used in both cases. In all previous work reviewed, Bacterium pullorum was described as producing a slight acidity in litmus milk in a week or ten days. In no place was an alkaline reaction obtained. One of the points of differentiation between Bacterium pullorum and Bacterium sanguinarum is the reaction produced in litmus milk, Bacterium pullorum producing a slight acidity and Bacterium sanguinarum an alkaline condition. In this work, the reaction of the milk (Table IX-lEBZ) on the 12 day reading shows but very little variation, the only variation being the degree of acidity produced. Some of the tubes showed no change from normal, while the greater share showed a very slight acidity. The tubes run in 1935 showed a much greater variation. Iiowever, it should ne remembered that the final readings in this case were made in 25 days instead of 18 days as in the case of the 1922 results. Due to the fact that some of the cultures showed decidedly alkaline and in 5 cultures, one of the tubes was contaminated, these cultures were re-inoculated into litmus milk and a second observation was made, as shown in the following table (Table K). Showing )hanges Produced in hilk by Bact.2ullorum. 1923 Strain 13 da (n .s O) 10 11 12 reading 1. i. i. i. 1 + l+ l+ I+ H' r+ l+ I+ l+ H" It l+ l+ H- 4. y i. i. t. t i + l+ If l+ H’ P* l+ l+ H' r+ l+ I+ l+ H- .4. 1S25 25 day reading 2. i + r.c. + + + + i i alk.alk. l+ l+ l* [1* H" + + i t. i i. t. i i. i. 4. 1+ H' I+ I+ |+ cont. It |+ I+ I+ [-1- cont. 4. + + ' 1922 ' Strain 13 day ' reading I I 29 i + , _ ' 50 + + , ._ ._ ' 51 + + , ._ _ ' 32 + + , _ _ ' 3" + + , _ _ ' 54 + + , _ _ ' ”5 + + , _ ._ I '76 - .- I ' 57 ~ - I ' 38 - - I ’ 59 - - I ' 40 - ~ I ' 41 - - I ' 44 - - I ' 46 - - I ' 47 - - I ' 48 - - I ' 49 - - I ' 50 - - I ' 51 - - I I {‘2 _ _ I ' 53 - - I I I 1925 25 day reading + + + .1 + + + + - cont. + + + cont. 1 i 1 + + i i .1 i i + cont. 1 + + alk. alk. - or + indicate slight acidity. r.c. indicates rennet curd. cont. indicates contamination. -40.. Table 1’. . ———— Reactions Produced in Litmus milk by Bacterium pullorum. Strain deaction Produced. 4 No change in 14 days. Later - Islight acidity. 5 No change in 14 days. Later - slight acidity. 10 Alkaline in 14 days 17 Slightly acid in 14 days 25 Slightly acid in 14 days 37 Alkaline in 14 days. later - reduction and curd. 59 Alkaline in 14 days 29 Slightly alkaline in 14 days. later - decidedly alkaline, with rennet curd. 36 Slightly alkaline in 14 days. 50 Neutral 53 Neutral These confirmatory tests Show that strains 10 and 27 still persist in alkali production while strain 53 which showed decidedly a kaline has changed and shows neutral. Strains 29, 56 and 59 in which one tube was contaminated before and where the second tube was neutral or slightly acid (strain 39) now show slight alkalinity. In fact after 25 days incubation these tubes showing alkali production in 14 days, now show decided rennet curd. The reaction of milk, than, is not always a reliable characteristic of Bacterium nullorum. .~' Special Fiochemical Tests. Dextrose dicotassium phosphate broth was inoculated with all strains and incubated at 37° 3. for 5 days. 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T' P "r ...n I 7 .-, - .- \ +' +- —;7 f. +_ . + ..7. .... p, ,.77 ... + - :77- 4- 77.; .— - — .— ..a ... —- ..77 -' ... ~— .. vv-n ~7 --r; a. ... n... —~ —. -— .... 7-. .... —. un- - — D- l . I _ a - .— -. _ ~ M77--."— 7 .-..777a—wv- 7‘ “—..-m77 1 I -\'- ._. . __ .. ...—7 ._.. 7 , .—-.--—-._.> ‘7 W- “.1 - ..7 7... ‘q—“aw..- ‘ ,- . w---wu~.~-—I~.~“~ a s—vu—s- .-7._~.~m.g—.-~—._.~e—77—~‘w77 _,.__._7.. 777.1- --—-..‘. - -_— 7-. .7._.7... - --...W “...-7...”. . ..-, 7~7~-....- . _ ...._ ... 7.7.7 ... . F‘ .- - q + + .— 7 q. .- 7 .-7- H. .-..nfi-v—u... ' -- 7 .m— v—u 7 “‘7 . .7»? .-.~; 7.7,, 777. 7777 "-4 1 I i“ I I I l I 7.7”“...V -44.. 13331713111 TA ‘1‘I ON {5 1UDIES dextrose The fermentation of this sugar appears to be fairly constant. A strong acid reaction was observed in all tubes, .both in the 1922 series and the 1924 series. Only one organism was slow in the formation of acid. Julture 44 in the 1924 series did not show acid until after 48 hours incubation.