110 866 __THS "‘i‘HES-IS {HE CULTIVATEON or BAC'EERIUM ABORTUS Dame} E. Hash”:- {924 .n... a . If?! V \lnv-A‘Ilrirulrpl. . 1 n . 1... ‘1. .11. 1. . . XI}. . w ..u ‘1 o... ...L...nurul. .,:|.a t...\u.. “1737..” . ...‘ 9‘ .I. Rinks’. .n..\u:fi.r..bufln.1.h..hk .LVuh“.\.lo‘..rl....l ...Vu Cal.) .. f!§‘ . .....t.ull|i \9L\.I...HJ.1. 5... .. ) I ‘L , h . y. .. [I P . .. .. 21!; ..c....:.. 3.. u... ... I: :k. .51.... 5 9.3;. . 21.113..- .9. . .:..L.. E. if?) .1.... .. r..........l...,..r .12.-. it 'E III! .oKl’. i! hxui vl1vlb| IIII'V.. [.\ ‘ I: E i. II I..- I .zv.l‘v-. II... ..\ D at . . J". . a L! t o . - . x. t , n I C It I. r . 1-. n . I r a 4. I. U. x . IA . L . A I . \.4" '4. THEE r1 '1 v - r .~ .~ —.- ,, .- - , , ,. 1, 1H1. VULTI'J'ATIUR 02‘ BAJ‘TJRI J11 ASCJJ’J‘JS THE )UL‘BIYATIUIJ 03 BAJ'L‘TLAI’fil 13‘ {Lil‘Ub‘ Submitted to the Faculty of the Lichiqan Agricultural Jollege in partial fulfillment of the requirements for the degree of master of eienee. Daniel E. ggeley June, 1924. fwfiEAS 'le‘T f" it JJ‘iL Introduction leview of Literature Method of Investigation Sources of Laterial Preparation of Media ”ethod of Plating Milk Method of Plating stomach content of Feti Method of Plating Organs from Infected Guinea Pigs methods Used for Incubating Jultures JXperimental Data 1. The Isolation of Beet. abortus from Fetal Katerial When Plated on Different hedia and Placed under Di fferent Gonditions of Incubation 2. The Isolation of Dec t. abortus from Kilk when Plated on Different Ledia and Placed under Different iLethods of tIncubation. Iart I. 5. The Isolation of ”act. abortus from hilk When Plated on Different hedia and Placed Under Different hethods of Incubation, Part II. 4. The Isolation of Daet. abortus from Iuinea- Pig Organs Jhen Plated on Different Ledia and Placed Under Different Methods of Incubation, Part I. rhe Isolation of Dact. abortus from Guinea- Pig Organs ahen Plated on Different Media and Placed Under Different Methods of Incubation, Part II. CI! 0 o. The Jultivation of Beet. ahortus on Different , ... ., 1 -w ,. hedia when Placed under Dilzerent nethods of Incubation. {VP V 9!.)\)2" 7. The Value of Adding Serum to Liver Agar for the Isolation of Beet. abortus. 8. The Effect of pH concentration of Liver Agar on the Growth of Beet. abortus. LO . The Value of clearing Liver Agar with Egg Albumin. lO. Ihe Effect of Incubation Aerobically Before Placing in an.AtmOSphere of 10 percent carbon Dioxide on the Sr wth of beet. abortus. 11. The Value of centrifuging as an Aid in the Isolation of Beet. abortus from hilk. General Discussion gunnery Acknowledgment References. 1'11"" " ' ’Y‘ T). ‘ I." )T’ I ' If|\F-‘ -1111 JL) :IaLI f;l..LI ‘1‘); .L.‘l.J..'._1.L.I. .41.- &.-/-‘.;.L._IO. m Il-Jl’ {’ ‘JUJIICU lhe isolation of Eact. ahertus presents certain difficulties. fhe early investi mitors, although convinced fic Ho that abortion was due to an undetermined but Spec agent, were unable to isolate the organi sh] It was not until suitable biolo;ica1 reeuirements were provided that the organism was obtained in rure cult W1? Io obtain successful m~e ults in the primary iso— lation of Bact. abortus, it is advisable to use Specially preeared media having a suitable pH concentration and necessary to incubate under ce, rta in conditions peculiar to this organism alone. several different media are used for the growth of Beet. abortus and several investigators have described methods for the cultivation of this organism. While these methods have been successful to a certain degree, they are not always wholly satisfactory, due to the amount of labor involved and the results obtained. It is the purpose of this study to eomnare the growth of hact. ahortus when grown on different media and when placed under different conditions of incubation in an effort to obtain the most satisfactory method for the cultivation of this organism. 123713.] UR 2.118. .13.": .133. fhe fact that animals cast their young before the full period of gestation was recorded in ancient times and . that abortion in cattle was contagious in many instances, and might be Spread from animal to animal in the herd, was _known to farmers and breeders in the early part of the nineteenth century. In 1826 Hutrel d'Arboval, a Frenchman, and in 1554 Youatt, an Englishman, concluded that abortion was often contagious and not due to environment or acci- dents. In 1878 Iehnert demonstrated this contagiousness by introducing into the vagina of pregnant cows the vaginal discharge and placental tissue of aborted cases, and thus causing abortion. (1). In 1886 hocard (a), investigating for the French government, made extensive bacter10103ical investigations in an effort to isolate a definite organism causing abortion. He obtained a bacillus and a micrococcus in pure culture, but with neither of these was he able to pr duce the disease. In 1897 Bang (5) published the results of his study of the etiolOgy of epizootic abortion in Denmark, in which he discovered the organism now known as Bact. abortus. He examined the uterus of a cow which had been slaughtered while showing premonitory symptoms of abortion. Between the uterine mucous membrane and fetal envelOpes an abundant 3. yellow, odourless exudate was found. In cover glass preparations from the exudate stained with methylene blue he observed a very small bacterium, apparently in pure culture. These were found 'singly and in clumps, free, and often intracellular. The following medium, which was originally designed by stribolt, was the one on which Bang first obtained an artificial culture of Bact. abortus} A solid medium containing 5/4 percent agar and 5 percent gelatin is prepared and nut into culture tubes in the ordinary way. aefore sowing,the solid gelatin agar should be liquefied by heat and cooled to 45° 3., after which about half its volume of liquid sterile serum in the raw state (species of animal not stated) is to be added, and mixed by shaking. The liquid medium is then inoculated with the seed material, and the tubes again gently shaken, and plunged into cold water to bring about rapid solidification. According to Bang, ”At the end of from two to four days' incubation there appeared a great number of very small colonies which develOped only in a definite zone of the tubes. This zone lay about half a centimeter under the surface of the nutritive medium, and it had a thickness of from 1 to l 112 centimeters; colonies vere not present above or below this. We had thus not to do with an aerobic bacterium which would have pushed its growth as far as the surface of the nutritive medium, and still less had we to do with an anaerobic fcmm which would have grown as far as the bottom of the tube. The under limit of the zone of growth lay exactly where the limit 4. of the growth of a strictly anaerobic bacterium shows itself, the necrosis bacillus for example. Ehis highly peculiar behavior of the abortion bacillus towards ox'gen made it at once apparent thatiwe had to do with a distinct Species? In Speaking of the cultural characters of this bacillus, Bang stated that his observations "appeared to show beyond any doubt that for the abortion bacillus in its behavior towards oxygen there are two Optima, first, a degree of oxygen tension in the nutritive