PHYSICAL ACTION OF SURFACE-ACTIVE CATIONS UPON BACTERIA by Edgar Welton Kivela 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 DOCTOR OF PHILOSOPHY Department of Bacteriology 1949 ProQuest Number: 10008353 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest, ProQuest 10008353 Published by ProQuest LLC (2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 4 8 1 0 6 - 1346 ACKNOWLEDGEMENT Acknowledgement is made to Dr* W* L* Mallmann without whose able assistance and guidance this thesis would not have been possible, and to Mr* E* S. Churchill whose aid in certain phases of the work was greatly appreciated* Table of Contents Page Introduction and Historical Note .••. •....... ..*•»•• 1 Experimental and Discussion 4 Bacteriostatic titre of Roccal ...... .............. ........... Increasing Tolerance 5 *..o. Increasing Resistance »*•••...................... 6 8 Reversal by Centrifugation and Washing 12 Reversal by Dilution and Shaking ••«*•••••••. » 14 Electrophoresis Studies **.................... 19 Agglomeration of Organisms by Cationics 26 Osmotic Pressure Studies Summary ..... .... 28 33 1. A new group of compounds of the quaternary ammonium type have received a considerable amount of attention as disinfecting agents during the last thirteen years. Their structure consists of an alkyl group, two methyl or ethyl groups with a benzyl or another ethyl or methyl group substituted on the nitrogen atom. There is another class of these compounds not included in this paper which are alkyl substitutions on the nitrogen atom of the pyridine ring. The salt of the quaternaries is generally a chloride or a bromide, although the latter occurs much less frequently in commercial compounds. The compounds used in this study were mainly alkyl dimethyl benzyl ammonium chloride known commercially as HRoccal% "Zephiran11, and f,B.T#C.M, and para tertiary octyl phenoxy ethoxy ethyl dimethyl benzyl ammonium chloride known commercially as "Hyamine 1622M or MPhemerol”. These two were chosen because they enjoy the most widespread distribution, and their activity as disinfectants is about the highest of any of the quaternary ammonium type compounds now on the market* These 1cationic detergents1, as they are called, were first described as disinfectants by Domagk (1935) having been previously used for increasing the fastness of dyed fabric to water. Since the compounds were readily available and relatively inexpensive, a great deal of interest was shown in their ability as disinfectants. As is true of most new compounds which show some promise, a large number of investigators became interested and a great many laboratory tests were conducted to try to determine of what value the compounds might be in the field. In these investigations was included a search for neutralizing agents which would completely nullify the action of these compounds. Baker, Harrison, and Miller (1941) conducted studies on the metabolic rates of organisms treated with 1cationics* and found that if lecithin, cephalin, or sphingomyelin is used to coat the bacterial cells, those cells are protected from the action of the cationics. They found that these lipids also protected the bacteria against the action of the anionic detergents such as the alkyl sulfates or synthetic soaps. During the study they mixed a solution of an anionic with a solution of a cationic to see if there was any increase in the strength of disinfection. They found, however, that the two compounds completely neutralized one another as far as bactericidal activity was concerned. They further found that ordinary soaps also neutralized cationics. This neutralization of cationics by anionics would naturally be expected since, as is denoted by their names, the cationic has as its active portion the large positive ion, and the anionic has as its active portion the large negative ion. When the two compounds are mixed, the positive ions and negative ions attract one another resulting in the tying up of both causing inactivation. It has since been shown that cationics and anionics react together in equal molar quantities. This was the first big step toward finding a suitable neutralizing agent for these compounds and also toward determining the limita­ tions of the conditions in which these compounds can be used. Since that time a great many neutralizing compounds have been 3. reported by numerous authors, the latest of which is the sodium salt of symmetric bis (meta-amino-benzoyl-meta-amino-paramethylbenzoyl-l-naphthylamino-4,6,8 -trisulfonic acid) carbamide as reported by Lawrence (1947)* A few years after the work of Baker et al. Valko and DuBoie (1944) were able to show not only neutralization of the germicidal effect of the cationics by the