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Jr'- \i of .1 on o . la‘ . . .3 x 4 v. d ..c x .\ f. \. :c. g u. no . h 0.. . .A o F \. \ ‘.I b u u a \J‘ o - 0...!I . .. . ... \ .. . 0‘ o . v. 24> 0-169 This is to certify that the thesis entitled A SW Q1 The Dieeecutien 0! 8 gen]: And The Sensitivity Of The e A Quaternary Antonius Compound. presented by Hornet H. Stilt!” has been accepted towards fulfillment of the requirements for H08 0 degree in ____B&°W101°U ijor professor Date ‘ 1522 1 ; _ 4 vv—i—C'fi wT—vr -ftv ‘ — ' B, e U _ t. a \ .‘ \ J .. I. ...,..A! B. , .4 I‘MLU 1:3 All: A STUDY ON THE DISSOCIATION or mm mm AND THE SENSITIVITY OF THE VARIANTS TO A QUKTERNARI AMMONIUM COMPOUND by Barnet M. Sultzer A THESIS Submitted to the Graduate School of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of MASTER.OF SCIENCE Department of Bacteriology and Public Health 1951 CONTENTS I. Introduction 11. Historical Rescue” III. Bacteriological Study of The Cultures IVL Hothode UBed to Induce Bacterial Dissociation Experiment 1. Experiment 2. Experiment 3. Experiment 4. V; Germicidal Sensitivity Determinations of Selected Variants of Sal. pullorum Culture Study Sensitivity Tests Results VI. Dieoueaion VII. Summary VIII.Literature cited Page 10 13 15 l4 15 16 18 18 21 39 Acknowledgement The author wishes to express his sincere appreciation for the generous assistance and guidance given by Dr.‘W; L. Mallmann. He also would like to thank M. Silverman for taking the photographs and I. Shkleir for donating the smooth immune serum. INTRODUCTION the phenomenon of bacterial dissociation or variability is a subject of both fundamental and practical significance. is a result, this phenomenon in all its diversity has been intensively studied for the last thirty years; however, much of the evidence which has accumulated has been Just inconclusive enough to warrant widely different interpretations and theories. Thus, the various changes in colonial and cellular morphology. biochemical behaviour. antigenicity, and virulence exhibited by the bacterial cell may be possible expressions of one of several fundamental conceptions. Such concepts include . (l) bacteria vary in definite trends or patterns , (2) the environmental conditions imposed select variants or nay directly result in variation. (3) bacteria Just as other organisns are subject to mutations, and finally, (4) the various ferns expressed by a bacterial species represent progressive cntogenetie transformation. commonly referred to as life cycles. Regardless of the theory one my adopt, the evidence to date indicates that dissociation occurs in virtually all bacterial species , and changes may be induced under suitable environmental conditions. Iith regard to the practical significance of bacterial dis- sociation. direct consideration can be made to the anomalies that frequently occur in the phenol coefficient results of quaternary alumina germicides. liaier and Muller in 1936 emphasised that dissociation of test organisms may result in a increase or decrease in sensitivenese of the bacterial to the test solutions. It seems apparent then. that the stability of the test organism in regard to dissociation must be considered if more accurate results are to be expected in the phenol coefficient tests. Mallmann in 1932 reported finding three stable colonial types in the Salmonella group. These three strains of gal. pullorum were found to be (1) a stable smooth type, (2) a stable rough type, and (3) a variable rough-smooth type. Dissociation incitants were without permanent effect upon the pure rough and smooth types, while true dissociation changes occured only in the variable type cultures. In view of these findings and other evidence presented in the literature , this study was lmdertaken with a twofold purpose in mind: to attempt to isolate a stable smooth strain of §_a_l_. pullorul, and to determine the sensitivity of smooth and variant forms of this organism to a quaternary ammonium compound. HISTORICAL RESM' Before pure culture techniques were developed, bacteria were regarded as belonging to an homogenous group, possibly of but a single species. In fact, in 1877, Nigeli propounded the doctrine of pleomorphism wherein all fission fungi were one type of cell. highly variable and capable of passing from one morphological type to another, as well as being capable of undergoing profound altera- tions in biochemical behaviour. The opposite extreme of monomorphisn became predominant with the subsequent development of pure culture methods by Koch and others. This doctrine proclaimed a constancy of species and fora in bacterial maphology. Despite the adoption of nonomorphisn by the great majority of bacteriologists, data accumulated ahich indicated that though most bacteria showed a remarkable constancy of form, morphological and physiological variation is not uncommon. In 1918, Baerthlein demonstrated that colony variability may serve as a criterion of cultural variability, for closely correlated with colony types are other important characteristics of the organism including cellqu morphology. piglent