STUDIES Off THE BACTERIOSTATIC AKD BACTERICIDAL ACTION OF STREPTOMYCIN AND SULFADIAZINE Off SALMONELLA PULLOHUM Kuan-how CJaaag A THESIS Submitted to the School of Graduate Studies of Michigan State College of Agriculture and. Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Bacteriology 1949 ProQuest Number: 10008278 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest ProQuest 10008278 Published by ProQuest LLC (2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 TABLE OF CONTENTS Page Material* and methods Results and discussion -— I. Streptomycin — — — --------— --- -— — — — -------------- 11 — is ----------------------- — -— — 16 1. The antibacterial activity of streptoagrcin against Salmonella pullorum and the influence of serum — .— — -— 16 2. The effect of age of culture and amount of inoculum on the antibacterial activity of streptomycin-------— ------------ 17 5. The effect of pH on the antibacterial activity of streptomycin —— — — — — — — — — — — — — is 4» The effect of some sugars on the antibacterial activity of streptomycin — — — — — — — — — — — 19 5* The effect of some reducing agents on the antibacterial activity of streptomycin — -— — — -20 6. The effect of some dyes on the antibacterial activity of streptomycin---------- — — — — — — — — gfe 7. The combined action of streptomycin and sodium sulfadiazine — --------------,g>s 8. The duration of antibacterial activity of streptomycin in the body of infected birds — — — — — 25 9. The therapeutic value of streptomycin against pullorum disease — — — — — — — — — — — 24 10. The effect of streptomycin on the course of the agglutination titer of infected turkey serum -— — 25 XI♦ Sodium sulfadiasine-------- -------- — — — 1. 2* S. 4* 5. 6* 7. — — — — --gy The antibacterial activity of sodium sulfadiasine against iS. pullorum and the influence of normal serum — -27 The effect of amount of normal chicken serum on the antibacterial action of sulfadiasine — — — — — 28 The influence of heating on the bacteriostatic activity of normal chicken serum and on its enhancing effect on sulfadiasine------- ---------- — — 28 The influence of storage on the bacteriostatic activity of normal chicken serum and on its enhancing effect on sulfadiasine-------------- — -- — ->-29 The combined action of infected ehieken serum and different proportion of sulfadiasine and normal -------------------------------- — -— 30 chicken serum The effect of the amount of serum of infected birds on the combined action of sulfadiasine -— 32 and normal chicken serum ---------------------- — — Sodium sulfadiasine level in chicken serum — — — — — — 53 TABLE OF CONTENTS (continued) 8* 9. Page The antibacterial activity of sulfadiasine in serum of normal chickens----------------- — --------- — 33 The combined action of sulfadiasine and normal chicken serum in the body of infected birds — — — — — 34 Summary Acknowledgements — — -------- — -- --------- --- — — — — — — — ---------- — — -— — 36 --.— 59 Bibliography ---------------------------------------------------- 40 Tables and figures HKflBW OF LITERATURE I. STREPTOMYCIN Streptomycin was first isolated by faksman and M s coworkers (1) from Strsptonyeea grlseus in 1944. Recently, Johnstone and Vaksman (2) reported that a new streptomycin-like antibiotic tentatively designated as streptomycin II, has been obtained from a new species Of Strcptomyces, Streptomyces bikiniensis, from Bikini soil • Since the discovery of this antibiotic many reports on in vitro and in vivo tests of streptomycin against various organisms have been published. Robinson, Graessle and Smith (5) found that streptomycin was active in vitro against a variety of gram-negative and gram-posi­ tive bacteria. The former included Eberthe 11a, Salmonella, Escherich­ ia, Shigella, Klebsiella, Irucella, Fasteurella, and Proteus; the gram-positive organisms were strains of Streptococcus hemolyticus, Staphylococcus aureus and Mplococcus pneumoniae. Boggs, Bronstein, Hirshfeld and Pilling (4) stated that the majority of the strains of EschericMa coli, Proteus vulgaris, Aerobacter aerogenes, staphylo­ cocci and streptococci were susceptible in the vitro to the blood concentration of streptomycin that oan be maintained in the average patient over a four-hour period when given in 800,000 unit doses. The blood concentration was 5 to 8 units per ml of blood. Keefer et al. (8) reported the results of treatment with streptomycin in one thousand cases. Streptomycin was most effective in the treatment of tularemiq, Hemophilus influences infections, urinary tract infec­ tions due to gram-negative bacilli, bacteremia and meningitis due to gram-negative bacilli* The results in typhoid fever, brucellosis and Salmonella Infections have been disappointing and inconclusive. Hlnshaw and Feldman (6) reported that experimental tuberculosis of guinea pigs may be arrested by prolonged treatment with streptomycin. This antibiotic did not exert a rapidly curative effect on clinical tuberculosis, although it did appear to modify the course of the disease in a favorable manner and exert a suppressive effect on previously progressive tuberculosis. Some experimental results on the antibacterial activity of streptomycin on different species of the Salmonella group have been reported. Vest, Doll and Edwards (7) studied the in vitro inhibition by streptomycin of 412 Salmonella culture, including 154 distinct serologieal types. Of the commonly occurring types, Salmonella paratyphi A and Salmonella typhi were the most sensitive, while Salmonella pullorum and Salmonella enteritidis were somewhat less susceptible. The remainder were store resistant. Sellgmann and Was- sermamn (8) found that 266 Salmonella strains representing 60 different types were sensitive to streptomycin in a concentration of 4 to 8 units. Oral or oral and subcutaneous administration of streptomycin resulted in the suppression of the normal fecal flora and the pathogens in ex­ perimental mice. After termination of the treatment the fecal flora and salmonellae reappeared. Foley and Snhenstein (9) reported that 57 strains of Salmonella covering 15 species, isolated from patients and carriers, were inhibited by streptomycin in concentrations rang­ ing from 0.004 to 0 . 0 6 4 per ml of medium. Slanets (10) found that streptomycin was effective in controlling S. enteritidis in mice as judged by fecal culture and by absence of contamination of vaccine for which the brains of the mice were used. Santivanes (11) reported that in the in vitro teste streptomycin had a definite bacteriostatic effect against Brucella abortus and Pasteurella avlcida* Its activity against S. pullorum. Salmonella typhimnrium and Salmonella hredeney was leas pronounced* Coles (12) found that Salmonella was comparative­ ly more resistant to the action of streptomycin than other susceptible organisms. Benson (13) reported that streptomycin reduced mortality from pullorum disease in baby chicks* The mode of action of streptomycin is not fully understood. The possibility that streptomycin might affect the oxidation-reduetion system in the bacterial cell has received considerable attention. Bondi, Diets and Spaulding (14) found that the antibacterial activity of streptomycin was reduced in the presence of various reducing agents such as cysteine, sodium thlglyeollate, stannous chloride, sodium bi­ sulfite, sodium hydrosulfite, sodium formate and sodium thiosulfate. They believed that the antibacterial action of streptomycin may be due to its ability to block some oxidative enzyme system. Denkelwater, Cook and Tishler (15) reported that streptomycin was inactivated by cysteine. The cysteine inactivation can be reversed by iodine. Geiger, Green and Waksman (16) found that the reducing agents such as cysteine and cevitamic acid diminished the effectiveness of strep­ tomycin. They proposed that the agents may block an active group in the molecule of streptomycin* Donovlckamd Bake (17) believed that the interfering action of sodium thioglycollate may be due to its role in reducing the oxidation—reduction potential of the medium. Fitzgerald and Beraheim (18) found that the oxidation of bensoic acid by nonpathogenic mycobacteria was inhibited by very small amounts of streptomycin. Since there was a parallelism between the ability of the drug to inhibit this oxidation and the growth of the organism, this inhibition may be one of the important mechanisms in the bacteri­ ostatic action of streptomycin. Recently they (19) stated that the inhibition of bensoic acid oxidation by streptomycin may not be due to the inhibition of the bensoic acid oxidase but to the inhibition of the formation of the ensyme. On the other hand, Tan Dolah and Christenson (20) studied the effeet of various oxidising and reducing agents on antibiotic activity of streptomycin and concluded that inactivation of streptomycin may not logically be ascribed to an oxidising nor reducing mechanism* In the study of the mode of action of streptomycin, Rhymer and Wallace (21) found that brain tissue possessed great ability to inhibit the action of streptomycin. Certain peptones, especially phytone from plant proteins also had this ability. Hobby and Lenert (22) reported that the presence of 1 to 5% or more horse or human serum enhanced the resistance of Str. hemolyticus, Streptococcus viridans, £♦ pneumoniae and certain strains of Straph. aureus. The same concentration of serum, however, had no effeet on the sensibility of Esch. coli, Eberthella typhosa, Klebsiella pneumoniae or A* aerogenes to streptomycin* Higher concentration of serum amy at times enhance the action of streptomycin on certain of these organisms. The changes in morphology of organisms due to streptomycin might be associated with the changes in their physiological function. Smith and Waksman (23) reported that the principal effects of streptomycin on the morphology of tubercle bacilli were loss of acid-fastness, in­ crease in granulation, and, in highly bacteriostatic concentrations, shortening of the rods. -4- The antibacterial activity of streptomycin against a given organism varies with different factors. action of streptomycin. Some of them might relate to the mode of The sensitivity of an organism to streptomycin was influenced by the age of the culture, else of inoculum, composition of medium, length of incubation period, different strains and even different cells, and other factors (28, 24, 28, and 22}. The effeet of pH value on the activity of streptomycin has been studied by many workers. Waksman, Bugle and Schats (26), Wo 1insky and Steenken (27), Berkman, Henry and Housewright (28), Abraham and Duthle (29) and Loo, et al, (33) found that the activity of streptomycin diminn ished with decreasing pH. Glucose has been found to reduce the activity of streptomycin (26). The interfering action may be due to the reducing property of glucose or to the production of acid from glucose fermented by the test organism. Geiger, Green and Wakaman (16) reported that the influence of various sugars on the potency of streptomycin depended on whether acid was pro­ duced by the particular organism tested in the sugar used. The possible mechanism of the reducing agents upon the activity of streptomycin has been discussed above. Sykes and Lumb (30), however, reported that using Each, coll as test organism, bile-salt-lactose agar as medium, the addi­ tion of glucose to the medium did not result in any apparent change in potency of streptomycin. When, however, nutrient agar was substituted for bile-salt agar a decline in potency was observed. If glucose was added to the solution of streptomycin instead of to the culture medium, the activity of streptomycin was reduced when assayed against Bacillus subtilis. In the tests with Esoh. coll, no reduction was observed either in nutrient agar or in bile-salt-lactose agar. They concluded that the effeet of glucose on the potency of streptomycin depended on the organism and the culture medium used. The experiments on the effect of salts upon the antibacterial activity of streptomycin produced conflicting results. Berkman, Henry and Housewright {28} reported that sodium chloride, potassium chloride, sodium sulfate, sodium tartrate, Soerensen*s buffer and ammonium acetate markedly decreased the activity of streptomycin against certain Btrains of Staph, areus, P. vulgaris, Shigella dysenteriae, E. typhosa, Bacil­ lus coreus, Bacillus anthracls and 13. subtills. The degree of antag­ onism was directly proportional to the concentration of the salts. Klein and Kimmelman (31) found that O.S% sodium chloride diminished the activity of streptomycin. Green (32) reported that salts of various acids protected gsch. coli against the antibiotic action of streptomycin. On the other hand, Loo et al. (33) found that the addition of phosphate to the streptomycin solution caused a marked increase in the size of the sons of inhibition. An enhancing effect was also shown by sodium chloride, acetate, bicarbonate and sulfate, potassium ehlorlde and bi­ carbonate, and lithium chloride and sulfate. Recently, Quan (34) re­ ported that the enhancing action of salt depended on the salt concen­ tration in the agar medium as sell as on that in the solution of the antibiotic. When the salt was added to the antibiotic solution the activity of the latter was increased. An increased salt concentra­ tion in the agar medium decreased the sons of inhibition produced by streptomycin and lessened the enhancing action of salts in the anti­ biotic solution. Domovick, Bayun, Canales and Pansy (35) studied the differential effeets of various electrolytes on the activity of streptomycin. Of the cations studied, sodium, lithium and potassium ions had little effeet on the ability of streptomycin to inhibit bacterial growth. Hagnssium and calcium caused the greatest interference with strepto­ mycin activity, imong the anions studied, acetate and pyruvate caused little if any interference, whereas, in increasing order, the following interferred with streptomycin activitys nitrate, chloride, lactate, phosphate, tartrate, citrate and sulfate. Treffers (35) reported that iodoaeetic acid, sodium floride, sod­ ium aside, merthiolate, cetyl pyridinium bromide, crystal violet and mapharsin increaed the inhibiting action of streptomycin on Salmonella dysenteries, Salmonella ambigua, Bach, col1, Staph, aureus and B . cor­ pus. Be believed that these chemicals acted as ensyme inhibitors. The experimental results on the effect of body fluids on the ac­ tivity of streptomycin were not conclusive. She difference in compo­ sition of different body fluids, or even of the same fluid from differ­ ent origins, probably is responsible for the discrepancy. Wolinaky and Steenken (2?) reported that streptomycin was not destroyed, nor were its bacteriostatic and bactericidal powers appreciably influenced by serous body fluid, pus, or normal tissue juices. Schoenbach and Chand­ ler (3?) found that the activity of streptomycin on a susceptible strain of Staph, aureus was not changed in the presence of serum or whole blood. &tm addition of fresh whole blood with or without immune serum did not augument streptomycin activity. On the other hand, the fact that the presence of serum did influence the sensitivity of certain organisms has been mentioned above (22). The reports on the combined action of two or more antibiotic f* and an antibiotic and a sulfa drug are conflicting* This is pro­ bably due to the fact that the numerous factors which influence the activity of individual drugs become much more complicated when sev­ eral drugs are combined* Klein and Kimmelman (38) reported that the combined action of sulfadiasine and streptomycin, or penicillin and streptomycin was greater than that of either drug alone* Klein and Balter (39) found that the in vitro combination of sulfathiasole, sulfadiasine, or sulfapyraxine and penicillin resulted in an increase in the penicillin titer only if both agents were in inhibitory con­ centrations* Mayaon and McMahon (40) reported that streptomycin, sulfadiasine and sulfapyrasine had definite value in the treatment of experiment animals infected with plague* The use of a combination of streptomycin and sulfadiasine did not seem to add to the effective­ ness of either* There are many methods of assay for streptomycin* Stebbins and Bobinson (41) developed a cylinder plate method using Staph, aureus as the test organism. The diameters of the sones of inhibi­ tion on the agar plate, produced by the antibacterial action of the sample assayed and standard streptomycin solution, were measured* Loo et al. (33) introduced a paper-disc plate method* was used as the test organism* B. subtilis Waksman and Beilly (42) used an agar-streak method for quick testing of antibiotic substances against a large number of organisms* Brice, Kielsen and Welch (43) developed a serial dilution method for determining the presence of streptomycin in body