medium less than that of the atmOSpheric air, and,second, the presence in the nutritive medium of a very high tension of oxygen, which, however, lies somewhat under 100 percent. Between these two Optima there is an intermediate zone in which the abortion bacillus grows badly or not at all." In 1901 Preisz (4) at Budapest isolated a similar organism from the vaginal discharge of a case of abortion. He made his inoculation directky on slanted ordinary agar made of meat infusion, peptone and salt,and after passing oxygen into these tubes be sealed them with wax and obtained a growth visible with a hand lens in three days. Inoculated into deep dextrose agar, the zonal growth appeared lying seven to fifteen milli— meters beneath the surface of the medium. Stab cultures of the organism rarely reached the surface after prolonged incu- bation. He considered that his medium was as favorable as the A.G.S. of Bang. He was able, by using alkaline pyrogallol, to produce a growth,and also by using acetylene gas. He believed that in these two instances growth resulted because oxygen ' was not wholly absent. He compared the organism to an anaerobe, but it differed in that it would also grow in pure oxygen. In 1908 howak (5), at the University of Krakau in Austria, (Fabyan) took up the study of this organism, being attracted by its very interesting biolozical characteristics. In studying pure material he found the method of Bang satis- factory, but where contaminations were present the growth might easily be killed out or masked. The broad surface which Petri dishes afforded was desirable to obtain isolated colonies. Nowak sought to obtain an oxygen tension less than air by the use of a closed chamber containing the actively growing culture of an organism like s. subtilis, a method which had already been employed in the removal of oxygen in tetanus cultures. He placed tubes inoculated, some with B.subtilis and others with @233. abortus, in a glass chamber with paraffin and placed this in the incubator. Satisfactory results were obtained when the relation of the surface of E. subtilis was in preper pro- portion to the volume of the chamber. If too little of 'Q. subtilis was “resent, no growth resulted; if too much, all the oxygen was absorbed and Best. abortus checked.‘he found one square centimeter of surface growth of Q. subtilis to fifteen cubic centimeters of volume the best preportion. Different varieties of 3' subtilis gave similar results. The tubes inocu- lated with suspected abortion material were first incubated twenty-four hours in the large chamber of the incubator in order to deveIOp any contaminations, and then incubated under the influence of g. subtilis,when typical colonies deveIOped in the clear areas. By gradually using less of g. subtilis, he trained gggt. abortus to grow in a normal atmOSphere. In pure oxygen two out of six cultures grew. Under compression he obtained good results from three atmOSpheres but no growth at six atmospheres, although when this latter tube was placed under the influence of B. subtilis colonies rapidly develoned. By his technic he was able to obtain many cultures from the fetus and vaginal discharge where other methods failed. In the United states the disease has been known for some time but it remained for Lacheal and Kerr (6) in 1E10 to isolate and identify Bact. abortus as being reaponsible for a part at least of the abortions in cattle in this country. By the howak plate method they were able to obtain cultures of Bang's organism in two out of four cases of abortion studied. In 1911 Holth (7) demonstrated that material from abortion cases inoculated on slanted serum agar gave no growth in two weeks' incubation, but if similar tubes were sealed with paraffin growth might sometimes be observed after six days. The eXplanetion offered was that the organism by using the oxygen over and over again reduced it to the prOper tension. In 1912 Giltner (8) called attention to the use of media prepared from pregnant uterine wall, fetal membranes, fetus, and amniotic fluid separately. In 1912 Smith and Eabyan (9) reported that £33£,abortus inoculated into guinea pigs produces a disease with characteristic lesions, and that the organism can be recovered from these Digs after a period of 11 weeks or more. By this method it was often possible to isolate hact. abortus from contaminated material when direct cultural methods failed. The greatest number of organisms were found to be con~ tained in.the Spleen and lymph nodes of the inoculated animal, while the bcme marrow, liver, kidney and lung, in order named, followed in this reapect. Ihe Nowak method of culturing was employed to recover the organism. In addition to g, subtilis, cultures were grown in symbiosis in a closed chamber with E. coli, g. megantherium and a staphylococcus. It was found that Q. coli, §,megantherium, and the staphy10coccus gave results similar to 2. subtilis. In 1918 Smillie (10) reported that cultures of £333. abortus could be regularly recovered from inoculated guinea pigs withih 3 to 4 weeks. ghe figures he obtained show that the number of living bacteria in the Spleen of the guinea pig was larger at that time than later, although the mzicroscOpic les ions tend to become more prominent as the number of bacteria decreases. Ordinary veal peptone agar tubed and slanted was the medium employed. Bits of tissue were rubbed over the entire surface of the agar, and the tubes were sealed with wax and incubated at 57° 3. colonies of 2333. abortus were observed on the agar slant after 5 to 10 days’incubation. Fabyan (11) and.schroeder (13) working independently demonstrated the presence of Bact. abortus in milk by guinea-pig _inoculation. In 1918 Evans (13) endeavored,to isolate the organism 8. from milk by the use of direct plating methods. She succeeded in isolating the organism from.milk of cows which had been inoculated with strains of that organism, but was unable to isolate the organism from animals which had aborted as a result of natural infection. In 1920 Stafseth (14) found that excellent growth of Beet. abortus could be obtained by employing spleen or liver media in place of beef. It was found that the addition of 1 percent dextrose Or of 1 percent starch improved the Spleen media, while the liver agar did not require the addition of carbohydrates. In 1920 Huddleson (15) reported his results on the isolation of Bact. abortus from milk. He found that the medium and its prOper preparation, the preper H-ion concentration, the employment of an agent which eliminated fast-growing organisms, and the method of incubation were the factors which must be con- sidered when isolating Beet. abortus directly from milk. Liver agar as described by btafseth was employed; glass wool was used in filtering, and.excessive heating'was avoided in its preparation and sterilization, for he (Huddleson) states "Huntoon (16) has shown that about half of the initial growing value of the media is removed by over-heating, and the use of cotton, cloth or paner in filtration." Optimum growth was obtained when the pH concentration of the media was between 6.6 and 6.4. Gentian violet in a saturated aqueous solution was incorporated in the media in sufficient quantity to give the dye a final