1 r.V\ '00 Q’I‘. .6.- u... o :x .3. a. r 3 {3 .2: .4: tr: 3. "3‘ {gr 1 II ,_K 5“. 3 . I 5: I“. .. ei‘: M5- . " 3c” .951 out". \- 1» '7 . Z . . . a. _._K L fies . x («J '- .th .b...... . any: o.-.w.M 9 a _. u as, U; «0.... $3.». anon-o Jov .gf ,. .. . so. ‘Ur ”an.“ .5... .. x Wu: t... nu.‘ .- L _ a . .9 ... . . 5?. ”3..” 2‘...“ a: ”g ..... S .._ an ‘ . es * a . . ‘ . s»: f - w boas . c. r. J... . . 9 s r .l at _ ,1 .. ... C “an“ ....~ 5?. ‘1“: ,.r\ II. Wua . ' I ‘ l l ‘ I \ l \ ‘. u i i ‘ J I . t d t I a V ' ' . II I I l l I I 1' ,. ' I | _. I J 1 .J. ‘4: , ¢ A l I a ' I l -A 1;: ' This u to certify that the -. thesis entitled ,1: EFFECT or some CHEMICAL AGHH'S upon THE exam or 31mm - . . ; ; BY m: USE or THE PAPER DISC, AGAR PLATE M‘s-tar“ , ~ . ' w n RMINED - ‘ _,_. 94 2 —. -4-__~..-._ _.__ - t L - I. In __ j ,3 |. l _ presented by ' 1 . 5 ‘ ' t ! ~, . . '.* t. . L . ', ‘jé‘ Osmone Hipolito } . :1 =1 :1 "1 V i " /V: ‘ L ' . 1_ has been accepted towards fulfillment ' p . ‘ ’11-»... 2‘ of the requiremcnts for - ' ‘ a M. 8. degree in Bacteriology _‘ . I :. . , Major professor 1. p 1‘1 r I ‘ ,I I F I I u , '. I Date Hey 20. 1955 . - g -'__‘ : x ‘ N . ’ r - , ! 0-169 . .~ _g ' ‘ . 1 1‘ v 2' '1 .’ .2. i 1 , 2 EFFECT OF SOLEE CHE‘fiICAL AGENTS UPON THE GROWTH OF BRUCELLL DETERMINED BY THE USE OF THE PAPER DISC, AGAR PLATE METHOD by Osmane fijpolito A THESIS Submitted to the Graduate School of Michigan State College of Agriculture and Applied Science in partial fulfilment of the requirements for the degree of MASTER OF SCIENCE Department of Bacteriology June, 1953 "rf/ 2/5’3 \ 5 <\-’-' This study was made possible by a grant from the Rockefeller Foundation, New York City. C»: Ir"! \J Huh (3 C ., ('9 V v TABLE OF CONTENTS I. Introduction ...........................oo II. Review of Literature ..................... III. ExPerimental Studies ..................... A. Materials and Method ............... B. Results and Discussion ............. 1. Agents Producing Enhancement Of GrO‘W-th oeeeeeeeeeeeOOOOOOO 2. Agents Producing Inhibition 0f Growth eeeeeeeeeesooeeeoee Iv. smary ......................‘O.......... Appendix eeeooenoeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeo Literature 011:“ eeseeeeeeoeeeeeeeeeeeeeeeeeeeee l 2 10 10 12 15 18 19 22 e I v I I . I I u C O O I O fl 0 n g . U . EFFECT OF SOME CHEMICAL AGENTS UPON THE GROWTH OF BRUCELLA DETERMINED BY THE USE OF THE PAPER DISC, AGAR PLATE METHOD I. INTRODUCTION Studies of the physiological prOperties of cells of the genus . Brucella are now being made as a means of determining the characteristics and properties of this important group of bacteria. A8 a matter of fact, there were no very extensive studies in this field until recently. Some difficulties, of course, have been found and the lack of a good medium of chemically defined composition seemed to be one of great importance. The behavior of this group of organisms differs according to the medium.used and it is rather common to observe remarkable differences in 1 growth of the same strain of organism.in different batches of the same nmdium. The question has received the special attention of a number of in- vestigators and as a result of such interest, an appreciable amount of information has been obtained. II. REVIEW OF LITERATURE The search for a suitable medium of defined composition which pro- duces good growth of Brucella was begun not very long ago. In 1919 Koser and Rettger (15) studied the utilization of nitrogenous compounds of definite chemical composition in culture mediums, but Brucella abortus failed to grow in all of those employed. Zobell and Meyer (143) in a study of the nutrient requirements of Brucella attempted to prepare a synthetic medium that would support growth. They stated that the mineral elements lig and either 313 or _I_(_ were definitely essential and E: was beneficial. The ammonium ion and certain amino acids furnished the E, and the _S_ demands were satisfied by sulfates and thio- amino acids. They were unable to find a medium in which Brucella would produce an appreciable turbidity. In another paper (141;) the same authors, utilizing a synthetic medium found that the Optimum osmotic pressure for the cultivation of Brucella was from 2 to 6 atmospheres and that the best growth occurred between pH 6.6-7.1“ The surface tension of the menstrmnnshowed a marked influence upon the reproduction and morphology of the subsequent cells. Finally, they concluded that “either a large inoculum of viable cells or traces of enrichment substances were necessary to insure the rapid multiplication of Brucella in synthetic medium". Wilson and Miles (140) describing the nutritive requirements of the genus Brucella stated that growth was better after the addition of nat- ural proteins to the medium. The Optimum temperature was 37°C. and pH be- t'eon 6.6.6. 8. .3- working with Bacto tryptose agar as a basic medium Kerby (13) found that the addition of nicotinic acid and thiamin hydrochloride in concen- trations of 0.30 and 0.25 mg. respectively, per liter of medium, enhanced the growth of Pl. abortus. bination of both substances. For routine use the author recommends the comp using a basal synthetic medium.of amino acids, glucose and inorganic acids, Koser, Breslove and Dorfman (16) made a thorough study of the acces- sory growth factor requirements of some representatives of the brucella group. (+) (+) (-) (-) (-) (-) Synthetic basal medium No. h Glycine .................... dl-alpha alanine ........... dldvaline .................. dl-leucine ................. l-lysine dihydrochloride ... l-arginine hydrochloride ... dl-serine .................. dldthreonine ............... d-glutamic acid ............ l-cystine .................. dI-methionine .............. l-hystidine hydrochloride .. l-tyrosine ................. dl-phenylalanine ........... l-proline .................. l-hydrcxyproline ........... l-tryptOPhane eeeeoeeeeeaese The composition of the synthetic medium.used was: Per liter 0.2 g. 0.5 g. 0.1 g. 0.1 g. 0.1 g. 0.1 g. 0.015 g. 0.015 g. 0.5 g. 0.15 g. 0.1 g. 0.2 g. 0.05 g. 0.1 g. 0.1 g. 0.1 g. 0.2 g. C’... I’.' In- KQHPO. ...................... 1.0 g. Ngso. ....................... 0.1 g. NaCl ........................ 6.0 g. Glucose ..................... 3.0 g. The pH of the medium was adjusted to 6.8-7.0 with N/l NaOH. It was sterilized by autoclaving at 15 pounds of pressure for 15 minutes. The significant accessory factors for growth were: thiamin, nicoti- namide, pantothenic acid, and probably biotin. Not all these factors are actually required by all strains. Riboflavin, vitamin Be, adenine, inositol and glutamine did not accelerate cell multiplication. The Optimum amount of sodium chloride was from 0.6 to 1.0 per cent. With 0.1 per cent or less, cultures failed to grow. Since the effect Of so- dium chloride is one of osmotic pressure, potassium.chloride, sodium sul- fate and potassium.sulfate can replace it. Koser and wright (17) confirmed the previous suggestion that biotin is needed by certain brucella cultures. Very small amounts of pure biotin supported growth. About 90 per cent of maximum growth was produced with 0.0001 pg. per m1. of medium.and about half maximum with 0.001'pg. Suc- cessive transplants were Obtained with 0.00001‘pg. per ml. of biotin methyl ester in the presence Of larger quantities Of nicotinamide, panto- thenic acid and thiamin. All the seven brucella strains required the pyrimidine but not the thiazole components of thiamin. Employing the synthetic medium.No. h.of Koser :2 31., McCullough and Dick (21) determined the qualitative and quantitative requirements of three strains Of Brucella in regard to thiamin, nicotinic acid, calcium pantothenate and biotin. The following are considered the Optimum.amounts of these factors for use as accessory factors for Brucella: thiamin 0.2‘ng. per ml., nicotinic acid 0.2‘pg. per ml., calcium pantothenate 0.0h‘pg. per ml., and biotin 0.001 pg. per m1. of medium. -5- Later the same authors (22) made a new investigation on Al recently isolated strains of £3. abortus. Strains requiring an increased carbon dioxide tension were not successfully grown in this medium. After accli- mation to the atmospheric conditions, growth of 50 strains was obtained. Thirty-three strains were carried successfully for 10 transplants in the presence of thiamin and biotin. Both nicotinic acid and calcium panto- thenate enhanced the growth Of some but not all strains. Doneddu (3) investigating the action 33.33212 of small and large amounts Of nicotinic acid on 21;. abortus and _B_t_'_. melitensis found that no influence was exerted upon the morphology and virulence Of such organisms but the substance markedly inhibited the growth of the same organisms. As a result of an investigation of the growth Of Brucella in a simple chemically defined medium, McCullough and Dick (25) obtained growth of seven out Of eight strains used. 'When potassium.nitrate was used as the only source of E no growth of any strain occurred. Roby (50) studying the growth requirements of the genus Brucella found that 25. abortus failed to grow in a broth medium prepared with casamino acids and necessary salts. The growth was enhanced after the addition of tryptOphane, nicotinic acid and glycerine, but glucose had a depressing effect. tAlsO there were no beneficial results from the use of organic carbon furnishing compounds. 1 The use of dl-leucine and d-lysine hydrochloride, either singly or together, resulted in prolonging the viability of the organism. The same author found that thiamin hydrochloride produced a very rapid multiplica- tion at the beginning but death rate was very high. Casamino acids medium plus leucine, lysine, thiamine, tryptOphane, nicotinic acid and glycerin resulted in a very good growth of organisms. Theznedium.employed revealed an increased tendency toward dissociation. The growth of all species of .6- Brucella in casamino acids equalled that in tryptose broth. maximum. growth was Obtained between the third and the fourth day and the number of viable cells was 5 to 8 x 10°. Tryptophane proved to be essential for _B_1:_. abortus and leucine was essential for _B_1;. melitensis. Polding (28) in l9h6 Observed that cells °f.§£f abortus in a dilution of 10-1 and 10-2 of a density equal to Brown's scale 5 grew on liver agar while those in a dilution of 10'3 and upwards failed to do so. He showed that the cause of such behavior was a non-dialysable inhibitor produced during the gentle hydrolysis of liver. The amount of heat during hydro- lysis and the condition ofiliver itself had a marked influence. Shwartzmann (37) investigating the nature of the residence of gram negative bacilli to penicillin,found that,upon the addition of methionine, methylsulfoxide and threonine,there occurs a marked enhancement of penicil- lin susceptibility of broth cultures of Brucella. The enhancement was apparently due to the ability of this amino acid mixture to reverse of- fectively the action of the antagonist present in the cultures. Methionine is essential for the enhancement of penicillin susceptibility. He found also that threonine and methionine sulfoxide facilitate the effect of methionine following a reciprocal quantitative relationship. McCullough 323;. (21;) Obtained .- good growth of a 21;. 