fluids, using Bacillus ciroulans as the test organism. Don- ovick, Hamre, Kavanagh and Bake (44) used a broth dilution method for assaying streptomycin* K. pneumoniae was chosen as the test organism* — 6— The degree of growth as affected hy the antibacterial activity of strep­ tomycin was measured by visible turbidity* Heilman (45) produced a specific method for the determination of streptomycin content in body fluids* The smallest concentration of streptomycin that completely prevented hemolysis caused by the presence of Bacillus megatherium was chosen as the end point* A colorimetric method for the deter­ mination of streptomycin was developed by Boxer, Jelinek and Leghorn (46)* Horne and Pollard (47) developed a qualitative colorimetric method for the identification of streptomycin* II. SODIUM SULFADIAZIHB In studying the therapeutic value of sulfonamides in pullorum disease of poultry, Severens, Boberts and Card (48} found that out of seven sulfonamides, sulfadiasine and sulfamerasine (in feed) were the most effective, judged by both mortality and rate of gain of the sur*viving chicks, ihere existed a close agreement between the amount of free sulfonamide in the blood and the effectiveness of the drug. That the use of sulfamerasine in drinking water or in mash is effective in reducing the mortality due to pullorum disease in chicks or poults has been reported by many workers (49, 50, 51, 52 and 55). Bottorff and Kiser (54) found that sulfamethazine, sulfadiasine or sulfamerasine caused a reduction in mortality among chicks. Seeently, Pomeroy, Ben** stermacher and Boepke (55) reported that sulfadiasine, sulfamerasine, sulfapyrazine, sulfaquinoxaline and sulfamethazine were effective in reducing the mortality from pullorum disease in chicks. Sulfadiasine sulfapyrasine, sulfaguanidine and sulfamerasine had little or no value in poults. Buddleson (56) found that the presence of sulfadiasine and fresh, normal rabbit serum in a culture medium or in the blood of an infected animal brought about a bactericidal action against Brucella. Sulfa­ diasine and normal or immune serum, used simultaneously, could termi­ nate experimentally produced brucellosis in guinea pigs. He believed that the bactericidal activity was due to a complex formed by sulfa­ diasine and the antibody-complement system. A method for quantitative determination of sulfanilamide has been developed by Bratton and Marshall (57). MATERIALS AND METHODS Streptomycin* solution Streptomycin (calcium chloride complex) 1.5 gm, which is equiv­ alent to 1*0 gm of pure streptomycin base, was diluted with 19 ml of sterile saline* One ml of this solution contains 50 mg of strepto­ mycin hase* Sodium sulfadiasine** solution One per cent solution of sodium sulfadiasine was made as stock solution and sterilised by passing through a D^ Hermann filter pad*** in a Seits filter. The solution must he made freshly on the day when the experiment is performed, since a precipitate will he formed on standing overnight. The desired dilution was then made from the stock solution* Bacterial suspension The strain of S. pullorum, used in this experiment, was isolated from a naturally infected chicken. Twentyfour-hour cultures on tryp- tose agar (Difco) slants were washed off with a diluting fluid con* si sting of 0.05$ tryptose (Difco) and 0.5$ sodium chloride* Its tur­ bidity was then adjusted to Ho* 2 Mac Parland nephelometer wtth the same fluid. One ml of this suspension contained approximately 35x107 bacterial cells. The desired dilution was then made and 0*1 ml of the final suspension used as inoculum unless otherwise mentioned. * Streptomycin used in this experiment was suppled theough the courtesy of Merck & 0©., Inc., Hahway, H. J. **Sodium sulfadiazine (Lederle Laboratories, Inc., Hew York) was kindly supplied through the courtesy of Dr. I. P. Huddleson. ***Hormann & Gk>., Brooklyn 2, H. Y. -11- Serum Blood of chickens and rabbits was collected aseptically by cardiac puncture, Turkey blood was obtained from the wing rein* The eolloeted blood waa allowed to elot at room temperature* The serum was then separated by centrifugation. Determination of bacteriostatic and bactericidal action in vitro The activity of streptomycin or sodium sulfadiasine against S. pullorum. was assayed as follows: Two-fold serial dilutions of the drug assayed were made in 5 ml of tryptose broth* A cer­ tain amount of normal serum was added to each broth tube when its enhancing action on the drug was studied* One-tenth ml of a suitable dilution of bacterial suspension was finally added. All tubes were incubated at S?°C and the degree of growth was recorded at intervals by the signs isms}* + and Ag (agglutination of the organ­ The highest dilution of streptomycin in which the bacteria failed to grow for at least 96 hours of incubation was defined as the bactericidal titer; while that in which the inhibition of growth lasted for less than 96 hours was designated as the bacterio­ static titer for the particular period of time during which the results were observed* The reason for designating a 96-hour incubation period as the dividing line between bactericidal and bacteriostatic activity was that the organisms which failed to grow at the end of 4 days rarely showed growth when the period of incubation was extended beyond that length of time* Sulfadiasine usually inhibited the growth of the organism for a short period of time* In order to study the enhancing action of certain substances on streptomycin and sulfadiazine, two sets of serial dilutions of the drug assayed were made in tryptose "broth. To one set, a cer­ tain amount of the enhancing substance which was previously proved to be unable to inhibit the growth of the organism, was added; the other set, to which this substance was not added, was used as control. The bacterial suspension was then added and the mixture placed in the incubator. Because the antibacterial titer of the drug against £♦ pull- orum changed at times, in spite of efforts to keep every condition con­ stant, it was necessary to include a control in each experiment. The same technic was employed in studying the effeet of certain substances which were antagonistic to the drug. For studying the effect of serum of infected birds on the action of streptomycin and sulfadiasine against S. pullorum, two-fold serial dilutions of such sera were made. To these dilutions, a certain amount of streptomycin or sulfadiasine, with or without normal serum, and bac­ terial suspension were added, and the mixture was then incubated at 37°©. The in vivo teats A. Streptomycin assay. Chickens and turkeys were injected intravenously with 0.5 to 1.0 ml, according to their body weight, of a suspension of a 24-hour culture of jS. pullorum (turbidity No. 2, UacFarland nephelometer). After one week, when the agglu­ tination titer was at the highest level, the birds were injected intramuscularly with streptoaycin daily (SO to 45 mg per kg of body weight) for two weeks. Blood samples were taken 1, 2, 3 and 6 hours after injection for determining the change in antibacterial activity "-IS- of streptomycin In the serum. The birds were then killed for isolation of the organism from various organs* B. Sulfadiasine assay* Chickens sere infected as indicated before. After one seek the birds sere fed with sodium sulfadiazine at the rate of 0*4 gm per kg of body weight daily for 3 days. chicken serum sas then injected intravenously. Fifteen ml of normal Blood samples were taken while the feeding of sulfadiasine was continuing. The bacteriostatic motion of the serum from these birds against S. pullorum was observed. determination of sodium sulfadiazine in serum A certain amount of serum, depending upon the concentration of sulfadiasine present, was diluted with 20 ml of 15$ trichloroacetic acid and water up to 100 ml. The mixture was then filtered. To 10 ml of the filtrate and to 10 ml of a blank (20 ml of 15$ trichloroacetic acid plus 80 ml of water), 1ml of 0.1$ sodium nitrite was added. Af­ ter standing for 3 minutes, 1 ml of 0.5$ ammonium sulfamate was added. Then after 3 more minutes, 1ml of 0.1$ N- (1-naphthyl )-ethylenediamine dihydro choir ide was added. The readings were taken in a lumetron. The concentration of sodium sulfadiazine in serum was determined by comparing the reading of an unknown sample with that of a known con­ centration of sulfadiazine recorded in a standard curve previously established. Isolation and identification of £. pullorum