I b l w I L' | K M ' HIWI 1 l l MFLUENCE OF GLUCGSE CGNCENTRATION ON THE GRQWTH OF STKEPTOCO-CCUS IFAECAQQ-ON E384, AND DISCQLCRATSON OF A NON-SYfiTHETEC MEDIUM 144 778 .THS Thesis far fho Dagm of M. S. MICHIGAN STATE COLLEGE {Zitaaéas *i-‘E‘Oe-éimsv Féi‘ééifi if? €955 T315875: This is to certify that the , thesis entitled ”T30 Itflnfifice 0? Glued”e cheawtr tine on fTT C“étfih a? ff~v # CoedVT f "b Ti? on III, _‘°"r">".:z)" L5. .. r“ g , 7 -:;j, ii. “'0 I " "‘71.." presented by Charles: '13. 1753:1315. . 11 en has been acce ted towards f lfillm t of the requirements for \v (a TI;(-|_V1I,'/‘Il.‘l,_ 0U. degree in 1.)». I“) l) . . Jr Maj professor t. m ,4. A” Date . (7-: - L144) 0-169 INFLUENCE OF GLUCOSE CONCENTRATION ON THE GROWTH OF STREPTOCOCCUS FAECALIS, ON pH, AND DISCOLORATION OF A NON-SYNTHETIC MEDIUM by Charles Woodbury‘gifield III A THESIS Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Microbiology and Public Health 1955 , Ian 33‘ A ACKNOWLEDGEMENT The author wishes to express his appreciation to Dr. W. L. Mallmann for his guidance and patience throughout the course of this work. Appreciation is also extended to Dr. H. J. Stafseth and Dr. E. S. Beneke for proof-reading the final draft of this the 8130 f .’ \ f ‘ I7: 5 ' . .(9 :? . ' . .. ‘ A- -J! t ‘ 3 t d INFLUENCE OF GLUCOSE CONCENTRATION ON THE GROWTH OF STREPTOCOCCUS FAECALIS, ON pH, AND DISCOLORATION OF A NON-SYNTHETIC MEDIUM by Charles Woodbury Fifield.III 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 Microbiology and Public Health Year 1955 Approved by Charles Woodbury Fifield III Using a non-synthetic medium consisting of 1.5 percent tryptose, 2.7 percent KHZPOH’ 2.7 percent K H130]4 and varying 2 concentrations of glucose it was found that increase in glu- cose concentration caused an increase in browning and an in- crease in active hydrogen ion concentration in the autoclaved medium. The products of the reaction, however, were not toxic to Streptococcus faecalis in the non-synthetic medium used. If the pH of the glucose containing medium.was adjusted after sterilization, equal growth was obtained in all concen- trations of glucose used. Likewise equal growth resulted in the medium autoclaved in the presence of glucose and in the medium to which sterile glucose was added after autoclaving, if the above consideration was met. Greater growth was obtained in the phosphate buffered medium than in the same medium in which the buffer was omitted. It is suggested that this is due to an enhancement of metabolic activity as well as to a buffering effect. Oxygen uptake of the resting cells was identical in all but the lowest concentration of glucose used. This con- centration affected 02 uptake the same as the higher concen- trations until it was completely utilized. Alkaline degradation products of glucose in the low con- centration used initiate growth as well as intact glucose. A recommendation for the use of 0.25 to 0.5 percent glucose in the described non-synthetic medium is given. TABLE OF CONTENTS INTRODUCTION‘ EXPERIMENTAL Preliminary Studies Growth Studies Oxygen Uptake Studies Growth of'§. faecalis in Glucose Degradation Products DISCUSSION CONCLUSION BIBLIOGRAPHY LIST OF TABLES TABLE Page I. pH and 11 ht transmission readings (610 milli- microns of the constituents and various combinations of the constituents of the base medium.and glucose before and after autoclaving at 121 C for 10 m1HUt08 e e e e e e e e e e e 7 II. pH and light transmission readings of the medium containing glucose before and after autoclaving at 121 C for 7 minutes 0 e e e e e e e e . e e 9 III. The growth of‘§. faecalis from minimal numbers in media autoclaved in the presence of varying concentrations of glucose. pH not adjusted after autoclaving e e e e e e e e e e e e e e 10 IV. The growth of.§. faecalis from.minimal-numbers in a medium.autoclaved Jointly with varying concentrations of glucose and in a medium.in which filtered glucose was used . . . . . . . . 12 V. Percent light transmission readings after 9 l/2 hours of incubation of.§. faecalis in media autoclaved with and without glucose . . . . . 13 VI. Growth of'§. faecalis from.minimal numbers in a medium containing 0.25 percent glucose degradation products as compared to growth in a medium.containing 0.25 percent glucose . . . 20 E T. , fer-JP" LIST OF GRAPES GRAPH Page 1. Growth of S. faecalis in phosphate buffered medium.