and slime production. fer- mentation reactions , serological reactions and virulence. Arkwright , 1921. recognised the significance of Baerthlein's work and described smooth and rough colony forms of the colon-tuphoid-dysentery group. Subsequent work by many others followed in voluminous proportion regarding the organisms belonging to this group and many others. Hadley published extensive literature reviews in 1927 and 1937 citing numerous instances of dissociation changes in various organisms. The colonial forms described fall into six general categories; namely. smooth(S), rough (R), intermediate (SR), mucoid (M), dwarf (D), and gonidial (G). The smooth colony is characterised by a round, even margin, and a convex smooth and glistening surface; the organisms are uniformly turbid in broth and produce a stable sus- pension in 0.85%uNacl solution. The rough colony is described as follows: the margin is irregular; the surface is flat, uneven, and granular; the orgnnisms produce a granular sediment in broth and clump spontaneously in physiological saline. The intermediate colonies are those that appear intermediate in form.between S and R colonies and usually change into one or the other. The mucoid.fcrms have a moist. glistening. mucous-like consistency; the colonies tend to coalesce. The dwarf forms are characteristically very small measur- ing less than 1 mm. in diameter and differing in respect to larger colonies of the same organism only in size. Finally, the gonidial forms are minute colonies representing,growth from filterable forms of bacteria; however, much disagreement still prevails as to the actual existence of these so-called filterable forms. ‘While dissociation of various members of the Salmonella group has been widely studied, comparatively little work has been forth- coming with Sal. pullorgm. In 1930, Plastridge and Rettger isolated. from.all appearances, a rough strain of Sal. pullorum from.infected adult fowl and young chicks. These same authors described in 1932 three principle colony types of this organism including the smooth, rough, and a somewhat intermediate form. Gauger (1936) reported the isolation of two rough strains of Sal. pullorum from.baby chicks; however, though these strains varied morphologically from typical a. smooth cultures , no autoflocculation in saline was exhibited. In 1959 Morgan and Beckwith observed that Sal. pullorun temporarily exhibited a nuceid type when grown at temperatures between 15° and 25° c.. but when cultured at 31° 0. the loss of the mucoid character or reversion t) the smooth type immdiately occured. The most extensive work on the dissociative character of is}; pullorun was reported in 1982 by liallmann. Although it is generally believed that the chief culture types (8 and R) are easily convertible one into the other, Iallmann presented evidence to the contrary. Working with stock cultures that had been kept in culture for many years, a total of 27 of these were found to be characterised by their smooth colony foraaticn. 13 were markedly rough and at no time showed smooth characteristics, while 16 of the organisms were variable R to S and 8 to R types. hplcying numerous well recognised dissociation incitants for considerable time periods including brilliant green beef extract broth as a chemical incitant, aging at various temperatures, rapid transferring, immune smooth and rough serm, bacteriophage, and animal passage, it was found that there were several strains which were unaffected by these methods, maintaining the original colony appear- ance of either smooth or rough. The 88 strains found variable as stock cultures were, on the other hand, easily changed to either 3 or R colonies depending on the type of incitant used. In support of this evidence, it was indicated that a large number of workers previous to 1950 found refractory organisms that did not respond to inciting agents. although in many cases they were disregarded in the final conclusions. Barber (1907) obtained abnormal strains of £92; 39;; that never reverted to the parent strain. . Clark (1910) was unable to convert avirulent diptheria bacilli to virulent types by means of animal passage or by sensitization with homologous immune serum. Buchanan and Truax (1910) in a study analogous to s and R types were mable to secure high and low acid producers of mm They attributed their lack of success to the fact that a pure-line culture has invariable characteristics and therefore variations. in their estimation. will occur in cultures of mixed lines only. Bchutse (1921) was unable to convert B type paratyphoids to the 8 form. In 50 rapid transfers DeKruif (1922) found a strain of the bacillus of rabbit septicemia that would not revert to the normal type. . Reiman (1925) could not revert R type paratyphoids to the 8 form by rapid transferring or 105 passages through nice. He believed that the change to an I type was characterised by a loss of certain proper- ties in the cell that cannot be regained. Jordon (1928) could not induce smooth types in two strains after 72 rapid transplants. In a study of pneumocoeci. Dewson