STUDIES OF THE EFFECTS OF OXYGEN ON MULTIPLICATION AND METABOLISM OF BRUCELLA By Ruth Evelyn Sanders A THESIS Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Sciencfe in partial fulfillment of the requirements'' for the degree of DOCTOR OF PHILOSOPHY Department of Baoteriology and Public Health Studies of the Effects of Oxygen on Multiplioation and Metabolism of Brucella I, II* Introduction................ . Experiments and Results A. Preliminary studies of me d i a............ B. Effaots of atmospheric gases..,.......... C» III* ....3 8 1, Multiplication.............................. 10 2* pH changes.......... 3. Glucose decomposition............. Metabolism........... ......13 ..11+ 15 1* Glucose ana thiamine......... 17 2* Pyruvate and lactate 17 3« Volatile acids................ 19 !+. Carbon dioxide and ammonia................ ..19 Discussion......... IV * Summary V* .................1 23 ...... Literature Cited............. .........27 28 STUDIES OF THE EFFECTS OF OXYGEN ON MULTIPLICATION AND METABOLISM OF BRUCELLA The emphasis which hag been placed on constituents of culture media has overshadowed the Importance of certain other faotors which influenoe multiplication and metabolism of mioroorganisms* Nutritional studies of the Brucella organisms have not solved the problem of pro­ duction of large numbers of cells in a liquid medium* An investigation of this problem resulted in a study of the effect of atmospheric gases on the growth of Brucella in tryptose peptone liquid media* It has been found that the oxygen demand of these organisms is extremely high and has been a limiting factor in growth in liquid media* Brucella cells have been grown in large numbers in a short period of time, in a simple and practical liquid medium In. an. atmosphere of pure oxygen* Studies of the nutritional requirements of the Brucella group in various base media have indicated that thiamine* nicotinic aoid, bio­ tin and pantothenio acid are either essential or stimulatory (17 , 21 , 22, 27, 28, 29, 30)* phane* Roby (3 6 ) included leucine or lysine and trypto­ McCullough et al* (30) reported that cystine, histidine, tyro­ sine, phenylalanine and tryptophane were essential amino acids and glycine, lysine, arginine, methionine, glutamio aoid, isoleuoine, as­ partic acid, serine and threonine were stimulatory. These authors also found Mg salts essential, Mn and Fe stimulatory, and one per cent glucose required for maximum growth. Glucose has often been added to various media to enhance the growth of Brucella* McAlpine (26) reported utilisation of glucose by aomo strains of Bruoella but not; all® Soule (37) found that two per cent glucose in veal infusion agar favored growth of all strains tested* McNutt and Purwin (31» 32) reported that aoid production from glucose by Bruoella depended on the quality of the base medium. That glucose utilication was not appreciably different in strains was noted by Zobell and Meyer (I4.0 ). Coleman et al* (6 ) studied the fermentation of monosaccharides by 39 strains of the abortus-melitensis group. Acid production indicated that arabinose was fermented b y all strains tested, xylose by all ex­ cept one, galactose b y 36 , glucose by 19 and levulose by 13* Aoid pro­ duction from rhamnose and lactose was not observed. Winslow _et al. (39) were among the first to show a great by aeration in the population of facultative anaerobes. increase With Escheri­ chia ooli the final population was increased five to ten times by aera­ tion. The increase was attributed to rwaoval of toxic waste products of growth and to increased oxygenation. Roby (36 ) noted a considerable increase in the growth of Bruoella oultures when aerated by agitation. Following preliminary studies of nutritional requirements, the ef­ fects of various atmospheric gases on multiplication of Bruoella in a practical and satisfactory liquid medium was determined. The metabo­ lism of the organisms when grown in an atmosphere of pure oxygen was briefly studied* EXPERIMENTS AND RESULTS A. Preliminary studies of media* Preliminary studies of nutritional requirements were conducted in 8 ” x 1" test tubes containing 15 ml. of medium. The twenty-four hour growth of a smooth strain of Bruoella frora liver agar slants was used for inoc\ilum. The cells were suspended in a diluting fluid containing 0*5 par cent sodium chloride and 0.05 P©r cent tryptose peptone. The cell count was determined by turbidity using a Libby photonreflectometer which was standardized b y plate counts. added 5 x 104 viable cells/ml. To each tube of medium were All cultures were incubated at 37°C. on a push-pull type of shaker machine operating at a rate of 75 strokes per minute. Viable count was determined after 72 hours incubation by plating suitable dilutions of the cultures on tryptose agar. Since practicality rather than definition was a prime requisite of the medium sought, Difco ’’Tryptose” peptone was selected as the most satisfactory basal medium (16). By adding various concentrations of sodium chloride it was determined that 0*5 par cent was optimum and this concentration was used in all media studied. justed to pH 6.7 with phosphoric acid. All media were ad­ As illustrated b y data in Table I, viable counts after 72 hours incubation in agitated cultures were Table I. Growth of Brucella in Stationary and in Agitated Cultures Viable Cell Count lCP/ml» Shaker Shelf Br. abortus Br. suis Br. melitensis Ojj. o.U 0 .5 5.0 8 .5 6.8 Medium* 2% tryptose, 0.5^ NaCl, pH 6.7; 15 ml. in 8 ” x l” test tubes. Inoculum* 5 x 104 cells/ml* Incubation: 72 hrs. ■ben to twenty times groater than in stationary cultures* When glucose was added to 1 per oent tryptose, the viable count of Bruoella aula was significantly increased (Table Il)» Stanier (38) noted that breakdown products formed in autoolaved solutions of glucose were toxic for the organisms in his study* Autoolaved