dilution of 1:10,000 as an agent to eliminate fast growing organisms. The growth of a large percentage of gram positive and a small percentage of a gram negative organisms is inhibited while the growth of Bact. r abortus w s not in the least affected. From experiments conducted, huddleson concluded that the initial growth of heat. abortus was due to an increased carbon dioxide tension, and, by incubating the inoculated medium in a closed chamber in which 10 percent of the air had been diSplaced by carbon dioxide gas, obtained growth in 24 to 72 hours. The results obtained from the direct plating method were identical with the guinea-pig inoculation method for de- termining the presence of 25231 abortus in milk. The chief advantage of the direct plating method is that it requires only four days to determine the presence of the organism, whereas the animal isolation method requires three to four weeks. In 1933 Eitch (17) reported thaiibeef infusion agar adjusted to a pH concentration of 6.8 to 7.8 plus 10 percent natu- rally sterile horse serum was xcellent for cultivating gagt. ibortus; the cultures developing in either an.atmOSphere of 10 percent carbon dioxide or 10 percent hydrogen. Ln 1922 Hagan (18) prepared cultures of hact. abortus as follows: The edges of the lower half of a Petri dish containing the inoculated agar was dipped into molten.paraffin and sealed to a sterile glass plate. )rowth appeared within a to 4 days when incubated at 37’ J. H. 10¢ ICE’JHOD 03‘ IIE'ESSI'IGA’JICI} 1'he literature reviewed gives several methods for the cultivation of 3333. abortus,but uhile these methods have been 'successful to a certain degree, they are not always wholly satisfactory, due to the amount of labor involved and the results obtained. However, the literature does not give a comparative value of the methods now in use for the cultivation of this organism, so this investigation was designed to give definite knowledge regarding this point, and to perfect the technic. It was necessary at the beginning of the study to eliminate those methods which required a great amount of labor in comparison with other methods which required less labor and gave equally satisfactory results. hang's original method renuired too long an incubation period so was not used; howak's method of growing in a closed chamber in symbiosis with a culture of 2' subtilis, and Hagan's method of plating on Petri dishes which were afterwards sealed separately, were not used because of the labor involved. Material from aborted feti, milk from animals which had aborted, and organs from guinea pigs which had been inoculated with material containing Bact. abortus,was plated on various media and placed under different conditions of incubation in order to study the effect of these various factors upon the isolation of the organism. The cultivation of laboratory strains 11. under the same conditions was also studied. A limited number of eXperimcnts were conducted to obtain data on the value of centrifuging milk as an aid in the isolation of the organism by direct plating methods. ”33-3335 0‘3 Inning-,3; Katerial for culturing was obtained from three sources: (a) In the diseased animal the Specific bacteria are found in the placenta and amniotic fluid, within the fetal intestine, sometimes in the tissues of the fetal organs, and in the wall of the maternal uterus. In abortion cases the material studied was obtained from the stomach of the fetus. The abdomen was seared with a heated knife, an incision made through the wall, and the stomach contents were removed and placed in sterile test tubes by means of sterile pipettes. (b) furious investigators have shown that Bact. abortus is often present in milk which is drawn from apparently normal cows, and if injected in guinea pigs will produce characteristic lesions. Eilk was used as a means of supplying the organism Bact. abortus for this study, both for guinea-pig inoculation and direct plating methods. The samples of milk were collected in sterile test tubes under conditions tending to exclude as far as possible all outside contamination. After washing the udder and flank approxi- mately 10 c.c. of milk was collected, having discarded the first milk. (c) Organs of guinea pigs, which had been inoculated with material containing hact. abortus, were also used as a source of material in this study. 3?-{1313A.LAL‘I’4‘>1J 0 .1‘ LLSDIA . Liver agar. The liver agar used was prepared as described by btafseth (14) and improved by Huddleson (15). An infusion was made from beef liver and 2 percent agar, 1 percent peptone, with 0.5 percent sodium chloride added. ihe medium was cleared by egg albumin, filtered through glass wool and adjusted to a pH concentration of 6.6. Gentian violet liver agar. Jentian violet liver agar was prepared as described by Huddleson (15). It consisted of the liver agar described above with the addition of gentian violet in a saturated anueous solution in sufficient quantity to give the dye a final dilution of l:l0,0CU. Beef infusion agar. Beef infusion agar was prepared containing 2 percent agar, 1 percent peptone, and 0.5 percent sodium chloride; it was cleared by egg albumin and adjusted to a pH concentration of 7.0. At the time of sowing, approximately 10 percent naturally sterile horse serum was added to the melted agar after cooling to 50° 3. Yeal infusion agar. feel infusion agar was prepared in the same manner as the beef infusion agar described above. 15. Uterine Wall and fetal me ibrane infusi3n agar. An in— fusion was prepared from the uterine wall and fetal me branes of a pregnant uterus, as mentioned by }iltner(8): 2 percent agar, 1 percent peptone, 0.5 percent sodium chloride was added and the medium adjusted to a pH concentration of 7.0. whis mezium is referred to in the following tables as ”uterine agar". Deetic digest agar. iwo percent agar was added to peptic digest broth which had been prepared according to directions given by hubvosky and Lyers (19). She medium was adjusted to a pH concentration of 7.0. LEEAM>OEIHmL IN}II"" One-tenth cubic centimeter of the milk sample to be cultured was placed on the surface of a solidified agar plate and evenly distributed by holding a sterile glass rod, bent at an angle of 90 degrees, against the surface of the medium and at the same time rotating the plate. LEBHOD OE PLAIING drULAJH .ULingio OJ ETTI The method used to plate the stomach content of feti was the same as followed in plating milk. MBIMQD UR BLAEIDG Odlahs Ennu IIPHJTBD GUIhJA 219 3 Using sterile instruments, the spleen and liver of ‘infected guinea pigs were removed and cut in sections which were streaked over the surface of the solidified agar plates. 14. neurons U630 r03 IthBafIhG JULTUJDS fhe cultures were incubated at 37° 3. under the followin; conditions: In closed jars after lo percent of the contained air had been diSplaced by carbon dioxide; in closed jars after 10 percent of the contained air had been displaced by hydrogen; in closed jars after in percent of the contained air had been diSplaced by nitrOgen; in closed jars from which 10 percent of the air had been extracted; in jars which were sealed only, nothing being added or subtracted. The carbon dioxide used was obtained from cylinders of commercial carbon dioxide; the same was true of the nitrogen used. The hydrogen used was prepared in the labora- tory by the action of hydrochloric acid on zinc. 15. in" ‘fig ?'