and Avery (1927) found one B strain that could not be reverted to the parent 8 type by animal passage. In the previous year Bronfenbrenner. Huckenfuss, and Kerb reported an avirulent strain of _B_._ pestis caviae which remained avirulent even r . . -A . w e ‘,. . I e . . - - . v . . r t ‘ . . . . s . v . s . . - -‘ I l w v . . . . , - ' u. ‘ 7’ -' - I ‘ , A A ‘ ‘ a , A ‘ . - . - . I . . after zoo rapid transfers, and concluded that this irreversible reaction is not a phenonenon of active hereditary innunisaticn but rather a result of irreversible variation. Julianelle (1928) employing rapid transferring in plain broth and broth containing supernatant serun did not convert B type strains of Friedlaader‘fi bacillus to their 8 types. Animal passage was like- wise unsuccessful. In the sane year Dulaney found that the reversal of stabilised rough forms of §_._ 331; was difficult. chd (1928) reported that , glossy colonies (8) of haenolytic streptococci derived fron natt colonies (l types fron fresh isolated cultures) were pernanently attenuated and could not be nade virulent by growing in the presence ‘ of undiluted normal human serum. The attenuated R colonies. further- more, returned to virulent cultures upon growth in seru. later publications also contributed significant data to the existence of highly stable forns. Leslie and Gardner (1981) working i with four phases of _B_._ Ertussis (two 8 and two R) reported that 'phass I", which appeared to be the lost advanced R, became 'firnly and irrevocably established' in some strains. 111 other strains were interconvertibls. In and l‘enyvessy in 1932 found extreme rough cultures of the influensa bacillus, characterised by thread fans and,twisted nycelim that renained unchanged for years. Their reversion to smooth colonies could not be accomplished by the use of any of the media lost favorable for propogation of smooth phase cultures. Only transdient success was obtained by animal passage - reversal to R from the 8 form being accomplished after a few passages on . . ' v - « .I . ,' ‘ ¢ ‘ w an? . 1 , . I l - . v .) v - - s . 1 . s' - I 4‘ - . A . . ‘ s ‘ ‘ ' s . - e . . . , . . n O . . . ‘ . r s. n T .. . _ . . . \ . . . e chocolate agar. Further evidence was presented by T’ung in 1940. By irradi- ation with gamma rays obtained from radon, a change of s to B forms of pneuaococcus was regularly induced, the change being so complete um it was 'impossible to revert the dissociants to the primary state. " the R form appeared suddenly. bean totally avirulent and irreversible. Animal passage was unsuccessful in reverting the organisms. Later in 1947. Kelner found ‘a strain of Chromobacterium violaceum, derived from a secondary colony formation on LiCl agar, that was stable for 33 months undergoing at least forty transfers on LiCl free medium. Won (1950) produced 'giant' cells of Pasteurella pestis by treatment with camphor that neither reverted nor dissociated after ten animal passages or fifteen medium passages. As previously mentioned, in testing quaternary ammonia compounds using the regular F.D.A. phenol coefficient. fluctuations in end titers frequently occur from day to day, even when the same individual performs the test. During the past several years many investigators have studied the quaternary ammonium germicides in this light, and the variations have been attributed to one or more factors related to the canpounds or to the test procedure itself. Inconstancy of the peptone composition of the culture media, unequal distribution of the bacteria in the test medium, the "carry--over'I of bacteriostatio amounts of the quaternary ammniun compound , and bacterial dissoci- ation of the test organisms have been considered the more important factors. Numerous modifications of the phenol coefficient method have been devised to overcome the first three factors mentioned, and vigorous controversy has arisen over the results obtained and theories proposed. The factor of dissociation. on the other hand. has received comparatively little attention. Kline]: and Ihbreit in 1947 stated that variations in the susceptibility of the indi- viduals comprising the population of the inoculum may account for viable organisms after short exposures to the quaternary ammonitm germicide. In oonne ction with this proposition is the reference to bacterial dissociation made by Iaier and Muller discussed above. BACTERIOLOGICAL STUDY OF THE CULTURES A.total cf 48 stock cultures of'Sal.gpullorum.'was received for study. Three of the strains. originally isolated in 1942, were obtained from the culture collection of the Department of Bacteriology and Public Health. lichigan State College. In... cultures had been freshly transferred.to tryptose agar slants. The remaining 45 strains ‘were obtained from the Poultry Diagnostic Laboratory, Department of Bacteriology and Public Health, Michigan State College. These cultures had been originally isolated from infected chickens or turkeys in July of 1960 and were received freshly transferred on nutrient agar slants. Immediately upon receipt, all the cultures were incubated for 48 