tryptose medium containing glucose (115°C», 15 min„) exhibited no toxioity for Bruoella organisms when compared to medium sterilized by filtration* Table II* Tryptose Stimulation, of Growth of Br* sula b y Glucose and Thiamine Medium Gluoose % % 1*0 1*0 1*0 1*0 1.0 1*0 2.0 1.0 1*0 0.0 0*5 1.0 0.5 1.0 2*0 llo 1*0 1*0 Thiamine mg. % 0.0 0.0 0.0 0 *5 0.5 0*5 0.5 2.0 5.0 Viable cell count 1 0 9/ml. 6 12 18 33 1±0 hO 35 31 36 Base* 0*5/6 NaCl; pH 6*7 Inoculums 5 x 10* oells/ml* Incubation* J2 hrs* on shaker. When the tryptose-glucose medium was supplemented by thiamine hy­ drochloride, a stimulatory effect was noted with all species of Bruoella* Representative data for Br* suis in Table II show the stimulation of growth by various concentrations of glucose and thiamine hydrochloride* Thiamine hydrochloride is more stable at a low pH (8)* It was es­ tablished that thiamine hydrochloride autoolaved in concentrated solu­ tion at pH 3 or sterilized b y filtration and added asoeptically to a m e ­ dium resulted in an increase in growth of Bruoella equal to that in the autoolaved tryptoso medium (pH 6*7) containing thiamine. The last pro­ cedure for sterilization was used for all media. The tryptose-gluoose-thiamine medium was supplemented with various other factors. As shown in Table III, multiplication was not increased by any factor tried. Counts of 35 * 5 * viable oells/ml. were con­ sistently obtained with Br. suis in medium containing one per cent tryp­ tose, one per cent glucose, 0.5 mg. per cent thiamine hydrochloride, and 0.5 per oent sodium chloride? higher concentrations of any of the nutri­ ents did not increase growth. An increase in multiplication in this m e ­ dium was demonstrated with eight strains of Br. suis, ten of Br. abortus and seven of Br. melitensis. Prom seven to nine mgs. gluoose/ml. were utilized by all strains and there was not a consistent difference in glucose utilization between strains or species of Brucella. Various carbohydrates were incorporated in one per cent tryptose medium to determine if there was utilization of the sugar and if there was stimulation of growth as was effected with glucose. Cultures in the carbohydrate media were compared to those in plain tryptose medium. An increase in cell count and a decrease in pH were criteria for oarbohydrate utilization. Br. suis utilized arabinose, xylose, galactose, and fructose (Table IV). The stimulatory effect of any one of these carbohydrates was not strikingly different from that of glucose. of Coleman (6). These results confirm those Table III* Effeot on the Grovrth of Br> suis of the Addition of Various Factors to the Base Medium. Substance Added Hone Uicotinio Acid Tryptophane Tryptophane Lysine Ca. pantothenate Alanine Alanine Riboflavin Leucine Leucine Leucine Cystine Gluoosamine Glucosamine Glucosamine Autolyzed Yeast Autolyzed Yeast Liver extract SnCl-g SnClg FeS04 FeS04 KC1 KC1 KC1 MgS04 MgS04 MgCCgHgOa)^ Cone* mg. % Viable Cell Count 10n/ml* 35 t 5 32 33 33 32 32 33 39 33 0.5 1.0 2*0 20.0 o*5 20.0 140.0 0 .5 10.0 20.0 5 0 .0 1 5 .0 5.0 10.0 20*0 2 5 0 .0 500 -0 hh 1.0 10.0 1.0 10.0 1 .0 10*0 100.0 1.0 5.0 10.0 31 22 2l+ 2h 22 23 26 22 25 2U 25 31 28 3k 32 32 27 ho 1.0 Base mediumi 1% tryptose, 0,5% NaCl, 1% glucose, 0.5 ^g* % thiamine* Inoculumj 5 3t 104 cells/ml. Br* suis* Incubation* 72 hours on shaker. Table IV. Effects of Carbohydrates on Growth of Br. Suis. Carbohydrate added pH Viable Cell Count 10fi/ml« None Gluoose Arabinose Xylos e Galaotose Pruotose 8 .1 6 .1 6 .5 5.9 7.2 7.5 5 35 31 33 32 21 Rhamnose Cellobiose Lactose Sucrose Raffinose Staroh Inulin Sorbitol Mannitol Duloitol 8 .1 8 .1 8.5 8 *ij. 8 .2 A O 8 .2 8 .2 8 .1 8 .2 h 5 5 5 7 7 6 6 5 h tryptose, 0«5 mg. % thiamine HC1, 0.5/6 NaCl; pH 6 .7 Carbohydrates * 0*5% Incubation* Ij. days on shaker. Base medium* B, Effects of atmospheric gases» The growth of Bruoella cultures In large volumes of the tryptose- glucose-thiamine medium was not comparable to that In the same medium in test tubes. Attempts to attain more efficient aeration in large culture containers resulted in a study of the effects of atmospheric gases on multiplication and metabolism of Bruoella, Subsequent Investigation proved that growth of Brucella in this liquid medium was limited by the supply of oxygen. When the demand for oxygen was met, the medium which was more than adequate under for­ mer culture conditions did not contain suffioient nutrients to produce a maximum population. There was simultaneously in progress at another laboratory stud­ ies on the nutritional requirements of Bruoella and growth in aerated broth cultures. Reports of this group indicate their success in pro­ ducing large numbers of cello in a short period of time (11, 12, 13 , 30). In a medium similar to that disoussea here, maximum yields of Br, suis in large volumes were 55 x 10° cells/ml, and in small amounts 100 x 10° cells/ml* Rahn and Riohardson (3k) reported that the oxygen demand of vari­ ous species of organisms varied from 0,5 to 2 x 10” to mg, oxygen/cell/ hour for Streptococci to 10 to 80 x 10-B mg, oxygen/cell/hour for Ba­ cilli and that the demand did not depend only on the size of the cell® He determined that one per cent Baoto-peptone medium was depleted of oxygen when bacteria had multiplied to about 2 to 10 x 10s cells/ml. The supply of oxygen by diffusion and by convection currents in sta­ tionary cultures was not sufficient to meet the demands of facultative anaerobes® They observed that the rate of multiplication of all anaer- obo3* They observed that the rate of multiplication of all aerobes studied in one per cent Baoto-peptone was the same until the oxygen was almost completely exhausted but that tolerance for anaorobio con­ ditions varied with different groups of aerobic organisms. Rahn and Richardson (35) studied the population growth of aer­ obes wit h an optimal oxygen supply. Streptococci and