*.\1‘.r; " '1!!- ias-J.)..4..L.u.u..LAJu JILLA (l) Ihe Isolation of best. abortus from fetal Eaterial Jhen Plated on Different Ledia and Placed under Different )onditions of Incubation. Ihe stomach contents of a fetus aborted in the )ollege eXperimental herd was plated on veal agar, beef agar, and gentian violet liver agar. Plates in duplicate of each of these media were placed in closed chambers containing 10 percent carbon dioxide, 10 percent nitrogen, and 10 percent hydrogen; in a close jar from which 10 percent of the contained air had been extracted; and in a sealed jar to which nothing had been added or subtracted. Table I shows .he amount of growth recorded at the end of 60 hours' incubation at 5V° J. Growth had not appeared upon any of the veal plates at the end of this incubation period. Qhe beef agar plates showed growth when incubated in carbon dioxide, hydrogen, or in'a 10 percent vacuum. The growth on gentian violet liver agar was very abundant, as is shown by the table. 16. mswesosmmanon npsoaw yo assess .smeaooon amped: on QpBOMw 03 N + A W — — I. I _+++_++++. ++++_ ++++ _ A {— u 1+++1+++ — — J A 17 .++++_*+++_ ++++_ ++++. _ — Al heme nobflm pmaows swapsom u . u L. u L _ _ _ . . _ I” I _ I H I _ + H + . + H + . I n I . + H + _ \ Edhmfl mmflom _ _ _ _ . _ . . . . . _ “OH MSHR waw Mmmm_ r h h h h n h u h h h h _ _ _ _ _ _ I. I . I . I _ I _ I . I . I _ I . I . I _ I . Edhofl omhom . . . . . _ _ _ . _ _ . WOH deR Hmmd meb . _ . _ ti _ a . . _ t . _ some”, . a2. . Ease, no? am no? as no? not was . egos . capoam¢. deacon. .QOprndosH mo onapfidson pseammmaq amass seesaw was made: psmammwfin so copmdm can: Heaaepwu profi song aspaopw mnm¢a .aer Mo sarong exp macaw (2) The Isolation of fact. abortus from milk Jhen Plated on Different hedia and Placed Different Methods of Incubation. Milk from Animal 80531 was gentian violet liver agar plates, beef plates, plates, and placed under conditions of Part I. plated on a an d 17. Under series of peptic agar incubation as in Hrperi- ment (1). At the end of 60 hours} incubation the average number of colonies of Bact. abortus which had developed on duplicate sets of plates is shown in Table II. Shows the Isolation of Beet. TaBLE II abortus from Lilk When Plated on Different hedia and Placed under Different Kethods of Incubation. r , ' v , . I I 3.3.”. 00531 I 105 002,105 N, 10$‘H2 '10} Vacuum Ioealed Jar L I F I , I r , - . I , ' a. 1. Liver ' I - v 44 6 O 0 v C : Agar , ' ' ' v I , L ' Beef agar and ' ' I ' .. q I ' Horse serum I ' ' O ' O I O : O I I 1 L ' Peptic digest ' ' I 7* ' ' agar : O ' 0' ' O I O : O I I _.f_ L— J ' 3.3.Q. 805B1 I ' I , : I I y ‘L a - - ' ' I r Y 'J. I. .1le81. ' ,. I 5 I 85581. I O r 10 , U ' O I O I I y L I 'Beef agar and ' , ' I I ' ' ' Horse serum : 50 ' 0 ' O I O I O ' I I 1 1 I I_ I . I I f ' '38'01310 Dlgest ' r 2 ' O ' O I O ' O ' I agar I i ' ' I ' R.R.Q.= light Rear Quarter 3.3.Q.= dight front Quarter I},7, = Gentian Violet 18. Solonies had develoaed only in the jars containing carbon dioxide and nitrogen, with the greatest number develon- ing in the aresence carbon dioride. The gentian violet liver agar plates in all cases showed the greatest number of colonies. (5) fhe Isolation of 2333. abortus from hilk when Plated on Different hedia and Ilaced Under Different Kethods of Incubation. Part II. in experiment similar to that under '3), except that different meiia were employed, was conducted in an effort to obtain additional data. Gentian violet liver agar, plaiu liver agar, uterine agar and peptic digest agar were employed. fhe average number of colonies of 3323. abortus which had develoeed on three sets of duplicate plates at t1e end of 60 hours' incubation is shown in Table III. Shows the Isolation of fleet. abortus from Lil: Jhen Plated 0n Different hedia and Placed under gifferent Methods of Incubation. I I r I r r I '3.a.o. 80531 '10; oog'iofi u '10;:Hg'105 Vacuum'fiealed Jar' I I I I I I I I I l I I I I .G.V.Liver . 2&0 . 190 . O . O , c . I Agar I l I I I I 'Liver Agar ' 210 ' 180 I O ' O 1 O ' I I I I I :Uterine Agar L'bntanimta'i 100 Z O ; 0 : O I IPBPtic Digest I 200 I 0 I 0 I o I 0 I I agar I I I L I I I I I I I I I [120310 Q0 80531 I I I I I I I I l I I I I [GO VoLivar I I r I r I I I I ' agar I 500 l 500 I O ! O I O ' :Liver agar : 460 : 580 I O I 0 : O : :Uterine Agar I 530 j 115 1 o I o I o : I' I I I I I I IPeptic Digest I 310 I 155 I o I o I o I l Jigs-3r I I I I I I d.3.Q. = dight Rear Quarter ' 3.3.?. = dight Front Quarter 3.7. = Gentian Violet 20. As in the case of the previo's experiment, colonies of Bact. abortus developed only in the jars con- taining carbon dioxide and nitrogen, and, as before, the greatest numbers develOped on the plates under the influence of carbon dioxide. Hilk from the rig‘t rear quarter gave the highest number of colonies on the gentian violet liver iar agar plates in each , , while milk from the right front quarter gave the highest number of colonies on the plain liver agar. (4) The Isolation of fleet. abortus From Guinea-Pig Organs when Ilated on Different Ledia and Placed Under Different hethods of Incu- bation. Lart I. Spleens from 5 guinea pigs showing characteristic lesions of abortion disease were plated on gentian violet liver agar, beef agar, and peptic digest agar. The plates were incubated under conditions similar to those given under experiment (1). The growth recorded at the end of 60 hours' incubation is shown in table IV. TAB Li) I '1 Shows the Isolation of Bact. abortus from }uinea Pigs When Plated on Different Media and Placed Under 21. ‘ - - ~ ‘r- - --- ‘--‘- Different Methods of Incubation. ' '10; 302' 10,6 N '10» He'lO,$'Vamnm 'Sealed Jar I I I I I I I T T T j r r {spleen G. . 944 L 1 L _: ' I}.V.Liver Agar I ++++ I - I +++ I ++++ I + I I I I I I 'Beef agar and 1 ++ ' _ ' _ ' _ ' ~ ' Horse serum ' ' ' ' _I_ I I I I L 'Peptic digest ' ' ' ' I I I I I L i 'Spleen G.P. 9&5 ' ' ' ' ' _L .L L I L f; :}.L.Liver agar : +++ : - 1 - ' - : +++ I - I :Beeg agar and : t I ' Horse serum ' ‘ ‘ ' ' ‘ I I I I I _ I 'Beotic digest ' ' ' I ever +++ I - I - I ++++ I +++ D I I I I I '0 1 (j. I I I I I 'epleen q.P. 9.6 I i ' ' ' I I I I I I IG.V. Liver agar . - , - , - , - , - 'Beef agar and ' ' ' I I ' Horse serum ' ‘ I ' v ' I v I - 'Peptic digest v - v _ I - I + I ++++ I agar I I I I I :1 10 Po 3.v. -IJ Erowth - Am unt of Growth Guinea pig Gentian Violet. Jorresoonding to Number Recorded. 1 each of the guinea pigs had been inoculated with material from an abortion case in the Jollege Experimental Herd which was caused by the injection of a laboratory strain of £333. abortus. As was to he eXmected, the organism was recovered with greater case than in the case of abortions caused by natu- ral infection. colonies of heat. abortus develooed on plates in all jars except that containing nitrogen. Che average number of colonies develcbing in the jars containing carbon dioxide, in the jar with a‘lO percent vacuum, and in the sealed jar being nearly equal. However, the colonies develoning in the jar containing carbon dioxide were larger in diameter than those in the other jars. The peptic digest agar proved a very good medium for the isolation of hast. abortus it bein? the onl” medium to ._...