FACTORS INFLUENCING THE GROWTH OF 53—11015; 115.. Dvsammgg 0N .m- " " LABORATORY MEDIA Thesis for the Degree of M. S. MICHIGAN STATE COLLEGE Caronn Cox Kilian 1942 mass FACTORS INFLUENCING THE GROWTH OF SHIGELLA DYSENTERIAE ON LABORATORY MEDIA by Carolyn Cox Kilian A THESIS Submitted to the Graduate School onMichigan State College of Agriculture and Applied Science in partial fulfilment of the requirements for the degree of MASTER OF SCIENCE Department of Bacteriology and Hygiene 1942 THESIS Contents Introduction Historical review Experimental Generation time Peptone Hydrogen ion concentration Buffer Sodium chloride Dextrose Beef extract The optimum medium The toxicity of differential media now in use Discussion Summary Literature cited Page 003050: 10 ll 12 12 12 13 13 15 17 19 Introduction In.mest laboratories, dysentery bacilli are detected in fecal samples by smearing plates of different kinds of media. In some instances the smear plates are made from relative enrichment media seeded with the fecal sample. In all cases several media are used, because it has been found that frequently only one of the several media used shows the presence of dysentery bacilli. The fact that the use of enrichment media or the use of several plating media increases the number of isolations indicates that many of the dysentery bacilli present in the fecal sample do not survive and repro- duce in the culture media. It is assumed in this study that a highly efficient plating medium.would support the growth of all dysentery bacilli, and there would be no necessity of using so many different media. The critical life in the growth of an organism.lies in the period following the first cell division. If the environ- mental conditions are so unfavorable that the cells can not adjust conditions to their needs, growth ceases and the cells die. For an isolated cell to reproduce, it is essential that the medium.approach ideal growing conditions as nearly as possible. It appears from.a review of the literature on media for the isolation of dysentery organisms that these media may not offer a favorable environment for the reproduction of the primary young cells. It has been demonstrated by Salter (l), - 2 - Sherman and Albus (2), Stark and Stark (3), and Sherman and Cameron (4) that young bacterial cells are more susceptible to adverse conditions of their environment than are cells in the logarithmic growth phase. The last two workers showed that young cells of Escherichia 22;};may be killed by abrupt environmental changes within the natural range of growth of the organism, The factors with which they worked were change of temperature and change of osmotic pressure. Huntington and Winslow (5) studied characteristics of physiological youth and found a definite and orderly relationship of changes; metabolic activity increased first, cell volume second, and cell division rate third. These characteristics are also found in higher plants and animals in their physiological youth. Sherman and Albus (6) demon- strated that mature cells from.ald cultures assumed the characteristics of young cells before reproduction began. They hold the view that during the lag period the old cells undergo a biologic rejuvenescence which fits them for repro- duction. Darby and mallmann (7) demonstrated by practical tests that a medium designed to shorten the lag phase of coliform organisms would actually increase the percentage of samples found to contain these organisms. The object of this work was to develop an optimum.base medium that would promote the growth of dysentery bacilli during the lag and early logarithmic growth Stages, the period of physiological youth. Such an Optimum.medium could then be used as a standard to which could be compared the - 3 - selective enrichment and differential media now used and possibly serve as the base of a new diagnostic medium, There is no available information that would indicate that an ideal medium.could supplant the use of several different media, but the writer believes that the use of one such medium.alone or in series with other media would materially increase the number of isolations. Historical Review Hardy and his associates (8) made a careful study of four differential culture media used in the study of acute diarrheal