Lactobacilli, which did not oonsume large amounts of oxygen, were not affeoted by aeration and growth was deoreased by pure oxygen. The logarithmic portion of the multiplication curves of Pseudomonas flouresoens ex­ tended from the second to the sixth hour of incubation in. a station­ ary culture and from the fourth to the twelfth hour in an aerated one. The total growth of Bacillus subtilis was increased by aeration. Levina (21+) observed that 100 per cent oxygen was neither toxic nor inhibitory to spores or vegetative oells of B. subtilis and that 20 per cent oxygen was near the minimum, that would support normal growth. The effects of various rates of flow of air, oxygen, carbon di­ oxide and mixtures of these gases on multiplication of Bruoella were determined and the metabolism of the organisms in optimum conditions for growth studied. It was necessary to reconsider the minimum re­ quirements for constituents of the medium. 250 ml. of each medium were sterilized in one liter bottles fitted with a two-hole rubber stopper and glass tubing. The inlet tube for gas extended to within an inch of the surface of the medium. The rate of flow of each gas into a cul­ ture container was measured by means of a ''Flowrator” (Fischer and Por­ ter). All cultures were grown on a push-pull type of shaker and glass float tubes were placed in the bottles to intensify the movement of the liquid and distribution of gas into the medium. Cultures grown in a normal atmosphere in bottles -which -were fitted with cotton plugs served as controls* A single smooth strain of each species of Erueella was used in all further studies. Twenty-four hour growth from liver agar slants was used for inoculum. In most of the experiments the media were seeded with 1 x 1 0 R cells/ml. of medium. tervals. Samples were removed at 2U hour in­ Viable count was determined b y plating suitable dilutions of the cultures on tryptose agar and total count measured turbidimetrically from a standard curve established by direct cell counts. Glucose was determined colorimetrioally by a modified Benedict's procedure (U) and pH measured electrometrically. 1. Multiplication. Growth of the organisms was compared in three media which differed only in the concentration of tryptose. Figure 1 shows the multiplica­ tion of Br. suis in a normal culture atmosphere in which the bottles were fitted with cotton plugs, in a flow of air (85 ml ./minute) and in an atmosphere of pure oxygen in which the rat© of flow was U5 ml./minute* There was only a slight stimulation of growth by oxygen in one per cent tryptose, but there was a marked increase in total cell count in two per oent and three per cent tryptose. A comparable increase was ob­ tained in the latter two media when the rate of flow of oxygen was on­ ly 15 ml./minute* The increase obtained in the multiplication rate of Br. roelitensis in an atmosphere of oxygen is illustrated in Figure 2. The effect of oxygen on growth of Br. abortus (Figure 5) is some­ what different from that observed with Br. suis and Br. melitensis. When one per oent tryptose was inoculated with only 1 x 10® cells/ml. 180170160- Tryptose Glucose Thiamine NaCl 1.0# 1.5# 0.5 mg. # 0.5# Tryptose 3.0# Tryptose 2.0# 150 140 - 10®/ml 130 120 Atmosphere A - Normal B - Air 85ml/min C - Oxygen 45 ml./min. 110 Total Coll Count 100 90-* 80 70 SO 50 40 30 20 10 72 ig„ 1. 96 24 48 72 Age - hours 96 24 48 Multiplication of 3r. suis in Three Concentrations of Tryptose 11C 1.0# 2.0# 0.5 m g . # 0.5# Tryptose 3.0# Tryptose 2.0# Atmosphere A - Normal B - Oxygen 45 ml./min 6C Total Cell Count - 1 0 B/ral IOC- Tryptose Glucose Thiamine NaCl 4C 3C- Ap.o Pig* 2* Mu 1 t.1 ] ]r-nl ton of M r . of me I I t.ori m1 n Tryptossn In Throo C o u n n n t r n 11 on 8 110 - Tryptose Clucoae Thlaiplne NaCl Tryptose 3.0# 1*0# 3*0# 0.5 mg.# 0.5# 100 yo Atmosphere A - Normal B - Oxygen 45 ml./min 80- o 70- 3 60" o o h 50 30 10 - 48 5'® 72 96 24 48 72 96 43 72 96 Age - hours Multiplication of Br. abortus in Three Concentrations of Tryptose 24 and oxygenated, there was no visible growth in 72 hours. In two per oent and three per oent tryptose media, stimulation of growth by oxy­ gen was slight. In Figure b the rates of growth of Br. suis in a normal atmosphere, in a stream of air and In oxygen are compared. The air flew was of suf­ ficient volume so that the oxygen content was the same as the quantity of pure oxygen. To one culture, pure oxygen was furnished at a rate of 15 ml ./minute and in the other, air flowed at a rate of 85 ml ./minute. The increase that oocurred in the total count in the atmosphere of pure oxygen was not paralleled in air with any species of Brucella. Determinations of residual glucose in the media proved that an initial concentration of one per cent was an adequate supply if one per cent tryptose was used. However, in a medium containing three per cent tryptose, multiplication and metabolism increased so much that an in­ itial concentration of glucose of 10 mgs./ml. was virtually exhausted in 72 hours. In Figure 5 data are summarized for Br. suis grown in three media which differed only in the concentration of glucose. The total cell count was slightly higher in two per cent glucose than in one per cent or three per cent. The maximum amount of glucose decom­ posed waB 17«5 mgs./ml. Similar comparisons are shown for Br. abortus in Figure 6 and for B r . melitensls in Figure 7* These two species decomposed more glucose in medium of three per cent concentration, but maximum total counts were attained in the medium containing two per cent glucose* When either glucose or thiamine was omitted from the medium, the maximum total cell count in cultures of Br. suis never rose above 29 x 10® cells/ml. (Figure 8). Comparable counts were observed with both 180 ~ 170 160 140 Atmosphere A - Normal B - Air 85 ml./rain C - Oxygen 15 ral./min. 130 120 110 100 Total Cell Count - 10B/ml 150 Tryptose 3 »0 % Glucose 2 »0 % Thiamine 0.5 mg NaCl qoh 70~ 605C 4C Br. 96 72 48 Age - hours Co mpa ris on of Rate3 of M u l t ip lic ati on in Atmosphe res of Pure Oxygen and Air 24 Fig. 4, suis 200 Tryptose 3.0# Thiamine 0,5 m g .