use of an anionic, but were also able to show so-called 11reversal** of the action of the cationics. Their method, in brief, was to expose organisms to a solution of a cationic, then subsequently treat the cationie-organism mixture with an anionic, and by subculturing immediately they were able to recover viable organisms from a 1:3,000 dilution of Zephiran which will normally cause death of the organisms in a 1:15,000 dilution or greater. This finding created a great deal of skepticism concerning the use of these compounds as disinfectants, since one uses a disinfectant for the purpose of killing organisms, and to be able to, so to speak, bring them back to life defeats the purpose of the disinfectant. Naturally most of the workers interested in this field tried to repeat the work of Valko and DuBois. The author also attempted this but was unable to repro­ duce similar data to that contained in the publication. Upon inquiry he found that other workers were having the same difficulty. Klein and Kardon (1947) reported that they could not obtain re­ versal of the action of cationics by using anionics. They found that if the anionic is added to the cationic before the addition of the suspension of organisms, then the cationic is inactivated. 4. However, if the anionic is added soon after the addition of the suspension of organisms, it serves only to protect those cells which have not yet been acted upon by the cationic. Kenner, Quisno, Foter, and Gibby (1946) attempted to show reversal of another cationic, cetyl pyridinium chloride, by the use of body fluids* They exposed a culture of Salmonella typhimurium to the cationic, then injected the killed organisms into mice* After five hours they cultured the hearts of the mice but were unable to recover any of the organisms. Thus another method of reversal was attempted but failed to recover viable organisms* The author, after some preliminary work on various bacterio­ logical properties of some cationics, also carried on some work on reversal with some success, which then led to further experi­ mentation to try to determine what property or properties of these cationic compounds caused their rather peculiar behavior toward bacteria* This paper is a presentation of these studies. EXPERIMENTAL AND DISCUSSION In all the subsequent experiments except where specifically noted, a 10 per cent solution of the cationics was used as the stock solution, and this was taken as the Is 10 dilution, thus all the recorded dilutions are based on the anhydrous material. In the preliminary experimental work, a bacteriostatic titre was run on Micrococcus pyogenes var. aureus using Roccal 5. as the test compound. The experiment was carried out by making dilutions of the compound ten times stronger than the final dilution desired, then placing one ml of this dilution into 9 ml of F.D.A. broth. These tubes were incubated at 37°C. growth indicative of contamination. and observed for The sterile tubes were then inoculated with one loopful of a 24 hour broth culture of the organism and incubated at 37°C. The tubes were examined every 24 hours for evidence of growth, and subcultures were made from the tubes into sterile F.D.A. broth by transferring three loopfuls of the material from the agitated tube. The subcultures were also incubated at 37°C. for 43 hours and examined for growth. results are presented in Table 1. The It will be noted that these compounds are highly bacteriostatic causing inhibition of growth of the test organism in very high dilutions. table, numerous skips are present in the data* As is seen in the By skips in the data is meant the obtaining of a negative result where a positive result is indicated. Conversely a ,wild plus* may appear, which is a positive result in the data where a negative result is in­ dicated, The explanation of these results is based on the sampling error. In making the subcultures from the treated tubes three 3 nna* loopfuls of material were carried over into the sterile tube. This represents a very small portion of the entire volume of the treated tube. In considering the treated tube, we will find a few resistant cells in some of the lower dilutions. In the higher dilutions killing of the organisms is taking place thus decreasing the number of viable organisms per unit of volume. 