The treated and control birds were killed, and inoculations were made on SS agar {Difco}, Bismuth sulfite agar (Difco) and in tetrathionate broth (Difco) from various organs. The broth was Incubated at 57®C for about 20 hours; then smears were made on SS agar and bis­ muth sulfite agar. Colonies which resembled those of £. pullorum -14- were transplanted to lactose motility medium*. If there was any growth with no motility and no fermentation of lactose, transfers were made to single sugar troths (glucose, lactose, sucrose, maltose and mannite) and gramstaln made for the identification of S. pullorum. The course of the agglutination titer of the serum during infection and treatment The agglutination titer of serum taken from turkeys, infected intra­ venously with S. pullorum, was determined hy the test tube method during the treatment with streptomycin. The hirds were also tested before the treatment was begun and after the treatment was discontinued. ♦Lactose motility medium: Water Motility test medium (Difco) Beef extract (Difco) K2HP04 Andrade*s indicator Lactose 15- 100.0 % 1,8 0.2 $ 0.05 1.0 1.0 % i % i RESULTS AND DISCUSSION I. STREPTOMYCIN The antibacterial activity of streptomycin against S. pullorum and the influence of serum a In in vitro tests, the bacteriostatic and bactericidal action of streptomycin against an organism can be performed by various methods (39, 41, 42, 43, 44, 45, 46 and 47). The broth dilution method was used in this experiment due to its simplicity and conven­ ience. Two-fold serial dilutions of streptomycin were made in 5 ml of tryptose broth. One-tenth ml of bacterial suspension (350 cells per ml) was added. Table 1 shows that the bactericidal concentration of streptomycin was 0.157 mg’* in 5 ml of medium, or 0.031 mg per ml of medium for S. pullorum. This figure was somewhat hitter than that obtained by West, Doll and Edwards (7). They found that the amount of streptomycin, completely inhibitive for most cultures of S. pullo­ rum, was 4 to 8 units per ml of medium by the agar-streak method. Ob­ viously the difference in strains, culture media, method of assay and other experimental conditions could account for the discrepancy. More­ over these workers have also reported that the point of complete inhi­ bition varied from test to test with a given culture. The presence of normal chicken serum did not affect the titer, al­ though a little better bacteriostatic activity was observed (table 1-B) during the first 4 days of incubation. by other workers. Similar results were obtained Sehoenbach and Chandler (37) reported that the pres­ ence of serum or whole blood did not influence the activity of strepto•One mg of streptomycin = 1,000 units mycin. Wo H u s k y and &teenken (27) also found that the antibacterial action of streptomycin was not affected by serous body fluid, pus or normal tissue Juices. On the other hand, Bhymer and Wallace (21) re­ ported that different peptones shoved different ability to affect the activity of streptomycin. Bobby and Lenert (22) found that the pres­ ence of serum enhanced the resistance of certain organisms but not that of others. Obviously, the discrepancy is due to the different composi­ tion and characteristics of various proteins. Besides, the different amounts of various crystalloids present in various body fluids make the conditions more complicated. In order to study the effect of serum from artificially infected chickens on the antibacterial activity of streptomycin, serial dilutions of serum from the infected birds vere made. ®he bactericidal amount of streptomycin (0.157 mg in 5 ml of medium) and bacterial suspension vere added. Wo growth was observed at the end of 7 days of incubation as shown in table 2. This indicates that the presence of serum of infected birds did not interfere vith the antibacterial activity of streptomycin. The effect of age of culture and amount of inoculum on the antibacterial activity of streptomycin against S . pullorum Various amounts, 0.1, 0.5, and 1.0 ml, of culture of S. pullorum at different ages, 24, 48, 72, 96 and 120 hours vere inoculated into tryptose broth containing serial dilutions of streptomycin. $he re­ sults recorded in table S show that the older the culture and the smaller the inoculum, the higher was the bactericidal titer of streptomycin' {higher dilution). Apparently the older cells vere less active in their metabolism, and thus they were less resistant to the drug than the younger ones. The reason why larger amount of streptomycin was required to kill -17- a large number of organisms was probably that certain cells in the larger inoculum were more resistant to the antibiotic* The effect of amount of inculum on the activity of streptomycin was especially prominent in quantities between 0.1 ml and 0.5 ml of older culture. However, as regards 24-hour-old cultures, the sice of inoculum made no difference in the bactericidal titer of streptomycin. A likely ex­ planation might be that the vigorous younger cells were more resistant so that the wall or none law" can be applied within a certain range of amount of inoculum. The bactericidal titers of streptomycin for cul­ tures of various ages and amounts are shown in table 3-F. The effect of pH o n the antibacterial activity of streptomycin Seven lots of tryptose broth with different pH values, pH 4, 5, 6, 6.9, 8, 9 and 10, were prepared by adding sodium hydroxide or hydrocholoric acid to the medium, the original pH of which was 6.9. The measurement of the pH value was made with Beckman pH meter. Serial dilutions of streptomycin were made and bacterial suspension was added. The data in table 4 and accompanying figure show that at pH 4 and 10, the organisms did not grow, due to the unsuitable acidity, as demon­ strated in the control tube. ly destroyed at pH 5. mycin at that pH. The activity of streptomycin was complete­ The organism grew in all dilutions of strepto­ At pH 6, streptomycin maintained its bacteriostatic action in the lowest dilution (1:16,000) for 96 hours only. Strepto­ mycin showed its highest bactericidal activity at pH 6,9 and 8. bactericidal titer at these pH values was 1:64,000. tericidal titer was reduced to 1:92,000. The At pH 9, the bac­ Generally, on the acid side, the lower the pH value of the medium, the lower was the bactericidal titer (lower dilution) of the antibiotic. Similar rtpults have been reported by many workers (26, 27, 28, 29 and 55). The most likely explanation is that the basic property of streptomycin, which probably is essential to its activity, is neutralized by acid. Abraham and Duthie (29) assumed that the ionised forms of basic streptomycin com­ pete with hydrogen ions for position on the cell surface. the effect of some sugars on the antibacterial activity of streptomycin Glucose has been found by many workers (16, 17 and SO) to have the ability to diminish the activity of streptomycin, this nay be due to the reducing property of glucose, or to the production of acid from glucose fermented by organisms, or both, or some other reasons. Geiger Green and Vaksman (16) reported that dextrose, levulose and sucrose which yield acid during incubation diminished the activity of strepto­ mycin; while non-acid-producers, mannose, galactose, and lactose had little effect on the activity of streptomycin against B. subtilis. However, Sykes and Lumb (SO) believed that the effect of glucose on the antibacterial action of streptomycin varied with the kinds of or­ ganism tested and the culture medium used. In this experiment an at­ tempt has been made to find out whether there was any relationship between acid produced from the fermentation of sugars and the anti­ bacterial activity of streptomycin. Sour different sugars, glucose, mannite, lactose and sucrose, were incorporated in the medium in a concentration of one per cent. She first two sugars were fomented by S. pullorum; the last two were not. suspension were added as before. tion was 6.9. Streptomycin and bacterial She pH of all media before incuba­ She results recorded in table 6 and accompanying fig. show that glucose reduced the bactericidal titer of streptomycin from 1:64,000 to 1:32,000. She final pH of glucose broth after 7 days of incubation was 4.7. She decrease in titer might be related to the acid formed by the fermentation of glucose which lowered the pH value of the medium* Hfowever, the nature of the sugar also played a part in the al­ teration of streptomycin activity* As