hours at 37° 0. and subsequently stored at 8-100 C. for future reference. A.preliminary survey of'all the stock cultures use made to determine the particular colony characteristic of each strain. The media utilised consisted of tryptose broth.and agar and nutrient broth and agar. The tryptose broth was composed of 2% Bacto-tryptose and 0.6% Neel, while the fbrmula for the nutrient broth was 0.9% Baoto- Beef'Extract and.0.6%.Bacto-Peptone. For the preparation of the agar. 1.6% plain agar was added to the respective broth formula. All media were adjusted to a pH of 6.9 before sterilisation. The broth was dispensed in ten.m1. quantities and then autoclaved. The agar plates were prepared in the usual manner using 15 to 18 m1. of agar per plate. Tryptose brothnwas seeded first with each stock culture and the tubes were incubated fbr 24 hours at 57° c. Tryptoso agar plates 11 were streaked from the broth cultures employing a needle streak technique in which approximately 5 mm. of the tip of the inoculating needle, bent at a 30° angle, was dipped into the broth culture and then lightly streaked over the surface of the agar plate making ten to twelve individual streaks. All plates were incubated for 48 hours-at 57° C. and subsequently observed as to colony appear- ance. Observations of the agar plates were made using a dissecting microscope with a total magnification of 101:. The colonies were“ illuminated with oblique transmitted light. To insure against any contamination and eliminate, if possible. any subordinate types, each culture was repeatedly plated by picking representative colonies, subculturing in broth for 24 hours, and subsequently plating as described above. lhen cultured in tryptose media it was found that 39 of the stock cultures demonstrated typical smooth colonies. The remaining nine cultures were intermediate in form. lhen representative colonies of each smooth culture were subcultured in nutrient media it was found that all the strains except Nos. 796, 856, and 978 showed a definite change in colonial appearance which persisted on successive platings. Generally the surface of the colonies became granular and wrinkled, taking on a somewhat opaque cast. them these colonies were subcultured again on tryptose media the typical smooth forms reappeared. All nine intermediate cultures produced the same forms on nutrient agar as on tryptose agar. 11;. data are presented in Table I. It thus appeared that a temporary environmental effect had been 12 produced with the smooth cultures, and in actuality, the changed ferns found on the nutrient agar were not true variants. In any event. the three strains which remained the same on both media were considered to be more suitable fbr further study. Hallmann (1932) found such strains of £2}: pullorum to be characteristically more stable to various inducement methods. Prior to the dissociation studies, cultures Nos. 796, 866. and 878 were checked culturally, morphologically, and physiologically to insure their species type. Smears were stained according to Gram's method and fresh nutrient agar stock slants were made from the final pure-line strains. The data are presented in_Tab1e 2. The fbllcwing studies represent an attempt to evaluate the influence of dissociating agents on these three strains. 13 METHODS USED TO INDUCE BACTERIAL DISSOCIATION Experiment I various inorganic salts have been frequently reported in the literature as successful agents for producing rough fbrms in numeroue bacterial species. Hadley (1927) demonstrated R ferns in.members of the paratyphoid group by using lithium chloride. In this experiment,a plain.nutrient broth.was prepared to'which 'was added lithium chloride in a concentration of one percent. The pH was adjusted to 6.9 and the broth was tubed in ten m1. quantities and autoclaved. Each strain was inoculated into the broth tubes, which were then incubated at 37° 0. At various intervals, nutrient agar plates were streaked from each culture and the plates were observed after 48 hours incubation. ‘leekly transfers mere made using one loopful of’culture. The experiment was carried through a period of 80 days. The data are presented in Table 3. Initial platings‘were not accepted as indicative of a true variant. Typical colonies were picked.and subcultured in nutrient broth and replated. If the colony type remained true to ferm.as first observed, the variant‘was recorded. The results indicate that variants appeared in each strain by 24 days. Several variant forms from strain No. 795 appeared on the agar plates after 20 days and reoccurred inter- mdttently from that'time on. All of the forms were generally inter- mediate, having surfhces that were either granular and slightly veined or coarsely granular to somewhat wrinkled. The margins remained circular and entire. Strains Res. 856 and 878, on the other hand, exhibited a different 14 variant. ‘After 24 days exposure to lithium chloride, colonies which were smaller than the typical smooth forms, decidedly darker, raised, and granular became evident on plates of both strains. By 48 days this variant disappeared on