___.___.I > J show growth from the spleen of guinea pig number 996. In other cases the colonies, although as numerous, were not as large as those develOping on the gentian agar plates. (5) The Isolation of Beet. abortus Erom $uinea—Pig Organs when Plated on Different Ledia And Placed Under Different Methods of Incubation. Iart II. Spleens from 2 guinea pigs showing characteristic l;sions of abortion disease were plated on gontian violet liver agar, plain liver agar, uterine agar, and neptic digest agar and placed under conditions of incubation as given above. Ehe growth recorded at the end of 60 hours' incubation is shown in table V. .I‘ ‘7 1 ' +£341‘J13 I r“ “3“. I ‘ .Lo - p *w‘ _~,V _' -1,‘ I . ,4 showing the IbOlfielOfl or néct. aeortis -rom Juinea Bits when Plated on nifferent Media and i-laced Under uifferent fietbods of Incl’ation. T— I I I fl ‘1— I . .1c-,z; 302,10”; LI .105 HkIlOflS 'facumnIb‘ealed Jar. I I I I ' I I I I I I I I I I 15.318071 J'ol‘o CC? I I I I y , I I I I I I I '}.7.Liver axar I ++ I 1+ I - I _ . _ . l I I I I I I ' liver .gar ' ++ ' ++ ' — ' — ' _ ' I I ' I I I I ' Uterine Age" ' ++ ' ++ ' - ' - ' - ' I I I I l I I :Peptic digest : + I + I _ : _ I _ t 3‘ agar I I I I I ‘g‘ I Spleen }.P. 1007: : I : z I ' .}.'-f..'LiVer agar ' +++ ' ++ ' -- ' - ' - ' I I I I I I I I I I I I I I I liver agar I ++ I + I a I n I — I I I I I I I I I I I I I I I , Uterine Agar . +++ , ++ . - , — , - , L L __L I._ I L I I I I I I I I , Peptic digest , , , , _ , _ , I agar I ++ I + I — I I I - = K0 arowth Amount of Growth JorreSponding to Lumber Recorded ‘- II G.7. - lentian Tiolet 24;. 1"he guinea-pigs in this case had been inoculated with 5 c.c. of milk and the organism was not recovered as easily as in the case of the previous experiment. Golonies of Bact. abortus develoPed in the jars containing carbon dioxide and nitrOgen; the otier jars failed to give growth. The best growth in regard to number and size of colonies apneared on tho gentian violet liver agar plates which were incubated in 10 percent carbon dioxide. (6) The Jultivation of Beet. abortus on Jiff rent hedia Jhen Placed Under Lifferent hethods of Incubation. In order to obtain the comoarative value of the different media and the different methods of incubation on the cultivation of strains of Bact. abortus after isolation three laboratory strains were cultured on liver agar slants, beef agar slants, uterine agar slants and peptic digest agar slants. These were placed under conditions of incuba— tion similar to those of the previous experiments. The growth recorded at the end of 24 hours' incubation is shown in Table VI. shows the Irowth of Laboratory strains of hact. abortus in 34 Hours Jifferont hethods of on Different Ledia Jh _. en Placed Under Incubation. ‘1 - 7 , ' , . o ' ' 10,5 SCI; '10,.2 lI‘ '10,a hII 'lO,a lac uum 'Arabic ' ' Strain No.200 ' ' ' ' ' ' I I I I I I I I o I I I I I I I liver agar , ++++ , ++++ , ++++ , ++++ , ++++ , I —+» —: r» r - I I Beef Aga ' + ' ++ ' + ' + ' +++ ' I I I I I I I 'Utal‘il’la Agar '. 4.... I +4, ' + I ++ I ++ I I I I I I I I 'Peptic digest ' ++ ’ ++ ' ++ ' + ' ++ ' I agar ' ' ' I I I I I I I I I I I I I I (\ I Q ' I Qtrain 1‘0.165v‘ ' ' ' I I II I I I I ' Liver Agar ++++ +++ ' +++ ' +++ ' ++++ I I , I I I I I I Beef agar ’ + v + ' ++ ' ++ ' ++ ' 'Uterine Agar ' ++ ' ++ ' + ' + ' + ' I I I I I I I "I‘Iu- ' I &. ' ' I ' ' ' I”Pti° dlSBSb +++ ++ ++ + ++ ' agar ' I I I I , T I j— —Y—' —I_ T ' Idtrain “0080531 I I I I I I I—_ T r T r W l I Liver Agar I ++++ I ++ I ++ I ++ I ++ I ‘I: :— 7 7 7‘ r 'I“ I Beef Agar I + I + I + I + I + I F F I I I I I IUtcrlne Agar I + I + I + I + , + I I I U . (7 ‘ I IPeptio JIDBbt I ++ I + I + I + I + I I dgal‘ I I I I I I - = No Growth + = Amount of Growth corresponding to humber Used. (‘0 C“) . A study of ta ole TI shows that liver gqar gives the most luxuriant growth in cornerison with the other media; and that incubation in carbon dioxide “iv s the greatest growth in coenarison with the other methods of incubation. It is also interesting to note that the slants incubated aerobically gave better growth than those incubated under all other conditions excepting those incubated in the jar containing carbon dioxide. (7) The Value of Adding Serum to Liver Agar Eor the Isolation of hast. abortus. whe addition of blood serum is a means used by many workers to enric 1) their media, and, "for the cultivation OI Bast. abortus on vea l or beef in: us ion agar, 10 “eicent of serum is generally added. In order to determine the value of the aLdi ition of serum to liver agar the following experiment was conducted. I4 Petri dishe were poured with gentian violet it ver agar’and gentian violet liver a3ar plus verging mnou-ts of fresh natu— rally sterile bovine s rum. nilk from the right rear and right .0. . I - J.‘ - ',,- I I 3‘. I.“ .' J.'I,.., _,. 4. , iront I1a:ters 0L aniI.;al I.o. l was syread on. these plat nee s44. (D I 6‘8 V'I“q . - 1 4—1 4“ 11* . '2 “I 3 1‘ - '\ ~ - 1“, 1 7“ ‘ r - I . wv . leCH the plates Ker: Ii,uoated under lg ;3roe1t carbon diohide at 37° 3. for 60 hours. “t the end of Ilia Weriod the number of colonies of Jawt. abortus develOpin; on each ylate was counted II and recorded; ts‘lg III shows the average count per plate for a series of six incubations. TiBLE VII. Shows the Value of ndding Bovine Serum to Gentisn Violet Liver Agar for the Cultivation of Bect. abortus. T T ' . Kedia .Average count per plate, T V ' 'G.V. liver agar 4-5% Serum ' 5 colonies ' ' £— 3 'G.V. liver agar 4'le3 Serum ' 8 colonies ' ' J ' 'G.V. liver agar 4-20,? Serum ' 15 colonies ' V .4 3 'G.V. liver agar 4-30fi Serum ' Contaminated ' ' a __3 'G.V. liver agar (no Serum) ' 58 colonies ' G. V. = Gentisn violet. 58. on of 50 perzeut serum the plates F). ’. I ' .l“ ‘1 . T‘.’ - H'lbh toe audit 5- ‘ v O‘KH “a“ l‘ \ v , I L - '» --‘ becare contaminated, ,rooahlJ 113 to the fee violet was in too high a dilution to inhibit the great}; of the 1 the increase in the amount (’3' {.4 contaminating organisms; for mi of serum added per plate the dilution of the dye became cor- reapondingly higher. who (ate shoarthat the addition of bovine serum to gentian violet agar does not give an increase in the number of colonies developing from milk; on the other hand,the plain gentian violet liver agar plates give a much higher count. (8) The Lffect of pH Joncentration of Liver Agar on the Growth of fleet. abortus. In an endeavor to learn the effect which the reaction of liver agar has on the growth of hact. abortus a number of flasks of media were prepared having a pH concentration varying from 6.0 to 7.6. rlates were poured from each flask and milk from the right rear quarter of animal 805Bl was plated. In three series of plates the average number of colo- nies of Bact. abortus develOping per plate is shown in table VIII. ZL‘ABLETIII Shows the Effect of pH Joncentration of Liver Agar on the Isolation of East. abortus from Lilk. '14 tti 1.10! I l I 101' 1 'Pi concen re on 'o.0'6.~'6.4'6.6,6.8'7.0,7.