shown in table 4, the antibacter­ ial activity of streptomycin was completely destroyed at pH 5* In this experiment, streptomycin still retained its bactericidal titer of 1:32,000 even though the pH of the medium was as low as 4.7* Shis in­ dicates that some metabolic products, other than acid, have something to do with the activity of streptomycin, since glucose itself was inactive, as far as antibacterial activity was concerned, as shown in the control tube (table 5-B). On the other hand, mannite increased the bacterici­ dal titer of the antibiotic from (:64,000 to 1:256,000, although the final pH of the medium was reduced to 5*6* Shis farther proves that the nature of individual sugars or their by-products plays some part in changing the antibacterial activity of streptomycin* creased the titer somewhat. sucrose medium. Lactose in­ Little change in titer was observed in She changes in pH of the sugar media after inoubation with S, pullorum are shown in table 5-F. She effect of some reducing agents on the antibacterial activity of streptomycin It is well known that reducing agents, organic and inorganic, diminish the effectiveness of streptomycin. Two reducing agents, ascorbic acid and sodium thiosulfate, were used in this experiment. Table 6 and accompanying fig. show that the bactericidal titer of streptomycin was greatly reduced by ascorbic acid. The more of the vitamin added to the medium, the lower was the bactericidal titer of streptomycin. The addition of the vitamin in a concentration of 0.5 mg per tube (5 ml of medium) or O.I mg per ml of medium reduced the titer from 1:128,000 to 1:52,000 (tables 6-A and 6-C). One mg of ascorbic acid per ml of medium completely destroyed the bactericidal activity of streptomycin (table 6-B). On the other hand, the addi­ tion of sodium thlosulfate in a concentration of 4 ms per ml of medium had no effect on the bactericidal titer (table 6-E). In lover concen­ tration^ ag per ml of medium, however, the titer was somewhat Increased (table 6-F). There was much evidence indicating that the antibacterial action of streptomycin might relate to the oxidation-reduction system of the bac­ terial cell. Bondi, Diets and Spaulding (14) found that the antibac­ terial activity of streptomycin was diminished by anaerobic Incubation, and by the presence of reducing agents, and that streptomycin showed its greatest activity against bacteria which grew better aerobically than anaerobically. They concluded that the antibacterial action of strep­ tomycin may be due to its ability to block: some ensyms system, oxida­ tive in nature, which is essential only to the growth of susceptible aerobic bacteria. Donovick and Sake (17) proposed that the interfer- ing action of sodium thioglyoollate may be due to its role in reducing the cxidation-reduction potential of the medium. The fact that the inactivation of streptomycin by cysteine can be reversed by iodine (15) further indicates the relation between the activity of streptomycin and the oxidation-reduction reaction. On the other hand, Tan Dolah and Christenson (20) found that the oxidising agents, potassium per­ manganate and potassium periodate, were very specific in the elimina­ tion of the antibiotic action of streptomycin. One of the reducing agents, sodium hypophosphite in high concentration caused essentially - 21- complete Inactivation of streptomycin. Sodium thiosulfate had no effect* They believed that the inactivation of streptomycin may not have to do with the oxidising nor reducing mechanism* hut rather with a specific reaction or an interference mechanism. The results in this experiment support the latter theory. The effect of some dyes on the antibacterial activity of streptomycin Byes themselves possess bacteriostatic activity against certain organisms. An attempt has been made to see if there is any synergistic action between streptomycin and certain dyes. Two dyes, crystal violet and brilliant green, were tested for their bactericidal titer. The titer of the former against S. pullorum was 1:80,000 or 0.012 mg per ml of me* dium (table 7*6) and that of the latter was 1:380,000 or 0.003 mg per ml of medium (table 7-Fa ph co ^ m lo into O 0>o catomintotototo • • § o o o o o to •H g 02 02 «*■+•+ + ++* + d + +• + + + + + 3 CO o + to^^ininin»n 0 CO o i o* in in to to to to •d • • P d o o o f t 0 ort4 o P « 4 4 4 4 + 4 o d + + + + + + + iw ^ p p p p o r- o t- • a i to in to to to to • o om & o o rl O 02 •H CD o> p X o c- 0 0 +■+-+■ + + + C- 0 + +■ + i to ^ in io in in in to o P P i i i i i in in in to O P •d • £ d H to P p o p o 0 cJ o D- P p c- p t) o P O p o § rH 0 0 p rH 0 i i i i i i i l t i d p • 0 0 i I i i i i I I •0 0 o d ■p 3 w O d O O d 0 o O rH to P P Pi P rH P rH 0 rH 0 tD si*CO• H H i i i i i i I i CO• H i i i i i I i i 3 a a I t d a d o •h •H o II o to•i—•i rH o rH p d P • •H rH * •!-) a 02 o o 02 O a i i i i i i i i o CO CD I i i i i i 1 i d O « 0 2 ^ io 0 o rH h 0 2 V m p q o i rH rH rl rH r4 rHj d * d ♦ • •H o O •pH p O ft d 0 a o ^*5 • a PO •a d •H * £ 0 P p O o o o m 0 O 0 & p h a s p • ♦ p 0 d P -H p p 0 a d 0 d d 0 0 p O © P d d 0 o 000 p d o 0 dP o d ftp d ftp d P 0 CQ M PQ H CO PQ a serum on the antibacterial activity rH O u p d o o I r- 9 d fl t oi CQ 3 0 chicken 2. The effect of infected of Streptomycin to O ^P •• rH 02 o to IO 0•2m 00 x • O o nH d o fd ! I t l l t S I T ! f T t o 00 02 I I I t I I T t rH •• H O 0 60 60 60 4- 4- I <3 <$ + + 1* M f to •*do tOtO^'sp'tP^ tOtQ rH «5 0 o to o P + 4- 4- 4* -»■ 4 4* 4*to ^ ’tp "si*io to rH rH .0 O rH • + ! • ♦ + + + 400 ••o 44CO sp sp ’sp ,icp IO CO rH 10 d o *H rd O TJ 0 P O 0 d M • < rH 4 *4 -4 -+ 4 -4 -4 ^ O CO LO 02 4 4 -4 4 4 -4 -4 --H iH to to ^ ^ ID tD O 00 02 + + + + . + + 4 4rH •* t o t o ^ ^ ^ ^ i o ^ o H O tP C0 •« 0 + 4‘ 4 - 4 - 4 4- -»■ + rH d P o P 00 a> d O H ctf + 1 o CQ'tpC002t0O'3,C0 rH r-iw sp c- o> 0 2 ^ < a HrIH • 8 3 u) £5 o p * 0 0 d p d <+H 0 O p d W cd & H oq § •H " P & ctJ O d P d S O 0) o d H I I I I I I I I rH d 0 JoW •H D 1 O to sp co 0 2 rH H 0 2 Cn03«HCT>03^tO rH rH rH P O •H ** P P cd o © P *H Eh P P O © O si ^ p H rH 8 P 4 + 4 + + 4 + 4 4 + 44 t 4 44 + + + + + *.+ + + 4 4 + 4+ 4 + HWHWHwmm -+ 4 + 4 4 H 4 4 ■». +-+ + + + +• + 4 4 4 4 + + + 4 + + i G o o Table 3. The effect of age of culture and amount of inoculum on antibacterial activity of streptomycin (continued )f# fi-i 00 xfi co o C3 M H 64 0} c- O H *-• H u •H 2 0 & 64 02 tO H LO •• 0 rH .O E4 to to lO 43 02 o # ■ *0 p H0 + ++ + + + + + ^ rj*torn + + + + ++ + + + 4 + 4 + + + 4 + 4 + ++ + 4 4 4 + 4 + 44 < ^ ^^•^< + ^1 0 1 0 0 P 2 P 4 + 4 + 4 4 + + 4m tOsF^'tf'tf’^lOlO O 0 0 O 0 P 2 p + + + + + 4 + -f- Pi 0 0 0 P E4 o G OP 02 H «• rH H 64 • O to (O *4 rH i i i i i i i i 1 1 1 1 1 l 1 1 i i i i i i i i 0) 64 02 02 to •4 rH H tO H rH •4 rH i i i i i i i i 1 1 1 1 1 l 1 1 i i i i i i i i i!1 p •H 0 l U & i i +• 4 4- + + 4 ^ ^ io m i i i i i i i i £ CQ s§ C- Q> 02 O rH r-1 rH G o «H • P <2 (SOW P -H Ah SS § o 1 1 1+ + + + 4 to H* to to 0 t o 10^0002100^00 H H 0 2 ^ t “ 0>02sFtO • iH iH iH • P G 0 o « H *■ 2 0 p *2 0 O0 • p «H A h p o 0 PC O P H I 4 4 4 -4 4- 4 4 ^ ^ tP IP If) a o• a IO • H H 02 P 0 0 4> p P 0 P + + + + + + + I to *F *F ^ in 10 H 0 1 o UJ^OOOJtDO^CO 5 • O P >> 0 • 9 2 O o 0 *254> • P 2 G 2 o G H O 0 H • 1 + + + ++ + ^ ^ in to O 1 O tO -tfC O C V ltO O -^ C O H H03^<0 m • P 0 2 0 • p 0 0 ffil H H H a o •H » 4> XS 0 O 0 P i,H W H ,iOin 4-4 4 4 + 4 + 4 5t» H* H* H* lO lO o o EH CO the O cO 02 4* k H + 4 4 + 4-4-+ + + 4 + + ,+ + + 4 • 4 4 4 4 4 + 4 *+++++++ EH r- on of inoculum The effect of age of culture and amount activity of streptomycin (continued) 3. Table + f 4 + + + + f d H rH 1 © 'P d p O 0 ■d 1 0 £ p 4 4 + + + + + + •stf*si* sj< st* si* si* lO lO cd 4 + 4 4 4 + 4 I sj* -; CQ * S d +3 P CD CD p d o | P cd f t CQ « Oow • tD^OOOatOO^cO HW^40)W^O rH rH r-l rH rH rH g O •H » P *d cd O cq d P o £ H *H U a> ftU s •rH P Ti cd O CQ .P *H P* d O <1) Pi ftjG H O «QsJ*c0 0 2 c0 O s i »00 —tcu^t-cr>(x?^i£> 1 i H rH 1—1 rH o • P CQ P CQ • P O » p 0 © & P 1 td © •ct B & i © 0 o + + + 4 H + + t H 4H 4’Nt4H 4H 4H 4tOlO EH 00 H* O 00 02 •• rH + 4 4- H- H- + 4 * 4 EH S O t- rH •• H EH © 02 P rH 0 4 4 + 4 4 4 + to to P I H* H« H* H* H 4lO to *• rH P O © EH © to to o 4 4 + 4 4 + 4 to 02 p 1 H 4H 4H 4H 4^ to IO •• rH rH a EH H 00 ♦ 02 o 1 1 1 1 1 1 1 1 H 4rH •• H EH H4 CQ tO rH rH EH 02 02 to Eh 02 02 CO 1 1 1 1 1 1 1 1 rH EH to 1 — 1i— 1 + + + + + 4 + + * W 1 1 1 1 1 1 1 1 rH O u P £4 1 O O 1 t 1 1 1 1 1 1 1 1 1 1 1 1 1 1 rH 1 1 1 1 1 1 1 I i— 1 o 1 t O H 4C 0 0 2 c Q O H 4CO O rHCQ^C-C^O^tO rH rH rH rH 0 m •H 9 o o 0 to o •a 3 •H a P nj o o 0 © 0 0 s -p a • O H 0 fH 0 0 0 -P O © O rQ o 3 •P £ti 0 P4-CJ M ft CO CQ EH tO rH H 1 1 1 1 1 1 1 1 H o 1 t 0 H ,C 0 0 2 t 0 O H 4C0 o rH02H4f-CT>(X!H4tO rH HrHrH • P ♦ •H CJ P. 0 O 3 •a S •H •* 0 p Td 0 O o si O to S3 p ♦ p -H Ej p. 0 0 0 © ©p O © O p fH o 0 p P cd H CQ CO M P cd P h g o © P O i CO a cd o 5 •p 3 ftp ©H © .g P H$ p o CO g O d P Og P © gP ©P PP O cd pq g o p p Eh Eh Eh 03 02 si* CO CO tD I i • • •» • • 5 r-f P P 5