% NaCl A-Glucose 1,5 £ B-Glucoae 2 . 0 % C-Glucose 3.0 £ Oxygen Atmosphere - 13 - lO®/®!* 120 Count 100 Cell 30 Total 60 8.C- 16 160 14 cc ©10 6 .0 - rH 5.0 40 Multiplication 20 24 48 72 96 24 48 72 Age - hours 96 24 48 5* Multiplication, Glucose Decomposition and pH Changes in Cultures of Br. suis in Three Concentrations of Glucose 9C Tryptose 3.0 $ NaCl 0.5$ Thiamine 0.5 mg.$ A - Glucose 1.0$ B - Glucose 2.0$ C - Glucose 3.0$ 22 20 “ 8.5 16- 8.0 14 7.5 12C“ Cell lOO~ 80“ Total Count - IG^/ml Oxygen Atmosphere 60- 10 6.C- 40- 20 - 5.CMultiplication Glucose Decomposed 72 48 24 24 Ago - hours Fig. 6 e Multiplication, Glucose Decomposition and pH Changes in Cultures of Br. abortus in Three Concentrations of Glucose 72 96 96 Tryptose 3.0j£ NaCl 0.5$ Thiamine 0.5 m g . # A - Glucose l.Ojf B - Glucose 2 . 0 $ C - Glucose 5.0% 1 60- 120 12 7.0 100 10 - 6.5 Total Cell - 1 0 */m± 18 Count Oaygen Atmosphere 20 8.0 16 140 7.5 80 60 5.5 40 5.0 20 Multiplication 4.5 24 48 72 Age - hours 96 24 48 Fig. 7. Multiplication, Glucose Decomposition and pH Changes in Cultures of Br. melitensis in Three Concentrations of Glucose 72 96 180 170 160 Total Cell Count - 10®/ml 150 140 130 120 110 90 Tryptose Glucose Thiamine NaCl 80 Oxygen Atmosphere 100 3,0 0.0 0.5 0.5 3.0 2.0 0.0 0.5 3.0^ ,% 2 0 0.5 mg .% Q.5£ 70 60 50 40 30 20 - 10- Br. suis Age - hours Pig. 8. Ef f e c t of Omitting Glucose or Thiamine on Rate of Multiplication Br. abortus and B r . rnel it ena 5a « Thiamine hydrochloride added to "the medium in amounts greater than 0.? mg. per cent did not inorease the cell count. Thus, the medium which was the most satisfactory for all three species of Brucel la w hen they were grown in an atmosphere of pure oxy­ gen oontained the following constituents* Tryptose Gluoose Thiamine HC1 NaCl Distilled Water 3*0 gm. 2,0 gm. 0.5 mg. 0,5 gm. 100,0 ml. Total and viable cell counts for the three species in this medium are shown in Figure 9 and maxima from the curves are given in Table V. Table V. Maximum Counts under Optimum Conditions. Cell Counts 10°/ml. Br. suis Br. abortus Br» melitensis Total Viable 178 92 126 102 h5 62 Growth was observed in atmospheres of pure carbon dioxide and in mixtures of oxygen and carbon dioxide. The multiplication of all three speoies was suppressed by pure carbon dioxide, which was supplied at a rate of nine ml./minute. In mixtures of the gases, the amount of car­ bon dioxide was maintained constant and the supply of oxygen was varied so the ratios of concentrations were 2*1, 9*1 and 18*1. The data in Figure 10 show that the total oell oount of Br. suis in pure oxygen was higher than in any atmosphere containing carbon dioxide. In the mix­ ture of two parts of oxygen to one of carbon dioxide, the multiplica­ tion of Br. suis was approximately the same as in the normal atmosphere. With mixtures of these gases in ratios of 2*1, or greater, the multi- 130 160" Tryptose Glucose Thiamin NaCl 150 Oxygen 45 ml./min 140 A - Total counts 13 - Viable counts 170 3,0$ 2,0$ 0.5 m g . $ 0.5$ Counts-10 B/ml 130 120 110 90 Cell 80 70 60 50 40 30 1C/ 24 r.';„r, l .Vii. Br. melitensis abortus 9* 48 96 24 43 72 age - hours 24 48 72 Comparisons of Total with Viable Counts under Optimum Conditions 96 18d Tryptose Glucose Thiamine Na C l 5.0% 2.0^ 0,5 m.g.% 0.5% 0 2 ,C 0 2 - m l #/m l n 10Q Count - 1 0°/ml I4d Total Cell QCh 6 ChNormal 40- C0«-9 24 Tig* lo# 48 Age 72 - h hours ours 96 M u l t i p l i c a t i o n of Br. suis In A t m o s p h e r e s O x y g e n and C a r b o n D i o x i d e of Normal Atmosphere IOC 6C cent viable cells 9C~ 3C 20 1C Tryptose 1% Tryptose 2% Tryptose 3^ Ag e-h our s Fig. 11* Effects of Culture Atmosphere and Tryptose Concentration on Viability plication of* Br. abortu s v*as equal to that in pur© oxygen; a ratio of 18x1 was as satisfactory for Br* molitenBis as pure oxygen* The per cent of viable cells was calculated for each culture in all media tried* Representative data for Br* suis are shown in Figure 11 for a culture in normal atmosphere and one in pure oxygen, in media containing one, two, and three per oent trvptose. The per cent of via­ ble cells decreased as the concentration of tryptose in the medium was increased. Cultures grown in an atmosphere of oxygen showed in gener­ al a slightly higher per cent of viable cells in the two per oent and three per cent tryptose concentration than those in a normal atmosphere, 2, p H Changes. The pH changes in the media during the growth of Br, abortus, B r * melitensis and Br. suis are plotted in Figures 12, 13 and li|. These are compared in a normal atmosphere and in pure oxygen in media con­ taining one, two and three per cent tryptose, without glucose and with one and two per oent gluoose* The arrows on the graphs indicate the time at which the supply of glucose was virtually exhausted* In media without glucose all cultures became alkaline* In gener­ al, an inorease in the concentration of tryptose in the medium resulted in a higher pH, while an inorease in the concentration of gluoose had the opposite effect. "When the supply of glucose was exhausted, there ws.s a distinct rise in pH from nitrogen decomposition products. The decrease in pH in cultures of Br* melitensis was generally not as low as that of Br* abortus. Cultures of Br. suis remained neu­ tral or became slightly alkaline even though large amounts of glucose were decomposed in all media. The pH changes in media during growth of Brucella cultures emphasize the uncertainty of ascertaining oarbo- Glucose 0 % Glucose 1? Glucose 2 G 8.5 8.0 A - Tryptose 1% B - Tryptose 2,% C - Tryptose 5% Atmosphere — Normal Oxygen -♦Glucose < 1 m g ./ml t 48 72 96 Age - hours pH Changes in Cultures of 3r. abortus in Three Concentrations of Tryptose, with and without Glucose 24 ig. 12© 48 72 Glucose Glucose Q% 1 % Glucose 2 % 8.C- 6.5 6.0 5.5 A - Tryptose B - Tryptose C - Tryptose Atmosphere Normal — Oxygen G l u c o s e < 1 mg./ml 5.0 4.5 24 ig, 96 72 24 48 72 96 24 48 Age - hours pH Changes in Cultures of Br. melitensis in Three Concentrations of Tryptose, with and without Glucose 48 72 96 Tryptose 1 % Tryptose 2 % Tryptose 2>% Atmosphere Normal Oxygen -> Glucose < 1 mg./ml 4.5 72 24 hours Fig* ll;. pH Changes in Cultures of Er. suis in Three Concentrations of Tryptose, with and without Glucose 4b A cre 96 hydrate utilization by detection of acid produotion with the usual acid-base indicators. The correlation of pH with produots of metabo­ lism will be discussed later. 