33.11 strain in a chemically defined medium.in which thiamin (0.05‘pg. per ml.) and niacin (0.h.yg. per ml.) were stimulatory. Cystine, histidine, tyrosine, phenylalanine and tryptOphane were essential for growth whereas glycine, lysine, arginine, methionine, glutamic acid, isoleucine, aspartic acid, serine and threonine'were stimulatory. .Yeast nucleic acid stimulated early growth. Magnesium.salts were found essential whereas manganese and iron salts were stimulatory. The addition of glucose, thiamin and iron salts to tryptose broth increased yields 5 to 10 fold. .7... In an investigation on the effect of paraminobenzoic acid on Brucella _i_n m Cotton and Swopo (1) found that 2 mg./m1. of PABA carapletely in- hibited the growth Of the three species of Brucella in tryptose agar. The same species suspended in distilled water (I46 million organisms per ml.) were killed in 18 hours in concentration of PABA of 5 to 20 mg./ml. The sodium salt of PABA showed slower action. Based on this prOperty of paraminobenzoic acid the same authors in another experiment (2) tried the agent in the treatment of experimental brucellosis in guinea pigs. When the treatment began three days after infection they obtained 100 per cent sterilization“. When treatment was delayed for two weeks after infection.80 per cent of the animals were negative to cultural examinations. Investigating the nutritive requirements of £5. abortus strain 19, Gerhardt (14,5)prepared a medium containing mineral salts, four accessory growth factors, lactate, glycerol and a single _N_ source. 0f 28 aerobic strains of 21;. abortus, 31:. suis and 133:. melitensis studied, all but four grew in a medium in which the dl-asparagine served as nitrogen source. Yields varied up to 19 billion viable cells per ml. It was found also that l-glutamic acid or l-histidine may substitute for asparagine as the .11 source for growth of _B_r_. abortus strain 19. Gerhardt and others (6,7) reported that two carbon dioxide requiring strains of 2:. abortus grew in a chemically defined medium (with 10 per cent carbon dioxide atmosphere) when glutamic acid was supplied as the 2! source; l-alanine could replace glutamie acid in this respect. Schuhardt _e_t_ :1. (55) found that tryptOphane and cystine showed a high level of toxicity for £5. abortus strain 1257 in a medium containirg no other amino acids. Methionine and phenylalanine showed slight toxicity to the organisms, whereas none of the other 15 amino acids (glutamic acid, -a-' proline, histidine, arginine, lysine, alanine, glycine, serine, aspartic acid, hydroxyproline, threonine, tyrosine, valine, leucine and isoleucine) of the casein digest series proved toxic at concentrations well above those found in l or 2 per cent casein digest. Forty-two strains of EEP abortus showed markedly less tolerance for cystine than did 10 strains of .EE..=2£5. In another investigation Schuhardt E: 31. (52) found an antibrucella factor in certain lots Of tryptose used as a liquid medium. .EE‘.EEEE and and BE. melitensis cultures exhibited varying degrees of susceptibility to the toxic factor. Oxidized polypeptides or amino acids were suspected as the toxic agents. The oxidative dissimilation of 27 amino acids and related compounds bY.§£° abortus was studied by Gerhardt and others ( 6). They found that only 1-g1utamic acid and l-asparagine and d- or l-alanine were oxidized at appreciable rates by strain 19 of 2:. abortus. Schuhardt and associates (5h) studying the development of peptone toxicity for Brucella with aging and the correlation Of this toxicity with the probable oxidation Of cystine were inclined to conclude that a breakdown product of cystinexnight be the antibrucella factor in toxic tryptose and other peptones. Later the same authors (55) found that probably the cause Of toxicity of cystine is due to degradation after heating and production of elemental sulfur. I The role of d-alanine in the growth and variation of fig. abortus was studied by Goodlow and associates (9) and they reported that this compound, rather than the l-form was responsible for the limitation of growth of smooth-type cells and creation of an environment favoring pro- gressive establishment Of nonsmooth mutants with greater resistance to alanine. -9- Yaw and Kakavas (he) reported that the growth in Albimi broth or two non-carbon dioxide requiring strains and one carbon dioxide requiring strain of EE‘ abortus were inhibited by d-amino acids at levels at which the l-forms do not exhibit growth. Addition of d-phenylalanine and d- methionine caused the greatest amount of inhibition. -10.. III. EXPERIMENTAL STUDIES The present investigation was undertaken to study a large number of chemical agents as to their enhancement or inhibition effect on the colo- nial growth of E5. abortus in two agar culture mediums, namely tryptose agar and beef liver agar. Tryptose agar has been used for many years for colonial growth studies while beef liver agar, first described by Stafseth (58), has been employed for mass growth of Brucella. It has been known for many years that colonial growth of 2:. abortus will not occur when the surface of liver agar is inoculated with 2,000 or less living cells. A. Materials and Method. l. Cultures. A smooth type of 2:. abortus strain 2508 was used in all experiments but in some cases the following strains were used: '25. abortus 5028, BE. suis 1776 and BE. melitensis 2500. All of the cultures were from the collection Of the Brucella Laboratory, Michigan State College. 