P*H I I O O CO ad cd © 3 <=8= = ID d g H Og o •H cd p 5 CJ 5 P © g p p p © -P EH EH EH EH EH 02 si* CO CO 00 CO c£> 02 03 02 •* ♦•HP P H P ............ PPP pq » P © a) -P d o EH cd CO CD rH ■3 EH P fH O co g 3 O d si* C3002 CO O 02 si* f- CO 02 rH pH O ft P 1 S3 i i i i i » i +■ + 4“ 4- 4- + + tO tO LO LO to to to +• 4- 4- 4- + + f t f t LO LO tO tO tO i i 1 i t i i + + 4. f - t + DO LO LO LO tO tO LO f t LO LO LO LO 10 tO i i i i i i i to to to in to to to i t i i i t i to to IO VO to to to .ft to to i » * j i i i + + + 4- 4- -4 H to to IO to to to to ,ft to to o o EH CO ft O ce 03 of streptomyc H ft 03 O r- rH + + 4 4 + 4 + - + + + + 4f t LO IO LO tO CO tO H EH 03 © rH f t to LO f t •a p activity antibacterial the pH on of effect The 4. 4 4 4 + -H H ,. + 4 4 + 4 4 + 4 4 4 + to LO to to H o Table + 4 H w ft r& d co ft to ft o EH cd to © LO 03 o •• p H H a H 00 H 03 • i f t i—1 O i * i i to ft DO to 3 3 1 1 * ! ! •a w ft to + 4-4-4-4-44 to in to l o to to to p • w ft p + 4- 4-H- 4- -H I tO f t LO LO LO tO < rH < CL] to IO LO LO to to rH ft I ( 1 03 to pq + 4 4 + 4- 4- 44 05 tO -tj* LO LO LO LO o + + + + I 1 I LOto to to H EH to I I I I I 1 I rH rH «• rH ft 1 o CO « CO O -sF 00 rH CS C- CT*02 ID r lH H Si ' •H • O ft § >! CO < •a r •H " i — O o S25 p ft Cd 0 o ft ft p £h 01 CO • p o cd pq 4 ¥ 4 4- 4 4 4*1 CO tO V ^ LO LO ID H4€0 05to O ^ 05 00 ft CO 05 tO O ft00 r—fr-4r-4 rH rH rH C - CT> OW S3 cd o 03 o •H "■ P ft cd o to § ft '2 *H h ft ft ft P ft •H o 0) o S3 ftft H O 0) o si ftft H + 44 1 1 I I LOto to tO 03 f t C- 0> CM f t tD § •H «* P rrt c d o 05 ft-H in ft ft ft O © O S3 ft«ft tH (continued) 4 + + + + 4 + 02 H 1H* ^ H 1ID ID 4 4 4 4 -4 4 4 H 1H 4H 4H 4ID tD + + 1 * f 1- + 4 H 1ID LO IO CO CO CD 4 4 4 4 4 4 + 02 H 4H 4H 4H 4»D ID + 4 4 4 t + 4 H 4 H 4H 4-v4ID ID 4 + 4 .+ - + + + H 1LO ID ID CO co CO 4 4 4 4 4 4 4 02 H 4H 4H 4H 4LD ID 4 H 1H 4" ^ 4 4 4 4 4 + 4 H* LO ID LO CO CO CO 4 4 4 + -4 4 4 02 H* H* H* H 4ID ID 4 4 4 4 4 4 4 H 4H 4H 4^ ID ID EH LO CO o 4. ID ID ID*CO CO*CO LO oj 4 -4 4 4 4 4 1 -s« -H H 4H 4LD ID 1 H* H 4^ H 1ID LD of streptomycin CO Eh CO 3 02 rH EH $ O r! LD ID H CO o i activity n CO O *# D H F. At pH 9 4 4 4 4 4 4. 4 4 4 1 02 H 4H 4H 4ID ID 1 1 1 1 1 1 t 1 1 ( 1 1 1 1 1 1 1 t 1 1 t 1 t 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 H 4CO 02 CO O H 4<0 02 s 44C- cr> 02 H ' CO 1 CO H 4 ID ID rH EH 02 02 CO t 1 1 1 1 1 rH EH cO rH rH co i o iH m iH H rH • p w p • -P o Cd pq Incubation period, hours t Q 4 4 4 4 1 1 J H 4H 4LD ID + 24 48 72 96 120 144 168 •p •• rH O ■P H + + + + + CO <—! H* 02 S I J H* H* ID ID ID H »4 rH • H O EH H4 1 1 1 1 1 1 > CO co Incubation period, hours a 02 0 4 4 24 48 72 96 120 144 168 iD E. At pH 8 B Streptomycin antibacterial the of pH on effect The 4, Table 4* + + + + t + H 4ID ID ID CO CO CO 1 P o •H -P rd cd o to P to p 0 0 o D -H Ej 01 P r-C H * * O D. CO 2! 1f p w «H » C •H *H •H Ov . vO ec a P ( C Cb f i «d co P 3 o C H si «.< O * «*> TJ O ■H G 3 o a® Vh «M I CM *♦ rH ufojCtandgjas j0 uojynjjp t t f T ^ n ^ q n u v 0) P G £0 W &• I the antibacterial action of streptomycin H O U 1 JJ> o o 00 on sugars of some effect EH co 4 4 4 4 + 4 + in in in co co co o 02 ** H 5. + + + + + + 4 in m co co co co co rH cd P ♦H O b a o -p ft Q> p p © a 4 + 4 -4 4 + 4 in in CD CO CD cD CD p •H 4 4 + 4 + 4 4 in in CO CD co CO CD © OT rH o ✓"V s 3 •H rH + 4 + I- + t + in in in co co co kh EH 02 © 4 4 4 + 4. 4 4 in in in co co co CO rH P IO P •• P iH P t> Eh cd CD © 4 + 4- 4- 4 + IO in 4 in in in co co cd 02 o ••p rH rH EH CO rH 02 • i rH o rH feO + + + + + + + in in co co co co co © p rH 4- + + 4 4 t h 1 h * m in in «• H p + 4 + 4 + 4 4 in in CD CD CO co co CO CO P •H O !>» l 1 1 1 1 l 1 a o o p ft 4 + 4 4 4 4 1 in cO CD CO CD CD © u p 1 1 1 1 1 1 1 CQ • o • PQ EH 4 4 4 4 4 4 1 in CD CO co CO co ft a © a © p P P CQ + 4 + 4 4 + 4 LO tO CO cO CO cO co rH ft P •H O r*> r-f V/ © P •H d □ O o 0 ft a i i i i i i i I + 4 4 + 4 i in co co co co 1 1 1 1 I i i i i t i i t i I i i i I 1 1 1 1 1 1 1 i i i i i i i i i i i i i i 1 1 1 1 1 t I 1 rH EH 02 02 CO rH E H • UAJ H H 1 H D 1 o H 1CO02 CO O H P .• •H O P 8 H* 00 co H H rH 02 H* I*- CF> OW >; Table a 3 •H TJ © a EH I 4 4 4 4 4 4 4 in in to co co co co + + -4 4 4 4 + in in co co co co cd p •H 02 c- O H *• CQ The + + •+ f + + + in in to CD CO * = O -C H* co 02 co O co 02 Hi t - O' 02 CO H rH rH 02 C0 02 CD O H* 00 C- CT> 02 H4 CD H H H P o p O •H ~ tH Pi P P o © o P ftp H HI P P cd o © * P Tj cd o © £ H Pj P P P O Q) O P ftP ft O H H o of streptomycin + 4 4 4 4 + 4 •H ft! + 44 ¥ ¥ ¥ ¥ LO LO IO tO tO tO action antibacterial the on © H O sugars -P O cd some of OT © ft 3 d 1 a o © I* B ft © ft -P CO a « • • • t t- ^ id c- c- P P cd -h ft ^ ft IO ID tO tO to tO © ft •H 0 4 4 4 I- 4 4 t + 4 4 4 4 ft ft © O ID ID tO tO tO tO + 4- 4 - 4 4 CD IO LO ID tO tO a| to to to to to ID ID to to tQ to ft O ft 3 8 P •H 4 4 4 -4 + H I ID ID tO tO tO tO § 3 3 I J3 cd 4 4 4 4 4j ft ft a ft I tO ID ID to tO © OT •H © ft O O EH sji to to •4 H EH 03 w to •• rH EH tO H H •• 1—I I I I I I I I ft © ft m I f I t I I I I I I I 1 I 1 I I I I I I I 3 ft I I II I I I a •H O s I I I I I I I HHH g •H * p ft cd o OT P CO m © © © © JjOS H ®; OT OT OT W ' ft ft ft ft H H H H ft ft ft ft ft ft ft ft ft 00 W to O sjt 00 02 ^ C7> 03 to HHH ft o o o o o ft ft ft ft ft ft ft ft ft ft ft o *H — P TO cd o to ■f3 t ft h ft © 03 ^ to ct» cr» * • • * • co 8 0 1 cr> cr> ft © OT ft P • O ft >. OT ♦ S ft o o OT Kp ft • © ©P ft ft © ft p cd CO PQ O' id cy>c- cr> con ' • • • • • 4 4 p H H ^ 4 4 P H H ft! © cd Eh © 4 4- * 4 4 to ID ID o I ID ID ID ID tO tO 03 ■ft •• H H 0 EH H 00 » 03 O I I I I t I I H o + + + + 4 <3* ID ID ID ID tO tO EH © 4 02 tD H <9 ft sH p ID 3 effect O c - tO O 02 a cop o 3 c- o rH •• rH © OT The ft ft EH >_' 5. H -tf* ID ID tO tO to tO EH 00 00 O 02 •* r-| 3 •H Table ¥ 4 4 -I 4 4 4 ■u I § © ft © © © © © CO V\ incubation, CO t-1 Cv uofinxTP TBTJaxoeqTitnr in days activity of streptomycin 11 CO -p.P—I o P. n> » o 1 —1 o ft p £ t + t 1 + 4 4 + 4 + 4 1 CO ^ ID ID ID ID ID EH 00 ■rH O w 4 4 4 4 + 4 4 I CO ^ ID ID ID ID ID of o activity Eh 00 5i! 09 o CO •• rH 4 + 4 4 4 4 4 4 cO cO ^^ ^^^'tf rH EH oS E- o H •• rH antibacterial the on agents reducing The effect of some streptomycin 6. f t i + o t 4 4 + CO EH 03 CD H lO •* H § H » a o -p ft CD Table ♦ i P CQ f t + "cf* 'vj4 4 4 4 4 + 4 I + sf« xj* -ci1-s# ■■ct' EH 9 4 4 + 4 4 4 + ID £ £ 1 DO ^ ID ID LO IP in 03 p P a o p ft 1 1 1 1 1 1 4 4 4 4 4 4 4 1 CO -cH ID ID LO ID ID 4 4 4 + ID ID ID ID 4 4 4 4 ID ID ID ID + + + 4 4 4 1 1 xj< ID ID ID ID ID EH CO rH 4 4 4 4 4 1 1 + ID ID tO ID ID rH rH £> 1 O cO^COOScoOs^cO cO rH H 03 -cH f - Cd 03 H rH rH £ # • •H O ft £ O >! CO • a £ •H ^ p p o o CO p cd o to • P -H f t ft p £ £ £ cd 0) o 0) o p ft o P cd £ ftp M f t 03 CQ o 1 o rH cO-'tfoOCtfcOO'tfoO H 0 3 ^ r - cn 03 ^ co 'd rH rH rH £ •H * •H o • O cd f t £ CO >5 o a o £ •H P T5 o •H to p P cd O CO f t ft • P *H f t CD O p £ ft £ O 0 o ft o o P CO cd £ ftp H CO PQ H o u + 4 4 + 4 4 of $o activity EH CO CM CO rH + 44-4 4 4 4 ■t'COH4H4H4H,H,H4 CO p P O + 4 + +. 4 4 4 4 CQ H 4 H 4 -H4 H 4 H 4 H 4 EH H4 c - 4- 4 4 4- *4 o C- H •4 H 02 DO ^ Eh a 02 4 4 4 4 4 + 4 "d* o a a 0 —i EH o ) ft o d4 • • CO o o b I 1 I I I I I I a co ••02 o H p ft 0 0 P CQ P I DO 'd*'d* St*"d* ^ ^ E4 d4 •4 O ft £ o 4 + 4 4 I I I I I I I I H H 0 LO 1 P o 0 H CO d4CO 02 cO O d4CO H 02 d4C- CD 02 d4cO ft H H H 0H r f t ft ♦ O OT f t > ! O OT a •H o X! § ft OT p P • ft 0 •P ft f t O ft O 0 CQCQ PQ P ft cd o ot O - H fj ft ft ft O 0 O 0 ftft M O o CO to + 00 cq •• rH 4- -I 4 > tO cO ^ ^ ’s}1 sj* ^ >* -p o o CO C Q tH •* rH n •H -P O cd 4 + 44+tl-t cO cO ^ sjivj1sj4sj*xj« H cd •H o o u <0 -4 ■*• + -» r r + > 4 'd CO cO ^4* "sf1 tO P o cd P •H -P iw CD •* rH 0 o 3 to o CQ p to o ■• 0 H 0 0) P -P S3 o o O -p O tO H H a •• S fl 0 bO H cd H f + t t + t + t £ .03 CO '5it’ ^1* '>1^ > a ■p o O -P 0 04 0 si400 •H CO -si4 C - CD CQ -t}4 CD Q H H H $ m • P- 0 P 0 • -p o 0 CO g $■0 cd o to fldfj P Ph P 0 0)0 S3 i;n . 