diseases, the four media being eosinémethylene- blue agar, Endo's agar, desoxycholate agar, and sodium desoxycholate-citrate agar. They found the sodium.desoxy- oholate-citrate agar to be of superior value and the two sodium.desoxycholate agars used supplementarily to be of outstanding value. macConkey's agar was found to compare favorably with eosinémethylene-blue agar and Endo's agar but not with the desoxycholate agars. He found desoxycholate agar to promote the growth of all the usual intestinal patho- gens and common Gramrnegative intestinal bacilli and the desoxycholate-citrate agar to inhibit most of the nonpatho- genic organisms and, to a limited degree, some of the patho- gens. Inhis whole survey only three varieties of Shigella dysenteriae were found-~Flexner, Sonne, and Newcastle. No Shiga or Schmitz varieties were found on any of the media. The desoxycholate-citrate agar permitted the use of a heavy inoculation.. Plain desoxycholate agar was somewhat superior - 4 - to either eosin-methylene-blue or Endo's agar, especially for Sonne and Newcastle. In their investigation of preserving solutions for the recovery of dysentery bacilli from fecal specimens, Bangxang and Eliot (9) studied the efficiency of various plating media and reported the following: (1) Desoxycholate-citrate agar inhibits the non- pathogenic fecal bacteria, but it does not grow Schmitz, Sonne, Shiga, and some Flexner strains. (2) Desoxycholate agar is superior to eosin-methylene- blue agar, Endo's agar, and macConkey's agar. Though it is slightly inhibitive to some of the strains it eliminates all Gramapositive organisms. (3) Eosinemethylene-blue agar is slightly inhibitory to some strains. (4) MacConkey's agar showed no inhibitory effect on any dysentery strain tested. (5) Endo's agar showed no inhibitory effect on any dysentery strain tested. (6) Bromecresol-purple agar exhibited no selectivity. In these studies the amount of inhibition exerted by the medium.on the growth of the bacteria was based on the degree of growth on the plates. This method of measuring inhibition is a questionable procedure, particularly when there are no controls used, as was true in this study. The conclusions in these studies should therefore be considered on a compara- tive basis. - 5 - In a study on the differentiation and identification of bacillary incitants of dysentery, Coleman (10) confirmed the work of Hardy and his associates in that desoxycholate- citrate agar was preferable for isolating dysentery bacilli. She found that it restricts the growth of a relatively small number of Flexner strains and an appreciable number of Sonne strains and therefore recommended the useqof desoxycholate agar without citrate, MacConkey's agar, eosinemethylene-blue agar, or a modification of Endo's agar for a supplementary medium. The Michigan State Department of Health uses a medium called the S and 8 medium for the isolation of Salmonella and Shigella organisms. Though no reference to this medium was found in the literature, it is mentioned here because it was tested in the same manner as were the other differential media studied. To summarize the criticisms of all of the media now used for the isolation of dysentery bacilli, sodium desoxy- cholate-citrate agar appears to be the medium.used most suc- cessfully. Because it restricts the growth of a number of dysentery strains, different workers have recommended that another differential plating medium be used in conjunction ‘with it. Sodium desoxycholate agar without citrate, Mace Conkey's agar, eosinsmethylene-blue agar, and a modification of‘Endo's agar have been named as good supplementary media. - 5 - The apparent inefficiency of these differential media is indicated by the haphazard occurrence of positive cultures on only one medium. This is true also of other intestinal pathogens such as Eberthella and Salmonella organisms. The. reason for this random occurrence of positive cultures on the various differential media could be that the media do not support the growth of the bacterial cells during the most critical period of their deve10pment--the period of physiolog- ical youth. Only the less delicate, more resistant cells are able to survive and reproduce, and the other cells die before their presence can be detected. With the object of develOping a medium that would reduce this mortality to a minimum.the following studies were made. Experimental In order to formulate a medium.that would give maximum reproduction during the period of physiological youth, growth curves in broth media were determined using the technidue of Darby and Mhllmann (7). A twenty-four hour tryptose agar slant culture of Shigella dysenteriae, Shiga (No. 1033), was used as the source of organisms. To intensify the effects of the different media, the number of cells introduced into the broths was reduced to a minimum.