6 Consequently in sampling a lower dilution, one may obtain only a very few loopfuls of material from the whole tube which would yield viable organisms. Thus a worker will obtain a negative test from a certain tube on one day, a positive test the next day, and a negative test on the third day. In sampling a high dilution a worker may transfer from the treated tube a portion which contains no viable organisms and thus obtain a negative test where a positive test has occurred one or more times previously. These skips and pluses are present in practically all of the data on these compounds, and will be present in other tables in this paper. was run in duplicate. For this reason the experiment From the table one may observe that there is a continuance of bactericidal activity along with the baeteriostasis as evidenced by the growth in the subculture tubes. Thus, over long periods of time these compounds are bactericidal in extremely high dilutions. However, this is of relatively little importance since for practical disinfection it is desirable to obtain the bactericidal action within five minutes or less. Bacterial organisms are sometimes able to increase their tolerance to numerous chemical compounds and subsequently in­ crease their resistance to any one chemical upon constant ex­ posure to that particular one. An attempt was made to increase the tolerance and resistance of Escherichia coli to two cationics; namely, B.T.C. and Tetrosan, the latter having a 1,4 dichloro substitution on the benzyl group. Serial dilutions of these Table 1. The Bacteriostatic Titre of Roccal on Mjcrococcus pyogenes. Incubation Period in Hours Dilution 1:100,000 24 Be* 48 Be 72 Be 96 Be 0 - 0 - 0 0 - 1:120,000 1 + 0 1:140,000 1 + 0 1:170,000 1 + 1:200,000 1 1:240,000 Be 24 Be 48 Be 72 Be 0 + 0 0 mm 0 - 0 - 0 - 1 + 0 - 0 - 0 - — 0 — 0 - 1 + 0 — 0 - 0 — 0 _ 0 — 0 - 1 + 0 — 0 - 0 mm + 1 + 1 1 + 1 + 1 + 1 + 1 + 1 + 1 + 1 + 1 - 1 + 1 - 1 - 1 - 1:280,000 1 - 1 - 1 + 1 - 1 + 1 + 1 + 1 + 1:340,000 1 + 1 + 1 + 1 - 1 + 1 + 1 + 1 — 1:400,000 2 + 2 + 2 + 2 + .2 2 + 2 - 1:480,000 2 + 2 + 2 + 2 + 2 2 + 2 + 2 + 2 + * Be - presence or absence of growth in tube subcultured from original at time shown. Numbers indicate the amount of growth, a twenty-four hour broth culture control having a growth of 4* compounds were made in 3terile tubes using sterile water as a diluent. One ml of each of the resulting dilutions was placed into 9 ml of sterile F.D.A. broth, and the tubes were incubated for 24 hours at the end of which time they were examined for contamination. A series of dilutions of each compound was seeded from a 24 hour broth culture of E. coli. The tubes were then incubated at 37°G. for 24 hours, at which time they were examined for growth. The tube showing growth in the lowest dilution was then used to seed another series of dilutions. for 24 consecutive days. This procedure was continued daily At the end of this time the lowest dilutions showing good growth were used for speed tests to deter­ mine the rate of kill of the organisms by higher concentrations of the same compounds to which they had been exposed. This was accomplished by exposing, in triplicate, 0.5 ml of culture to 5 ml of varying dilutions of the compound and subculturing into sterile F.D.A. broth at one minute intervals for 10 minutes. A control run was made in the same manner using an untreated 24 hour broth culture of E. coli. The results of the first part of the experiment are recorded in Table 2. It will be noted that there is an increased tolerance of the organism to both compounds. On the tenth day of the ex­ periment a series of tubes containing dilutions of B.T.C. was seeded from a tube containing the organism grown in Tetrosan. Growth was obtained in a dilution of 1:80,000. Although on the same day the usual B.T.C. subculture showed growth in a 1:40,000 dilution, the growth obtained from the Tetrosan subculture showed Table 2 The Increase in Tolerance of Escherichia coli for B.T.C. and Tetrosan Tetrosan Dilution 1* 2 1:10,000 - 1:20,000 - - 5 - 7 - - - - ■ 12 - 13 - 16 - 23 - 24 - — - - - - + - 1 :40,000 1:80,000 1:160,000 + - + - 1:320,000 + + + + + + - + + + + + + + + + + B.T.C. Dilution 1 4 5 7 9 17 1:10,000 + 1:20,000 1:40,000 - - + - + + 1:80,000 - + + + + + 1:160,000 + + + + + + 1:320,000 + + + + + + * Numbers represent individual day in series of 24 daily transfers in which a change in the lowest dilution showing growth occurred. a definite increase in tolerance as compared to the tolerance ob­ tained on the first day with B.T.C. This would seem to indicate to some extent that the increased tolerance is not entirely specific for the one cationic, but may be for cationics in general. The results of the second part of the experiment are recorded in Table 3* It is quite evident that there is no increased re­ sistance of the organisms grown in the presence of the cationic to the germicidal concentrations of the same compound. There seems to be some decrease in resistance of the treated organisms as measured by the untreated controls. At first this was attri­ buted to the fact that some cationic was being added with the organism suspension