3* Glucose decomposition. It is shewn b y data in Figures 5» 6 and J that if an ample supply of gluoose was available, the Brucella organisms decomposed 18 to 21 mgs./ml. w hen grown in an atmosphere of pure oxygen in a medium of three per oent tryptose, 0 .5 per cent sodium chloride and 0 .5 mg. per oent thiamine hydrochloride. There was not a significant difference in the amounts of gluoose decomposed by the three species even though the total populations ranged from 92 x 10® cells/ml. for Br. abortus to 1?8 x 10® cells/ml. for Br. suis. In a medium of one per cent tryp­ tose in oxygen, however, Br. suis decomposed a significantly larger amount of glucose than did Br. abortus or Br. melitensis, and the three cultures did not vary widely in total cell count. To facilitate these comparisons, calculations were made of the glucose decomposed per unit number of cells. Data in Table VI are average values for Br. suis in in media which contained thiamine and an ample supply of glucose. As the concentration of tryptose was increased, less gluoose was needed for each billion oells produced. The total mgs. of glucose decomposed Table VI. The Effect of Tryptose Concentration and an Oxygen Atmosphere on Glucose Decomposition by Br. suis. Tryptose % 1 2 3 Glucose decomposed-mgs./ml*/l0H cells Normal Atmosphere or Flow of Air 0.32 0.08 0 .0 5 Pure Oxygen 0 .2 2 0.32 0.0 7 Multiplication Tryptose 1.0% Glu c o s e 1.0% Thiamine 0.5 rng.% NaCl 0.5% Glucose Decomposed. 10 - Count - l 0 8/nil Atmosphere: A - Normal B - Oxygen 6C“ 50“ 40“ Cell SO­ Total SO10 ~ 0 Fig. 3 5* 24 96 48 0 24 Age - hours E f f e c t of Oxygen on Decomposition of Glucose by Br. suis 48 72 96 in three per oent tryptose was much greater, as -was the total count, but the amount of gluoose utilized by each cell was less. More glu­ cose was decomposed per unit number of cells in an atmosphere of pure oxygen than in a normal atmosphere or in a flow of air. This differ­ ence was most marked in one per cent tryptose and is clearly presented in Figure 15• In general, Br. abortus and Br. melitensis decomposed 50 to 100 per cent more glucose per unit number of cells than did Br. suis. The influences of the concentration of tryptose and of the cul­ ture atmosphere were evident, though less marked with these two species. C® Metabolism. Experiments were designed to study briefly the metabolism of glu­ cose under aerobio conditions. Attempts were made to detect interme­ diate products and end-produots which might be expected from the oxi­ dation of glucose and which would account for the pH curves character­ istic of Brucella cultures. In a study of oxidations produced by gonoooooi, Barron and Miller (l) summarized that in the presence of atmospheric oxygen, gluoose was fermented to lactic acid which was oxidized to pyruvio acid, which in turn was oxidized to acetic aoid and carbon dioxide. Krebs (2 5 ) re­ ported that the preferential reaction of gonococci and staphylococci was an anaerobic dismutation of pyruvic acid to lactic aoid which was then oxidized* Barron and Lyman (2) showed that the dismutation of pyruvic acid observed by Krebs was independent of the oxidative pro­ cess, that under optimum conditions for oxidation pyruvic aoid was di­ rectly oxidized to acetic acid and carbon dioxide, and under optimum conditions for reduction it might be reduced to lactic acid or split by dismutation into acetic aoid and formic acid* In later 3tudies Barron and Friedemarm (3 ) showed that glucose was oxidized by a number of bacteria without previous fermentation and that in cases in which the glucose molecule was not oxidized it oxidizable as soon as the molecule was phosphorylated. Kliger and became Grossowicz (18, 19, 20) studied the role of niaoin and thiamine in metabolism of glucose. In medium containing both substan­ ces there was quantitative production of carbon dioxide from glucose or lactate b y Salmonella. In cultures of Staphyloooccus aureus two and a half times as much glucose was utilized as when niacin alone was present and the end-products consisted of about 1+0 per cent acetic ac­ id, 20 per cent lactic acid and slight amounts of pyruvic aoid. If thiamine was lacking there was active growth of Staph, aureus under aerobic conditions and partial utilization of glucose. was The reaction essentially glycolyticj pyruvic acid and lactic acid were produced. Grossowicz (ll+) determined that pyruvic aoid was an in metabolism of glucose by Neisseria intracellularis. intermediate Thiamine cata­ lyzed the metabolism of pyruvic aoid but did not increase growth. In respiratory studies of propionic-acid-bacteria Quastel and Webley (33) found an accumulation of pyruvic acid during oxidation of glucose, lactic acid, glycerol, and propionio acid by thiamine-defi­ cient bacteria. The amount of pyruvic acid diminished in the presence of thiamine, which also increased the oxidation rate of acetic acid by these bacteria. In an attempt to determine the direction of the breakdown of glu­ cose, quantitative determinations were made of pyruvic acid, laotic acid, acetic acid and carbon dioxide. The effect of thiamine on the amount of glucose decomposed and the role of thiamine in the utiliza— tion of pyruvio acid were studied. Glucose was replaced by pyruvate and lactate to determine if the organisms could utilize these substrates In these experiments the organisms were grown in 250 ml. of medium in one liter bottles in an atmosphere of pure oxygen as previously described. Samples were removed at 2J+ hour intervals for total cell count, pH and chemical analyses. All data are representative of teats in at least two preparations of each medium. Pyruvic aoid was deter­ mined by the method of Clift and Cook (5 ) as modified by Elliot et al. (7). The procedure of Friedemann and Graeser (9 ) was followed for the measurements of lactic aoid and the method of Friedemann (10) for vola­ tile acids. 