(control’ and in the non-buffered medium....................15 2. The pH of the medium without buffer and of the buffered control at various incubation thmes or so faecalis e e e e e e e e e e e e e e e e 16 3. Oxygen uptake of S. faecalis in varying concentrations of glucose at 36.5 C. . . . . . . 18 INTRODUCTION The formulae of many non-synthetic bacterial media used today have glucose concentrations in excess of 0.5 percent. These media generally are basically composed of a phosphate buffer and tryptose as a source of nitrogen. It has often been observed that the autoclaving of such media alters the composition in such a way as to have a deleterious effect on subsequent bacterial growth. This phenomenon has been referred to as the Maillard reaction, browning reaction, or caramelization and has been recognized as being extremely important in nearly all industries dealing with.materials of a biological nature. ' The present investigation describes studies involving the use of various concentrations of glucose as affecting (l) the growth of §treptococcug faecalis in a non-synthetic medium, (2) the oxygen uptake of the organism, and (3) the browning and pH of the medium. Monod,1 using a synthetic medium with mannite as the sole source of carbon and'ggcherechia coli as the test organism, showed that the total growth is a linear function of the concentration of the mannite, as long as the concen- tration does not exceed 0.5 percent. 2 In 1912 Maillard demonstrated that solutions containing amino acids turned brown when.heated with glucose. Hill and Patton3 suggested that optimal growth may not be ob- tained in microbiological assays due to the effects of the Maillard reaction. Using a casein hydrolysate medium.pre- pared for tryptophane assay and using Streptococcus faecalig g’as the test organism, they showed that better growth could be obtained in a medium.in which the glucose had been auto- claved separately and added aseptically to the previously cooled base medium. Identical growth also resulted when sucrose, a non-reducing sugar, was substituted for glucose. Patton and Hill“ found that a solution containing known amounts of L-tryptOphane and D-glucose heated at pH 10 showed more browning but less tryptophane loss, as determined by microbiological assay, than a similar solution heated at a lower pH for a longer time. They were of the opinion that the decrease in growth was not due to formation of growth inhibitors as products of the browning reaction but to actual destruction of part of the tryptophane, and that nutrients of both a vitamin and amino acid nature may be damaged during autoclaving with glucbse. Stevens and McGinnisS pointed out that the decreased nutritive value of overheated soy bean oil meal for chicks was almost fully corrected by adding supplementary methionine and lysine. These amino acids when added to properly heated soy bean oil meal, however, did not improve the growth of chicks. Lysine autoclaved for four hours at 120 C supple- mented overheated soy bean oil meal for chick growth as effectively as unheated lysine. Chen, Medler, and Harte6 found that highly flourescent substances were formed from.certain amino acids when heated with.Deniges reagent (para-formaldehyde and sulfuric acid). Graham, Hsu and McGinnis7 showed that flourescent substances were formed when glucose was used to furnish the aldehyde group instead of Denige's reagent. All fifteen amino acids tested showed flourescence. These investigators concluded that brown color formation, flourescence, and decreased amino nitrogen are associated with the destruction of methionine brought about by autoclaving in the presence of glucose. 0n the other hand several investigators have indicated that the heating of a medium.in the presence of glucose is essential for maximum.growth of certain organisms. Orla- Jensen8 reported this phenomenon after studying various streptococci and lactobacilli. Smiley, Niven and Sherman,9 working with a synthetic medium for Streptococcug salivarius, found that if glucose was added aseptically to the medium. after autoclaving, growth was greatly delayed or never initiated. It was suggested that a hydrogen acceptor was required in the initial carbohydrate metabolism. If such an acceptor were not present, carbohydrate metabolism.might INFLUENCE OF GLUCOSE CONCENTRATION ON THE GROWTH OF STREPTOCOCCUS FAECALIS, ON pH, AND DISCOLORATION OF A NON-SYNTHETIC MEDIUM by Charles Woodbury Fifield.III 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 Microbiology and Public Health Year 1955 Approved by Charles Woodbury Fifield III Using a non—synthetic medium consisting of 