in the case of the treated organisms, but was not with the controls. To eliminate this factor, both the control and the treated organisms were centrifuged, washed with saline solution, and resuspended in saline solution for carrying out the test. The results were not significantly different, which would prove the point that the resistance of bacterial organisms toward cationics cannot be increased. It has been found, however, that some strains of organisms are more resistant to these compounds. Mallmann, Kivela, and TUrney (1946) recovered a strain of E. coli from swabs taken from glasses which had been rinsed in a cationic solution. This strain proved to be resistant to the action of the usual bactericidal concentrations of cationics. Table 3 Rate of Kill by B.T.C. of Escherichia coli Grown in Presence of 1:20,000 B.T.C. Dilutions of B.T.C* Treated Culture Untreated Culture Exposure Time (rain.) 1:4,000 1:6,000 1:8,000 1:4,000 1:6,000 1:8,000 1 . . . » - _ - + + + - - — + + + + + 2 — — - — + - + — — — - - _ + + + - — + 3 ■+■ + + 4 + + 5 a. Note: The symbols + and - represent growth or no growth respectively in subculture tubes. Each dilution was run in triplicate and is recorded as such. + M Hucker et al. (1947) reported that the spores of Bacillus subtilis were killed by high dilutions of cationics in a short period of time. The author was in doubt as to the validity of these data when taking into consideration the structure of a bacterial spore. They have a very thick cell wall, low moisture content, and their metabolic rate as measured by respiration coefficient is very low. They are very well known for their high resistance to heat and all chemicals used for disinfection, and there is no reason to think that they would be more suscep­ tible to this disinfectant than they are to any other. The author thought that this reported bactericidal action might be no more than bacteriostatic action which was not reversed. Con­ sequently experiments were designed to try to remove the compound from the spores or to neutralize it sufficiently to stop its activity. When spores were treated with a cationic, then sub­ sequently treated with an anionic as a neutralizer, a few spores were recovered from their inhibition, but the apparent percentage kill was still exceedingly high. It was finally decided to attempt the removal of the excess cationic by centrifugation of the treated spores followed by repeated washing with sterile distilled water. This procedure gave a high percentage of recovery of the spores, far greater than that obtained by neutralization using an anionic compound. In preparing the spore suspensions, B. subtilis was grown on the surface of plain agar at room temperature for a period of seven days. At the end of this incubation period the growth was 13 harvested in distilled water and the resulting suspension was heated to 80°C* for 10 minutes for the purpose of destroying the vegetative cells and the heat-susceptible spores* The suspension was immediately cooled and preserved by storage at 10°C, for sub­ sequent use. It may be of interest to the reader to know that physiological saline solution was tried first, but at room tem­ perature the saline evidently furnished enough material to stimulate the spores to change into the vegetative state, which causes some difficulty in the experiments^ since the vegetative cells are much more susceptible to the action of cationics than are the spores. In conducting the experiments, 10 ml portions of the spore suspension were treated in duplicate with a 1:1,000 dilution of the cationic compound making a final dilution of 1:2,000. To determine the initial number of bacteria used, control tubes were prepared by adding 10 ml portions of the spore suspension in the same manner as the medicated tubes. Another control to show the effect of no further treatment was made by adding 5 ml of the spore suspension to 5 ml of the 1:1,000 dilution of the cationic, giving a final concentration equivalent to that of the treated tubes. This latter control was allowed to stand at room tempera­ ture until the termination of the experiment when it was plated in the same manner as that used for other tubes. The treated tubes and the first control tube listed were handled by centrifuging the duplicate samples until the spores 14 were thrown down* The supernatant fluid was then removed, 20 ml of sterile distilled water were added to restore the suspension to its original volume, the sample shaken vigorously to resuspend the spores, and standard plates were made with plain nutrient agar. The suspensions were again centrifuged, the spores resus­ pended, and plate cultures made for two more rinsings. are presented in Table 4* The data From these