(lO to 50 organisms per'milli- liter). It has been shown by many workers that it is more difficult for a small number of organisms to establish them- selves than it is for large numbers of organisms. Growth rates were determined by taking counts of the number of - 7 - organisms at different intervals of the growth curve. In each growth curve effort was made to have only one variable, the factor being tested. The base medium for the different broths was made up in one lot and portioned out in 200 ml. quantities, to which the variable was then added. Duplicate flasks containing 100 ml. each were prepared of each broth. Except in the growth curves in which the optimum.concentraticn of a con- stituent was tested, the base medium.had the following com! position: Peptone........... 2.0% Sodium.chloride... 0.5% DextroseOOOOOOOOOO 0.2% K 04.000.000.00. 0.4% 2P0 0.00.0000... 0.15% This base medium was selected because it had worked so well for Darby and Mallmann for growing E, coli, an intestinal organism closely related to Shigella dysenteriae. The tryp- tose agar plating medium.was selected for the same reason. The numbers of organisms in the broth.media were deter- mined at 0, 2, 4, 6, and 24 hours after the seeding. These counts were determined by plating two onesmilliliter quan- tities of the following dilutions: Hours after seeding 0 1 ml. 2 1 ml., 1-10 4 1 ml., 1-10, 1-100 6 1-100, l-lT, and 1-10 or l-lOT 24 1-10013, 1-1M, and 1-101' or l-lOIvI - 8 - The dilutions were made in standard milk dilution bottles equipped with.Escher stOppers. Plates were poured with buffered tryptose agar (pH 6.8) and incubated 48 hours be- fore the colonies were counted. Generation 9.1-2.2: The generation time of an organism has been defined as that amount of time which elapses between the fission of a cell into two new cells and the subsequent fission of the newly formed cells. The generation time can be computed by a formula developed by Buchner, Longard and Riedlin (11). This formula is g - t log 2 "IEg‘b -logj§ when t = time B = number of bacteria at the beginning of time period b = hummer of bacteria at the end of the time period 3 a generation time A short generation time is indicative of an environment which enhances reproduction in contrast to a long generation time indicating an unfavorable environment. Since the most critical period in the growth and reproduction of bacteria is the very early part of the lag phase, which occurs after the introduction of those cells into a new environment, it 'was assumed that the medium which gave the shortest genera- tion time during this period would support the growth of the more delicate cells and would be the best medium. Since all cultures were found to be in the early logarithmic growth phase four hours after the broths were seeded, the genera- tion time from.zero to four hours was chosen as being the - 9 - index to the efficiency of the media, the broth having the shortest generation time during this period being the most favorable medium. Peptone: The source of nitrogen is especially important in a nutrient medium, not only as to the kind of peptone but also as to the concentration. 