4) P 0) -I « rH ,P 3.O B •H flr •H •‘ O u, if* P activity I ♦Oi -rj Ci. o < V ti U1 3 +I* o ■H —} tit o t3 m o reducing agents on the antibacterial of streptonycin la HIH 3 T) £ 5* Ci -rl r—* a .3 S 3 C •H Effect of some -H O r^v uofintTP CM •H H O f fl + 444 + + I I H4H4H4H4H4H4 P fl o EH P H O fl +3 fl 1to + + + + ++ + + 1 -> CD of streptomycin H EH CO CO + f f-M-* 4> H O EH 00 H4 00O eh C~ a -I- 4 + 4 4- 4- 4 4 C O H ,H,‘'d,H«H4H' o H Eh H •• rH © P 0 rH P EH to 03 + 4 + 4 + 4 I + H4 H< H* H* 'sh IO m*X ! H y i EH H4 to I I I I I I I I CO •• rH EH p © A P CQ • 02 I I I I t I I I CO rH pq tO rH £ fl I I I I I I I I p LO •« 0 H y p cd y © © EH © to o LO LO p o I I I I I I I I p 03 •4 h£ rH a rH a EH CO 00 O rH 03 • ♦ H4rH o o I I I I I I I I «ft rH Eh H4 I t I I I I I I CO to •• rH EH 03 03 CO I I I I I I I I ft* rH EH EH to <£> I I I t I I I I •• rH effect I I I I I I 1 I © a O p H 03 a rH a Eh rH 6 o -p p © fl P + 4 I I^ w o H♦# H CO o • © LO to O H Cd +3 © I t I I I I I I 03 •4 P rH rH I I I I I I I I rH rH H 10 D 1 O The t + rH action antibacterial the on dyes of some Smm w * 7. I | | ^ ^ H4H* H* o 5 Table 4 4 4 4 + ft O O tO 00 03 to O HOJ^C'-CDCQ'^cd r H r lr l rH 0 « •H • O P >5 © • a 0 o o CQ !3! -p p • © © -P P fl o 0 P cd £ l CQ PQ 1 o tO^COWtOO^CO p rH r—|CQ>vfi£-0'>(X!'=j: y p fl cd p fl CQ o pq § •H •» -P TS fl) O to fl fl fl o © o 0 P£3 H Eh O CO 03 CO H «• H 4 + + + .+ t + + *► + * * + -a- a- 3 H - C O ^ s jt s i* ^ ^ '^ O to of H action antibacterial the The effect of some dyes on streptomycin (continued) 7. O 01 Eh O to 03 CO •» H ■fl q + + +H- + + + 3 CO stf ^ s* stf ^ stf •* a 3 ■H •P 0) | | ^ ^ si* -si* si* a I © Eh A to O 3 to P rH •• A H o (D H rH a •H EH H O • CO O •« H a to & O o 1 1 1 1 1 1 I IO q t© 1 t 1 1 I I 1 1 to • o ii EH O 0} 03 •• H 1 I f 11 i l l 1 1 1 1 1 1 EH O rH 1 t <*h O q o rH ❖ •rl » I i H* si* H* H* H •»■ + H + + ^* ^* s# ^* 03 p A Pr q •H © s a O p p © P P IQ Q . EH si* 03 O rH 1> •» H +++*++ 1 1 ^ S* stf S# EH 03 to rH IO •• rH a i © EH A tO p IO IO P 03 •• A H © © *9 3 p -rtf 1 ♦ + + + + » o 3 ^ * s i« ^ ^ « 1 f H- ■«- + 1 1 CO si* -Si* A © a a © © EH 00 o o • ^i1 03 rH «• u rH H pp 1 1 1 1 • 1 1 aa rH rH Eh O ♦ • O « * * CO to O «• rH 1 1 1 1 1 EH 03 03 CO •* H 1 * t 1 1 1 1 1 EH to rH H •• •H 1 1 f 1 1 1 1 1 p i 0 t O H < C 0 O 2 tO C W 0 0 1 H 0 3 ^ *C -0 > 0 3 ^ t0 rH rH rH 9 o -p rH * © rH • O • 3 -rH P > to O • p rH cd P p P rH O • O Eh ■«5t« O o co t PH § *h H rH -H a §o p (4 CD © Ph W) u O cd i—i 0$ I rH Eh O Table H O Ph I Pg ml 3 O EH O CO •si* O CO 03 p a • & .© <9 £ o tD s j*C 0 0 3 t0 O s i« C 0 1 H 03 H* t - 0> 03 stf to H rH rH q o © H q © •H Phi © bl © P q fe © O a •H • o q 3 03 P nd q p «H © O © • A *H U © iH Ph rH P P «H © © © o CQ q p p pqp * pq 1 EH O 00 03 + H- 4 4- + 4 tQtOH*H*H*H*H*'l* H Hi* of action the antibacterial The effect of some dyes on streptomycin (continued) 7. Table 4 4 +• + 03 t- O H•« iH Eh u o EH 00 fal H 03 i a H• • 03 H H O • e Eh O• O 03 to "d* o O (•0« o ♦ H o CO O 03 (0 to H I I Eh 03 03 to *• H P H 3 O Hi* to crH + + +■+ + + I 1 tO *1* "d**d* EH 03 a 0)(0 H 0) to 0) 0 P P Ph tk£ H B 5 4> P EH J3P al to (0 o o •H LO fid f id H 03 0 © * • H •H H o o +3 43 u •d* 4* 4* ♦ Hh 4-Ht + 4-03 "d*-si*»d*xsl*-Q*-«1* EH EH p <0 03 H•* 00 H 1 I I I I I I I I 0 P Eh p O P to(0 H••P 2 P o rH 0 o 0 .H cd U Hi*EH 43 I I I I I I 1 P © O C4Or 0 • aH Ph bD rH rH EH • P 1 1 1 1 I I 1 0 toO O *• d* 0 • H rH H H Eh •H o Ph 1 1 1 1 ) 1 1 PQ 03 03 •• • rH l*t Eh o H H I I I 1 C I I 0 EH •H to I o H H >: • * a H o +3 P 0 (0^0003(00^00 (3) 1 H03^t-0>03^*(D Ph o +3 p a ) CQ H 0 • a Ph • •H U • CQ P O P O t>!■P 0 • a P P «H • +a >d O o cd 0 cd o w £3 +3 •H • P *H Ph P H 0 a) H •P p Ph •H O +3 Ph ir o X) ->H H3 a. (0 o ua 0 3 « tfL q H rH rl o rH d P • o ft to • 0 £ o o to Sss p ft • 0) 0) p Xi p o p r CO ,s £ o P * P 4. t I H-CO IO I I I « I I I I I I I ! I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I l I I I I I I I I I I I H 03 rH I LO ** rH a) © £ £ £ £ EH p P to I LO £ £ 03 o o «* cd cd rH a) 0) £ P o 0 o p tD •• rH 02 p to H rH rH O H 03 P • p *d • o cd ft £ o fc ^H to p * £ 0 CO p «d to £ O (0 p ♦ £ P P ft a> • P £ £ £ P •d o o O *• at 6-t CO O 02 CO *• iH + f 4 -+• -n. -| » to to ^ IO to IO IO EH CO 02 Oi rH rH ♦ + + + + t • 1 to ^ IO toio to EH 4 * 4 * +• ■* * 1 to to to lO to to CO CD •• rH Eh 03 O t- H•• H H H CO IO «• H 0) 0 P P EH p <0 ■P -P to ID 03 ••PO P o H td cd 0 o E* o o CO •p p 02 H♦♦ w iH aa H to iH 02 • EH • O to o CO *• H EH 02 02 tO rH Eh 10 H H H EH 02 r- to •• iH * 1 1 1 1 1 1 1 * * * * * * * * 02H*H‘H,tOtDlOtO ■* + ■*+•-*+ * + HlHTl o o 0 OT cd o 01 3 !-p 0) « P -H p p • P 0 0 0) 0) p p O 0 O O -p 3 C/3 eg fS 02 EH ♦* H O O H IO •• H * 4 4 * 4 + 4 1 1 02 02 tO -st*IP tO + 4 4 4 H- + * I 02 02 tO rl* tO rH O O to a 00 o rt 1 •H cQ^C0 0 2 cDO'd'dO 3 O -P 13rH H 02 ^ C- Ot 0 2 CO H H H cd • «H •H •d • cd P fl OT O •H 3 •H • O P OT -P S3 OT • S! 9 ? 0 • -P 0 0 O © O p tJ o s 8 8 9 Fig. for table c Tne combined action of streptomycin and sodium sulfadiazine IsSCCOT Antibacterial dilution 1: 1*000T 1O000T otreptorrycin plus c mg sodium sulfadiazine 1 i2000T streptomycin plus 0.2!? mg sodium sulfadiazine 1:100CT Base line (snowing growth) Sod. sulfadiazine 1 — — ■ 1 2 - 3 1 + 5 ’ t, i-eriod of incubation, in days of •f + + + ♦ body t ot o41^ s P^ the o t o 03 ♦+++♦+ t ot o' 4 *« 4 *s p^ •»+■!-*■ + + t Ot O4 »4 < 4« 4« b O M M M-* + ^ bO bp W) M ^ 41 ♦ < J< « 3} 02 02 02 02 Table 9. The duration of antibacterial infected chickens activity of streptomycin in 02 02 02 02 o G © 2 t o o •H p W» bD * + +• -*■ <4 < $4 *^ P4 *< 4* ©2 02 M bp ♦ 4- f *0 4> 4 *4 < 02 02 o o t o H t o t t )W )+ •• * -I -+ < 4* 44 *4 *4 *4 * ©2 02 + H* -h »4 «4 * <4 <4 4 1 4 02 02 G 0 P W> bO bp M + + 4 «rl M b p b p b p + -*■ < 4 « 4 < J4 4 14 1 U t ot ot ot o 0 < | J < [ p 02 02 02 02 P i dt ot ot ot o4 * W) bp bp bp bp W bDbO M M W <4 «4 «4< 4 *4 *4 * <4 <4 ©2 r H g p -d p-«d 0 o 0 o •H p 0 ft 9 G 0 fJ G a 0 u 0 O ftp § fl ftp t £ >4* C O0 2t oO H 02 ^ P t * -< J >02 H o •H * •H to to %P COP£ 02 02 g o o « H * * P *rl P 0 o t o^ 0 00 2t oO iH©2 4 < t -C T >0 2 - «c 000 P p u G g G 000 G ftp H a o Pts 0 O P-H 0 h O 0 ftp *4 O >» •d o fl © fl o 00 tp CO o 0Q CO »• d 9 0 fl ft 0) d fl 01 0-1 o £ •H > fl O © fl O © rH co d © fl 4 ■

bD Mbf © rH I EH d © o ■H fl O d o fl *d cd O f l vH Fh d d o © _g ftfl 8 d o •H fl bp H) U) +> * 03 oa to to W) b£> •* < 4 4 *4 -4 4* 03 03 03 03 03 tO 4* 4* bp bObObO 4 4 4 4 4* 4* 03 03 03 02 bp bD W) t© i* M <4 4 4 <4 41 ^ 03 03 toto tuO"t + + .4*4*4* 4* 03 03 bO W)bD bO + 14 44444* 10 to to to W) MbDbO-l' . 4 4 4 4 4* 4* 03 02 02 03 o a bo bo w bp bp ♦ to 3 4 4 4 4 44* rH ‘ to to to to to W) tap bO 4. -I. 4 < 4 4* 4* + 03 03 tO 4* bp bO bO bD -I- -f 4 4 4 4 4* 4* to to to to *) bObObObD-t4 4 4 4 4 4* 03 03 03 03 tO bpM bp fc© bp * to to to to to bp bp bpU)-f + bO W 14 4 4 4 4 tO 4* 4* 4* bp W)bD W) bp b£ 4 4 4 4 4 4 tO 4* 4* 4* 4* 4 4 4 4 4 *4 * The duration of antibacterial activity infected chickens (continued) of streptomycin in €0 o 02 to H £ ■8 •p o © £ © •P O lO C O 03 H I I 1 t I fH (0 4* CO 03l£> O H 02 4* t- 01 02 cd •p £ O1 oo 00 rH ID t tO tO 4* 00 03 10 O H 03 4* C- Oi 03 H to A • * P 01 O s0 £01 •p a • ©£P i £©p •p 0 ■P 01 00 PQ >» Table bO bp hD bp +• + O 4* & o A « (O £ O •H +3 pg“ C Oo C O ,Q *H £ W A M S o •H • -P «tJ 00 O «> ,3 -H £ 3 © Q £ o •H • ■P *CJ <0 o 01 Xi-rt £ gag £ o ■P tj a) o w ,Q *H £ £ £ £ © © o P P bD bO W) bD M bf U bfl W 4 *4 4 4 4 to to ^ ^ 10 43 bO bC <4 tO tt)tt)hO M M W 4) Ph a> <4* p to to 02 # O £5 • O S M M bfl W <4 ^^ <4^ <4<3 <4"=4 t I to’•J*00 02 to O rH 02 t- 0> 02 tD CO 02 tO O H 02 t# t-0> 02 fH o tO H 02 00 02 tO O 0> 02 H t o-^co 0 2t oO H 02 tH C-0> 02 H O l O iH PI •H O >» • • P (0 O P Pi a) Ph P CO 01 g 9 • S P 0) O to aS PC| P P o “ •H Pd -d o CO ■gti. S 2.ii H fi Pi *ph d*• o ~ «H P TJ PI •H m ,2 «rl P 4> -H P .H P o as o m do at>* 3 o P M as o w P u 00)0 s** O p8) do n oP P a? Po the 10. The duration of antibacterial activity body of infected turkeys (continued) of streptomycin in o to MMMMbObf <4 <4 *4 "4 *4 <4 to *0 CO ■thto O C O #• IO 3F H t ot ot o ^ t o W) bO bO bD b) « 4 4 <4 <4 to to ^ ^ ^ to •H p o © T» o 4 * W) bp bD bO bD tat 4 4 4 4 4 4 to tO ^ ^ to •• © © •H bD taObD bD b£ <4 <4 <4 <* t ot o^ t f * t o o 02 to H jpjMPJW 02 tO "4 IO U> bO bO W) bO M 4^ 02 tO 4* CO 02 tO O H 02 4» 0- CO 02 I