data it can readily be seen that there is a definite revival of the theoretically killed spores as shown by the comparison of the bacterial count of the first rinse and the treated control. Furthermore, this revival increases with the greater number of rinses. In the above experiments it was noted in every instance that the bacterial counts of the various dilutions plated failed to follow the normal pattern, e*g*, the low dilution plates gave a lower total count than did the higher dilution plates, although the number of colonies were within the normal (30 - 300) number of colonies permissible for counting. This could only mean that the simple process of dilution and shaking was further reversing the action of the cationics* Following the above observation, an experiment was made to determine whether or not this reversal could be demonstrated by a simple dilution technique. The tubes were prepared in the same manner as in the previous experiment;- namely, 10 ml of twice the desired concentration of the cationic were added to 10 ml of the spore suspension* One ml samples were drawn at two minute intervals Table 4 The reversal of bacteriostatic effect of lt2.000 B.T.C* on spores of Bacillus subtilis by centrifugation Treatment No rinse None 1st rinse 2nd rinse 3rd rinse 151,000 100,000 150,000 1 0 0 590 1,900 2 0 0 160 2,700 16, for 20 minutes, placed in 99 ml of sterile distilled water, and shaken 50 times. One ml and 0,1 ml were plated and 1 ml portion was placed into a second 99 ml sterile distilled water blank and shaken as before* One ml of the dilution was then plated, A control was made by adding 5 ml of sterile water to 5 ml of spore suspension and plated before and after the experiment to check any change in bacterial population due to other factors which might enter into the experiment. set of results. Table 5 gives a typical The data show that no germicidal action occurs after the first two minutes exposure. The bacterial populations remained practically constant for the entire 20 minutes of ex­ posure, In Trial 1, in a concentration of 1:2,000 practically all of the spores were recovered. In Trial 2 where the cationic concentration wa3 1:1,000 a kill of 50 per cent was apparent. When the concentration was increased to a 1:500 dilution (Trial 3) the kill was increased to approximately 90 per cent. Data are presented in Table 6 showing the effect of dilution upon the number of colonies appearing on the plates. It is interesting to note that although the 1:1,000 and 1:100 dilutions came from the same flask as represented by the 0,1 ml and 1,0 ml portions plated, the plate count from the 0,1 ml portion plate in practically all cases yielded a greater number of colonies than did the 1 ml portion. This would indicate that the dilution resulting from mixing in the nutrient agar tended to eliminate Table 5 The reversal of bacteriostatic effect of nB,T«C«H on spores of Bacillus subtilis by dilution and shaking Number of bacteria per ml Exposure time Trial 1 1:2,000 Trial 2 1:1,000 Trial 3 Initial count 310,000 770,000 2,320,000 2 minutes 149,000 310,000 206,000 4 minutes 136,000 384,000 206,000 6 minutes 256,000 204,000 196,000 8 minutes 218,000 428,000 160,000 10 minutes 272,000 276,000 118,000 12 minutes 140,000 224,000 141,000 14 minutes 248,000 168,000 126,000 16 minutes 264,000 254,000 124,000 18 minutes 240,000 134,000 114,000 20 minutes 278,000 168,000 158,000 1:500 Table 6 The reversal of bacteriostatic effect on spores of Bacillus subtilis by agar plating after treatment with 1:500 dilution of MB.T.C«fl as demonstrated by comparative counts obtained with 1 ml and 0.1 ml dilution agar plates Sample Number Number of colonies appearing on the plates 0.1 ml 1*0 ml 1 14 206 2 33 206 3 40 196 4 45 160 5 11 118 6 63 141 7 18 126 8 63 124 9 122 114 10 66 158 19. bacteriostatic action. This would be in addition to the reversing action of the agar as reported by Quisno et al. (1946) and Sherwood (1942). Much work has been reported on the electrophoretic mobilities of bacterial cells; some in regard to their pathogenicity (Frampton and Hildebrand, 1944)> some regarding the stage of growth of an individual organism (Moyer, 1936a), some regarding the effect of cations in general upon organisms (Moyer, 1936b), and most recently some on the effect of surface-active agents upon various bacterial cells (Dyar and Ordal, 1946). Powney and Wood conducted a series of experiments reported in three different articles on the effect of detergent solutions upon the mobility of oil drops. Included among the detergents was dodecyl pyridinium chloride, a cationic. They found that 0.0001 per cent would reverse the sign of the net charge on the particle, while 0.01 per cent gives the