plates from No. 856 and in another eight days the same thing occurred'with culture No. 878. Instead, a new fern considerably rough in character, appeared in both instances. Refer to figure 1. It may be noted that typical smooth colonies for each strain per- sisted throughout the entire 80 day period. Refer to figure 2. Epsriment 2 Various toxic substances and dyes when used in sublethal concen- trations have been reported as successful dissociation incitants. hallmann (1932) utilised the dye brilliant green obtaining R variants 'with several fig}, pullorum strains. This method was followed in this experiment. The medium.was prepared by adding seven m1. of a one percent aqueous solution of the dye to one liter of nutrient broth. The pH was adjusted to 6.9, the medium was dispensed in ten.md. quantities and sterilised in the autoclave. Strains Nos. 795, 866, and 878 ‘were seeded into the broth and the tubes'were then incubated at 87'0 C. NutrientLagnr plates were streaked at various intervals. The data are presented in Table 4. 'leekly transfers were made in fresh broth. The experiment was carried on fer 81 days. Intermediate variants appeared from each strain after 12 and 18 days and persisted throughout the rest of the experiment. Typical smooth colonies were observed in each strain up to 32 days, after 15 which they were no longer noted. Figure 8 is a photograph of the intermediate strain derived from culture No. 795. With cultures Nos. 856 and 878, the same type of intermediate variant that appeared after exposure to LiCl also appeared in this experimnt. However, at no time were any rough colonies observed. It may be noted that with brilliant green as the inciting agent, a shorter time interval elapsed before variants were observed. Experiment 8 many workers have commonly observed variants in old stock cultures. Thus, methods of aging have been utilised to recover these forms. Three different broth media were employed in this experiment at incuba- tion temperatures of 20° - 25° 0. Nutrient, tryptose, and liver infusion broths were prepared in the usual manner. Tubes were seeded with strains of §_a_l_._. Elorum being studied. Nutrient agar streak plates were made at various intervals; the total incubation period being a month. The data are presented in Table 5. Strain No. 795 exhibited intermediate colonies after 16 days in nutrient broth, eight days in tryptose broth, and 30 days in liver infusion broth. Strain No. 878 demonstrated intermediate colonies after a month on nutrient broth, 18 days in tryptose broth, and a month in liver infusion broth. On the other hand, strain No. 856 remained smooth after a month's incubation in each mediun, and also typical smooth colonies prevailed in each culture for the entire length of the experiment. One defined rough ferm was observed with strain No. 795 after a month in tryptose broth. This colony closely resembled those rough colonial types exhibited by the spore-forming bacilli. The surface was very coarsely granular and the edges were irregular. However, an interesting phenomenon was noted when such a colony was subcultured in plain nutrient media. It was found that after 48 hours on the agar plate both typical smooth colonies (although in the minority) and the rough variants described appeared. Several successive platings revealed the same thing. lhether these colonies were unstable variants as describedby Dcskowits (1956) with §_a_l.. aertrzche, or merely mixed strains, could not be determined. Experiment 4 Irradiations by I-rays, gamma rays, and ultra-violet light have been reported as successful dissociating incitants. Haberman and Ellsworth (1940) demonstrated the dissociative effects of I-rays on Staphylococcus aureus and Serratia marcesoens, producing many variant forms. Graingcr (1947), also using x-rays, produced intermediate colony forms from smooth cultures of fitnhosa. Ultra-violet light has been shown to produce biochemical variants. This type of irradiation was used here in an attempt to induce changes in the colonial morphology of the selected cultures. ‘ Twenty-four hour nutrient broth cultures of strains Nos. 795, 858, and 878 were smeared on a four square inch surface of a nutrient agar plate with a sterile cotton swab. Two plates were made for each strain in the manner described. The first plate, with the cover removed, was exposed at a distance of four inches from a 15 watt 9! germicidal lamp for a period of 30 seconds. The second plate was eqosed in the same manner for a period of 60 seconds. A third plate was streaked by the needle technique and left unexposed as the control. The plates were incubated and then observed. Two variants that were selected for the germicidal sensitivity 17 tests described below were also exposed to the ultra-violet light. The experiment'was repeated twice for all strains with the same results. The data are presented in Table 8. No colonial variants or reverted ferns were produced. 