0f the twelve different pep- tones tested, Armour I, Armour's Siccum, Protecse Peptone 3 (Difco), and Tryptose (Difco) were found to yield good results, as shown in the tabulated data. Peptones Armour I and Armour II were submitted by Armour and Company for bacte- riological investigation and are not on the market as come mercial peptones. Table 1 presents the results of the growth curves run for the purpose of selecting the most satisfactory peptone. Since in the first series of peptones Protecse 1 and Armour's Siccum gave better results than did Witte, Bacto, Stearn, or Neo, these two were selected for further study. In the next series Armour I and Protecse 3 were found to be better than Armour's Siccum, Armour II, and Protecse 1 and 2, so these two peptones were studied again by comparing them with Tryptose and Tryptone. Armour I proved to be the most favorable peptone and Protecse 5 the next most favorable. These peptones were selected on the basis of short generation times from.aero to four hours after the seeding. To deter- mine the significance of small differences in the index generation times, a few of the growth curves were repeated several times, and in each instance the results were the same as the first growth curve. TABLE 1 The Influence of Peptones in a BaseiMedium* on the Early Growth of Shigella dysenteriae ' Generation time Number of bacteria per ml. in minutes i Kind of ; peptone Time of sampling in hours Timeigfhgfimgling 0 2 4 6 24 0-4 0-2 2-4 4-6 Armour Siccum.42 43 540 3600 304M 65 3600 33 44 Protecse 45 53 320 1800 512M 85 516 46 48 Neo (Difco) 40 46 170 230 251M 114 602 64 277 1 Stcarn's 33 60 130 1220 166M 120 139 37 64 Witte's 49 50 180 370 216M 130 4100 66 116 Bacto(Difco) 54 44 170 2500 157m 142 62 :31 Armour Siccum 24 31 500 7200 470M 55 328 30 30 Armour I 18 30 530 4700 530M 48 161 29 38 Armour II 36 36 500 4500 160M 63 31 38 2 Protecse 25 16 410 6000 280M 59 25 3O (Difco) Protecse 2 35 60 900 6800 260M 51 157 30 41 (Difco) Protecse 3 23 60 750 4200 410M 48 85 33 48 CDifcc) Armour I 57 220 3900 79000 470M 39 61 29 28 Protecse 3 54 120 2500 33000 270M 43 103 27 32 3 (Difco) Tryptose 61 130 1400 20000 350M 53 109 35 31 (Difco) Tryptcne 67 140 1500 14000 280M 53 112 35 37 (Difco) '%iso medium.consists of: NaCl 0.5%, dextrose 0.2%; K2HP04 0.4%, KH2P04 0.15%; peptone 2%; pH 6.6 before sterilization. Table 2 Influence of Armour I Peptone Concentration in a Base medium? on the Early Growth of Shigella.dysenteriae Number of bacteria per m1. ’Generation time 0.4%,1KH2P04 0.15%; pH 6.6 before sterilization. Concentration in minutes of Armour I Time of sampling in hours TIE§;§:o:::pIing o 2 4 6 24 0-4 0-2 2-4 4-e per cent \ I 0.5 19 18 310 5500 35M 59 29 2 1.0 24 15 670 24000 230M 49 22 2 2.0 18 16 1000 32000 250M 42 20 2 3.0 19 20 820 15000 370M 44 1800 22 fie medium consists of: WWW—dextrose 027i 4 - 10 - After it was found that Armour I was the most desirable peptone, it was necessary to determine the Optimum concentra- tion in which to use it. As can be seen in table 2 the most satisfactory concentration proved to be 2 per cent. This concentration of peptone gave an index generation rate of 42, a rate significantly shorter than those yielded by lower and higher concentrations of peptone. Hydrogen ion concentration. Earlier workers believed that dysentery organisms could not survive in an acid mediums The failure of certain preserving solutions to permit the isola- tion of the dysentery organisms substantiated this belief. Bangxang and Eliot (9) reported that;though Dudgeon assumed that pH was the factor of paramount importance in preserving) the viability of dysentery organisms in stools, they pre- sented data that indicated that the adjustment of the speci- men to an alkaline reaction was not the only factor concerned in preserving the viability of dysentery bacilli. To determine the Optimum pH for the growth of Shigella dysenteriae, flasks of broth were prepared and adjusted to pH values of 6.4, 6.6, 6.8, 7.0, and 7.2. All broths were adjusted to the desired pH by the addition of normal NaOH or H01 until the desired reading was obtained on the Beckman pH meter. All pH values given indicate the pH before the broths were sterilized. A pH of 6.6 to 6.8 was found to be the optimum value for the test organism, To determine the possibility of the Table 3 Influence of the Hydrogen Ion Concentration of Amour I Broth* on the Early Growth of Shigella dysenteriae Generation time in minutgs Time of sampling Nmnber of bacteria per m1. PH Time of sampling in hours in hours 0 2 4 6 24 0-4 0-2 2-4 4-6 6 . 4 49 250 10000 2201' 19m 31 51 22 2'7 6 . 6 38 260 8800 3551' 26m 31 43 23 22 6 . 8 .60 276 10000 5151‘ 55m 32 55 23 51 '7 . O 65 260 8800 260T 410M 34 60 23 7'7 '7 . 