2. Mediums. The mediums used throughout the investigation were: tryptose agar and beef liver agar. The effects Of two agents were also studied in Albimi's brucella agar and in trypticase soy agar. 5. Chemical agents. For convenience Of study the chemical agents used were divided into the following groups: I. Amino acids II. Vitamins III. Carbohydrates IV. Nucle0proteins, purines, pyrimidines -11- V. Inorganic salts, acids and bases VI. Miscellaneous chemicals h. Method. In the present investigation a paper disc, agar plate method similar to the technique described by LOO and others (19) was used. The surface of Petri plates containing either tryptose or liver agar ‘was inoculated with 1 ml. of a.1iquid containing approximately 10‘ cells/ ml. As soon as the liquid covered the surface of the medium the excess 'was removed. If the plates were incubated on a flat surface, the inoculum produced from 500 to 800 separate colonies. Filter paper discs (SS 7h0 E, 12.7 mm. diameter) were used as sup- port fcr the agent in the experiment. These disc pads absorb moisture from the medium very rapidly and for this reason they must be placed on the agar plate just before adding the substance. The disc was placed in the center of the inoculated agar surface and 0.1 ml. of the agent was added to the disc. Unless otherwise mentioned all the agents used were prepared as l per cent solutions. The plates were incubated at 57°C. and the readings recorded after 2h, h8 and 72 hours of incubation. The diameter of the colonies around the disc was measured and compared with that Obtained in control areas on the same plate. In the case of agents producing enhancement or inhibition of growth the diameter Of the area around the disc was recorded together with other data. Although it was known that one of the agar mediums, namely tryptose, contained sufficient nutrients to support colonial growth of 2:. abortus, it was thought that the diffusion of the agent from.the paper disc into the agar medium might increase the growth and thus reveal a substance in ‘which the medium was deficient. In the case Of'liver agar it was hoped that one or more substances could be found which the medium required to produce colonial growth. TABLE I CHEMICAL AGEHTS STUDIED Agent No. Amino Acids Sol. pH 1 dl-valine + 2 dl-threcnine + 5 dl-methionine + h d-lysine monohydrcchloride + 5 dl-phenylalanine + 6 l (+) lysine monohydrcchloride + 7 l (+) arginine monohydrcchloride + 8 dl-serine + 9 l-glutamic acid ‘ + 5.5 10 l-tyrosine - ll dl-isoleucine + 12 l-histidine monohydrcchloride + h.01 l5 l-aspartic acid - 1.07 1h l-hydrcxyproline - 15 1—tryptophane - l6 glycine + 17 d-leucine - 18 dl-norvaline + 19 l (+) arginine + 20 l-hydroxyproline + 21 l (+) cysteine hydrochloride - 1.7 22 d-methionine + 25 ldmethionine + 2h dl-crnithine monohydrcchloride + 25 dl-djenkolic acid - 26 betaine + 27 ethionine + 28 dl—crnithine + 29 dl-citruline + + - soluble - - insoluble Sol. - Solubility in water TABLE II CHBJI CAL AGENTS ST UDIED Agent No. Vitamins Sol. pH 50 Nicotinic acid + 3.h5 51 Nicotinamide + 52 Paraminobenzoic acid - 5.68 55 Riboflavin - 5h Biotin e 35 Ascorbic acid 2.75 56 Thiamin hydrochloride + 57 i-inositol + 58 Vitamin 136 hydrochloride + 3.11 59 Vitamin A acetate (2) hO Alpha tOOOpherol 141 Vitamin B 12 (3) AZ : 2 methyl-naphthcquinone (vitamin K2 - # (Menadione) Merck + 3 soluble Sol. ' Solubility in'water - I insoluble (1) - 2.5 ps- (2) - 105 units (3) - 1.5 lie. TABLE III CHEIICAL AGENTS STUDIED Agent NO. Carbohydrates Sol. h5 Dextrin + hh Sucrose + h5 l-arabinose + L6 d-galactcse + h? d-xylose + hB mannose + h9 d-manitcl + 50 d-levulose + 51 lactose + 52 i-eurythritol + 55 adonitol + 5h dulcitcl + 55 d—ribose + 56 d (+) raffinose + 57 glycerol + + - soluble - - insoluble Sol. - Solubility in water TABLE IV CHEMICAL AGENTS STUDIED Agent No. Nucleoproteins, Purines, Sol. pH Pyrimidines 58 Sodium desoxyribonucleate + 59 Xanthine - 60 Uracil - 61 Adenine sulfate ,- 2.92 62 Guanine hydrochloride 63 Gluthathicne + 6h Sodium.nucleate * + ' soluble - - insoluble Sol. ' Solubility in water. TABLE‘V CHEMICAL AGENTS STUDIED Agent NO. Inorganic Salts, Acids and Gases Sol. 65 Lead acetate + 66 Sodium hydroxide N/l solution + 67 Hydrochloric acid N/l solution + 68 Sodium phosphate, di-H + 69 Sodium phosphate, mono-H + 70 Sodium pyrcphosphate + 71 Sodium hydroxide N/lOO solution + 72 Hydrochloric acid R/lOO solution + + - soluble - = insoluble Sol. ' Solubility in water TABLE VI CHE‘AICAL AGENTS STUDIED Agent NO. Miscellaneous Biochemicals Sol. 75 Pimelic acid + 7h d (+) glucosamine hydrochloride 75 thicuracil 76 Sodium taurocholate 77 Urea + 78 Laked blood (rabbit) (l) + 79 Hemoglobin + 80 Hemin + 81 Phenylacetic acid 82 2 thio 6 oxypyrimidine (Deracyl) - + ' soluble - - insoluble (l) - see table II SOle Solubility in water -12- B. RESULTS AND DISCUSSION 1. Agents Producing Enhancement of Growth The procedure employed revealed few agents that enhanced the growth of Brucella abortus on tryptose agar. The four agents that enhanced growth to some degree were arginine, mannitol, uracil and nicotinic acid. The results in colony size are shown in Table VII. Arginine and uracil caused a greater increase in the size Of the colonies than the other two agents. For this reason the forementioned agents were studied further. a. Growth enhancement action of arginine and uracil. In order to determine the most suitable concentration of arginine for promoting growth, different amounts were added to tryptose agar and inoculated as previously mentioned. The size of colonies that develcped were measured and compared with those on a control plate without arginine. The data (Table VIII) showed that the best results was Obtained with 0.1-0.2 g./ 100 ml. of medium. With this concentration colonies could be detected in less than 211 hours and this observation confirmed the importance of arginine as enhancement agent for cultures of _}_3_r_. abortus (Fig. 1.) NO enhancement of growth was obtained when _B_r_. 3.1.13.1 strain 1776 and 21;. abortus strain 5028 were used as test cultures. With 25‘ melitensis 2500 only a slight enhancement of growth was obtained in tryptose agar as well as in beef liver agar. Arginine was added to the chemically defined mediums used by Koser, Breslove and Dorfmann (loo mg./l.) (16), McCullough 23 3_1_. (82 mg./l.) (214) and Rode, Oglesby and Shuhardt (100 mg./1. of medium) (51) but no mention was made as to its ability to promote enhancement of growth. TABLB'VII some PRODUCING me man or Gama OF .133. morons ON TRYPI'OSE AGAR Diamet f Agent 8 hrs. 72 hrs. 1 (+) Arginine monohydrcchloride 0.15-0.25 0.9-1.2 Control 0.10 0.7-0.8 1 (-) hairline 005.006 100.102 Control 0.1 0.7-0.8 UT‘Oi-l 0.5-0.6 102-1014 Control 0 0.5-0.6 a (-) Manitol 0.25-0.30 0.9-1.1 contrOI 0.15-0020 008-009 Nicotinic acid 0.10-0.15 0.8-0.9 contrO]. 