1.5 percent tryptose, 2.7 percent KHZPOH’ 2.7 percent K HPOH and varying 2 concentrations of glucose it was found that increase in glu- cose concentration caused an increase in browning and an in- crease in active hydrogen ion concentration in the autoclaved medium. The products of the reaction, however, were not toxic to Streptococcus faecalis in the non-synthetic medium used. If the pH of the glucose containing medium.was adjusted after sterilization, equal growth was obtained in all concen- trations of glucose used. Likewise equal growth resulted in the medium autoclaved in the presence of glucose and in the medium to which sterile glucose was added after autoclaving, if the above consideration was met. Greater growth was obtained in the phosphate buffered medium than in the same medium in which the buffer was omitted. It is suggested that this is due to an enhancement of metabolic activity as well as to a buffering effect. Oxygen uptake of the resting cells was identical in all but the lowest concentration of glucose used. This con- centration affected 02 uptake the same as the higher concen- trations until it was completely utilized. Alkaline degradation products of glucose in the low con- centration used initiate growth as well as intact glucose. A recommendation for the use of 0.25 to 0.5 percent glucose in the described non-synthetic medium is given. TABLE OF CONTENTS INTRODUCTION EXPERIMENTAL Preliminary Studies Growth Studies Oxygen Uptake Studies Growth of.§. faecalis in Glucose Degradation Products DISCUSSION CONCLUSION BIBLIOGRAPHY LIST OF TABLES TABLE Page I. pH and 11 ht transmission readings (610 milli- microns of the constituents and various combinations of the constituents of the base medium and glucose before and after autoclaving at121Cf0r10m1nu1368 00000000000 7 II. pH and light transmission readings of the medium containing glucose before and after autoclaving at1210f0r7minutes..........’.. 9 III. The growth of §. faecalis from minimal numbers in media autoclaved in the presence of varying concentrations of glucose. pH not adjusted after ant001aVing e e e e e e e e e e e e e e 10 IV. The growth of‘§. faecalis from.minimal-numbers in a medium.autoclaved jointly with varying concentrations of glucose and in a medium.in which filtered glucose was used . . . . . . . . 12 V. Percent light transmission readings after 9 l/2 hours of incubation of‘§. faecalis in.media autoclaved with and without glucose ' . . . . . 13 VI. Growth of S. faecalis from.minimal numbers in a medium containing 0.25 percent glucose degradation products as compared to growth in a medium containing 0.25 percent glucose . . . 20 LIST OF GRAPHS GRAPH Page 1. Growth of‘§. faecalis in phosphate buffered medium.(control) and in the non-buffered medium....................15 2. The pH of the medium without buffer and of the buffered control at various incubation times Of§0faecalis 0.0000000000000016 3. Oxygen uptake of §. faecalis in varying concentrations of glucose at 36.5 C. . . . . . . 18 INTRODUCTION The formulae of many non-synthetic bacterial media used today have glucose concentrations in excess of 0.5 percent. These media generally are basically composed of a phosphate buffer and tryptose as a source of nitrogen. It has often been observed that the autoclaving of such media alters the composition in such a way as to have a deleterious effect on subsequent bacterial growth. This phenomenon has been referred to as the Maillard reaction, browning reaction, or caramelization and has been recognized as being extremely important in nearly all industries dealing with.materials of a biological nature. ' The present investigation describes studies involving the use of various concentrations of glucose as affecting (l) the growth of §treptococcug faecalis in a non-synthetic medium, (2) the oxygen uptake of the organism, and (3) the browning and pH of the medium. Monod,1 using a synthetic medium.with mannite as the sole source of carbon and.