2 65 149 3500 871' 1981 42 100 26 25 ”Froth cons sts of: Amour I peptone 2%, NaCI 0.5%, dextrose o. 2%, K2904 o. 4%, anroé 0.15%. A Table 3a Influence of the Hydrogen Ion Concentration of Tryptose Broth* on the Early Growth of Shigella dysenteriae ifieneration time number of bacteria per ml. in.minutes Time of sampling pH Time of sampling in hours in hours 0 2 4 6 24 0-4 0-2 2-4 4-6 6.4 70 160 2500 SOT 112M 47 100 30 27 6.6 58 175 5000 91T 230M 37 75 25 28 6.8 48 145 4100 7ST 240M 37 75 25 28 7.0 69 160 3800 65T 224M 41 99 26 29 *fifOth consists of} firyptose 2%, NaCI 0:5%: dextrose 0.2%, .KZ'HPO4 0.4%, KHZP04 0.15%. - 11 - optimum pH varying with different peptones, five different peptones were studied, and in each instance the optimum.pH remained between 6.6 and 6.8. The influence of pH was less marked with Armour I than with any of the other peptones tested. Tryptose, for instance, gave an index generation time of 37 minutes at pH 6.6 and pH 6.8, while pH 6.4 gave an index of 47 and pH 7.0 gave an index of 41 minutes. Buffer. Salter (1) found that K2HP04 in a concentration up to 1 per cent accelerates the rate of growth of Bacillus communis (Escherichia 921;), causing a production of the maximum.number of organisms in a shorter time. Perry and Hajna (12) recommended a mixture of 0.4 per cent KéHP04 and 0.15 per cent KH3P04 to give a constant pH; this mixture was also used and recommended by Darby and Mallmann (7) in their study of media for coliform organisms. That phosphates are important in the nutrition of bacterial cells is indicated by the high phosphorus content of bacterial ashes. The pH of the broths tested was controlled to some extent by the use of the phosphate mixture recommended by Perry and Hajna. Broths containing this mixture gave shorter generation times during the lag phase of growth and larger total numbers of organisms at the end of 24 hours incubation than did the unbuffered broths. The buffered broth gave an index generation time of 51 minutes, while the unbuffered broth gave an index of 58. Table 4 The Influence of a Buffer Mixture in the Base Medium“ on the Early Growth of'Shigella.gysenteriae Nmber of bacteria per ml. fi:;r§tg3:e:m° Buffer Time of sampling in hours “fig-nah? oaragpling 0 2 4 6 24 0-4 0-2 2-4 4-6 one 32 35 600 10000 130M 58 900 29 29 esent 39 36 1000 12000 270M 51 25 33 *Base medium: Armour I peptone 2%, dextrose 0.2%, naci 0.5%; adjusted to pH 6.6 before sterilization. - 12 - Sodium chloride. Mboney and Winsl W’( 13 ) found glucose to be inhibitive to Salmonella pullorum.in an aerated culture unless NaCl was present. Darby and Mallmann (7) found the addition of NaCl to increase the growth of'Eggh,‘ggli in broth media. Because of these findings the influence of NaCl on Shigella dysenteriae was determined. It was thought that the addition of the phosphate buffers might alter the osmotic pressure enough to warrant a change in the usual NaCl concentration, so growth rates were determined in broths containing 0.0, 0.5, 1.0, and 2.0 per cent NaCl. Table 5 shows 0.5 per cent NaCl to give the shortest index generation time. Dextrose. To supply a readily available source of energy, dextrose was added to the base broth in various concentra- tions. Broths containing 0.0, 0.2, 0.5, 1.0, and 2.0 per cent dextrose were tested. There was not much difference in the results yielded by 0.2 and 0.5 per cent dextrose, but higher concentrations seemed to lengthen the lag phase. §§§f_extract. Salter (1) found that 0.3 per cent beef ex- tract added to a peptone water solution gave much greater growth of B, communis than the peptone water solution with- out the beef extract. It should be noted that there was no phosphate in the base broth used by Salter. The effect of beef extract on Shigella dysenteriae ‘was determined by making broth media containing 0.0 and 0.3 per cent beef extract and testing them in the usual Table 5 Influence of'Sodium Chloride Concentration in the Base medium? on Early Growth of Shigella.gysenteriae Generation time Concen- Number of bacteria per ml. in.minutes tration Time of sampling in hours Time 0f sampling of NaCl ogre 0 2 4 6 24 0-4 0-2 2-4 4-6 per cent 0.0 21 16 360 34000 200M 59 26 18 0.3 39 36 1000 12000 270M 51 25 33 1.0 33 37 800 6700 240M 52 722 29 39 2.0 31 19 160 1400 230M 101 39 38 lBase medium.consists o??