0. 10.0015 006-008 O - No detectable colony TABLE VIII EFFECT OF DIFFERENT CONCENTRATIONS OF ARGININE IN TRYPI'wE AGAR 0N MOMENT CF GROWTH OF BRUCELLA Diameter of colonies (in mm.) Grams/100 ml. 55 hrs. he hrs. 72 hrs. 002 0010-0015 0.6-0.7 1.0-1.1 Control 0 0.3-0.1; 0.9-1.0 0.1 0.10.0015 0.6-0.7 1.0-1.1 Control 0 0.10-0.15 * 0.7-0.8 ' 0.01 0 0.15‘0020 0.8-0.9 contrO]. O 0.10.0015 007.008 0.001 O 0e10’0e15 Oe7-‘Oe8 Control 0 0.10-0.15 0.7-0.8 O - No detectable colonies As one can see from.the data in Table VII, uracil was one of the most important growth enhancement agents. In order to find the optimum concentration for promoting growth the agent was added in different con- centrations to tryptose agar. The results obtained are recorded in Table. IX. According to these results the best enhancement effect was obtained when 0.05 g. of the agent was added to 100 ml. of medium. In comparison with arginineg‘uracil gave better results with a .. low”: concentration. Autoclaving of the medium containing uracil or arginine did not change the growth enhancement effect of either agent. In order that the effect of uracil on the growth of 2:. abortus could be measured in a liquid medium, an experiment was carried out on the fol- lowing basis: Medium G1 ./100 m1. Tryptose 2.0 NaCl 0.5 Dextrose 0.5 Thiamin 0.0005 Dissolve by stirring. Filter through paper. Adjust to pH 6.8-6.9 with 2 per cent phosphoric acid. The medium was divided in several portions to which the following amounts of uracil were added (0.01, 0.05 and 0.10 g./100 m1. of medium). One portion of the medium tO'which no uracil was added was used as control. The mediums were distributed in culture tubes in 101ml. amounts. After sterilization the tubes were inoculated with 3 x 105 cells of 2:. abortus and incubated at 57.0. TABLE II EFFECT OF DIFFERENT CONCENTRATIONS 0F ,URACIL IN TRYPTOSE AGAR 0N ENHANCEMENT 0F GROWTH OF BRUCELLA Grams/100 m1. Diameter of colonies (in 111111,), 21;, hrs. L8 hrs. ’ 72 hrs. 0.05 0.2-0.3 0.2-0.3 0.9-1.0 0.1 0.2-0.3 0.2-0.3 0.8-0.9 0.2 0 0.3-0.1; 0.8-0.9 Control 0 0.1-0.2 0.6-0.7 To obtain a viability count, plates were made LLB hours after the first turbidity was recorded. The results can be seen in Table I. The best result was obtained when 0.01 g. of uracil was added to 100 m1. of the medium and it is interesting to note that with this amount the num- ber of colonies is nearly twice those found on control plate. 0n the other hand, with 0.10 g. of uracil per 100 ml. of medium the number of colonies was greater than those obtained with 0.05, that is, just the Opposite found in solid medium. Arginine and uracil were also added to Albimi's brucella agar and trypticase soy agar and the surface inoculated with a suspension of _B_1_'.. abort.u_s;. These agents were unable to enhance growth on either medium. b. Growth enhancement action of laked blood and sodium sulfite. Pre- vious results from the use of laked rabbit blood in paper discs, as pre- viously described, have shown that the solution obtained from laked erythro- cytes enhanced the growth of 21;. abortus in beef liver agar. In order to evaluate its influence, experiments were carried out with laked rabbit blood in liver agar medium. Seven m1. of blood were collected by intracardiac puncture placed in sodium citrate solution and centrifuged to remove the plasma. Distilled water was added in the proportion of four ml. for each m1. of original blood to lake the blood. The solution was divided in two portions and one was filtered through a D8 Hormann pad. The laked cell solution was added to the liver agar medium in the proportion 2, l, 0.5 and 0.2 ml. to each 50 m1. portions of medium at the time of pouring the plates. The data are recorded in Table XI. The results of the experiment showed that when sufficient laked rabbit blood was added to beef liver agar medium colonial growth was produced. TABLE II. ENHANCWENT EFFECT OF URACIL ON 2%. ABORTUS Amount of uracil in the medium 10' dilution 0.10 11h colonies 0.05 192 colonies 0.01 262 colonies Control 1h? colonies TABLEIKI cams moms moor or LAKING BLOOD IN LIVER AGAR MEDIUM 0N pg. memos gg,) in Dilution 2h hours fiB hours 72 hours Non filtered 2:50 0 0.10-0.15 0.6-0.7 1:50 0 0 0 0.5350 0 0 0 0.2:50 0 0 0 Filtered 2:50 0 0.10-0.15 0.6-0.7 1:50 0 0 0 Ooh-.50 O 0 0 0e2350 O O 0 Control No laked blood 0 0 0 -15- Sodium sulfite, used in the same concentration as the other agents, that is, 0.1 ml.¢0f a one per cent solution, produced a remarkable en- hancement effect upon the growth of 2:. abortus in beef liver agar medium. In 72 hours the diameter of the area of growth was 20 mm. (Fig. 2). 2. Agents Producing Inhibition of Growth As one can see from.the examination of table XII, several agents were found to produce inhibition of growth of 31;. abortus. Vitamins £2 and A gave excellent results and deserved a more detailed study in the present investigation. many of the substances were found to be inhibitors, due to the very low or very high pH change around the disc. This was Specially true of sodium.hydroxide and hydrochloric acid normal solutions. For this reason the pH of some agents as well as the solubility in water were recorded together with other data. - Nothing can be said about the ability of such agents to diffuse through the medium.but it is probable that a high significance can be afforded to this factor. a. Growth inhibiting action of vitamins K andgga. It is known that vitamin E exists in some organisms and is essential for growth of the Johne's bacillus. In 19h} Ter Horst and Felix (59) found the marked antifungal action of 2,3 dichloro-l,h-naphthoquinone and the idea occurred that this effect