§§cherechia 22;; as the test organism, showed that the total growth is a linear function of the concentration of the mannite, as long as the concen- tration does not exceed 0.5 percent. In 1912 Maillard2 demonstrated that solutions containing amino acids turned brown when heated with glucose. Hill and Patton} suggested that optimal growth may not be ob- tained in microbiological assays due to the effects of the Maillard reaction. Using a casein hydrolysate medium.pre- pared for tryptophane assay and using Streptococcus faecalig §_as the test organism, they showed that better growth could be obtained in a medium in which the glucose had been auto- claved separately and added aseptically to the previously cooled base medium. Identical growth also resulted when sucrose, a non-reducing sugar, was substituted for glucose. Patton and Hill“ found that a solution containing known amounts of L-tryptophane and D-glucose heated at pH 10 showed more browning but less tryptophane loss, as determined by microbiological assay, than a similar solution heated at a lower pH for a longer time. They were of the opinion that the decrease in growth was not due to formation of growth inhibitors as products of the browning reaction but to actual destruction of part of the tryptophane, and that nutrients of both a vitamin and amino acid nature may be damaged during autoclaving with glucOse. Stevens and McGinnis5 pointed out that the decreased nutritive value of overheated soy bean oil meal for chicks was almost fully corrected by adding supplementary methionine and lysine. These amdno acids when added to properly heated soy bean oil meal, however, did not improve the growth of chicks. Lysine autoclaved for four hours at 120 C supple- mented overheated soy bean oil meal for chick growth as effectively as unheated lysine. Chen, Medler, and Harte6 found that highly flourescent substances were formed from certain amino acids when heated with Deniges reagent (para-formaldehyde and sulfuric acid). Graham, Hsu and McGinnis7 showed that flourescent substances were formed when glucose was used to furnish the aldehyde group instead of Denige's reagent. All fifteen amino acids tested showed flourescence. These investigators concluded that brown color formation, flourescence, and decreased amino nitrogen are associated with the destruction of methionine brought about by autoclaving in the presence of glucose. On the other hand several investigators have indicated that the heating of a medium in the presence of glucose is essential for maximum.growth of certain organisms. Orla- Jensen8 reported this phenomenon after studying various streptococci and lactobacilli. Smiley, Niven and Sherman,9 working with a synthetic medium for Streptococcug salivarius, found that if glucose was added aseptically to the medium. after autoclaving, growth was greatly delayed or never initiated. It was suggested that a hydrogen acceptor was required in the initial carbohydrate metabolism. If such an acceptor were not present, carbohydrate metabolism might be blocked. Evidently the breakdown of glucose supplied the necessary hydrogen acceptor. Evanslo reported that glucose breaks down into many compounds when oxidized under alkaline conditions. Products of carbohydrate metabolism such as pyruvic and lactic acids as well as acetaldehyde were among the products which ap- peared as the result of the alkaline breakdown. Englis and Hanahan11 demonstrated that phosphate plays an active part in initiating various changes in glucose on autoclaving. Not only was the glucose altered but a distinct discoloration of the solution occurred. Dubos and Davis12 reported that autoclaved glucose gave rise to toxic caramelization products which inhibit the growth of small inocula of tubercle bacilli. EXPERIMENTAL In experiments dealing with the effect of heat on a medium, it is important to include not only the tbme and temperature of sterilization but the approximate total time the medium.is subjected to heat. The autoclave used in these experiments was a 17" x 26" single wall type made by the American Sterilizer Company and was in perfect working order. The time required for the temperature to reach 121 C from.room temperature was six minutes. The time of sterili- zation was measured as soon as 121 C was reached. Eleven minutes elapsed after the period of sterilization before the medium.was removed from.the autoclave. The term, "joint sterilization", is used frequently in this thesis. It is a term.adopted from Orla-Jensen8 to mean that the sugar was autoclaved in the presence of the other constituents of the medium. The base medium.used in the following experiments, unless otherwise stated, consisted of: Bacto-Tryptose 1.5% Kzsroh 0.27% KHzPOh 0.27% The organism.used was Streptococcus faecalis B 33 A. The temperature of incubation in all cases was 37 C. Preliminary Studies The ingredients of the medium including glucose were autoclaved separately and in various combinations in 100 m1 amounts in 250 ml Erlenmeyer flasks at 121 C for 10 minutes. The hydrogen ion