t I < < < 3 it o 4 * JMfipjf I It o4 *4 *t o bO 3 H to to 02 At Q & >» Table rl •• H to © •3 to 4* CO 02 to o H 02 4* C- O Cj s $ a H tO 4* CO 02 tO O r-H 02 4* C- O* 02 to » o o o pH y •H O • • P © 1 •a 0 © O o p a & p • © © u P O P u p © J P C Si O © a 0 •H •* P <© 01 o w P*H 3 © o ti O ^ys rH CM CM rH u o -^ s u t ^ tv[29v Infected Fig. A-2 The of Reinfected course agglutination titer 'O r*\ C\J Days CD CM rH rH CO rH a a V R uox^BUfq.nxS^V O CM rH rH Infected Reinfected on the Treated with streptomycin Fig. A-3 The effect of streptomycin course of agglutination titer Days CK vO <\l CM CO rH rH j e - m uonwiT^niSav CM Infected The effect Treated with streptomycin Fig, A-h CM Days Infected of Treated with streptomycin of streptomycin on the course agglutination titer Days H O q •P 1 S o p H O 0 a 3 cd $ 5 N cd •H •P cd H S 4 4 CO CO 44 H 64 O 02 CO 0- •• H 0 P EH J3 o +3 CO H jeJ CO »« c H 0 0 E4 O 0 CO ■P to *4 H rE 64 O H « •«O rH £4 O 02 to •• H £4 O H•» 02 H a> H § IO 1 o ts)rH 0 •H • *d • 0 P 44 0 • rH Pi 2 0 S 0 0 1 4 -4 4 4 4 4 l + 0 2 tO ^ 4 + 02 tO-4* 4 4 4 W W W 4 02 tO tO © 0 1 -4 -4 02 02 tO +4 4 4 4 JCJ a cd 0 o P 02 tO tO cd 4 ■P i—1 0 to 4 4) •P 4 4 iH 4 ♦ 4 02 4 I I 1 + tO 0 to 4 4 4 02 tO I I -f 02 tO 0 o 02 tO tO 4 02 tO ) I I I 02 4-j 1 4 tO 4 1 < to 4 4 t 4 02 4 .q S + + + 02 02 tO 4 1 t +02 ^ I I I -H I I I I 1 4 I I 02 l 1 I I I CO st* CO H 02 ^ CO <*}' 00 02 CO rH • • ■P P Sod 0) 4 4 4 I 4 02 tO 4 4 4 02 JO tO I I 02 •4 + 4-4* 02 tO'sf 4- to H + WWIO £ cO^tf 00 rH 02 ^ m i o I—I 0 *3 O *H ** O •H • (8 0 ® ,Q *H f i 2 P S» 0 0 O 3 p.^3 ,3 *h P •P *Ci H 02 + f -P +J >d cd o w §*§ •rt .13 P a o «H •» ■p -d 0 O W I & ,Q «H P J3 Fh J3 0 4) O S af3 H O ft 1 P o o EH O CO 02 0> H 1—1 cd •H f-i 0 P O cd P •H 44 a cd 00 O 3 3 4* H 0 0) 1- + 1 1 H- 02 CO + I 1 + +02 10 -cP CO 02 to 0 ft "t H Oi'tPC- O •H o N rH 0 •H « *b « 0 P P O + I ^<<0(0 10(0(0 0 44 H O 02 10 ♦4 H 0 a K o p H 'lO tO tO tO tO tO tO H EH O to H 44 rl EH O CO rH Eh O H* rH EH O 02 H Eh O rH rH •4 0 P P p P o 0 0 I I tO (O (O (O (O tO I I 02 tO tO to tO to 4- H- O P H a 02 • o 44 + + H* + I I I I ID lO CO tO t + 4 + I I I H-lO to to tO ♦■ + + 44 I I I I I to to to •• I I I I I I H< H* •f -+• Eh in #» rH 0 P •H 0 p 0 •H *d O 0 ■H P rH 0 2 0 rH 0 •a •d H o c§ P! I I I » I I xtf to H* CO 02 to O H* CO I H 02H- o> w-tpto rlHrl O H P o •H *' a P h 0 0 P H iH cd to a $ 03 rH o © © •Q £ r* P P P .P X3 © O cd © © © o o p p I + 4 - 4 - 4 - 4- + I LO CD CO CO CO CO I I I + 4- + 4- + I CO CO CO CO CO I + 4 1 - + I -cj* to CO CO I o I I I I I I + + H. + I -sf to to to I 4- 4- 4” I ^ to to rH Eh O H t I I I I + + I 'Cp lO rH EH to •% I I l I I I I I rl § •H N p cd © 1 o t0 < tfC 0 0 3 c 0 O 'c t< C 0 H W 'tf r - O X M ^ c O H r lr l H • • P to «H o 3 © o P a (0 i-1 s • cd 3 (0 « 0 H p o d R © o O © fS 5 (8 a •rl iri o cd •H *H Xi rH © o p u o H s • n VJ to• H• o .p o R "A •• 0) p © •rl Eh O 03 «H O EH O CO H o N d cd H *t 4- H- f 4- + I IO cO cO co CO cO M Eh O 03 to Eh O CO IO • • H Cj 5 I s g © m p o *H ■ p d © o © X> -H p p p p o © o © P H P4 2 P N © •H d © u H p 03 • Q I 4 4 4- 4 - H- I t 1 lOtOcOcOcOCOcO EH O 03 03 H ♦« H 4 t 4- f 4 + + I 03 lO CO CO CO CO CO £h O <5? CO »« H t 4 - 1 - 4 — 1- +- 4+ IO CO co CO CO CO 1 + + 4- 4* 4- T 1 tO CO cO cO CO CO EH O 03 to •• rH EH O CO H •• H © © p jo p p p p ,P jp a o © © © © EH O GO •• H o o p p EH O rH -• • O O 44 s -4- + H- -+ 41 1 1 to CO CO CO CO 4- H- H- 41 1 1 1 tO CO CO CO 3 4- ~+ 1 1 1 1 1 1 IO CO rH EH O 03 •• I I 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 H EH O H •• rH Eh to •• rH 2 P © P © © •H d o © •H *H .P rH o £ H TO •© R d P o o TO a 9 p 5 © © s. cO'H'COOScOOrHCO I H 0 3 > c f O - 0 > 0 3 ’«d*cO H H H o rH # * P P © O ■rl • P © p d © o © • ,Q -rH P P P P P O o © o (3 -f- -*— »- -I- -** I tO IO tO tO tO tO tO f 4 1- + + *- T I tO tO tO tO tO tO tO +■ + *1* 4- 4 + + I tfl) to to to to to to + + + *f + 4 I I to to to to to to ® to P © o -H o rH a P g n P

03 H•4 O + ♦ ■»• t ID 03 H•ft 4- 4- H* T •cf -tfto H 0 H *§ EH P O co •P CO H o 03 10 ■«* ID •• 00 rH 0 0 EH o O p ID CO •f t •H rH *H EH H O 3 H 63 H IU ^ I D • ft O I I* 0t o H EH O 03 H• I I ft03 © fQ EH rH • (1)*d•po -9 o cd P H CO CO P O «H * +3 X) co o 0 rO •H R gfcg I 03 ^ I D P P O O P p O o 0 0 0 0 O O rH O + ft I to*# P P CO •ft H H 9 •s L OH ■0 • o O EH ■•cf Pt H ft I CO CO EH O 03 •ft rH I I IN EH 1 t I 03 d EH ID 1114* •ft H •H P O H 0 a H & ^ CO 02 1 H 03 -*Pt>- •H N p cd 0 •H •d o cd ■H .P O o rH • • P « CO O P «H ft ft ft* 4 0 0 a a S 5 EH O CO H •ft H "S p Eh O 44 o O 0 0 0 0 • p aa ID H •• oo rH H •cf •• EH O 03 •ft H till I I I I 1111 I I I I Eh till ID *» rH 0P ‘ol •H 63 p H 0 $ ** f I I I 03 rH •H M •d O 0 P Ti CO •d a cd o w 0 3 « , 0 «H P «p r P •d 0 0 o g S g 0 to p5 £ « 1 I 03 ftp O o P d N0 EH •rl O H •d 0 rH•ft P 0 ft *| I ID ID 1 ID ID Eh O 03 to •• rH EH O CO •ft rH 0 I EH O tt* CO •ft H po 'd p00 0 O $ P d p M o •3 O H•• H q H 0 0 P rH Pi •rl « i H©3 '* 0 3 o Ncd H •H • • 0 0 93 p3 CO rH «• .p CO P H ft ft ft H EH EH 3 P 0 O 03 t•o • fi o •H .P o •d a) pn* 60 PQ 0 )e- • p •d cd «H H *d cd P u CO H P O P CO S CO 0) CO cd « EH H 0 d •H N • • H ID •ft rH 9 3 Pi o t o03 tO tO H* IO tO EH © • Eh rl ft*ft ft CO •ft H EH O 03 ft •+ 1t- t •o • © tOnP "d< ID H g O H § ■H* ^ id to rH EH EH O ^P CO co EH O CO 03 H •ft rH ♦ -#-«*• CO 4- ft t ID <0 1 Eh H 8 ♦ 4- < Hp'cflDtO i 0 •H 4H 43 rH O ft 0 P p 0 O P •H * 0 p xi 03 O P0 h a 03 • • a m P fd R 0 o 3 O 0 & CO ^ CO 03 H03 a .O *H P O U Q> Pi.P The influence of storage (in refrigerator for 24 hours) on the bacteriostatic activity of normal chicken serum and on its enhancing effect on sulfadiazine 14. I ( to to EH O t ot o^ ^ co 03 »t ot oi n + + 1 I to to H *• H p I 03 to IO t ot oh*H4 H (D CO ti a) X 03 to ^ I I I to IO o Cd I I I IO © I 0 H 0 P 1 cd 0 N 0) •H 5 • d I I i I T) & *H a •H P O i— 1 IO (211 e f-l Q a p CO CD P Cm m UJ d H Pr CD < ti 1111 N cd •d td rH till 03 03 t/3 • to 03 I I I I I- tO H4CO 03 I H 03 ^ t*o rH 0 O + (\J ^ Tf -sjlT# If) tO tO tO tf) ID tO tO + + + + +| If) tO tO tO + to 10 of + + + + + IO to to to 0) m o> o + + + I- + si' If) to to to t ot o -«- + + .*- + H1If)to to to t ot o *rl & O © -P o © a # 0-1’ + <5 «=$«a{to to ^ to to GO tO Tp CO w ^ o> ^ * t o rH H a & o *H fi O •H •* ■p *d to •• b£ H a otooH 3 TO *H O W) to 1 a t o o TO Sft to ©© £ £ PP -P +» i i i i i H H i i i + + 02 © ©© oQ p +5 aa to•rH• oO O CO •« bfl bfl bp H> W 0 TO CM -si* O •• H O (O IO to If) to I I I -*- tl 03 < 02 I 1 rH U) MW) tt U o« H3 • ©a pu •P• 3 © © CO to (-1H + + f f ♦ 02 tO tO s* O 04 bO hO +» +- ■*- H ID + + : <} «q «s}|Q t o-Si* ( 0 © 2t o o 04 I feO W) W) +■ ^ 0 ^ N tO tH < D H I Sh O •H • ■p «o • P n ow ,Q*H k 554b) 053 0 to o> to 03 ^ ^iio 03 M o A O c tfi § H m 03 H 3 •d $ t 03 I © H 04 H • • P 3 (0 O P n 49 >d >* © 'S 3 4-9 • o a 03«tf o H to S bp 4- + 4- 4 o O & 3 ” * «o o w & HU P 3 g Pi.3 O 0) a N 0) •H ■d cd <3 __i p! 00 (0 p H Pi Q P (I) 0) >» 0) .y u 8 •d © 49 O <1) « PQ O 03 H ID pH o to tD 03 H o CO 03 H rH © © o p p y* p P to 49 ■p H P X! o a O cd cd 03 © e to O o rH 49 49 o b£H to a S H H H pH • • O O o 00 H O ■4 4 4 4 wieto^ b Ob p4-4-t . q ‘l •H o S N H cd ¥3 «H « P TJ , 49 cd P (0 »d H p © P to 4> S to • o p 0) 3 • 49 <9 3 o * H • +3 eg o W O-H 3 P 3 P O f l >o £ “ ** 10 10 10 to to to to to ID o o § p o TO Q TO S3 TO TO M to •H TO A a TO p *a 1H < H § TO H *d to © <0 •P *H TO*b TO cd a p < •rl to«d ou 1 P ° 3 P P-P pop O -H O S -P TO 3 «is •P P TO TO«H TO TO P ^ 9 3 ©4 O TO TO T O # 10 tO tO ID TO TO >» I | TO X H •0 TO •P TO *d TO •P to TO TO TO TOf-i •*- I CO *#«D 09*0 H©2^*t-0» -J* o •rl P <0 Jh I& P O TO up O TO 1 tQ tO 4* IO (0 £ © +• + t + tO tO ^ IP © •H P o OS +• f to to "Sj*to a> £ o rH a) s u o £ «tl< © Ah IQ Ah o d o •H ■P O cd rH Cd s_ © A o N H 1 © © £ H 02 ^ C- « •rl CO rH tO "# CO 02 »d • © P Ah © •H P O fj © d O P Tj © O w ,Q *H d © • p P d •9 •d o O © g Pi © • &8 p •H N d © © tH X •d o © •rl Ah fit rj a P wH 0 © 3 •s u 'd K © o © m co tst A Eh O to to •E- +■ 4* + +- + in the 03 EH serum o> + 41 00 4* 4* 4- +- -I— I- + -4- H- + + 4- ■+ 03 10 ^ ^ 4* 4* 4* EH M M + + + + <4 <4 4* 4* 4* 4* O 03 tO to chicken normal and tO M - H - 4 « + +| + <<<^ t o4 * S Ib 0b p tO fn to * 0) +3 53 4} PQ <2 . to "tf*00 03 to 00 H 03 4* f- 0> tO H to ^ 00 w to oo H 0 3 4 * t*> 0>tO 19. & O •H P Table The combined action of sulfadiazine body of infected chickens O + + + -I h + O ©_ o pp a O •H ** a o M P -H U P U 3 A 10 P * 4*0 P O M Ed a vu o3 i H ftxj