maximum charge. Dyar and Ordal (1946) showed that about 0.003 per cent cetyl pyridinium chloride was necessary to neutralize the charge on a bacterial cell. versed. Above this concentration the charge is re­ Electrophoretic mobility, using a technique similar to that used by the foregoing workers, was used to show the removal of surface-active agents from the surface of bacteria by washing procedures. A simple electrophoretic cell with a depth of 600 microns was used. The distance between the electrodes was 3«4 cm. To keep from encountering various currents within the electrophoretic chamber of this type, all determinations were made with the microscope focused at a distance of 400 microns below the bottom of the coverglass. This depth was chosen because it has been shown by other workers that in a 600 micron cell the most stationary levels are at 200 and 400 microns from the top of the cell. The lower level (400 microns from the top of the cell) was chosen for observation because the spores are relatively heavy and tend to settle toward the bottom of the cell. A con­ stant potential of 20.0 volts was used for all tests, and a current of 1 milliampere was never exceeded. In making the electrophoretic tests, the time for a single spore in the electrical field to traverse 200 microns in one direction was observed and recorded. The poles of the cell were then reversed and the time for the same spore to travel back to its original position was determined* This was done for the purpose of nullifying any currents, convection or other>wise, which may have been present at that level in the prepara­ tion. Five determinations on each preparation were made in this fashion, the 10 time intervals were averaged, and the mean was used for the calculation of the rate of speed of the spores in the electrical field. In carrying out the tests, 10 ml of twice the desired con­ centration of the cationic were added to 10 ml of the spore suspension. After mixing thoroughly, a small aliquot was removed for the first treated test. The remainder was centrifuged, the supernatant fluid withdrawn and discarded, and 20 ml of sterile distilled water was introduced. This was shaken vigorously to evenly disperse the spores throughout the solution. After a second aliquot had been taken for testing, the suspension was again centrifuged, the supernatant fluid discarded, the 10 ml of distilled water added to resuspend the spores. The concen­ trations of the cationics were 1:1,000, 1:2,000, and 1:4,000. The mobility rate of the untreated spore suspension was deter­ mined as a control. The data for the above experiment using Roccal as the cationic are given in Table 7* Similar experiments were con­ ducted using B.T.C. and Tetrosan as the surface-active cationics, and also using as test organisms E. coli and M. pyogenes var. aureus. The data for the last mentioned organisms are not given because they present identically the same picture as that pre­ sented in Table 7* In making the above experiments, no attempt was made to regulate the pH by use of a buffering system since preliminary experiments showed only negligible differences between mobilities in the buffered and the unbuffered solutions. Furthermore the presence of a buffer salt so increased the conductivity of the Table 7 The electrophoretic mobilities of spores of Bacillus subtilis when treated with "Roccal" followed by centrifugation and washing with distilled water Treatment Mobilities-^/sec. in 20 volt field Dilute.on of Cationic 1:2000 1:1000 1:4000 No wash + 18*6 + 16.7 + First wash - 16.9 - 16.9 - 20.3 Second wash - 25.5 - 25.9 - 19.8 None - 19.0 - 19.4 - 19.4 7.0 The symbols + and - represent electrical charge on spores 23. solution that difficulty was encountered in keeping the electrodes free from gas which changed the potential across the cell. The data show that when the organisms were treated with 1:1,000 solution of the cationic the spores changed from a negative mobility of 19 microns/sec, to a positive mobility of 18 microns/ sec. After one washing the positive charge was lost by removal of the cationic from the surface of the spore* showed a negative mobility of 16.9 microns/sec. The spore now A second washing increased the negative mobility to a point greater than the negative mobility of the untreated spores. This latter finding was obtained several times but the author is unable to explain its cause. These data confirm the bacteriological finding that dilution will remove the cationic compounds from the surface of the spores. The fact that it is possible to remove the cationics from the surface of organisms is rather surprising since it is a well-known fact that the cationics are very difficult to remove from any intact