18 GERMICIDAL SENSITIVITY DETERMINATIONS OF SELECTED VARIANTS 0F SAL.PULLORUM Culture Study; Two cultures of El. pullorum were selected for this study. Culture No. 795 demonstrated both typical smooth and intermediate colonies on nutrient agar after 32 days exposure to brilliant green nutrient broth. Culture Ho. 856 exhibited smooth and rouyu colonies after 48 days exposure to one percent lithium chloride. Representative colonies of each form were picked and subcultured in nutrient broth. Successive platings were made to insure the initial stability and purity of each colony type designated. Smears were made and stained according to Cram's method. The biochemical behavior, motility, agglutinability with 5:1. pullorum smooth immune serum, and reactivity in physiological saline were tested for each culture. Data are presented in Tables 7 and 8. It should be emphasised that this preliminary culture study was undertaken to make sure that the smooth organisms isolated were identical not only in colony form, but also physiologically and serologically with the original cultures and species. The variant forms were studied to determine any differences in the nature of the organisms bosides the colony appearance. Cultures Nos. 796 S, 795 SR, and 856 8 gave results representatiw of 8:1. pullcrum organisms; i.e., dextrose and mannitol were fermented with acid and gas while lactose, dulcitol, sucrose, arrl maltose were not. Furthermore, the cells were gram negative, non-spore forming rods, and morphologically typical of what is most frequently described. Each culture gave a strong positive reaction with smooth immune seruum and formed stable suspensions in physiological saline. Culture No. 866 R, on the other hand, differed. Ihile acid and gas were produced in mannitol, only acid was produced in dextrose. Maltese was also reacted upon with acid resulting. The organism appeared to have lost the typical gram's staining characteristic, 1.... while most of the cells were gram negative, some were tending to become gran positive. liorphologically, the cells were uniformily short rods arranged in packets. 1 granular suspension and sediment were apparent in 0.85% laCI. When the salt concentration was reduced to 0.21%, a more stable suspension was apparent, although some fine suspended granules were still present. Furthermore, in dilutions of 1-100 and 1-aoo of positive smooth immune ..run, the agglutin- ability was somewhat less. It my be noted here that the antigenic suspension of this culture was prepared using a 0.21% HaCl solution. Arkwright (1921) found that rough organisms sometimes formed more stable suspensions when the salt concentration was reduced, An attempt at reverting the two variants was also undertaken to detemine whether the forms typical of the parent strains could be recovered. nutrient broth cultures of Nos. 796 SR and 866 H were made and at 18 to 20 hours, transfers were made into fresh broth. This method of rapid transferring was continued and nutrient agar streak plates were made from each tube. After four such transfers, strain No. 856 R reverted completely to typical smooth forms. Culture No. 795 SR reverted after nine rapid transfers. As described in Experiment 4, ultra-violet light had no effect on these cultures. Unfortunately, time was not available for making ‘ > O C ‘ . $ . u - . C . s . ‘ - . » u ‘ e S a u ‘ I ‘ A y . . , s 9 I a _ . v e . I . ‘ V , 1 . . . , ' . . ; ' O . s e , . e a o a . , a a complete study of the reverted forms, although gun's stains demonstrated typical gran-negative rods. Sensitivitj Tests: The procedure utilised in testing the germicidal sensitivity of the selected cultures consisted of a modification of the P.D.Ao phenol coefficient method. fen ml. of nutrient broth was seeded from the stock slant and incubated for 24 hours at 37° c. The broth culture was then centri- fuged at 2&0 R.P.H. for 20 minutes. The supernatant broth was discarded aul the cells were suspended in an equal quantity of saline and reoentrifuged. This washing procedure was repeated, after which the cells were finally suspended in ten m1. of saline. In the case of culture No. 856 R, a 0.21% Nacl solution was used at all times in this procedure. The final suspension was shaken for 10 to 15 minutes to insure the dispersal of any clumps. Since some fine granules persisted with culture lo. 866 R, this suspension was filtered through sterile Whatman filter paper No. 1 into a sterile test tube. Total cell counts were nde of each cell suspension with a Petrauf-Hausser cell counter. Each suspension was adjusted in volume with the appropriate sodium chloride diluent to give a count of 320 million cells per ml. One-half ml. was used as the inoculum for each medication tube. Nutrient agar plates were streaked from each cell suspension ani incubated for 48 hours. These plates served as a control for the colony stability. The cresclic compound employed and sold under the trade name Lysol, 21 consists of soap, cresylic acid, and crtho hydroxydiphenyl as the active ingredients in a total concentration of 59%. The Lysol was used in seven aqueous dilutions ranging from 1-300 to 1-600. l‘he quaternary ammonium compound, sold under the trade name of Phemerol Chloride, consists of an aqueous solution of a para- tertiary-octyl-phenoxy-ethoxy-ethyl-dimethyl-benayl-ammoniump chloride-monohydrate. It‘was used in dilutions ranging from 1-10,000 to 1-70,000 and l-30,000 to 1-90,000. Dilutions of phenol (1-90, 1-100, l-llO) served as the standard control. The test was run as otherwise prescribed by the P.D.Aa method. Results: The data are presented in Tables 9 and 1c, and represent results repeatedly obtained for'each culture tested against each compound. using Lysol a phenol coefficient of 5.0 was obtained with each smooth and variant culture, indicating no difference in sensitivity to 'this cresolic compound. Considerably different results were obtained with the quaternary ammonium compound. 