concentration was measured before and after autoclaving using a Beckmann Model G pH meter. The amount of discoloration was measured with a Beckmann Model B.Spectro- photometer using a wave length of 610 millimicrons. Table I. The amount of glucose, when used, was 2 percent. The results (Table I) indicate that a solution of pure glucose showed a little, if any, discoloration after auto- claving. This is in agreement with the results of many workers. The formation of small amounts of acid lowered the pH considerably. A solution of 1.5 percent tryptose remained relatively stable as to pH and discoloration. A combination of the phosphate buffer and glucose was stable as to pH but showed very slight discoloration. A solution of glucose and tryptose showed a significant drop in pH but almost the same discoloration as the tryptose alone or the solution of glucose and buffer. Considerable discoloration was evident in the glucose, buffer, tryptose solution. Growth Studies Growth curves obtained by using the minimal inoculum technique were employed to determine the effect of the hits r .em .mo em.e :a.e mmopaasa + socasm + smoosae .mm .No - no.0 mm.o enoughae + omoosaw .Hm .mo mw.o mw.o omOBQhaa + magnum .em m.oo :~.e ma.e omoosac + seamen .00 .mm mo.“ 0H.h omOthae m.oo .om om.: om.o . omoosflo .. us ma.e ma.e flopsemmoemv assess .ooa .ooa mm.e mm.e sons: eoaflapmaa WMwwwmewwma ommwwmawmwwme wcH>sHoop34 wwa>mH00pnd soapsaom pnmaq R woman R aopum mg eachom mm mmBDZHZ OH mom OHNH ad GZH> Qz¢ mBamDaHBmzoo awe mo AmzomUHEHQAHS OHQV mOZHaddx ZOHmWHSmZ¢mB EmcHQ 93¢ mm H mflmwe browning reaction on the growth of‘§. faecalis when various concentrations of glucose were used. Ninety-nine ml of the base medium together with the glucose were placed in 250 ml Erlenmeyer flasks. In each case the pH and light trans- mission readings of the medium were recorded before auto- claving at 121 C for 7 minutes and again after the medium had cooled. A wave length of 610 millimicrons was used for the light transmission readings. The pH of the medium was not adjusted after autoclaving. The results in Table II show that the higher the glucose concentration the greater the drop in pH-and the darker the medium. Two drops of a 16-hour culture were placed in a dilu- tion bottle containing 99 ml of sterile physiological saline. Four-tenths ml of this suspension was pipetted into a second 99 ml saline dilution bottle and from.the latter one ml was transferred into each flask containing the medium. The seedings of each culture were between A9 and 56 organisms per ml. Duplicate plates were made from each sample at the time of seeding and after 2, h and 6 hours of incubation. All plates were incubated 2H hours. Table III. From.the results shown in Table III, it was not possible to ascertain whether the decrease in growth in the higher concentration of glucose resulted from.the browning or Maillard reaction or from the acidity produced from.the breakdown of glucose by the autoclaving process. mMHDZHE N mom OHNH 84 02H> mHH3 MHBZHOH Qm>¢qooebd EDHsz 4 2H mmmmzbz QdSHZHE 20mm mHAH mum mumqa 21 DISCUSSION The results in Table I show that a combination of the phosphate buffer and glucose was stable as to pH but showed slight discoloration. This is in keeping with the work of Englis and Hanahan,11 although they reported more discolora- tion. This may be attributed to the fact that they used a higher concentration of phosphate and a longer heating period. Englis and Hanahan11 showed that phosphate is a factor in glucose breakdown and discoloration during heating. A reaction between glucose, phosphate buffer and tryp- tose seemed to enhance browning. Before an explanation of this phenomenon could be attempted, a considerable number of chemical analyses would have to be undertaken. There was no notice of a direct or inverse relationship between drop in pH and the extent of browning. Many laboratories are in the habit of raising the pH of a medium.above the specified level with the result that after autoclaving the desired level is reached. This prac- tice eliminates sterilizing the ingredients separately or adjusting the pH aseptically after autoclaving. Usually the medium.becomes darker. Obviously, if a medium.drops in pH and becomes discolored, it is reasonable to assume that some ingredient or ingredients have been altered. In many 22 cases this is not important, for growth is affected little, if any. However, in physiological studies and in differ- ential media, especially those depending on a carbohydrate as