~,7.4,7.6 , A... l— I l a] y I F Y j— r ' fi Average number OI . D‘ .5.2.5verev3v5'5'2 ' ’\ colonies ' I ' I I ' ' , , , (D [\ As shown by the table, colonies develOped over the entire range of pH values, the highest counts occurring at 6.6 and 6.8, the difference in count being very small. slants were also prepared from the above flasks and several strains of Beet. abortus were grown under different conditions in an effort to obtain additional data on the effect of the 3H concentration. btrain D-l had been recently isolated from a fetus aborted as a result of natural infection; strain 1665 had been rhi- recently recovered from an abortion case in the colleg \D perimental Herd; strain A3 was taken from the first trans- plant after isolating from milk; strain GP was aken from the first tranSplant after isolating from the Spleen of a guinea pig; and strains LUCAS, 670, and 200 had been grown in the laboratory for some time. These different strains were incubated at 57° C. under the following conditions: aerobically, with tubes sealed with sealing wax, and under 10 percent carbon dioxide. Table IX shows the amount of growth recorded at the end of 24 hours' incubation. O bhows the Jffect of p: ‘ 0n the Jultivation of Laboratory strains. of Bact. sbortus I concentration of liver Agar 30. IPH SOHOBD-l r r T T T I ' trgtion '6.0 6.2 ' 6.4 ' 6.6 ' 6.8 ' 7.0 7.2 ' 7.4 7.6 ' I T I “II I— I I I I \ 1 I I I I I I I taggiéglg-l' - - ' + ' ++ Y ++ I ++ + I + + I 5:; ~ # v‘: 3‘ ‘IL ‘r _f I IStrain D-ll I I I I I I I Sealed I+++ +++ I +++ I +++ I +++ I +++ +++ I ++ ++ I ItGSt tubesI I I I I I I 'Strain 1665 + ++ ' ++ ' ++ ' ++ ' ++ + ' + + ' I Aerobic I I I I I I I I 1" r I I 7 I r I ,StraflllobB, , , , , , , ,Sealed , ++ ++ , ++ , +++, ++ , ++ ++ , ++ ++ , ,test tubesu L L‘ , L L , Istrian I I I I I I I ' LUGAB ' ++ ++ ' ++ ' +++' +++ ' +++ +++ ' +++ ++ ' I Aerobic I I I I I I I I I r T T r T I .Strain 670' + ++ ' +++ ' +++' +++ ' +++ +++ ' ++ ++ ' I Aerobic I + I I I I I I I ‘ 2 I I I I I I I ,btrain 00' + ++ I ++ I ++v ++ I ++ +$ I ++ ++ I , Aerobic , , , , , , I I 0 w I I I I I I I IStran}: An ' + ' ++ ' ++' ++ I _ _ ' - _ ' Aero ic _ _ k L , ‘ ra n P ' 'btj I AL '+++ +++ ' +++ ' +++ ' +++ ' +++ +++ ' ++ ++ ' [10/0 '402 I I I I I I I I r 7— r r r r 1 I ‘ 3' I I I I I I 'lgi Eggx I+++ +++ +++ . +++ . +++ , +++ +++ , +++ +++ l I U -- I 3— w‘; 3 J —% 5 '— I Ib‘train 5}? I I I I I I I I10} 102 I + + I ++ ++++I ++++v ++++I ++++I ++++I +++ I - = No Growth + = Amount of Growth JorrOSponding to number Aeoorded. Growth appeared over the entire range of pH concentration in all cases are pt the recently isolated strains when grown aerobically. However, the best average growth for all cases seems to occur at the pH concentrations of 6.6 to 6.8. (9) Ihe Value of Jlearin; liver Agar with 33“; Albumin. In order to know the value of clearing liver agar with egg albumin two flasks of media were prepared, one of which was cleared with 10 percent powdered egg albumin before filtering,.while the other was not cleared. Plates were poured from each flask and milk from the right rear and right front quarters of animal No. 2 was plated in duplicate. Table X shows the count of Bact. abortus per plate after 60 hours' incubation in an atmOSphere of 10 percent carbon dioxide. dhowing the Value of Jlearing Liver Agar for the Isolation of Bact. abortus from hilk. TT T'— I‘ I I I liver Agar I Liver Agar Jleared with I I Milk 80531 I h0t Jleared I Egg albumin I I' I I T I I d.j. Quarter ' O ' O ' 0 ' C ' 3.3. Quarter ' o ' o v 93 I 170 , Slants were also name from be above flasks and se eied with diffe re1t lao)r atory strains and placed under different conditions of incubation. a rain h- had been recently isolated from a fetus aborted as a result of natural infection; strain 89531 had been re- cently isolated fr m an aborted fetus from the Jollege Exocri- mental .alerd; and strains LUCAS and 2300 had been ‘;r0'x‘~.’n in the laboratory for some time. The slants were incubated aerobically and in an atmosphere of 10 eercent carbon dioxide. Cable X $10113 t1ae amount of growth recorded at the end of 84 hours’ incubation. Shows the Jalue of M10 aring Live er Agar with he: Albumin for the Cultivation of Lal oratory strains of 2 4- . I bacu. abortus. ' ' . 'T . . .. T , , liver agar , leer Atar ‘leared with I T I Not Sleared , Egg Albumin , r . A 47 ' “T , ctruin o-l , t ' - + ++ I aerobic I I , T . F Y T , Strain 80531 , , , + ++ I aerobic I I I I r r .T N 1“." U I btrain LUUAQ I ++ . ++ , . aerobic I I I r ‘ T T r +++ +++ . aerobic I I I r“ . I j ' , Strain D-l , +++ , ++++ ' , I I 10/0 00‘ ' I i ' I Y r r I btrain 80531 t , I I 0,10 U02 ' l ! + 3 Amount of lrowth Sorresnondinq to I I ‘ .umber fiecordel 55. The above tables indicate that liver agar is much improved for the isolation and cultivation of Bact. abortus by clearing with egg albumin. This may be due either to the fact that beneficial products are ad'ed or that injurious substarnes are subtracted from the media by the egg albumin. (10) The Effect of Incubating Aerobically Before slacing in An Atmooal re of 10 Percent Garbon LiOxide on the }r nth of Bacterium abortus. It has been the custom of investigators to incubate, aerobically, for a period of 12.hours,p nla es cultured 1ith hact. abortus material before placing u1der conditions more favorable for the growth of this organism. Sontaminatinr colonies nwli h had develooed at the end of this period were marked, thereby showing clear areas upon which the colonies of Bac t abortus ” uld develop 1hen in- J O eubated again. In an effort to determine the effect of this preliminary incubation period upon the growth of heat. abortus, milk plates were prepared from the right rear and right front quarter of imal ho. 2M1 tee in duplicate from each quarter were placed dm ectly in an atm sphere of 19 percent carbon dioxide and a core reapondin: number of plates from each quarter were incubated aerobically for a period of 36 hours before placing in an atmosphere of 10 percent carbon dioxile . Both sets of plates were incubated in carbon dioxide for a period of 3 days. The number of colonies obtained per plate is shown in table XII. TAB 3”“ XII Shows Lffect of 1olding Plates Aerobically before Jlaci 13 in an atmosphere of lo percent Barbon C.io:-;ide on the Isolation of hact. abortus. I l I I ' ' 3.2%. Quarter ' 1.3. t'ttuarter ' I I I I ' Direct in 002 '~ 73 ' 92 ' 315 ' 264 ' I I I I I I I —I_ r T r I' ' Aerobic 56 hrs. ' O ' 0 ' 92 I leg I I I I I I I '1 Fell winf the same procedure as given above,a second series of plates was made. One set of plates was placed (lirec tl5',7 in an atmosphere of in percent carbon dioxide,and a second set was incubated aerobically for a period of 13 hours before being placed in carbon di ride. The number of colonies deveIOpin3 per plate is shown.in table XIII. TABZE XIII. shows 3f: ct of holdin3 Elat (J 3.. '("3 O (‘4 ((23 [.4 H k: before Placing in an Atmosphere of 10 percent Garbon Dioxide on the Isolation ofl act. abortus. I I I I : I 3. R. Quarter I L. R. Tuarter I 7 T I r g T : Direct in 302 I l : 2 : 97 I 144 : ' f t F . . ' :Aerobic 12 hrs.: 0 : 2 z 3 l 30 , ' I I In order to obtain additional data, eight samples were collect- ed from the right rear quarter of animal NO. 2 and plated and incubated as above. Sable ZII’shohs the results obtained. in. ‘w'w'W v r .inl).u.1 4.x. ’0 Shows Effect of Holding Slates Aerobically before Placing in an AtmOSphere of 10 percent carbon Dioxide on the Isolation of fact. abortus. I T— I— I I I T T T I F‘ I damele Lo. ' 1 ' 3 ' 5 ' d ' 5 ' 6 ' V ' J'zotal' Av.’ I I I I I I I I I I I I I I I I T I r I I I I I direct in JUI ' l ' l ' 18 ' O ' 1V0 ' 130 ' O ' 60' 350 ' 43 ' I I I I I I I I I I I I— I I I —T I’ I I I I I Aerobic 12 hrs.‘ 6 '15 ' O ' G ' C ' ' 2 ' 14' 55 ' 4 ' I I I I I I I I I I I Ehe above results show that,on the average,a 3reater number ‘ of colonies of Sect. abortus dechOp on plates which are immefi- ately placed in an atmOSphere containin; 10 percent carbon dioxide than on thos plates which are first incubated aerOeically and later placed under the influeflce oifcarbon dioxide. (11) The Value of Jentriiuging as an Aid in the Isolation of Bacterium abortus from 111k. In an attempt to de-crmine the value of centrifuging as an .L aid in the isolation of hact. abortus from m'lk, the following experiment was undertahen; One tenth cubic centimeter of milk was plated from each 0 1 sample collectei, after which the same sample a £0 (1') centrifuged for two hours at 2000 revolutions per minute. After centrifuging, plates were made from the cream, middle milk, and sediment from from each sample. She four ‘eries of plates from each sample U were then incubated in the same closed chamber in order to give identical conditions of incubation to each plate. Uhe medium used was gentian violet liver agar; the plates were incubated under the influence of 10 percent carbon dioxide. q f) ‘ fine milk was obtained from three animals, SOlGOtoh becau se of the difference in numbers of Beet. abortus per cubic centimeter in their milk. From 1 to 200 colonies per plate wece isolated from the ri ht rear ouarter of animal NO. 1; from 1 to 400 colonies M1 per plate were isolated from the rizht front ouarter of the same animal; from 1 to 8 colonies per plate were isolated from animal he. 2; while no colonies developed on any of the plates from animal number three. fhe count of Bact. abortus per plate is shown in tables :7, X' , XIII, and XVIII. TgBLE XV. Shows the humber of Colonies of Eact. abortus developing n -5. I _rom Silk from the Rifht Rear Wuerters of Animal 30. 1 before and after Centrifuting. I I g , Before Centrifusing ; After Centrifu in? , I I I I I I I .Whole Hilk,0ream,£iddle Hilk,3edimont , I I I I I I . Animal $1: I 19 I o . o . 4 I I R.R.quar-I \ I I I I I ter I I I I I I " I 23 I O I O I 13 I I " I 113 I 5 I 3 I 3 I I " I 158 I 17 I 25 I 2 I I " I 185 I O I 5 I O I I " I 140 I O I 7 I 13 I I I. I e~ _¢ +1 ' II 3 lo I O I O I O I I " I 25 I 5 I 1 I '0 I I e I I 1‘ =4 ' " I 5 I O I 21 I 4 I I ¢~ e 7! e— we I " I 21 I O I O I O I I ~¢ : :7 1A ~a ' " I 24 I 0 I O I O I ‘3 I f ‘r e w? I " I 8 I O I 0 I 0 I I —: ‘r 4. a 1‘1 I " I 1 I O I 0 I 0 I 3 f I ? 1 “'1 I " I O I 0 I 0 I 0 I —% er e: e e 1 I " I 6 I 0 I O I O I I :— ? ? ? *1 I " I 15 I 0 I O I O I I 7 ? Af I *1 I II I 0 I 0 I O I 1 I I 1 r e 1 fi I II I O I O I 0 I O I I I I I I I 'Totel j_ 749 ' 25 1‘ 58 17—40 r I I I I I I average Ii“ 42 ' 1.4' 5.2 ' 2.2 ‘T I I I I I I —‘—IL 7 I 1’ I "'Y 0 C I I I I k I before 0' l lkISediment $111 To. 1 ortus developin ‘ ab entrifu 1 r1 u 1' r ‘0- Lnimel of Beet. *fter L; a ing. I us- r W IUITI . olonies L. .L F! ‘J TABEE f ForWard Quarter of 87 t fumber o n q Ifihole MilkICreamIHiddle I 'Animcl Fl' J— if I l d after centri T 73 .Ls ‘. Y1 J. ‘ 1' '\ I .- Before Centrifuging Shows the rom't 'n.F.ouar-' ter I I .5.- .1. I 9w II 74.5 '7 26 68 410 W' 7 7.5T I I T128T 77 1,757 1oz T II II verage ' l :1 r'Total T & TLBLE XVII Shows Humber of Colonies of Beet. abortus developing from Hilk from Animal Ho. 2 before and after Centrifuging. I I I I Before Centrifuging. After Centrifuging . T I r I .1 I I IWhole HilkICreamIHiddle HilkISediment I I— I I I I I 'Animel EZI I I I I 'RQRQQULZLI'" ' I I I ' ter ' 2 ' O ' O ' O I I .L I L I I I II I 8 I 0 I O I 0 I I 5* 1 I __l_ __I 'n.F.qusr~' ' I I I ' ter 'W O ' O I O ' O ' ' 5 1 3 I I I II I O I O I O I O I I t 3- J L ' 'L.R.quar-' O I O ' O ' O I I ter I I I I I I L 4 L J I I II I O I O I O I O I I I e—% if 3 I 'L.R.quar~' ' I I I I ter I l I O ' O ' O ' g 4 E L l I I II I 1 I O I 0 I 0 I ' Total ' 12 ' O I O ' O ' l. L L _L I i TLBLE XVIII Shows Iumber of Colonies of Boot.abortus developing from Hilk from Animal No. 5 before and after Centrifufing. 1 hr . ' . Before Centrifuging , After Centrifuging , T I I T 1 . ' I ,Yhole Hilk,Cream,Hiddle Hilk,Sed1ment , I— I W I T I ' Anifiul #5 I I I I I '3.3.querter' O I O I O I O I L I I I I I ' " I 0 ' o I o I o I I I I I I ___I 'R.F.quarter' O I 0 I o I o I I I I I I I I II I 0 1 O 3 O ' O ' I I I I I I 'L.R.quarter' O I O I O ' O I I __ I I I I I I II I O I O I O I O I I I I - I I _I 'L.F.quarter' O I O I O I O I I I I g I I I II I O t O ' O I O ' _L L J L; I I ' Total I O I O I o I o I L L L L J I As is shown in table XV, 18 samples from the right rear quarter of animal No. 1 gave a total of 749 colonies on the plates from mi‘k not centrifuged; 25 colonies on the plates streaked with cream; 58 colonies on the plates from the middle milk; and 40 colonies on the plates streaked with sediment. I Table XVI shows 17 samples from the right front quarter of the same animal with a total of 1,757 colonies on the plates from milk not centrifuged; 128 colonies on the plates streaked. with cream; 68 colonies on the plates from the middle milk; and 78 colonies on the plates streaked with sediment. Table XVII snows 8 samples from animal Ho. 2 with a total of 12 colonies on the plates from milk not centrifuged, while all plates made after centrifuging failed to Show growth. Table XVIII Shows that 8 samples from animal ho. 3 failed to show growth either before or after centrifuging. From the above data it appears that centrifuging milk does not aid in the isolation 0f.§i§£° abortus. The total number of colonies developing on plates from milk not centri- fuged was 2,518, and the total number of colonies developing on the three series of plates madeaifter centrifuging was but 597. From each individual sample the tables Show that in 50 out of 51 cases the number of colonies developing from the mi k before centrifuging was far greater than the total number developing on the three series of plates from the milk after centrifuging. Figure 1. Figure 1 represents a photographed Petri dish showing the growth of Bact. abortus on peptic digest agar when incubated in an atmOSphere of 10 percent carbon dioxide. The colonies were isolated from the spleen of a diseased guinea pig. 42. Figure 2. Figure 2 represents a photographed Petri dish showing the growth of Bact. abortus on gentian violet liver agar, when incubated in an atmOSphere of 10 percent carbon dioxide. The colonies were isolated from milk. 43. 