— Armour I:peptone 2%fl dextrose 0.2%, KgHP04 0.4%, and KH2P04 0.15%; pH 6.6 before sterilization. Table 6 Influence of'Dextrose Concentration in the Baseiuediumi on.Ear1y Growth of Shigella dysenteriae Concen- Number of bacteria per m1. Geniiazigfittime trgtion Time of sampling in hours inohozgmpling d°x1r°5’ o 2 4 6 24 0-4 0-2 2-4 4-6 per cent 0.0 84 110 860 7100 180M 71 301 41 39 0.2 82 110 920 10000 420M 68 278 39 35 0.5 82 110 980 11000 340M 67 278 38 34 1.0 76 95 690 3300 310M 75 361 42 53 2.0 85 95 440 1800 220M 102 722 54 59 LFise medium.consists 3?: Armour‘I;peptone . ,‘fiECImfiTEST‘ KQHPO4 0.4%, KH2P04 0.15%; pH 6.6 before sterilization. - 13 - manner. Since there was no appreciable difference given by the broths containing 0.0 and 0.3 per cent beef extract, it 'was thought that its inclusion in the base medium would not be justified. Levine (14) used K3HP04 instead of beef ex- tract in a modified Endo's medium which he found very suc- cessful. It appears that it is not necessary to use phos- phates and beef extract in the same medium. The,optimum.mediumd From.the data presented it appears that a medium.with the following composition would give the Opti- mum conditions for early growth of Shigella dysenteriae: Armour I peptone..... 2.0 % K 04............... 0.4 P0 OOOOOOOOOOOO... 0.15 Neal-0?...OOOOOOOOO... 0.5 Dextrose............. 0.5 pH 6.6 before sterili- zation ‘ The amounts of phosphate given are those amounts recommended by Perry and Hajna (12); however, it might be more practical in this medium to adjust the phosphates to give a pH of 6.6. It was believed that this medium.would shorten the generation time during the period of physiological youth to a minimum, The. toxicity 93 differential media now _i_r_1 Egg. To compare the efficiency of the various differential media used for the isolation of Shigella dysenteriae to this so-called optimum.medium, broths were made according to the formula of each.medium except that no agar was added. It was assumed that agar is only a solidifying agent and does not Table 7 Influence of Beef Extract in the Base medium* on Early Growth of Shigella dysenteriae Number of bacteria per ml. “mun“ “m D f in minutes ee Time of sampling extract Time of sampling in hours i 102:9 0 2 4 6 K 24 0-4 0-2 2-4 4-6 per cent 0.0 37 350 11000 220T 420M ‘ 28 37 24 28 0.3 30 340 8700 34T 280M 29 34 25 61 1Ease medium.cons stso : Armour I peptone 2.0%,Na01 0. 5%, K2HP04 0. 4%,KH2P04 0. 015%; pH 6. 6 before sterilization. Table 8 Comparison of Various Differential Media with Armour I Broth* for Early Growth of‘Shigella dysenteriae Generation time in,minutes Time of sampling Number of bacteria per m1. medium. Time of sampling in hours in hour; 0 2 4 6 24 0-4 0-2 2-4 4-6 Endo as 40 320 1500 less 78 1300 40 54 than 10T MacConkey 35 3'7 65 250 20014 258 722 157 60 S and S 36 37 245 1500 270M 87 3600 44 47 Armour I 44 61 640 5600 460M 68 278 39 41 ! *Armour Iibroth consists of} Armour‘I’peptone‘E. fiaCI 0.5%, KZ‘HPO4 0.4%,2KH22P04 0.15%; pH 6.6 before sterilization. - 14 - enter into growth reactions of organisms. The growth rates in these broth.media were compared with the growth rates in the Armour Itmediume The results of this study are presented in table 8. None of the differential media promoted growth SO‘W811 as the Armour I medium, Listed in order of in- creasing toxicity, they are Armour I, Endo, S and S, and HacConkey. The desoxycholate media could not be included in this study because of inability to obtain some of the ingre- dients. To confirm the preceding results, a technique simu- lating actual working conditions of these media was employed. A saline suspension of the organism was diluted so that the estimated number of organisms per milliliter was approxi- mately 50. One milliliter of this suspension was placed in each of 25 sterile petri dishes, and five plates were poured with each agar medium, Again the Armour I medium gave the largest count, 43, thus indicating a high.mortality of cells on the differential media. Endo's medium, with a count of 33 organisms per milliliter, was less toxic than any of the differential media tested, though.MacConkey's agar was nearly as good, as shown by the count of 29. Desoxycholate-citrate agar and the S and S medium, with