($able XIIJ Gas formation did not appear until after 7 days so the organisms in question apparently at the time of the test was undergoing one of its typical variations, which will be discussed later. Jultures 11 and 47 failed to show any gas formation on dextrose in either of the series of tests. (Table XI) In the 1924 series, culture 41 failed to show gas formation and‘culture 10 showed gas production, which is just the Opposite of the 1922 series. In general, the quantity of gas produced was much less in the 1924 series than in the 1922 series. Very frequently, only a bubble of gas was Vroduced. In these same tubes acid production was delayed. In cultures, 50, 51 and 52 where only a slight amount of gas was produced, the acid produced was slight and disappeared in 7 days. Mannite. Ihe fermentation of mannite, l he dextrose, appears to be fairly constant. N0 variations were observed in the acid production in either series. Acid production was strong in ’1 24 hours and remained constant through the 21 days of observation. The gas production did vary. In both series of tests cultures 11 and 47 failed to produce gas. In the 1924 series culture 41 in addition to 11 and 47 failed to produce gas, which is similar to the results obtained on dextrose. The amount of gas produced in the 1924 series, as in dextrose, was limited in amount and frequently confined to only one of the two tubes used, and this tube showing perhaps only a bubble of gas. The time of appearance of the gas varied. Instead of appearing in 24 hours, as occurred in the 1922 series, it frequently would not appear until after 48 hours incubation. Levulose. The fermentation of levulose was quite variable as compared with dextrose and.mannite. In the 1922 series, gas production was variable as to quantity produced and time of oroduction. However, in summing up, all culture showed gas production with the exception of 11, 41 and 47. In the 1924 series, a decided variation was noted, cultures 4, 8, 9,10, 11, 15, 14, 22, 23, 26, 27, 29, 32, 35, 57, 47, 50, 51 and 52 not showing any gas in 21 days incubation. In those cultures showing gas, the amount was generally confined to a bubble. The formation of acid was eSpecially constant in the 1922 series. In the 1924 series, although the cultures showed acid, the amount of acid produced waried somewhat. Gultures 4, 8, 9, 10, ll, 12, 15, 14, 19, 40, 41, 44, 46, 47, 48, 49, 50 and 51 showed less acid production at first than the others, but in 7 days all tubes showed about the same reaction. iknniose. Due to the cost of this sugar, only 0.1 percent was used instead of the regular one percent as used with the cheaper sugars. As the amount of sugar was limited, the fermentation was less marked, particularly the acid fermentation. The variability of this sugar was similar to levulose in that some strains produced no gas while others produced considerable. Jultures 28 and 47 were the only strains showing absence of gas in the 1922 series. Strain 11, which has been.negative on all sugars discussed, showed a bubble in one tube. This might be an error as the 1924 series showed no gas. In the 1924 series, an extremely large number failed to produce gas. Gultures 6, 7, 8, 9, 10, ll, l5, 16, 17, 20, 21, 22, 25, 27, 28, 29, 50, 51, 54, 55, 40, 41, 44, 47, 49, 50 and 51 failed to produce gas. Where gas was produced, it was con- fined generally to a small bubble. Frequently no gas was pro- duced until after 5 days incubation had elapsed. Acid production was decidedly variable in both series. In 1922, cultures 16, 27, 28, 50, 51, 52, 54, 55, 56 and 59 failed to show acid. It is interesting to notice that the cultures that failed to produce acid in 1922 with the exception of 2 orggnisms produced gas in 1924. This is just one example of how these organisms vary. Acid production, when ~resent, was generally slight, so the absence of acid has no diagnostic value, even though it were a constant preperty of the organism affected. The fact that gas was produced in a number of cases where