1"irst of’all, each culture was more highly sensitive to this compound than to Lysol. Secondly, each strain and its variant differed as to their resistance to Phemerol Chloride. Culture No. 795 8 gave a phenol coefficient of 400. Culture no. 795 SR showed a phenol coefficient of 500. Culture No. 856 8 showed a phenol coefficient of 650, while culture No. 856 R gave a coeffi- cient of'550. It appeared then that strain No. 866 was more sensitive than strain No. 795. It may be noted that the resistance to the phenol standard controls was quite constant, as was the case with the expressed resistence of the test organisms to the cresolic germicide Lysol. DISCUSSION The first objective of this study was to attempt an isolation of a stable smooth strain of 351. pulley, or in other words, an organism which nintained its smooth colonial characteristics despite being exposed to various dissociation methods. Forty-eight stock cultures of Sal. pulloru! were initially studied, and of these, three were found to produce the same smooth type of colony whm grown on tryptose and nutrient agar. While ultra-violet light had no effect upon these cultures, variants were produced by exposure to lithium chloride and brilliant green. One strain remained stable when aged in different media at room temperature for a period of a month. The other two strains exhibited several variants. In reality, the problem of stability is of a relative nature. Thus, one can present a new environment to an organism and observe a change in the colonial morphology upon initial plating, while another culture would not show an observable change until after a month ’s exposure, or still another would not vary at all for a year or more. lolfram(1951) using _8_a_l_. pullorum cultures also obtained from the Poultry Diagnostic Laboratory of Michigan State College, found a number of variants after only three days incubation in plain tryptose broth. 0n the other hand, the strains this writer used were considerably more stable, although, eventually variants were produced with various incitants. It was further found that by a method of rapid transferring in broth, a typical intermediate am rough variant previously induced reverted to colonies identical in morphology to the original smooth forms. Thus, the variants pro- duced were not of a very stable nature, although they did maintain their particular characteristics in stock culture and when grown in preparation for the germicidal sensitivity teste. The second objective of the study was, as mentioned in the Introduction, to determine the sensitivity of the variants to a quaternary ammonium compound. In these determinations the experi- ments were designed so that a comparison in sensitivity could be drawn between the variants and the original forms to a cresolic as well as a quaternary ammonium compound. The inoculum'was standardised in each instance as to the number of cells introduced into each medication tube, and sodium chloride suspensions of the organisms were used to discount any possible interference by the peptone content of the culture medium. Brewer (1943) and others considered the phospholipid content of peptones to be an important factor in interferring with the germicidal action of quaternary ammonium compounds. In the case of the phenol standards and the cresolic compound, a constant resistance on the part of all the test organisms was apparent. However, this was not the case with the quaternary ammonium germicide. The intermediate variant of one strain, from.the results obtained, was more sensitive to the germicidal action of'the quaternary ammonium compound than the original smooth culture of the same strain. The rough variant of the second strain, however, was more resistant than the smooth culture of the same strain, Thus, both an increase and decrease in sensitivenese was observed by different variants of the same organism. These results serve to emphasise the possible action of bacterial dissociation in producing fluctuating end titers when quaternary ammonites compounds are tested according to the phenol coefficient method. Whether a truly stable organism would show a consistant level of sensitivenese to a quaternary ammonium compound, while reasonable to expect, could not be determined in this'study since such an organism was not isolated. 26 SMART l. A.dissociation study was made of'three strains of gal. pullorum that were originally smooth in colonial morphology on stock culture. 