respective counts of 25 and 13, were the most toxic media tested. Since only one strain of Shigella was used, and inas- much as there are other pathogenic members in the group, it was thought advisable to test the other members' growth on the Armour I medium, Growth.rates were determined for six Table 9 Viability of Shigella dysenteriae in Various Differential Media and the Armour I Medium ‘Average Medium. Plate Counts Plate Count Armour I 43 46 39 38 49 43 Endo 36 30 34 35 31 33 macConkey 35 31 25 26 29 29 Desoxycholate~Citrate 27 16 31 29 24 25 S and S 12 13 14 12 l3 l3 Table 10 Comparison of Armour I Mbdium* and Plain Nutrient Broth for Early Growth of Dysentery Organisms Plain Nutrient Broth Armour I Medium Stggin. Number ofIbacteria per ml Number ofIbacteria per ml Time of sam linggin.hours Time of sampling in hours Organism 0 2 4 6 24 O 2 4 6 24 Newcastle 84 87 84 690 130M 76 84 320 6700 640M Shiga 43 105 1600 33T 160M 44 140 2200 42T 410M Flexner 72 75 0 - - 88 140 3300 150T 340M Strong 105 160 2100 17T 230M 105 150 2000 16T 770M Hiss 83 210 2300 SST 220M 96 280 7300 200T 540M Sonne 130 600 21T 750T 150M 120 630 17T 760T 380M ”Armour I medium consists of: Armour I peptone 2%: a01 0.5%, dextrose 0.2%, K2HP04 0.4%,.KH2P04 0.15%; pH 6.6 before sterilization. Table 10a Comparison of Armour IIMedium* and Plain Nutrient Broth for Early Growth Rates of Dysentery Organisms Plain Nutrient Broth Amour I Medium Strain Generation time in.minutes Generation time in.minute of Time of sam ling in hours Time of sampling in hours (Organism 4% 0-4 0-2 2-4 4-6 0-4 0-2 2-4 4-6 eweastle --- 2400 ~-- 40 115 830 62 27 Shiga 46 93 31 28 43 --- 21 28 Flexner --- 2100 --- --- 46 180 26 22 Strong 56 200 32 40 260 226 32 40 Hiss 50 90 35 23 38 79 25 25 Sonne 33 55 23 23 34 50 25 22 ‘Armour I medium.aons.sts of: Armour Ipeptone2%L NaCI 0.5%, dextrose 0. sterilization. 2%, KzHPO4 0.4%, KH2P04 0.15%; pH 6.6 before - 15 - other members of the group of dysentery organisms in the new'medium.and in plain nutrient broth. The growth of the Newcastle, Shiga, Flexner, and Hiss strains was much better in the neW'medium.than it was in plain nutrient broth. The Strong and Sonne strains seemed to grow as well in one medium as in the other. It was interesting to note that Flexner could survive only 4 hours in plain nutrient broth, whereas in the new medium.it gave a count of 340,000,000 cells per milliliter at the end of 24 hours incubation. Discussion of Results In the data which have been presented, the growth rates of Shigella dysenteriae, Shiga, in media of various compositions have been compared. Those media which pro- moted the early growth of the organism were selected as being the most desirable media, and by the process of elimination a formula was derived for a medium that was believed to be optimum.for the organism” It was found that the type of peptone used materially influenced growth during the early growth phases. Armour I peptone was found to give the shortest generation time, but Protecse 3 and Tryptose were also good. It was found that the addition of NaCl and dextrose increased the value of the medium. The optimum.concentration of NaCl was 0.5 per cent and that of dextrose 0.2 per cent. The phosphate buffer mixture recommended by Perry and Hajna proved to shorten the lag period of growth and make the addition of -16... beef extract unnecessary. By studying one variable at a time a base medium was formulated to give the shortest generation time with minimal inoculation. When the base medium.was compared with the various differential media now commonly used, it was found that these media did not support growth as well as the base medium did. Endo's, MacConkey's, sodium desoxycholate- citrate, and S and S agars were investigated. The writer believes that the variations which occur in isolating the organism from.5 or 6 media are due at least to a large extent to the inhibition of the organisms by these media, making isolation improbable unless the cells are present in large numbers. It is realized that the work presented in this thesis has been based on the assumption that a medium which will promote the growth of dysentery bacilli during the lag and early logarithmic growth phases will also permit the survival of an increased number