acid was not pro- duced indicates that the sugar was attacked and that the acid production was masked or neutralized by ammonia or alkali production from other sources. Galactose. This sugar was also used in amounts of 0.1 percent instead of the usual 1 percent. The fermentation of galactose in the 1922 series compares very favorably with mannose. All strains produced gas except cultures 11, 47 and 52. In 1924 the gas production like the rest of the 1924 series, showed much less gas where gas was produced and a large number of cases where no gas was produced. Jultures 4, 6, ll, 29, 40, 41, 44, 47, 50 and 52 produced no gas. Acid production was quite regular on this sugar in both series. In 1922, cultures 8, and 18 did not show gas production, while in 1924 all strains produced acid. Arabinose. Arabinose was used in 0.1 percent amounts. This sugar was used in only the 1924 determinations. Here the gas pro- duction was decidedly variable. Strains 6, 8, ll, 9, 14, 15, 16, 17, 18, 47 and 52 failed to produce gas. f this sugar had been run in the 1922 series, it would probably have acted as mannose, galactose and the others, producing gas in all of the strains. .8 Acid production was quite constant, as all strains produced acid in approximately equal amounts. Rhamnose. Jhamnose or iso-dulcite was used in 0.1 percent amounts and was used in only the 1924 determinations. This carbohydrate, according to Soldberg and Hadley, is always fermented with gas and acid. strains 5, 12, 15, 19-55, 57, 46 and 55 produced gas; however, only one set of determinations was made and these were made in the 1924 series, at which time practically all of the cultures were very slow in producing gas and as pre- vio'sly stated, many organisms refused to produce gas at all, although this prOperty was not constant. Only 5 strains, cultures 56, 44 and 51 showed absence of acid formation. However, none of the strains produced very much acid. In every case the acid disappeared after the third or fourth day. Glycerine. }1ycerine, according to Hadley, Joldberg and others, is not attacked by Bacterium pullorum. In both the 1922 and 1924 series, glycerine was fermented with acid production. The acid production never occurred until after the 7 days incuba— tion, and occasionally not until the 14th day. In the 1922 series, all strains produced acid while in the 1924 series, strains 7, 14, 15, 17, 24, 26, 28, 29, 51, 52, 55, )4, 56, 57, 40, 41, 44 and 46 failed to show acid in 21 d ys. This might be explained by the fact that the broth used in the sugar fermentations in 1924 had a titre of pH 7, while in 1€32 the titre was an 6.8. ‘4 The glycerine broth was sterilized in both cases in ca the autoclave at 15 pounds preS‘ure for 20 minutes. It may be p ssible that the glycerine was broken down into compounds, that Bacterium pullorum is able to ferment with acid production. A study of the effect of heat on glycerine is planned for future studies in order to check these results. The glycerine was not distilled before using. Adonite. Adonite w s used in 0.1 percent amounts. The results obtained on this carbohydrate confirm previous wort. Ho acid or gas was produced in any strains. Raffinose. jaffinose was also used in 0.1 percent amounts. This trisaceharide was constant on all strains, producing neither gas nor acid in 21 days incubation. A Dulcite. Dulcite was used in 0.1 percent amounts. In the 1922 series, strain 55 caused slight acid production, but in the 1924 series, all strains showed negative acid and gas. Inulin. This polysaccharide was used in 1 percent amounts. It was sterilized by autoclaving at 15 pounds pressure for 20 minutes, due to the fact that inulin sometimes carries re- sistant Spore formers. All tubes were kept at room temperature for a Week to eliminate any contamination that mizht develOp. In the 1922 series, strains 11, 15, 14, 15, 16, 17, 18, 20, 25 26, 27, 28, 29, 50, 51, 52, 55, 55, 58, 46, 47, 49, 51, 52 and 55 showed slight amounts of acid. In many cases only one of the two tubes inoculated showed acid production. In the 1924 series no strains showed acid