1. Glucose and thiamine, Figures l6 , 17 and 18 are graphic representations of results from experiments designed to study the effect of thiamine on multiplication, glucose decomposition and pH changes. The addition of thiamine to tryp­ tose did not altar the total cell counts or pH changes* When gluoose was added, but not thiamine, there was a significant increase in rate of multiplication of Br. abortus and Br. melitensis and in these cul­ tures six to seven mgs. gluoose/ml. were decomposed. In the culture of Br. suis llj. mgs. glucose/ml* were decomposed but there was not a significant ohange in the total cell count. When the medium oontained both glucose and thiamine, maximum populations were obtained. The thia­ mine effected an Inorease of the decomposition of glucose which was most marked with Br. abortus and Br. melitensis. 2. Pyruvate and laotate. There was evidence of a slight accumulation of pyruvic acid from Multiplication Glucose Decomposed A - Tryptose 2,0% 0,5% NaCl B - A + Thiamine 0,5mg,% 20C - A + Glucose 2,Qf%0 D - B + Glucose 2,0% Oxygen Atmosphere Total Cell Count - 10°/ml 8C- 18 8,5 16 8 .0 - 14 7.5 •A, B 7.0- 6 0- 6.5- 10 5 0- 40- 30- 5.5 -D o4.5 24 Pig. 16. 48 72 48 72 96 24 Age - hours Effects of Thiamine and Glucose on Multiplication and Metabolism of Br. abortus 48 72 96 24 96 Multiplication A - Tryptose 2.0 % NaCl 0.5% B - A + Thiamine 0.5mg.# C - A + Glucose 2 . 0 # D - B + Glucose 2 ,0# 20 - 18- QO- 16 70- 14 7.5 60 12 7.0- 50 10 Total Cell - 10a/ml. 8.5 Count Oxygen Atmosphere Glucose Decomposed 6.5 - 40 6 .0 - 30 5.5 20 4.5- 10 48 72 96 Age - hours Fig. 17* 24 48 Effects of Thiamine and Glucose on Multiplication and Metabolism of Br. melitensis 72 96 Multiplication Glucose Decomposed 14C- 20 - 18 8.5 16 8.0 -A,B 100“ 8 0- Cell Count - 1 0 e/ml 120 14 7.5 12 7.0 10 6.5 Total 60“ 6.0 5.5 Tryptose 2.0% NaCl 0.5 % A + Thiamine 0.5 mg.% A + Glucose 2,01 B + Glucose 2.0? 5.0 Oxygen Atmosphere 4.5 •A,B It, 72 24 48 Age - hours Effects of Thiamine and Glucose on Multiplication and Metabolism of Br. suis 96 glucose when the organisms were grown in a medium without thiamine (Table VII ). The amounts measured in cultures of Br. suis, however, were within the range of experimental error of the determination. Though only small amounts of pyruvic acid were measured in a medium without thiamine, none oould be detected in cultures in medium con­ taining thiamine. Table VII, Fyruvio Aoid Detected in Cultures of Bruaella, Species Medium Days | 3 1 1 2 1 h Pyruvic Acid mgs,/ml. Gluoose lf + Thiamine 0.0 0.0 0• 0« 0 0 0.1 0.0 0 .1 0 .0 Br, abortus Glucose n + Thiamine 0.1 0.0 0 J 1 0 .5 0.0 0 .0 0 .7 0 .0 Br, melitensis Glucose n + Thiamine 0.6 0.0 1.3 0 .0 1 Br, suis 1 J 4. 1.5 0 .0 0.0 Base medium* tryptose, 0.5/£ NaCl; pH 6,7, Gluoose* 2/&j Thiamine* 0.5 mg.$. Atmosphere* Oxygen. Analyses for lactic acid showed that none was present in cultures of any of the three species of B rue el la when examined daily during six days of incubation. The decomposition of pyruvate, with and without thiamine, and de­ composition of lactate were determined in media in which these sub­ strates replaced glucose. The acids were neutralized with sodium hy­ droxide and added to tryptose broth beforo autoclaving. The relatively low cell counts in pyruvate media ranged from 15 x 10° eells/ml. for Br. abortus to 50 x 10° cells/ml. for Bi% suis. The addition of thia­ mine did alter multiplication except with B r . melitensis and with that speoies the stimulatory effect was slight. The data in Table VIII show that pyruvate was deoomposed by each speoies and its decomposi­ tion was most rapid in cultures of Br. suis. The slower rate of uti­ lization by Br. abortus and Br. melitensis was implied by the accumu­ lation of pyruvate from gluoose in the absence of thiamine. The pH in all cultures in pyruvate media increased to 8.2 to 8 .5 . In the lactate substrate there was virtually no multiplication of Br. melitensis, growth of Br. abortus was slight until the third day, but growth of Br. suis approaohed that in a glucose medium. The quantity of lactate utilized correlates with the population in the cultures. Results in Table VIII show that considerable amounts of lactic acid wero deoomposed by Br. suis but none by Br. melitensis. Br. abortus began to decompose lactio acid after the second day. 5* Volatile acidsTo verify the application of Friedamann’s procedure for identi­ fication of volatile acids to the conditions of the experiment, small amounts of acetic and propionic acids, added to uninoculated medium, were distilled and titrated. Each of the three speoies of Brucella was grown in a medium con­ taining three per cent tryptose, two per oent glucose and thiamine# Numerous analyses did not indicate the presence in any culture of ace­ tic acid during four days of incubation in an atmosphere of pure oxygen. Carbon dioxide and ammonia. In early studies of metabolism of Brucella, McAlpine and Slanetz (2 6 ) measured large amounts of free ammonia produced by all strains in plain broth. In glucose medium, strains which used glucose pro- Table VIII, Decomposition of Pyruvate and Lactate by Brucella, Br. suis Medium. Br. abortus Br. melitensis Days 1 2 h 3 1 .2 1 3 2 3 k 1.5 1.3 1.5 1.9 1.6 2.0 0.0 0.0 0.0 1 Pyruvic acid decomposed mgs./ml • Pyruvic Acid M + Thiamine 2.3 2.7 2.8 2.9 2.9 3.9 2.9 2.9 0.3 0.3 0.6 0.8 0.7 0.7 0.8 1.0 0.9 0.9 Laotio acid decomposed mgs./ml. 2.1 Lactic Acid % lii.l+ 8.7 18,1.1 ,3% NaClj Base medium* 3 tryptose, 0 Pyruvic acid* 3*7 mgs./ml. Lactic acid: 19J+ mgs./ml. Atmosphere* Oxygen, pB: 0.6 6 ,7 . 