surface, and for that reason have been considered bene­ ficial as sanitizers since they leave a germicidal film on the utensil or surface which has been rinsed in a cationic solution. Furthermore, their use on dyed fabrics for water fastness would be of little avail if the compound was removed on the first washing. In light of the above results on the removal of the cationics by dilution and shaking, further studies were conducted on E. coli. Eberthella typhosa. and M. pyogenes losing the dilution technique as a method of reversing the action of the cationics* Dilutions of 1:5,000, 1:6,000, and 1:10,000 of the cationics were used with an inoculum of over 10,000,000 organisms per ml. In no instance, by either shaking or centrifuging, could the organisms be re­ covered after an exposure of three minutes or more. In most cases one or two colonies were found on the plates containing the 1:100 dilution of the cationic-organism suspension after an exposure of one minute. In one case a count of 1700 per ml was obtained after the exposure of M. pyogenes for one minute. This dropped to 300 in two minutes, and then to zero in three minutes. This, then, would seem to indicate that vegetative cells are killed or sufficiently hampered by the action of the cationic so that their recovery by the above methods, namely, washing or centrifuging, is not possible. An application of the latter experiment was made under practical conditions while collecting some of the data for a paper on sanitizing dishes by Mallmann, Kivela, and Turney (19A6). While taking swabs of glasses treated with 150 p.p.m. of a cationic, the swab bottles were shaken vigorously 30 to 50 times to thoroughly fluff the cotton of the swab immediately after the swab had been taken. This was done for two reasons. First, there is a possibility that the concentration of the germicide in the swab where the organisms are trapped among the cotton fibers may be higher than the concentration of the germicide in the surrounding solution since the compound is dispersed mainly by diffusion. Thus in ordinary swabbing techniques where the swab is not shaken until it reaches the laboratory there is a possibility that the organisms in the swab have been subjected to a relatively high concentration of the germicide for a longer period of time. The immediate shaking of the swab until it fluffs washes most of the organisms out of the swab and disperses them into the solution where the concentration of the germicide is negligible. If one wished, he could plate the swab samples in bacto-oxgall medium described by Klarmann and Wright (1943) for the supression of the bacteriostatic activity of the cationics. However, the author does not think that this is necessary since the concentration of the germicide is so low that any bacterio­ static effect would not be great enough to kill any of the sur­ viving organisms. It will be recalled from the first part of this paper dealing with bacteriostatic titres that even with an organism as sensitive to the cationics as M. pyogenes no dilutions greater than 1:400,000 showed bacteriostasi3. Let us now con­ sider the amount of cationic carried from the glass into the swab bottle. Assuming that 0,5 ml would be absorbed on the swab, there would be a 1:20 dilution of the 150 p.p.m. made when the swab was placed in the 10 ml of solution in the swab bottle. This is far too low a concentration to cause bacteriostasis* The second reason for shaking the swab is to remove the cationic adsorbed on the surface of the organisms as was shown possible 26 in the laboratory experiments cited, and thereby effect a reversal of the action of the compounds on those organisms which are still viable at the time the swab is taken• The results showed no difference in the count between the shaken and unshaken swabs, which again demonstrated that the organisms present on the glasses were evidently killed before the action of the cationic was reversed* A report was given by E, C. McCulloch (1947) stating that the cationics caused agglomeration of bacterial organisms* Two possible explanations for this were considered by the author in light of the electrophoretic studies which had just been made. First, the concentration of the cationic might have been such that the charge on the organisms was neutralized thereby re­ sulting in their agglomerating or clumping. However, as shown by the work of ^yar and Ordal (1946), a 1; 35,000 dilution will neutralize the charge, but the higher concentrations which are used in the field would give the organisms a positive charge which would interfere with the agglomeration* The second ex­ planation was the possible spontaneous agglomeration of the organisms as their charge is changed from the