production, perhaps because the titre of the broth was pH 7.0 instead of pH 6.8 as in the 1922 series. starch. The starch broth was wrepared in the same manner as the glycerine and the inulin, using the same amount of the carbo- hydrate. In the 1922 series, strains 5, 24, 27, 28-50, 51, 52, 55, 54, 55, 56, 57, 58, 40 and 41 showed slight acid produc- tion. In most cases, the tubes showed a slight acidity on the second day which disappeared in a day or so. In the 1924 series, all of the strains showed negative cid production, which shows that this preperty of attaching starch, if such is the case, is transitory. haltose. Due to the fact that lactose sacdharose and maltose are very susceptible to heat, these broths were prepared by filtering the sugar through a Berkefeld filter and adding aseptically to sterile broth. The susceptibility of maltose was demonstrated quite early in these studies. haltose broth fermentation tubes were prepared by sterilizing in the autoclave. These tubes upon inoculation showed slight acid and gas production. Using the same sugar, the broth was prepared without heating and none of these tubes showed gas or acid formation. - 51 Lactose. Nith lactose, acid.vns produced in strains 6, 8 and 11 in the 1922 series. In the 1924 series, strains 15, 51, 56, 41 and 50 showed acid. with the exception of strain 56, only one of the tubes in each set showed acid. baccharose. In saccharose, strains 6, 9, 10, 11, 13, 15, 16 and 29 showed acid production in 1C22, while in 1924 strains 6 and 56 showed acid. strain 56 showed pronounced acid production. Dextrin. The dextrin broth was prepared by autoclaving for 20 minutes at 15 pounds pressure. A one percent solution was used. In the 1922 series, acid production was shown in strains 15 31, 52, 55 54, 55, 37 58 39 and 41. 9 9 9 14, 15, 15, 27, 29 In the 1924 series only strain 56 showed acid. Zylose. Xylose was used in 0.1 percent amount and was sterilized by autoclaving at 15 pounds pressure for 20 minutes. This carbohydrate was used in only the 1S24 series. host of the strains produced no change, but strains 4, 5, 6, 12, 15, 14, 17 and 18 showed slight acid production. Hadley, Goldberg and others state that Xylose is always fermented with acid forma- tion. I (:1 lo I 11-11111. :3 r0 nurse Iu ‘ r311: 311132015 -{ISFUIJSIBLu 1 ,- d 5' Lu THE I {11"“Jumi «AS 1’ «MN ‘1TI(1\I 11.3.23 .33 01" 15135."){1111 PULL}; Ihe Inconsistencies of Gas Production of.hacterium pullorum upon Permentable Sugars. Bacteri:u pullorum, as a careful study of Tables XI and XII shows, does vary considerably in its fermentative preperties. ‘fhe accomnanying table (Table XIII) is a list of the 1S24 de- terminations showing those strains that failed to produce gas. A careful study of this table will show that although all of these sugars were inoculated at the same time, incubated at the same time and all, still a strain will attack dextrose vigorously and then refuse to attack mannose, or it will attack maanose and not levulose. If an organism would at one stage of develoement attack all sugars of similar molecular structure and then at another stage not attack any of these same sugars the complexity of the problem would not be as great and an eXplanation might be advanced. On the six fermentable sugars studied, only a few strains were constant. Strains 11 and 47 are anaerogenic types while strains 5, 46 and 55 are aerogenic types. All of the remainder of the 47 strains showed some variation. Experiment 1. 10 Letermine Whether the Medium For Invigoration Has Any Influence on Gas Oriduction. Three media were selected for investigation, namely, plain agar, liver agar and infusion agar. The cultures were transferred every day for a week, when they were transferred + + + + + + I + + O + + + + + + + + + + + + 0p H S» m2 + + + + + + I + + I + + + + + + + + + + + + meflka 1. I I I I I I + I I I I I + I I I + I I I I omega?” + I + + + + I + + + + + + + + + + + + + + + mmgfimaw + I + I + + I + I I I + + + + + + + + + + I 05038 + I. + I I + I + I l I + + + + I. I. + + I I I. mwogq a.” + I I I + + I + + + + + + I + I + I I + + I @835H ,5 . mm mm Hm om we we be as we as ea as an an a: me an :e an an on me gauge 1 deadepa edflcuge TIM + + + + + + + + + + + + + + + I + + + + + + m p H gm 2 + + + + + + + + + + + + + + + + I + + + + + + mmofimg I I I I I I I I I I I I I I + + I I I I I I + @821wa + + + + + + + + + I I I I I + + I + I I + I + m aging + + + + + + + + + + + + + + + + I + + + + I + mm 3.0 mfiww I + + + I I I I + + I I + + I + I I I I I I I mmoflfldfi I I + + + I + + + + + + + I I + I I I I + + + 0 wow; QH new 1mm mm em mm me an on ma eH ea 0H me we me me fie ea a e a e m ammen eoaeepn maaeepn , n .mmwaen wmmfi one ma mhwmen mfipwpsmfihmm moan Edao~flmm Ed mopemm mo Seaweddoam mac 2H mmfiocopmfimsooQH HHHN Handed to dextrose and mannite extract broth. The data in Table XIV show that with dextrose,the three different invigorating media check in gas production, but on mannite considerable variation is evident. Although a variation occurs, still the data do not point to any one medium as giving 001 Istant re ults. It appears that invigoration, as far as the medium is concerned, plays no part. h-tperiment 2. 1'0 Determine the Influence of Invigoration on }as Production. xperi wnt 1 shows that invigoration apparently plays no part in determining gas production. To determine the effect of lack of invigoration, transplants were made directly to dextrose and mannite extract broth from the stock gelatin cultures wh ic nhad not oeen transferred for several months. The results obtained (Table XVI where no invigoration was used were also variable; the dextrose showed gas where the organism was aerogenic, but in the mannite no gas was produced. Further study here would be of interest, although the results obtained on mannite after invigoration showed variation,and the fact that variations also occurred before invigoration would tend to show that invigoration plays no part in deciding gas pro <. uction. HA {3 %‘¢3rmuent 5. Influence of hind of hrotl: on. s Eroduction. Hadle] states tZIat frenuently when an ac; Ogenic strain refuses to produce gas in extract broth it will produce it in infusion broth. ts I.ith ex tract (I? At the thee of the 1222 series of te broth two sets of infusion broth containing dextrose and mannite respectively were also run. TABLE XIV H3 The deletion 0 Gas Production to the Invigorating medium Mannite jxtract Broth strain Elain Agar liver Agar Infusion Agar Gas Acid Gas acid Jas acid 47 - - + + - - + + - - + + 41 B - + + - - + + - - + + 11 - - + + - - + + - - + + 10 - - + + B - + + 15 - + + Dextrose Extract Broth 47 - - + + - - + + - - + + 41 10 10 + + 5 10 + + 10 15 + + 11 - - + + — - + + - - + + 10 5 10 + + 1’ 10 + + 15 B + + IYI ‘ 1A.}; XV Le The Effect of lack of Invigoration on Gas Prouuction Dextrose Extract aroth.Mannito Extract Eroth strain Gas acid Gas Acid 4'7 - - + + ~ - + + 41 5 O + C - + 11 - — + + - - + + 10 510 ++ -- ++ The variatiuns obtained are shown in Table X71. Invigora- tion used for these tubes was similar to that used for the ex- tract broth. The time between running the two sets of tests was approximately two weeks. The results, as shown in Table X71 do not show any marked difference. It will be noted that on dextrose, the amount of gas produced was slightly greater on infusion broth than on the extract broth. Although strain 41 showed gas on infusion broth and not in extract broth, still strain 52 was exactly the 0pcosite. On ma nite, gas production was very frequently negative on infusion broth,while on extract, it was positive. From all outward ap earances the extract br th was the better. This might not be borne out in repeated studies, which should be carried out. In summarizing all the results obtained, it up ears that gas production in Bacterium pullorum is a variable characteristic, and,further, it appears that it is some inherent factor that we know nothing about at uresent. Table XVI Relation of Infusion Broth and Extract Broth in Determining Gas Production. Extract Broth Infusion Broth dextrose mannite dextrose mannite ‘— l B B - - B - - - 2 5 5 B B 20 10 - - 7 SO 10 - - 1 B B - - - - - - 2 5 5 10 10 5 B B B 7 