the differentiating substance, it is of the utmost impor- tance. Table I shows that a solution of buffer and tryptose remains stable after autoclaving as to pH and discoloration. If glucose sterilized by filtration was added to this auto- claved solution, the resulting medium.would have, for the most part, the same composition as before sterilization. If the medium.was refrigerated until used, the glucose would be present as glucose and not as the breakdown products of glucose. The practice of adding sterile carbohydrate to an autoclaved medium to prevent breakdown is not new. The results presented here serve only to strengthen that argu- ment. The fact that joint sterilization of various concen- trations of sugar affects pH and discoloration of the medium is shown in Table II. The mechanism of browning is compli- cated and, as yet, is not clearly understood. It has been postulated that the hydrogen ion concentration increases be- cause Of a combination of the aldehyde groups of the carbo- hydrate and the amino groups of the nitrogenous substance which results in setting free a corresponding amount of carboxyl groups. 23 Table III represents the results of growth measurements in a medium in which the pH was not adjusted after autoclaving. There was a marked decrease at four and six hours in the total number of viable cells in the media that contained the higher concentrations of glucose. When the pH was adjusted after autoclaving so that all media were the same (pH 6.85), the growth in all concentrations of glucose was the same. Table IV. Likewise there was no difference in growth be- tween the medium autoclaved jointly with glucose and the medium autoclaved separately. At the time of turbidity the bacterial density was significantly the same for all glucose concentrations as shown by Table V. The base medium without glucose showed less growth at six hours but was comparable to the media containing glucose at 9.5 hours indicating the presence of an available carbon source in the tryptose; but in all probability, it was not as readily available as glu- cose, at least during the lag phase. 9 Two facts are Obvious: (1) the products Of the browning reaction are not toxic but the difference in growth in the media not adjusted as to pH was due to a higher initial acidity as a result of heating the glucose and base medium together, and (2) low concentrations of glucose, much lower than recommended for most non-synthetic media, will support the growth of §. faecalis up to the time of turbidity as efficiently as the higher concentrations. Monod1 found that a straight line relationship existed between total cell numbers of‘§.'ggli and the concentration of glucose used if that concentration was below 0.5 percent. He stressed the fact that this relationship would not hold un- less there was but one source of carbon. It is presumed that his counts were made at 2h.hours. The longest incubation time of the experiments described in this paper was 12 hours. It is the opinion of many investigators that the lag phase is the phase most affected by a medium. By using a minimal inoculum, the marginal differences in growth rates in the various media are increased. The absence of the phosphate buffer from the base medium caused a considerable decrease in growth even at ten hours as shown in Graph 1. The results presented in Graph 2 show that the buffer reaches its maximum at about twelve hours after which the pH drops rapidly. It is evident that the non-buffered medium has some buffering capacity although not as great gs the phosphate buffered medium, especially at ten hours. Since there is greater growth in the buffered medium, even at seven hours it would seem that the increase in growth must be explained on some other ground than a mere neutralization of the active hydrogen ions. The results of Mallmann and Gallolh proved this point. They used 25 representative organisms from.many groups and showed that the addition of potassium.or sodium phosphate to nutrient media enhanced physiological activity by playing a part in the direct metabolism of the organism tested. 15 Slanetz and Rettger observed that when a solution of KZHPOH and NaHzPoh was added to a plain peptone medium, so that the concentration was 1 percent, and inoculated with various members of the Enteric group, it exerted a regula- tory effect on the hydrogen ion concentration, but in the same medium to which glucose had been added the buffer ex- erted little check on the pH drop. They obviously failed to realize that the limit of the buffer was probably reached, due to the acid from glucose, before their first reading at 2h hours was taken. Darby and Mallmann16 found that when 0.u percent KZHPQL and 0.15 percent KHZPOH were employed in a lactose-tryptose medium.for'g.