44. Figure 5. Figure 5 represents a photographed Petri dish showing the growth of Bact. abortus on gentian violet liver agar When incubated in an atmOSphere of 10 percent carbon dioxide. The colonies were isolated from milk. 45. Figure 4. Figure 4 represents a photographed Petri dish Showing the growth of Bact. abortus on gentian violet liver agar when incubated in an atmOSphere of 10 percent carbon dioxide. The colonies were isolated from milk. Figure 5. Figure 5 represents a photographed Fetri the growth of Fact. abortus on gentian violet when incubated in an atmosphere of 10 percent The colonies were isolated from the Spleen of guinea pig. 46. dish showing liver agar carbon dioxide. a diseased 47. GIIIZR L”) DI SCUfjéIOlT. Experiments have been outlined dealing with different methods of isolating and cul ivating aact. abortus. The first part of the experimental work gives data in regard to the isolation and cultivation of 8act. abortus from infected Katerial bv means of various media and different methods of incubation. The second part of the experi- mental vork gives data showing, (a) the effect the manner of preparation of liver agar has on the isolation and cultivation of duct. abortus; and (b) the number of colonies of .act. abortus isolated from milk under different methods of plating. The first five tables give a comparative value of several media for the isolation of Bact. abortus as well as a comparative value of various methods of incubation. From a study of these five tables it appears that gentian violet liver agar surpasses other media used for the isolation of Bact. abortus. Fentic digest agar gave .4. Satis iactorv results, but the method of preparation of this medium is more complicated than is that of liver agar. Plates incubated under the influence of 10 percent carbon dioxide g: ve the best growth. The sixth table gives a comparative Value of several media and the comparative value of various methods of incubation in regard to the cultivation of previously isolated strains of tact. abortus. Liver agar gives the 48. best growth in comparison with the other nedia. In the case of the recently isolated strains, the use of carbon dioxide gives the maximum growth; for the older strains satisfactory results were obtained by culturing on liver asar and incubating aerobically. Tables VII to XI inclusive give data in regard to the preparation of liver agar. These results show that the addition of serum to liver agar is of no value in the isolation of Bact. abort s; that the organism develOps over a range of H concentration of 6.0 to 7.6 with the best results obtained at a concentration of 6.6 to 6.8; and that in.the preparation of liver agar it is necessary, to obtain satisfactory results in the isolation and cultivation of Bact. abortus, to clear the media with efg albumin before filtering. These results confirm the work of Kuddleson. (15) Tables III to XIV inclusive give data in r gard to methods used in incubating plates in carbon dioxide. The results show that the sreatest amount of growth is obtained when the cultured plates are placed directly under the influence of carbon dioxide; the older method Was to place the plates under aerobic conditions for a period of 12 hours before placing under a carbon dioxide tension. Tables XV to XVIII inclusive give da'a in regard to the technic of isolating Beet. abortus from milk. FiftT-one samples of milk obtained from four animals 49. were plated in an effort to determine the effect of centrifuging milk as an aid in isolating. The total nudber of colonies develoPing on the plates made before centrifuging being 2,518 While the total num- ber of colonies developing on the three series of flutes mude after centrifuging being but 397. These results indicate that milk should not be centrifuged when one is endeuv ring to isolate duct. ;bortus, but that direct platingq Should be made in order to obtain the most successful results. ;13113Y. Gentiun violet liver ugur proved the best medium .r studied for the isolation of Duct. abortus. Cultures incubated in a closed jur in.which 10 percent of the contained uir had been disnluced by carbon dioxide gave the most luxuriant growth in com- ' 1-"- purison With other methods of incubation. The cultured plutes should be placed igmediutely under the influence For the cultiVution of strains of duct. abortus Which have been in the laboratory fer some time gnd grow aerobicully, slants of liver agar rive the bes growth in Be hou s'incubetion. In the case of recently isoluted struins it was found thut liver deur incubated in u closed jer contuininr 10 percent curbon dioxide ; geve the best growth in 2d hours'incubetion. ?or the most successful erowth of duct. abortusL liver dear should be adjusted to a p? concentration of 6.6 to 6.8; it should be cleared with egg albumin du f‘l ‘ing its preparation; and the serum should not be udded. In the isoletion of duct. ubortrs from mi k by direct pletinr methods, the milk should be eluted directly as obtained aid not entrifueed in an effort to throw the orgunisme out of surrension. AJLLOJLHDGMJLT. The writer wishes to acknowledge his in- debtedness to Mr. 1. Forest Huddleson, mr. Robert L. Tweed, and Dr. Ward Giltner for suggestions and assistance received during this investigation. (1) (7) (8) (9) (10) (ll) (12) 13* F ”3" ‘Tfl‘ts -LA: ....-l.:.1& IJJo U. Fabyen, Hurshal. 1912. A Contribution to the “ thosenesis of B. abortus, Bang. dour. of"ied. Res., Vol. XXVI, p 441. Hocerd.* 1886. Recueil de fled. Vet., 669. Bang, 8.* 1897. .ei‘“~ur f. Shiermed., 241. v * ETC-3183. 117.80. 1905. Centrelbl. f. Buht., l lbt. Orig., XKXIII,190. Uowuh, Jules.* 1908. hnn. de 1' Inst. Festeur, XXII, 541. Hue eel and I3rr. 1910. B. ortus of hung, the 0 use of Contdgious Abortion in Cattle. rJour. of Inf. Dis., T01. VII, p 469. Eolth, Helfdun.* 1911. Berl. tiererztl. fichnschr., XXV, 686. Giltner, Kurd. 1912. Infectious nbortion in Cattle. Proceeding of 1m. Vet. hed. iss'n., p. 345. Smith, Theobald, and Febyun, Inrshell.* 1912. Centrulblstt fu SexteriOIOSie, ibt. 1, Vol. 61, p. 549. Shillie, E. U. 1918. An Improvement in the Iethod of Isolation and Lecovery of decillus of Cattle gbortion Through Guinea pigs wr~ r-vrr Jour. th. Ied., Vol. LnAIII, p. 585. Fubyun, Hershul. 1915. A Eote on the l‘resenee of B. abortus in Cows' 311k. JOUJ‘. ::ed.. 1198., 7'01. :‘CNIII, p. 85. Schroeder, E. C. 1915. in :zperimcnit with Zen and Heated Cows' Xilk and Its Lesson. Proceedings of the th innuul Conference of the imerican led. Iilk O’dl sion, p. 210. (15) (19) 3! U! (N o Evens, Lliee. 1918. Further Studies on Beet. abortus and Related Bacteria. Jour. Infect. Dis., Vol. XXIII, p. 554. uut1~€th E. J. 1920. 1 Few Notes on the Isolation and Cultivation of Beet. abortus with Special Eeferenee to Liver and Spleen Hedie. Tech. tul. Ho. 49, Iich. Lgr. Expt. Station, part II. Huddleson, I. 301 rest. . 920. The IsoL tion of 8ucteritm abortus f'on Lilk. Tech. .nfil. lo. 49, Iflxfli.ifiqr. Irp. Station, part IV. Huntoon, F. I. 1918. POrmone Hedium. Jour. Inf. 31s., Vol. XXIII, p 169. Fitch, C. 3. 1922. The Cultivation of Bucteriitn abortus Bang. Jour. of Inf. 318., vol. III , p. 235. Hagen, J. n. 1922. The Yulue of heat -Iilled Cultures for the Prevention of the decillus abortus Inoculation Disease of Guinea fiigs. r",r‘ Jour. of :31X0.Ked., Vol. IIIVI, p. 711. Dubovsky, Bertha and ”e7er, K. F. 1922. In prerimentel Study of the Kethods Available for the Enrichment, Demonstra- tion and Isolation of 8. botulinus. Jour. Inf. Dis., v01. III , p. 199. n sfwarified. . .é. .__.. Flu-rt... . sir: u! 17.2.1.5. .241 well" 'fiilflllflflllllml IW 3 1 2 9 3 0 3 VER 08 4 SI TYI | NW 982 9 ARIES "M