20 15 5 B 1 B B - - - - - - 2 5 5 - - B - - - 7 10 10 10 - 1 B B - - - - - ~ 2 5 5 - - B 5 - B 7 8 10 10 - 15 15 - 15 l B B B 5 - - - - 2 5 5 20 40 B B B - 7 10 15 4O 5O 15 15 10 5 1 B B 5 5 B - - - 2 10 5 50 CO 10 5 B B 7 50 10 SO SO 25 25 10 10 l B B - - - - - - 2 5 5 - - B B - - 7 10 10 5 5 10 15 - - Table XVI continued. Extract Broth Infusion Broth dextrose mannite dextrose mannite 11 1 - - - - - - - 2 - - - - - B - 7 B B - - - B - 12 l B B B B B B - 2 5 10 30 60 B _10 - 7 10 15 4O 60 15 50 - 15 l B B - - - - - 2 3 B 40 B B B u 7 10 B 4O 10 10 10 - 14 1 B B - - - - - 2 5 5 5 10 B 5 - 7 10 10 10 50 2O 5 - 15 1 B B B B — - - 2 3 5 IO 10 B B - 7 10 10 2O 2O 10 15 - l6 1 B B - - - ~ - 2 10 5 15 20 B 5 - 7 29 10 2O 50 15 2O - 17 l B B - - B B - 2 10 25 B - 10 IO - 7 2O 50 10 10 50 40 - 18 1 B B - B - B — 2 15 15 2O 50 5 2O - 7 2O 2O 4O 40 20 25 - 57b - XVI continued. Extract Broth Infusion Broth dextrose mannite dextrose mannite 19 1 B - ~ - - - - 2 10 5 25 25 B 5 - 7 15 115 40 5O 2O 2O - 2O 1 B B - B - - - 2 10 5 15 IO 5 5 - 7 15 15 2O 15 50 2O - 21 1 B B - - - - - 2 B 15 B B - B - 7 10 50 5 5 10 15 - 22 1 B B - - — - - 2 B B - 3 B B - 7 5 5 5 5 15 2O - 25 1 B B - - - - - 2 10 5 B B B B - 7 20 IO 20 IO 15 15 - 2d 1 - - - - - ~ - 2 10 B - - B - - 7 IO 15 5 15 20 2O - 25 1 B B - - - B - 2 B B - - 5 5 - 7 5 5 5 5 50 15 - 26 1 B B - - - - - 2 20 10 B B B B - 7 2O 10 15 B 20 2O - 57c - XVI continued. Broth Infusion Broth mannite dextrose mannite U1 bd OJ Li ‘0) Li] Cd as +4 -q as +4 [q as +4 Ci as q ()1 w cu bJ as +4 [q as t4 ()1 bowed on to to l4 q Lu 0‘! a no +4 q m as B 3 B B - :3 15 IO 15 - - - B - - B 3 10 B - 20 ”O 50 15 - - 3 B B - - B 5 5 - B B B B B 3 B 20 25 B - - 3 - - 20 10 5 15 - _ - 3 g - E B 15 10 - 10 15 B B - B - - - - B 5 - B - 50 10 15 15 - - 57d - Table XVI continued. Extract Broth Infusion Broth dextrose mannite dextrose mannite 55 l B B - ~ - ‘ ‘ ’ 2 B B B 10 B B - - 7 B 5 5 10 10 15 - - 56 1 - - - - - - - - 2 5 5 B - 10 B - - 7 5 5 15 15 40 20 - - 57 1 B B - - - - - - 2 B 5 B B B B - ~ 7 5 10 2O 2O 50 5O - - 58 1 B B - - - - - - 2 B 5 10 B - - - - 7 5 10 50 IO 10 15 - 10 59 l B B - - - - - - 2 B 5 - - B B - B 7 B 10 5O 50 10 15 9 B 40 1 B B - - B - - e 2 10 10 B B 10 5 ~ - 7 15 10 10 IO 40 40 - - 41 1 - - - - - - - - 2 - - - - 3 3 - - 7 - - - - 5O 2O - - 42 1 - ~ - - - - - - 2 - - - - 10 15 - - 7 20 B 40 40 5O 5O - - - 57a - Table XVI continued. Extract Broth Infusion Broth dextrose mannite dextrose mannite 46 1 a B - a 3 .. ._.; 2 B B 15 40 B - - 7 5 5 5O 60 B - - 48 l B B B B - — - 2 15 15 5O 2O 10 5 - 7 25 25 5O 5O 5O 2O - 49 1 B B - - - - - 2 10 10 B B B B - 7 IO 15 2O 2O 10 10 - 5O 1 B B - - - - - 2 5 B 15 15 B B - 7 15 10 2O 10 15 10 - 51 1 - B — .. .. .. .. 2 B B - - B B B 7 10 5 10 15 15 15 B 52 l B - - - - - - 2 B B - - - - - 7 B B B - - - _ 55 1 B B - - B - - 2 15 10 - - 10 B 2, 7 15 10 B B 50 'O - AZ} ‘rLlJ’ilnniL. : l-‘U‘JER 015‘ 'fx‘a...’ItJUS 33-1.5111} OF 1 "1 *' ' r- “*‘v': rn’ 4J1} IL .-IIJ‘ZJ -PUT-«Jakf ‘..\\}J.'. In l€.32 at the time of the first sugar fermentation studies, antigens of the 47 strains studied were prepared. Ihese ware prepared by washing the growth f on a 48 hour liver agar slant culture with nhysioIOjical salt solution which had 0.5 oer3e nt whenol added 11es OantiVG. Uhese antigens were t1- en standardized to a ncphelometer reading of l. A strong aquutinating Bacteriug pullorum serum was selected and run against the 47 antigens separately, using dilutions of 1-50, 1- 100 and 1-200. feedings were made in 24 hours. The results obtained are given in mahle XVII. T1BLE XVII laglutinating Bower of Various Strains of Bact. pullorum. Strain Control 1-50 1-100 1-200 E Strain Control 1-50 l-lOO 1~200 Ho. : Ho. 4 cloudy 4 4 4 E 25 cloudy 5 4 5 5 " 5 4 4 E 29 " 4 - 4 5 " 2 5 5 E 50 " 5 5 1 7 " 5 4 4 E 51 " 4 1 1 8 " 5 5 5 E 52 " 4 4 4 9 " 2 5 4 E 55 " 4 2 2 lo " 5 5 4 E 54 " 5 5 z 11 settled E 55 " 4 4 4 out 2 5 2 : : 57 n 4 4 4 12 cloudy 5 5 5 : : 58 " 5 5 1 15 " 4 5 2 : : 59 " 2 1 l 14 " 5 4 4 : : 4o " 2 1 1 15 " 4 4 4 : : 41 " 4 4 1 16 " 5 5 4 : : 44 " -+’ ~ - 17 " 4 4 4 E : 46 " 4 4 4 18 " 1 5 4 : : 47 settled 19 " 5 4 4 : out - - - 2o " 5 4 4 E 48 cloudy 4 4 4 21 " 4 4 4 E 49 " 4 4 4 22 " 5 4 4 E 50 " 5 4 4 25 " 2 4 4 E 51 " 5 2 1 24 " 5 4 4 E 52 " 4 4 4 25 " 4 4 4 E 55 " 5 4 4 26 " 5 4 5 E 27 " 5 5 5 E These tests as shown by the table indicate a variation in the agglutinating nower. Both strains 11 and 47 settled out in the control tubes and made it nearly imoossible to determine whether any agulutination occurred. Jonsiderable variation was also evident in these cultures showing aggluti- nation. The variation was so pronounced that cross-arzlutination studies were made. The antigens were prepared as before and birds were immunized to cultures 4, 5, 5, 7, 8, 9, 10, 12, 15, 41 and 50 resoectively, two birds being used for each culture. Only one injection was made, using 1 c.c. of a 24 hour suSpelsion in phjs1010§ical sclt solution. fhe method of setting us the test was as follows: Ten dilutions were used. hincty-five hundredths of a cubic centimeter was pipetted into the first tube and five-tenths of a cubic centimeter into the other nine tubes. Five hundredths c.c. of undiluted serum was added to the first tube, making the total one c.c. and giving 1-20 dilution. After shaking, 0.5 c.c. of this 1-20 dilution was transferred to tube 2, giving a 1-40 dilution. Using the same pipette, 0.5 c.c. was trans- ferred from tube 2 to tube 5 and so on, giving dilutions of 1-80, 1—150, 1-520, 1-640, 1-1280, l-2560, l-512 and 1-10240. The 0.5 c.c. amounts per tube were used,as the quantity of serum available was limited. This amount of antigen allowed 21 maximum number of dilutions to be used. Before adOpting this znnount, experiments were carried on to determine the accuracy as compared with 1 c.c. and 2 c.c. portions. ho difference existed provided small diameter Hasserman tubes were used. The tubes were incubated at 37° 3. for 48 hours before reading. The data obtained are given in the accompanying graphs. (Fig. ) As also shown in the ace npanying graphs, all the antigens were active against all the sera except cultures 11 and 47. These two cultures are, according to hadley and dettger, Bacterium pullorum Beta. Although these w re the only two Eeta type organisms used, and althoujh their antigenic power is apoarently low, still the data are insufficient to state that the Beta type will not agglutinate Alpha type seam. The graph of culture 11 serum shows very poor agglu- tination prOperties on all antigens. 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M.m_.w_-nm,u. ., .,M ._ ,w d.__..;» mnpmach n__ _._7: l l ,. ..L. ‘- . _TL _U :U T... “O _U \ figs w. , ..u H. d m H” _ ._..“ m .- v . N . _F. a m o . m . ... H ... n d .0, ”a D a . ..... a an. H . a ... _ NH”- . . . _ — J .-_.» J21- 4 , . , 1V - . '17 J:., ,!. r- ..- _ . . n A. .o.. . .. _. u . . U .me mfimfififiqisgfiwjaaflmn_go . .._H. _ ..H __ _. . :; “ fiovflgw 41?.91 samuumdea M1 H: n: . . , __ : .. __ . .V I...» . .3. H ._. . . I . ... ..- ., . ... «r . . .... - V _. .. +. m: H J. _. a ”a .. ,. _ . v . a. w . . , . we .. . m. . . _ 1m. . _._ m_ . it-.. . L W. -. ...: .-.... ... ....1...» ....z. . .... .- .. .._.... , . LL .. _ -.. rt l. .... . . . - 62 - SLlj‘ LL :L.~d-5Y . Of the 47 strains studied, tw distinct types were found which diifer in.their gas produci1 lg powers. One strain attacks dextrose and mannite with gas and acid formation, while the other produces acid and no gas. These results confirm the work of Retbg er and Hudley. Cros s agglutination of strains failed to show any marked differences with the exception of strains :Tos. 11 and 47, which .1 were anserogenic strains. Because of t}:e fact that onlv two n strains or the enuerosenic type were studied, the above results oet.ined in the cross-c celutingtion tes sts ere ins uszicient for drawing any conclusions. 