might be linked with the structural similarity of this compound to that of vitamin K. After this observation vitamin E and related compounds have been extensively tried on different kinds of bacteria. Studies of this agent on_gycobacterium.tuberculosis have been carried out by Piso (27), Illand 33 £3. (12), Kimler (1h), Pichat and Minjat (26), and others. TABLE II I AGENTS PRODUCING INHIBITION OF’GROWTH 0? BR. ABORTUS 0N TRYPTOSE'AGAR ‘ L Inhibition in 72 hrsfiifi Agents Inhibition in1h8 hrs} _ Colony size Diameter Colony size or lemio a°1d 0 0e2-003 2207 Control 0.1 0.7-0.8 Paraminobenzoic acid 0 0.1-0.2 22.7 COHtl'Ol 0 0e7'0e9 Hystidine monohydrcchloride 0 0.h-0.5 22.7 Control 0.2-0e3 007-008 l-aspartic acid 0 0.2-0.3 22.7 Control 0 007‘Oe9 Guanine hydrochloride 0 0.1-0.2 h2.7 Control 0 0.7-0.9 Nicotinic acid 0 0.1-0.25 Control 0 0.6-0.7 dl-threonine 0.1 0.h-o.6 contrOI 0.10-0015 0e7'008 Niacin 0~ 0.1-0.3 22.? contrO]. 0.10-0.15 0.7-0e8 Adenine sulfate 0 0.1-0.2 22.7 Control 0 0.6-0.8 Vitamin B6 hvdrochloride n " ’ “ ' “ ’ -16- Sohwartzman (56) in a study of the antibacterial preperties of h, amino 2, methyl-l, naphthol hydrochloride found that this substance pos- sessed marked antibacterial activity against gram-positive and gram-nega- tive organisms. The compound is strongly effective against gram-positive organisms in the presence of broth, casein hydrolysate, and blood serum and against gram-negative organisms in synthetic medium. The activity against the latter organisms is antagonized in broth medium. Pratt gt =1..(29) suggested the use of vitamin K5 (2, methyl-h, amino- 1, naphthol HCl) as antimicrobial medicament and preservative for pharmaceu- ticals, food and beverage due to its ability to inhibit the growth of pathogenic and saprophytic bacteria, yeasts and fungi, as it has low toxicity. Mule' (25) in 1946 reported that vitamin Eflm showed antibiotic action in liquid as well as solid medium.for a large group of bacteria, Brucella included. Maestrone (20) studying the association of paraminobenzoic acid with oloramphenicol in the therapy of experimental brucellosis of rats reported that these substances, in daily doses of 50-200 mg./kg. of 1 iving weight do notprotect white rats against the acute experimental infection byegg. melitensis and EE‘ abortus which kills controls in 3—6 days. In preliminary tests vitamin K2 (2-methyl-naphtoquinone) was found to produce a considerable inhibition of growth of 2:. abortus. In order to . evaluate the extent of inhibition a dilution method was used in liquid culture medium. From a l per cent solution, dilutions were made in tryp- tose broth. To each dilution and a control tube was added 15 x 106 cells of 25. abortus. Similar dilutions were used for El. suis and 2:. melitensis. The tubes were incubated at 37°C. and growth was recorded by + signs. -17- The data (Table XIII) showed that complete inhibition was observed in 72 hours in dilution of 1.6 x 10"“, 8 x 10'", and 2 x 10" for £5. abortus, _I_3_l_'_. suis and 135-. melitensis, respectively. b. Growth inhibiting action of Vitamin 33 Based on the knowledge of the anti-infective prOperty of vitamin _A_, it was decided to try this agent in different forms to see its effect upon the brucella group. As one can see by the examination of Tables XIV and XV and Fig. 3 vitamin A exhibited a very marked inhibition upon this group of organisms. The best result was obtained with the greatest concentration of the agent (0.1 ml.) and after L18 hours of incubation. There was no significant difference among different ester forms of the vitamin, but the concentration used as well as the time of incubation were important factors. Reviewing the literature concerning the prOperties of this vitamin no reference was found as to its inhibition power toward Brucella. Wolbach and Howe (Lil) stated that ”specifically lack of vitamin _A_ causes atrOphy of the epithelium with substitution of a stratified kera- tinized epithelium forithe normal epithelial structure. Due to this effect on the mucous membrane of the mouth and the salivary glands, there is a great susceptibility to infections". The mechanism that underlies the decreased resistance of animals deprived of vitamin A is not known. ”There must be some defect in the defense mechanism of the tissues - in the efficient functioning of the reticulo-endothelial system itself or in the conditioning or access-ry action of the humoral antibodies - but of thenature of that defect we have as yet no knowledge" (1.10). TABLE XIII moor or 2453an NAPHTHOQUINONE (VITAMIN Ii.) ON THE cams OF ransom Incu- ” Dilution bation q. -5 (hours) Species 10 2116‘ 1316‘ sxld‘ 1.6110 3.2::16" Control 2h Br. abortus - - - - - + + ha - - - - - ++++ ++++ 72 . - - - - - ++++ ++++ 2h ~Br. suis - - - - ++ ++ ++ us - - - - ++++ ++++ ++++ 72 I - - - - ++++ ++++ ++++ 2h Br. melitensis - - - - + ++ ++ hB - - +++ +++ ++++ ++++ ++++ 72 - - ++++ ++++ ++++ ++++ ++++ - - No growth + - Growth No. of + signs indicate intensity of turbidity by eye. TABLE XIV EFFECT OF VITAMIN A ACETATE (loa UNITS/0..) ON GROWTH OF 31;. ABORTUS, BR. SUIS AND BR. MELITENSIS (PAPER DISC METHOD) Inhibition zone in mm. Species Amount 2b hrs. h8 hrs. 72 hrs. Br. abortus 0.02 ml. 28 (strain 2308) 0.0h ml. 23 0010 ml. ’40 Control 0 O 0 Br. suis 0.02 ml. 0 25 20 (strain 1776 B) 0.10 m1. 