0.9 7.3 12.2 0.0 duood very slight amounts of ammonia. He was unable to show that Br, abortus, whioh produced large amounts of ammonia, could utilize glucose in a peptone medium. Sinoe it has bean shown in this study that all strains of Bruoella are capable of extensive utilization of glucose in optimum culture conditions, it was of interest to observe the effect of this utilization on peptone metabolism. Quantitative measurements of carbon dioxide and ammonia were employed as rough estimates of carbohy­ drate and peptone decomposition. Metabolism was compared in a tryptose medium without glucose and one containing two per cent glucose. Cultures were grown in 250 ml. of medium in one liter bottles on a shaker machine in an atmosphere of pure oxygen. The gas was directed from the culture container first through a bottle containing one liter of 0,01 N sulphuric aoid and second, through one liter of 0.3 N potas­ sium hydroxide. A 25 ml, aliquot was taken from the standard acid d a i l y and titrated with standard sodium hydroxide. A 50 aliquot of the potassium hydroxide was removed each day and after precipitation with barium chloride, the excess titrated with hydrochloric acid using phenolphthalein as the indicator. Ammonia in the culture was determined by steam distillation of a sample into dilute boric acid and titration with 0,01 N sulphuric acid, A culture sample was aerated by a stream of air into 0.03 ^ potassium hydroxide to measure dissolved carbon dioxide. The cultures were examined daily for four days and final tests were made on the seventh day of incubation. Quantities of ammonia and car­ bon dioxide were computed as the amount produced in each 214. hour period; the values for the seventh day were calculated as one-third of that ac­ cumulated in the three previous days. Calculations included determina­ tions on uninoculatod media. The data shown in Figure 19 as rates of ammonia and oarbon dioxide produotion emphasize the preferential break­ down of carbohydrate over peptone decomposition. Without glucose in the medium the rates of metabolism were highest during the first 2I4 hours of incubation and followed a similar pattern in all three species. Ammonia production was greatest in the oulture of Br. suis; this was the only species of Brucella in which the pH of the culture medium increased sufficiently to release ammonia in meas­ urable amounts in the collection bottle of standard acid. In medium containing gluoose, the carbohydrate breakdown, as meas­ ured by carbon dioxide produced was greatest during the second and third days of inoubation in all species. From Br. suis 3 .I4 gms. carbon diox­ ide were collected in a seven day period, from Br. abortus 5*0 gms., and from Br. melitensis i|.8 gms. ture was only 0.1 to 0.2 gms. The carbon dioxide dissolved in each cul­ The ammonia production indioated that Br. suis attacked peptone during the first day and reached a peak on the second day. However, no ammonia was detected until the third day in cultures of Br. melitensis and until the fourth day in Br. abortus. Thus glucose exerts a ’sparing action* on peptides ’which is most obvious with B r . abortus and Br. melitensis» The presence of ammonia in cultures correlates with pH in all me­ dia. Figure 19 shows that from the time of initiation of growth of Br. suis there was production of ammonia and an increase in alkalinity. The rise in pH produced by Br. abortus and Br. melitensis occurred si­ multaneously with the appearance of ammonia in the culture media. ■rtus Olucose 2 DISCUSSION Studies of the effects of atmospherio gases on multiplication of the three speoies of Brucella emphasize that controlled culture atmos­ pheres are equally as important as medium constituents, A continoua supply of oxygen to a culture in an adequate medium had a pronounoed stimulatory effect on multiplication, but the mechanism by which this was accomplished was not explained. When oxygen was supplied to a cul­ ture in a flow of air, the total cell count was not greater than when air was available through a ootton plug. It was established that car­ bon dioxide, in the concentration which exists in air, was not inhibi­ tory, A stimulation of multiplication by a mixture of carbon dioxide and oxygen in a ratio of 18:1 was equal to or approached that of pure oxygen. However, Br. abortus exhibited a tolerance for carbon dioxide much greater than did Br. suis or meiltsnsia. In view of the re­ quirements for carbon dioxide of this species for isolation from patho­ logical material (1 5 ) this finding was not unexpected. In an oxygen atmosphere maximum total cell counts of each of the species of Brucella were obtained in a medium containing three per cent tryptose, two per oent glucose, thiamine and sodium chloride. In media containing less than these amounts, multiplication was limited by an inadequate supply of nutrients. It seems possible that largeramounts of medium constituents did not support further multiplication because of the physical factor of the high concentration of molecules in the immediate surroundings of each cell in the early period of growth. It is feasible that populations higher than those reported here for Br. suis might be obtained by replenishing nutrients in the culture medium at intervals during incubation. Multiplication of Dr. abortus did not equal that of Br. melitensis or Br. suis