1 The morphology of the Beet. pullorum chunged considerably when grown on liver sear medium. Gus production on the fermentsble sugars varied consider- ably. Experiments carried out to determine the cause of this verietion failed to show any results. Lotion on the non-fermentuble sugars with few exceptions WJS quite constant. Biochemical tests were also quite constant with the exception of the hydrogen sulphide test. Reaction on litmus milk varied considerable with some teins. Host strains, however, were quite constant. U0 psiticulur strain ‘wi esred to be varie ole at all times. Practically all of the strsins seemed to vary at one time or another. Acknowledgements I wish to acknowledge the helpful suggestions and cOOperation extended to me by Dr. Stefseth, Dr. Giltner and other members of the Department. 1. 5. 4. 5. 6. 7. 8. 9. 10. 11. 12. ffi'.‘ "U‘T'TT’NT‘C‘ 11.4111.-- ‘. ”L. JJJL) O Endley, Elkins and Cdldwell. The Colon-Typhoid Intermediates as Causative Agents of Diseases in 91rds. R. I. Bul1. 174. Hey 1918. Rettger. Fetal Septicemid in Young Jhickens. H. Y. Med. Jour. 71, p. 803. Rettger. Fntsl Septicemia in Young 3hickens. M. Y. Ked. Your. 75, p. 267. ?ernot. Investigutions of di eases of pozltry. Oregon std. Bull. 64, (1900) § In . 11:8. ‘-I‘€311”"ir1n'r"»r ‘36: [‘O’C t 1°01" "' ome dj sea 0 m 0"" ‘fo‘w‘n a. r111 1re-f] _ . ..L "d ..'. 1’ .. '._i . v.16.) LI {-3 '4... CL. 1 S. La. ngri. fixpt. Stu. Bull. 108, (1908 p Rettger and Stonehurn. Bucilsry Ihite Diarrhea of Yound Chicks. Storrs Conn. its. Bull. 60, (1909) Moore. Coccidium as Guise of white Dicrrhes. U. S. D h. Bull. Ciro. 128. Hadley. V Studies in Avian Coccidiosis. Cent. Eekt. Orig. I, 1909, p. 548-555. dettser. - 0 Further Studies of Futsl Bepticemis in Young Chickens: on fihite fiisrrheu. Jour. fled. Res. 21, 115-25 Jones. Fetal Septicemis on 3scill¢ry Ihite Diarrhee in Young Shichs. Rept. of I. Y. 8. Vet. fell. 1090-10, p. 111 Gque 0 Notes on varicn Infection with Sect. Pullorum in the Domestic Fowl. Jour. Jed. Res. 24-491, (1911) Jones. (1915) An Outbreak of an neute Disease in Adult Fowls Due to 9nct. Pullorum. H. Y. s. Vet. Coll. Rept. 1911-12, p. 140. 13. Jones. (1913) Value of fluoroscopic inglutindtion Test in Detecting Fowls thdt Edrbor 9cct. pullorum. Jour. Led. 2?es. 27, 47 -é79. 14. Jones. (1911) The Telue of the IncrosCOpic igglutinstion iext in Detecting Powls that Eurbor 9ict. pullorum. 1.7. Y. to ‘Vet. 3011. :0 ‘\to lgII- “I64, p. Iago 15. iettger, Kirkp; tr ck end Ltoreburn. (1912) 9dcillsr"lH1i e Fierrhes of Youn: Chicks. ... ”UOZ‘I‘S JO-L11. !:tL'.. Bull. 74;. 16. Retteer, Zirko trick and dtoneburn. (1914) Bdcillur: -hite Dicrrhe; of YOT‘f Shicks. Ltorrs, Sonn. etc. Bull. 77. 17. Safe. (1914) On ”he Eidfinosis of Infection with 91st. pullorum :he 9omestic 30.1. Ides. gts. 9ull. 149. 18. Tettger, Zirlqhi tricl: uni JC nes. (1915) Eccillnry white fidrrheu of Youns Chicks. Steers Conn. Std. 9u11. 85. 19. Smith and Ten9roeck. (1915) 4 lots on the Re :t'on Between 9dct. 1.1110rum(Rettger) and Beet. Sell 117. 11.1. (701133. frat. :168.J1. 54:7. 20. Eettger end Iorer. (1916) floaterstive Study of Esoterinn Tullei um end 9d cterium sturvuiwtruun. fig“ A '7 00%).. o J. .CuL. :ie-‘J. c) 443 0‘1 21. Her end fiullumher. (1917) in Ir wtr dermal Tes ior vast. prlloru 1 Infection in --u. u 1t 1‘ 0‘." r. e. 3. i. 9u11. 517/ 22. Go 11be~~. (1917) _ _ _ 1 tud" 01 th;_”er1enting rroperties of 9ect. pullorum end Beet. 8:21:1111; run. Jo -.-. 71.01}. 7:33;. ”$3703. 51 "' 205. 25. Zulsov. (1919) '. ’1 ‘."“~ . L. v 7' ”'3 .- , 1 :3 + n Jenpdritite :tudJ 01 9dct. pullorum end 91cc. C‘rsrirsrflm. your. Ini. Dis. 055. up [._.I O} , 5 -162. Berry. (1917) studiefs (M1’91c1111r3'1321tc Eic119xa1 of tLeW<31€ tic Soul. Thesis for h. J. decree, 1ich. Lir'l. College. 171111613 . (1917) Infections Jensed by Vest. pullorun in ”flu 1J Fouls 3. I. ata. Bull. 172. Esdley,C1ldwe11 1nd Te1th. (1919) 91ctcri310'1c 1 Jotes on 9sct. 0L110tW‘ U TO :11“ o 11.0 I . 4'. , 6 7“ Q .x1a1ev,;1k-ns 1nd o1lawe11. (1918) ’$118 'J‘OlOIl-i:ri‘ ‘ 310161 :11 UI'L: “7“” “DC-L1 11;“th 1’s DGIJFLVi-uii‘fe i. ”(‘11 Of -i€@999 in ir . I. The Tui1t3phoid Bacteria. -1. I . 13-1111 . l"4.. LC. Us Rettficr, Lier1tM‘i E 1nd Cord.(l919 fiq o nucill1ry u W1 e Sierrhes of Younj Chicks. VII. Ltorrs Joan. 9u11. 101. ...I i \ MICHIGQN STQTE UNIV LIBRRRIES IIIllllllllllllllilllIHllllHl|||l||||ll||lI||||1Hll||||||2|l|| 31293015914512