0 51 31 Control 0 O 0 Br. melitensis 0.02 ml. 0 25 15 (strain 2500 0.10 ml. 0 ho 30 Control 0 O ‘ O O = no detectable colonies. TABLE XV EFFECT OF DIFFERENT VITAMIN A COMPOUNDS ON CRONTH 0F 23. ABORTUS (PAPER DISC METHOD) Concentration Zone of Name of the compound and amount inhibition 1. Vitamin A acetate 109A§;% 0.3 ml. 20 mm. 2. Vitamin A crystalline 0.1 ml. 18 mm. (Alcohol) 3. Vitamin A ester 21104 0.1 ml. 19 mm. (concentrate) 1=1o6 units per gram. -18- IV . S [1131le .A study was made on the behavior of nearly 80 chemical agents upon the growth of Brucella by the use of the paper disc, agar plate method. Enhancement or inhibition of growth was obtained with only a small number of agents, some of them.in high degree. In order to evaluate the effect of sunh agents, the size of colonies as well as the diameter of inhibition or enhancement around the disc was measured and compared with those in control areas in the same plate. In the group of agents producing enhancement of growth arginine and uracil gave the best results for Brucella abortus in tryptose agar medium. Laked blood (rabbit) and sodium.sulfite enhanced growth of the same or- ganism either in tryptose agar and in beef liver agar. Among the agents producing inhibition of growth vitamin K; and vitamin A gave excellent results. The concentration of such agents was a decisive factor. The diffusion of the agent through the medium seems to be an impor- tant factor as well as the water solubility and pH. many substances in- hibiting growth did so due to the very low or very high pH around the pad. No one agent was found to produce enhancement of growth of the three species of the genus Brucella in tryptose agar as well as in different kinds of mediums. The paper disc agar plate method proved to be a cheap, easy and suf- ficiently accurate method of measuring the ability of many substances to influence the growth of organisms of the genus Brucella. F150 10 -19- Showing zone of enhancement of growth of Brucella abortus produced by 0.1 m1. of l per cent solution of arginine in tryptose agar. Incubation period LB hours. -20- Fig. 2. Showing growth of enhancement effect of sodium.sulfite upon growth of Brucella abortus on beef liver agar. Incubat on perIod 96 hours. Fig. 3. -21-‘ Showing nine of inhibition of Br. abortus produced by 0.1 m1. of 10° uni'EE/g‘T‘; 0 vi" . A on growth of Brucella abortus. Incubation period 72 hours. 1. 2. 3. h. 5. 7. 8. 9. 10. ll. -22.. V. LITERATURE CITED Cotton, 0. M., and R. E. Sw0pe. Brucella chemotherapy. I. Studies on the effect of paraminobenzoic acid on Brucella £3 vitro. Amer. Jour. vet. Res., 9: 16h-168, 19h8. Brucella chemotherapy. II. Para- minobenzoic acid in the treatment of experimental brucellosis in guinea-pigs. Amer. Jour. Vet. Res., 9: 169-172, l9h8. Dcnedu, 0. The action in vitro of small and large amounts of nicotinic acid on'tfie‘Engnisms of the typhi-paratyphi-coli and brucella group. 801. Ist. Sierot. Milan., 22: 239-2h5, l9h3. Gerhardt, P. The nutrition of brucelleaec growth in simple chemically defined media. J. Bact. 56. l7-2h, lgba. The metabolism ofamino acids and related compounds by bruceIleae. Unpublished Ph.D. thesis. University of'Wisccnsin, 19h9. Gerhardt, P., H. B. Levine, and J. B.'Wilson. The oxidative dis- simillation of amino acids and related compounds by Brucella abortus. J. Bact. 60: h59-h67, 1950. Gerhardt, P., L. A. Tucker, and J. B. Wilson. The nutrition of brucelleae: utilization of single amino acids for growth. J. Bact. 59: 777-782, 1950. Goodlow, R. J., L. A. Mika, and W} Braun. The effects of metabolites upon growth and variation of Brucella abortus. J. Bact., 60: 291-300, 1950. Goodlow, R. J. and others. The role of d-alanine in the growth and variation Of.§' abortus. Arch. Bioch., 30: h022h06, 1951. Hoyer, B. H. Some aspects of the physiology of brucella organisms. Brucellosis. American Association for the advancement of Science. 1950. Huddleson, I. F. Brucellosis in Man and Animals. Revised edition. The Commonwealth Fund,,New York. 379 pp, l9h3. Illand, C. N. Effect of antibacterial analogs of vitamin K on M cobacterium tuberculosis. Farm. Sci. Tec. (Pavia), h: 2&3'577. I939. Seen in afistract only. Kerby, G. P. Nicotinic acid and thiamin hydrochloride as growth promoting factors for Brucella. J. Bact., 37: h952h99, 1939. Kimler, A. The_1_!_1_v‘itr.o analogs of vitamin K on ljyccbacterium tuberculosis var. hominis, strain H37 RV. J. Bact., 50: 1:59-57: s :9; 0 15. 16. 17. 18. 19. 20. 21. 22. 23. 27. 28. 29. -23- . Koser, S. A. and L. F. Rettger. Studies on bacterialllutrition. The utilization of nitrogenous compounds of definite chemical composition. J. Inf. Dis., 2h: '301-321, 1919. Koser, S. A., B. B. Breslove and A. Dorfman. Accessory growth factor requirements of some representatives of the brucella group. J. Inf. Dis., 698 1111-1214., 1914.1. Koser, S. A. and M. H. wright. Further experiments on accessory growth factor requirements of the brucella group. J. Inf. Dis., 71. 86-88, 191120 Libby, R. L. A modified photronreflectometer for use with test tubes. Science, 93: h59-h60, 19hl. Loo, Y} H. 33 a1. Assay of streptomycin by the paper disc plate method. ”I: Bact., 50: 701-709, 19145. Maestrone, G. P. L'associazione dell'acido paraminobenzoicc com i1 cloramfenicolo nella terapia della brucellosi sperimentale dell ratto. Arch. vet. Ita1., 3s 389eh18, 1952. McCullough, N. B. and L. A. Dick. Physiological studies of Brucella. I. Quantitative accessory growth factor requirements of certain strains of Brucella. J. Inf. Dis., 71: 193-197, 19h2. Physiological studies of Brucella. II. Accessory growth factor requirements of recently isolated strains of Brucella abortus. J. Inf. Dis., 71: 198-200, 19h2. Growth of Brucella in a simple chemicalIy defined medium. Proc. Soc. Exp. Biol. Med., 52: 310-311. 19h3o McCullough, W} G. et al. Studies on the nutritional requirements of 3.3:. suis. J. B'Ec't'T, 53: 5-15, 19m. Mule, F. Antibiotic action of vitamin K. Policlinico (Rome), Siz prat., 53. 653.655, l9h6. Seen in abstract only. Chem. Abs. Lbs 10799 h, 1950. Pichat, P., and B. Minjat. Action of certain vitamins on Koch's bacillus _i_n vitro. Compt. rend. soc. biol. 1143: 508-509, l9h9. Seen in abstract only. Chem. Abs. h3: 5097 f., 19h9. Piso, I. Synergistic antibiotic action of p-aminobenzcic acid and vitamin K in experimental tuberculosis. Farm. Sci. Tec. (Pavia) h: 273-278, 19h9. Seen in abstract only. Polding, J. B. Some peculiarities in the germination of Brucella. Jo Comp. Patho, 56‘ 215-256, 19146. Pratt, R. 22.31. Vitamin K5 as an antimicrobial medicament and preservative. J. Amer. Pharm. Assoc., 39: 127-13h, 1950. 