of organisms on a solid medium. It was thought that a broth medium.that would enhance repro- duction in the most critical period of the growth curve would permit the survival of a larger percentage of viable cells when used as the base of a solid medium. The higher plate counts obtained with the new medium from the same suspension of organisms than with the differential media indicate that the assumption was justified. It is hoped that in the future some selective agent can be added to this base medium -17- that will make it a highly efficient diagnostic medium. Summary 1. Armour I peptone was found to be superior to Armour's Siccum, Armour II, Protecse l, Protecse 2, Protecse 3, Witte, Bacto, Stearn, Tryptose, and Tryptone peptones in a broth medium.for the early growth phases of Shigella dysenteriae. 2. A concentration of 2 per cent Armour I was found to be the most desirable concentration of peptone, closely followed by Protecse 3. 3. The pH at which optimum growth was obtained was 6.6 to 6.8. 4. The addition of potassium phosphate buffers produced shorter generation times during the lag phase of growth and larger total numbers of organisms at the end of 24 hours incu- bation. 5. Sodium chloride in the concentration of 0.5 per cent gave more desirable results than 0.0, 1.0, and 2.0 per cent. 6. It was found that the concentration of dextrose could be varied from.0.2 to 0.5 per cent without causing any signifi- cant difference in generation times, but higher concentra- tions increased the generation times appreciably. 7. The addition of beef extract appeared to have no influence on the rate of growth during the lag and early logarithmic phases. 8. The medium having the following formula was selected as being the most favorable medium.for the growth of Shigella - 18 - dysenteriae: Armour I............ 2. 0 per cent KMPO O O O O O O 0 O O O O O O O O. 4 figPO: O 0 O O O O O O 0 O O O O O O l 5 N80 1 O 0 O O O O O I O O O O O O O O 0 5 Dextrose............ 0:2 pH before sterilization: 6.6 9. Generation periods in the neW'medium were found to be shorter than in.Endo's, MacConkey's, and S and S agars. 10. Plate counts obtained from a saline suspension of Shigella dysenteriae were higher on the Armour I medium.than on.Endo’s, S and S,‘MacConkey's, and sodium desoxycholate- citrate agars. 11. The growth of the Newcastle, Shiga, Flexner, and Hiss strains was much greater in the Armour I medium.than in plain nutrient broth; however, the Strong and Sonne strains showed as good or better growth in plain nutrient broth as in the Armour peptone broth. - 19 - Literature Cited Salter, R. 0. Observations on the Rate of Growth of Bacillus 921;. Jour. Infect. Dis. 24: 260, 1919. ' Sherman, J. M3, and Albus, W. R. Physiological Youth in Bacteria. J. Bact. 8: 127, 1923. Stark, C. N., and Stark, P. The Relative Therman.Death Rates of Young and mature Bacterial Cells. J. Bact. 18: 353, 1929. ' Sherman, J. M5, and Cameron, G. M; Lethal Environmental Factors within Natural Range of Growth. J. Bact. 27: 341, 1934. Huntington, Evelyn, and Winslow, C.+E. A. Cell Size and metabolic Activity of Various Phases of the Bacteria Culture Cycle. J. Bact. 33: 123, 1927. Sherman, J. M3, and Albus, W. R. Function of Lag in Bacterial Cultures. J. Bact. 9: 303, 1924. Darby, C. W., and Mallmann, W. L. Studies on.Media for Coliform Organisms. Jour. Am. W. W. Assoc. 31: 689, 1939. . Hardy, A. V. Differential Culture media in the Study of Acute Diarrheal Diseases. U. S. Pub. Health Repts. 54: 287, 1939. na Bangxang, E., and Eliot, C. P. An Investigation of Preserving Solutions for the Recovery of Dysentery Bacilli from.Fecal Specimens. Am, J. Hyg. Sect. B. 31: 16, 1940. 10. 11. 12. 13. 14. - 20 - Coleman, M. B. The Differentiation and Identification of Bacillary Incitants of Dysentery. A. J. P. H. 30: 39, 1940. I Buchner, Longard, and Riedlin. Centralbl. f. Bakteriol., 1887, ll, p.1. Perry, C. A., and Hajna, A. A. A.Modified Eiijkmann Medium. J. Bact. 26: 419, 1933. Mboney, G., and Winslow, C.-E. A. The Metabolic Activity of Various Colon Group Organisms at Different Phases of the Culture Cycle. J. Bact. 30: 427, 1935. Levine, max. J. A. P. H. A. 8: 886, 1918. .9. . ..¢t~..()¥a.y¢.l. quy ’J‘VVJ‘. 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