under any conditions studied. er multiplication It is possible that a great­ cf Br. abortus could be obtained by some additional nutritional factor and a different atmosphere. That a small inoculum of Br. abortus did not initiate growth in one per oent tryptose media in pure oxygen indicates that the atmospheric requirements of this spe­ cies differ from those of Br. suis and Br. melitensis. There were many observations of differences in the three species in multiplication, glucose decomposition, and pH changes in different media and atmospheres. The amount of glucose deoomposed per unit num­ ber of cells was less for Br. suis than for the other two species. This indicates less efficient utilization of energy frcm glucose by Br. abor­ tus and Br. melitensis. An oxygen atmosphere was optimum for perform­ ance of the oxidative mechanisms as indicated by a decrease in the glu­ cose required per unit number of cells. The concentration of tryptose in the medium was also a factor in glucose decomposition. As the con­ centration of tryptose was increased, the glucose deoomposed per unit number of cells was less. This effect, greatest with Br. suis, may be correlated with the rate of production of ammonia which was an estimate of the breakdown of nitrogenous substanoes. The metabolism of glucose by Brucella when grown in an atmosphere of pure oxygen is a rapid and complete oxidation of the carbohydrate. Carbon dioxide is ths major end-product. Addition of thiamine was not required for decomposition of glucose, although, when thiamine was pres­ ent, the rate of decomposition of glucose and the extent of utilization of the energy, as measured by cell production, was accelerated* In the absence of thiamine, however, only minute amounts of pyruvio acid were detected in the culture media. There was no evidence of the formation of lactic acid or acetic aoid from glucose. Basic variations in the oxidative enzyme systems in the three spe­ cies of B rue el la were observed when the organisms were cultured in py­ ruvate and lactate substrates. The rate of utilization of pyruvate by Br. suis was significantly higher than by Br. abortus or Br. melitensis; its utilization by all strains was only slightly accelerated by thiamine. The decomposition of lactate by Br. suis was rapid and 18 mgs./ml. were utilized to produce more than 100 x 10° cells/ml. There was no decomposition of lactate by Br. melitensis and virtually no multiplica­ tion. The growth of B r . abortu s in lactate medium was initiated after 1+8 hours incubation. During the third and fourth days of incubation, 12 mgs. lactate/ml. were decomposed. It is evident that the enzyme systems of Br. suis responsible for peptone metabolism perform more efficiently in the media studied than do those of Br. abortus and Br. melitensis. The accumulation of ammo­ nia from decomposition of nitrogenous substances in media without glu­ cose results in a rise in pH to 8.0 to 8.6. The pH of the media con­ taining glucose in which Br. su is was multiplying remained neutral or slightly alkaline as a result of continued peptone breakdown simulta­ neously with carbohydrate utilization. A low pH was measured in cul­ tures of Br. abortus and Br. melitensis and even though large amounts of glucose were decomposed, carbon dioxide was the only end-product of metabolism which could be detected. Br. abortus and Br. melitensis pro­ duced an initial decrease in pH in the medium during the time that more carbohydrate was decomposed and no ammonia was formed* After two to three days of incubation, utilization of carbohydrate was slower, pep- -26- tono metabolism was initiated and the pH in the culture medic, increased as ammonia accumulated. SUMMARY The multiplication and metabolism of the three speoies of Brucella have been studied in a practical liquid medium which will, in an opti­ mum atmosphere, produce large numbers of viable cells in a short period of time. The rate of multiplication, total cell count and glucose me­ tabolism ivere greatly increased when these organisms were grown in an atmosphere of pure oxygen. The medium which was most satisfactory for all three species contained three per cent tryptose, two per cent glu­ oose, 0,? per cent sodium chloride, and 0,5 rog. per cent thiamine hy­ drochloride, Maximum viable oell counts were observed on the third or fourth day of incubation. The tolerance for carbon dioxide in the culture atmosphere was greater with Br, abortus than with Br, melitensis and Br. suis. The pH changes during growth wsre followed in various media and •were influenced by the concentrations of tryptose and glucose® Large amounts of glucose were decomposed by all species of Brucella and utilization was aooelerated b y thiamine. The oxidation of glucose was rapid and complete, as indicated by a brief study of end-products of metabolism. Pyruvic acid was detected in culture medium laoking thia­ mine, but a significant accumulation of acids, in the preseno© or absence of thiamine, was not demonstrated® The difference in enzyme systems of the three species of Brucella was indicated by a variation in multiplication and utilization of py­ ruvate and lactate substrate. Peptone decomposition in cultures of Br. suis proceeded at a significantly higher rate than in cultures of Br. abortus and Br. melitensis in which there was a marked preferential break­ down of glucose. ACKNOWLEDGMENT The author wishes to express her appreciation to Dr. I. Forest Huddleson for guidance in these investigations* -CH- LIT M A T U R E CITED 1. Barron, E. S. C. and Miller, C. Phillip, Jr. Oxidations. I. Studies on Biological Oxidations produced by Gonococci. J. Biol. Cham., 97, 691-715 (1932). 2. Barron, E. S. G. and Lyman, Carl M. tions. XI. The Metabolism of pyruvic acid by animal tissues and bacteria. 3. J. Biol. Chem., 327, li|-3-*l6l (1939). 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