30. 31- 32- 33- 37. 38- 39. ho. lilo h2e h}. .eb- Roby, T. 0. A study of the growth requirements of the genus Brucella. Unpublished. M. S. thesis. Michigan State 0011938, 1914110 Rode, L. J., G. Oglesby and V. T. Schuhardt. The cultivation of brucelleae on chemically defined medium. J. Bact., 60: 561-668, 1950. Schuhardt, V. T. et'gl. An antibrucella factor in peptones. J. Bact., 58: 1-8, 197:9". Schuhardt, V. T., L. J. Roie, and G. Oglesby. The toxicity of certain amino acids for brucelleae. J. Bact., 58: 665-67h, 19h9. Schuhardt, V. T., gt;gl. The development of peptone toxicity for ‘ brucelleae with aging and the correlation of this toxicity with the probable oxidation of cystine. J. Bact., 60: 655—660, 1950. Toxicity of elemental sulfur from brucelleae. J. Bact., 63: 123-128, 1952. Shwartzmann, G. Antibacterial properties of h-aminc-2-methyl, naphthol hydrochloride. Proc. Soc. Exp. Biol. Med., 67: 37 -5780 19148e Shwartzmann, G. Studies on the nature of resistance of Granlnegative bacilli to pencillin. Antagonistic and enhancing effects of amino acids. J. Exp. Med., 83: 65-88, l9h6. Stafseth, H. J. Studies in infectious abortion. II. Technical Bulletin No. h9. Michigan Agricultural Experiment Station, 1920. In Huddleson (ll). Ter Horst,'W. P. and E. L. Felix. 2,3-dichloro-1, h-naphthoquinone. A potent fungicide. Ind. Eng. Chem. 314. 1255-1259, 1919. Wilson, G. S. and A. A. Miles. Topley and'Wilscn's principles of bacteriology and immunity. 3rd edition. The'Williams and Wilkins Co., Baltimore. 2 vols., 19h6. 'Wblbach, S. B. and P. R. Howe. Tissue changes following deprivation of fat-soluble A vitamin. J. Exptl. Med., 112: 753-777, 1925. Yaw, K. E. and J. C. Kakavas. Studies on the effects of d-aminc acids on Brucella abortus. J. Bact. 63: 263-268, 1952. Zobell, C. E. and K. F. Meyer. Metabolic studies on the brucella group. VIII. Nutrient requirements in synthetic mediums. Jour. Inf. Dis., 51: 3hh-360, 1932. Metabolic studies on the brucella group. Mysfcocheiical requirements in synthetic mediums. Jo Info Dis., 51. 361-381, 1932. ACKNOWLEDGMENT The author wishes to express his sincere thanks to Dr. I. Forest Huddleson under whose lofty inspiration, constant supervision and unfailing interest this investigation was undertaken and to whom the results are herewith dedicated. Grateful acknowlegement is also due to the workers at the Brucella Laboratory of the Michigan State College, especially to Drs. EVelyn Sanders and Marvis Richardson. The writer deeply appreciates the financial support of the Rockefeller Foundation and the "Universidade Rural do Estado de Minas Cerai.‘ (Brazil) which made it possible for him to complete this investigation. EFFECT OF SOME CHEMICAL AGENTS UPON THE GROWTH OF BRUCELLA BY THE USE OF THE PAPER DISC, AGAR PLATE METHOD by Osmane Hipolito AN ABSTRACT 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 MASTER OF SCIENCE Department of Bacteriology Year 1953 ApproveM-‘M M Osmane Hipolito ABSTRACT The investigation was undertaken with the purpose of observing the be- havior of nearly 80 chemical agents upon the growth of Brucella. The agents studied were: I. Amino acids; II. Vitamins; III. NucleOproteins, Purines, and Pyrimidines; IV. Carbohydrates; V. Inorganic acids, salts and bases and VI. Miscellaneous chemicals. A paper disc, agar plate method, similar to the technique described by Loo and others (1) was used. Plates of tryptose agar and liver agar medium were inoculated with 1 m1. of a suspension containing 10‘ cells/ml. of a culture of Brucella abortus strain 2308. In some cases cultures of EE' suis and Br. melitensis were also used. After removal of the excess of liquid and complete dryness of the plate surface, filter paper discs (SS 7h0 E. 12.7 mm. diameter) were used to receive 0.1 m1. of the agent. Unless otherwise-mentioned all the agents were tried in a 1 per cent solution. The plates were incubated at 37°C. and the readings recorded after 2h, A8 and 72 hours. The diameter of the colonies around the disc was measured and compared with that obtained in control areas on the same plate. In the case of agents producing inhibition or enhancement of growth the diameter of the area around the disc was recorded together with other data. In the group of agents producing enhancement of growth arginine and uracil gave the best results for 2:. abortus in tryptose agar medium. Laked blood (rabbit) and sodium sulfite enhanced the growth of the ammo organism in tryptose agar and in beef liver agar. Among the agents producing inhibition of growth vitamins A and K2 gave excellent results. The concentration of such agents was a decisive factor. The best result for vitamin A was obtained when 105 units was used in the Osmane Hipolito paper disc. The diffusion of an agent through the medium.seems to be an important factor as well as the solubility in water and pH of the solution. Many substances inhibiting growth did so due to the very low or very high pH around the pad. I No one agent was found which produced enhancement of growth of all three species of the genus Brucella in the several different kinds of agar mediums used. The paper disc agarlplate method proved to be a cheap, easy and suf- ficiently accurate method of measuring the ability of many substances to influence the growth of organisms of the genus Brucella. REFERENCE (1) Loo and others. Assay of streptomycin by the paper disc plate method. J. Bact., 508 701-709, 1914.5. .IA .. 1' a“ J . . . ..-: I P f: '7. be. 3 . l ,. e A». a [32:1 a J's-a .- ..‘WW'T ‘9fitha' I o I - . It ' ' . s ‘yyu - ,. MICHIGAN STAT l E 31293 NIVERSITY LIBRARIES IIIIIIIIIIII 015 2879 fl 0