‘ggl; 161, the buffer caused a much greater growth in the late logarithmic phase and a slightly greater increase during the lag phase. The results of the oxygen uptake studies on.§. faecalis are similar to those of Gerard13 on S. 1232a. The oxygen uptake of the resting cells is not dependent on concentra- tion of glucose. The system may be regarded as an enzyme versus glucose reaction. The curves representing the various 26 sugar concentrations fell on the same position on the graph until about 25 minutes when the curve representing 0.025 percent glucose returned to the endogenous rate. It is as- sumed that the available glucose had been utilized. There is, then,at least within the range of concentrations studied, no threshold concentration needed for respiration. Had there been a noticeable difference in oxygen consumption with the various concentrations of glucose it might be reasonable to suspect a similar difference in multiplying cells, however the fact that the oxygen uptake was the same for the four highest concentrations does not necessarily indicate that multiplying cells would be affected to the same degree. Since Smiley, Niven and Sherman9 noticed that jointly autoclaved glucose was necessary for the growth of‘§. gglivarius but that the alkaline degradated glucose initi- ated growth equally as well, it was a matter of interest to learn whether degradated glucose autoclaved jointly or separately would affect the growth of‘§. faecalis in the non-synthetic base medium.used in these studies. Since there seemed to be no difference in growth in these experi- ments, it remains to make analogous studies using a syn- thetic medium. 27 CONCLUSION 1. A combination of glucose, phosphate buffer and tryptose in solution was discolored when sterilized, to a greater degree than other combinations of the ingredients of the medium used. 2. Increase in glucose concentration caused an increase in browning and an increase in active hydrogen ion concen- tration in the autoclaved medium. 3. The products of the browning reaction were not toxic in the non-synthetic medium used. If the pH of the glucose containing medium.was adjusted after sterilization, equal growth was obtained in all concentrations of glucose used. Likewise equal growth resulted in the medium.auto- claved in the presence of glucose and in the medium.to which sterile glucose was added after autoclaving, if the above consideration was met. h. Greater growth was obtained in the phosphate buffered medium.than in the non-buffered medium. It is suggested that this is due to an enhancement of metabolic activity as well as to a buffering effect. 5. Oxygen uptake of the resting cells was identical in all but the lowest concentration of glucose used. This concentration affected 02 uptake the same as the higher con- centrations until it was completely utilized. 28 6. Alkaline degradation products of glucose in the low concentration used initiate growth as well as intact glucose. 7. A recommendation for the use of 0.25 to 0.5 percent glucose in the described non-synthetic medium is given. 1. 2. 3. h. 5. 6. 7. 9. 10. 11. 12. 13. 15. 16. 29 BIBLIOGRAPHY Monod, J. l9hl. Academic des Sciences, Comptes Rendus, 212, 771-773 0 Maillard, L. C. 1912. Academic des Sciences, Comptes Rendus, 15g, 66. Hill, E. G., and Patton, A. R. l9h7. Science, 1 5. h81. Patton, A. R., and Hill, E. G. l9h8. Science, 102, 68. Stevens, J. M., and McGinnis, James. 19H7. J. Biol. Chem., 121, (+310 Chen, J. L., Medler, J. D., and Harte, R. A. 19h8. J. Amer. Chem. 506., 10—, 3114-50 Graham, W. D., Hsu, P. Y., and McGinnis, J. l9h9. Science, 110, 217. Orla-Jensen, A. D., 1933. J. Soc. Chem. Ind., 52, 37h- 379. Smiley, K. L., Niven, Jr., C. F., and Sherman, J. M. 19,430 J. Of Bac'tog fig m5’u-530 Evans, Wm. Lloyd. 1929. Chem. Revs.,‘é, 281-315. Englis, D. T., and Hanahan, D. J. 19h5. Jour. Am. Chem., él. 51-51» Dubos, R. J., and Davis, B. D. l9h6. Jour. Expt'l. Made, _819 11.09-14.23. Gerard, R. W., and Falk, I. S. 1931. Biol. Bull. Marine Biol. Lab., 60(3), 213-226. Mallmann, W. L., and Gallo, F.. 1930. Mich. Acad. Science, lg, 617-639. Slanetz, C. A., and Rettger, L. F. 1928. J. of Bact. 12. 297-317. Darby, C. W., and Mallmann, w. L. 1939. J. of Amer. (I ”'TITIIN’WENQIijfflflnijflflfljflIfs