2. Intermediate and rough variants were found to occur with the application of certain dissociation incitants. S. Ultra-violet light had no effect on the colonial morphology of smooth, intermediate, or rough cultures of the same organism. 4. No observable differences were found to exist in the sensi- tivity of smooth, intermediate, and rough cultures of S31. pullorum to phenol and the cresolic compound Lysol. 6. Both an increase and decrease in the resistance to a quaternary ammonium compound Phemerol Chloride was observed to occur with variants of Sal. pullorum, i.e., the intermediate form was less resistant and the rough form'was more resistant than the typical smooth culture. TABLE 1: Colony Appearance of Stock Cultures of Sal.pullorua on Tryptose and Nutrient Agar Culture Number 13-; 13-2 13-3 770 775 775 781 703 795 822 827 829 830 843 856 860 866 867 889 873 874 878 879 880 892 896 897 909 911 937 940 942 945 949 961 950 982 983 969 980 990 1005 1008 1033-2 1035-1 1040-3 1049-2 1028 8 - Smooth SR - Intermediate Tryptose Agar m w cocoon Uncommoooooomoommmooco U) giuss: u: :3 will I! mmcocococorocommmmmmmmmmmmmmmmmmm Nutrient Agar SR SR SR SR SR SR SR SR 8 SR SR 8R SR 8 SR SR SR SR SR SR 8 SR SR SR SR SR SR 8R SR SR SR SR SR SR 8R 8R SR SR SR SR SR SR SR SR SR SR SR bl 28 .o o...» a. are 3 ”53333 .33 33 .. 4 mac one v.34. I S hm O -nt 1.5 f 0H0 each». .. 3 a 2.» u 3 a one .. 3 m we. 83 H3 $.38 so» W .31? -29.: Ends 23d. EB - lflB ghondsm .ku do .2933 388 go 33.23336 Hoodonoasmm no. noouooonfio: a... 3m; 29 .593 SS Soc 3 .2... 2332» auto: . swoon u a 3.33.235 .. mm spouse u a _ . m.m m.m m.m m.» mm.m mm.m mm.m mm.m mm.m m m m m m m a new do do Mum m5 um um.” mum and mm.» m m m m m a m 9.8 mm; a $4 .. m m mum 55 $2M mm; m m m m m m we. 8 «a A S. on we 3 mo. 8 on 8 S 2 a lo a 1838 Sofia s enouomnm me when Hennmauo canpnoo 233m 45 no .8329 £33 oooooaom no .3820 Sofia 3.82 .8 co nodes 3 an .Apcap ocean pneuaauap meeau u« some showcase» haxoen.s e opoaooauooon 7 mm goooaa : a a» mu _ mm mm mm mm mm mm.m mm.m n mm.m u m e a a was mu mm mm mm mm mm mm mu.m mu.m mu.m m m a a a one as as mm mm mm mm as uu.m mm.m mu.m mm.m m m m a one no on so on , no so on a» on W we «a o o N I; lay hooaoo nopanz * enouomflm Mo when ammuwuao chevHoo auLOflHom.Hem mo uenepnso mucosa oopooaem no mocha pnmwaamam Mo panama «e num on) 3} ”8”me“ “MM” Show .53 noose no? ...—:qu going ..Hdm no 33333334 3 as 35 TABLE 9: The sensitivity of Selected Cultures of §Elf Eullorun to Lysol Culture Colony Dilution. Transfer Minute: Phenol Coefficient (5) (10) (15) 796 3 1-300 - - - 1-560 - -» - 1-400 - - - 500 5 1450 ,l - - W = 1-500 ,( - - 1-660 l I / 1-600 / K K 795 SR 1-500 - - - 1-350 - - - I.“ o u a 5m 5 1-450 - - - 166' ’- 1-600 / - - 1-550 / / - 1-000 / ,l - 866 3 1-300 - - - 1-350 - - - 1-‘00 - - " 500 _ 5 1.450 / - - TOT " 1-600 / - - 1-550 / ,1 - 1-000 ,1 / - 856 82 1-300 - - - 1.350 - - - 1-400 - - - 500 - 5 1460 - - - "I‘M‘ - 1-500 / - - 1-550 ,1 ,1 - l-eoo / K / Phenol Standard 1-90 - - - 1.100 / - - 1-110 / ,l - -. .d to Phemerol TABLE 10: Culture Colony 796 8 796 SR 856 S 856 - SR Phenol Standard Dilution 1-10 .000 1-30.000 1-30 .000 1-40 .000 1-50 .000 1-60.000 1-70.” 1-30 .000 1-40 .000 1-50 .000 1-60 .000 1-70 .000 1-80 .000 1-90 .000 1-30 .000 1-40 .000 1-60 .000 1-60 .000 1-70 .000 1-80 .000 1-90 .000 1-10 .000 1-20 .000 1-30 .000 1-40 .000 1-60 .000 1-60 .000 1-70 .000 1-90 1-100 1-110 Transfer Minutes (5) ‘k‘k‘kI I I I \‘K‘k‘K‘kl I \\\\I I \‘kIIIII \‘k I (10) \‘k‘kI I I‘ \X‘K‘K I I I \‘k‘kl I I I I \‘Klllll \I (15) \‘K‘Kllll \‘k‘kIIII \‘I\\III 36 Sensitivity of Selected Cultures of _S_o._l. Eullom 40.0.9.0; "—156 50.000 65 .000 55.000 Phenol Coeffi oient 400 500 650 550 .Q 37 Figure 1. Rough colonies of §_u_l_. Bullorum on nutrient sgsr after 48 hours. Culture 110. 856 st 0 magnification of 3.3. I' a) ‘I 1.2:; Figure 2. Typical smooth colonies of gel. gullorm on nutrient user after 48 hours. Culture 110. 856 at a. magnification of 3.8. 38 I. I F'igure 5. Intermediate colonies of 80.1. Bullorun on nutrient czar after 48 hours. Culture No. 795 at s. magnification of 3.3. 1. 5. 4. 5. 6. 7. 8. 10. 11. 39 LITERATURE CITED Arkwright. J. 1.. 'Tariation in Bacteria in Relation to Agglutination Both By Salts and By Specific Serm'. J. Path. and Bact.. 24: 36.1921. Baerthlein. x.. 'Ueber bakterielle Vlrltbllitzt. inbesondere sogenannte Batterienmutationen: Centralbe. f. Bakt. u. Parasitenk.. 3:1. 11:11. 3.6. S. 369. (As cited by Hadley (1927).) Barber. 11.4.. I'On Heredity in Certain Microorganisms'. Kansas Univ. $0. 31111.. 4. : 1.1907. Bronfenbrenner. J.. 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'- . . . x y . . A . . 71 4 I . \l a 1 A I n. I L 4 . J . u o I . . . u s \a — u v . 0 1 p. ' . . . y _ _ s e I . . . t I I I _ . r . . . . I I . e . r. 0 . . . u .... . . . . .. . ~ (., ,_ . x _ 7.. A I I u . «I v v . J . p .. ‘ . . v . . . _ . . . ‘ . u . 1 . v . \ .. . I I l . v I . . .., . . . g . . . .. . r c o a . ~ a a v .. MICHIGAN STATE UNIVERSITY LIBRARIES I {IN 3 1293 I I III II lllllll l O 3146 1894