negative to the positive. In addition to this possibility Klarmann and Wright (1943) reported the possibility of the migration of bacterial organisms to glass surfaces. This was refuted by Klimek and Umbreit (1943) who used techniques identical to those of Klarmann and Wright but used tubes of material other than glass. In 27. carrying out the following experiment, the possible migration to a glass surface is taken into consideration. To determine whether or not agglomeration occurred experi­ mentally, tests were run on E. coli, E. typhosa. Ps. aeruginosa. and M. pyogenes. Twenty-four hour broth cultures of the above organisms were centrifuged to remove the broth, and were then resuspended in sterile water by vigorous shaking to assure breaking up of all clumps which might have been formed by the centrifugation. These suspensions were then added to varying dilutions of three different cationics; namely, Roccal, Hyamine, and B.T.C. Glass coverslips were placed in the dilutions of cationics before the organism suspensions were added to facilitate the clumping of the organisms upon a glass surface. After the addition of the organism suspension, the coverslips were removed, placed upon a clean slide, and examined using dark-field illumination. The following results represent the three cationics collectively since no differences in their reactions were noted. Pseudomonas aeruginosa - clumping occurs up to a dilution of 1:4,000. No clumping in dilutions of 1:5,000 or greater. Escherichia coli - clumping occurs up to a dilution of 1:2,000. No clumping in dilutions of 1:4,000 or greater with the exception of 1:60,000 which shows slight clumping in 24 hours. Eberthella tvphosa and Micrococcus pyogenes - no clumping in any dilutions from 1:1,000 to 1:100,000* It will be recalled that in a previous portion of the paper dealing with reversal, it was noted that when working with dilutions commonly used for disinfection purposes, namely, 1:1,000 to 1:7,000, no reversal of the cationic action could be obtained with vegetative cells even though it was shown electrophoretically that the compound had been washed from the surface of the organisms by shaking, which process was suffic­ ient to effectively disperse any clumps present. This, then, would seem to show that the organisms had either been killed before the clumping occurred, or the more logical observation that the compound adsorbed on the surface of the organisms is still continuing its action even though the organisms are clumped. It is of interest to note that in carrying out the above experiments the dark field illumination showed those organisms that had been killed or immobilized by the cationic to have a much higher refractive index than normal organisms or those organisms present in the solutions which had not as yet been immobilized. In light of the studies cited on electrical charges that the apparent adsorption of the cationic compounds on the sur­ face of the organism, an attempt was made to correlate these with a possible mode of action of the cationic surface-active agents. Hotchkiss (1946) reported that the cell contents of organisms had been found in the supernatant fluid after the 29. organisms had been acted upon by surface-active cations, and he believed that this was due to the denaturation of the cell wall which thereby destroyed its selective permeability and allowed the cell fluids to escape into the suspending fluid. The author thought that, in addition to the cell wall breakdown, the osmotic pressure exerted by these compounds might increase the amount of these cell fluids in the surrounding medium. Thus a study of the osmotic pressure of a surface-active cation was made, using Roccal as a typical compound. The osmotic pressures of various concentrations of the cationic were determined by the indirect method of freezing-point depression. Solutions of the cationic varying from 0.5 gm. per liter to 10.0 gm. per liter of solution were used, and the results plotted as in Figure 1. In arriving at the final values, the following formulae were used: M ■= 1000 K - J' AT W where 'M1 is the molecular weight of the solute, fK* is a constant of 1.36 degrees, 'w* is the grams of solute dissolved in 'W1 grams of solvent, and ^ T 1 is the decrease in the free zing-point temperature; P= £ 0 M V which is the standard vapor pressure formula, but which, in this instance, is equal to the osmotic pressure in atmospheres. 30. The osmotic pressures of solutions containing less than 0.5 gm. of cationic per liter of solution were not used because of inconsistencies in results, nor were those of solutions containing more than 10.0 gm. of cationic per liter of solution since difficulty with foaming was encountered. The results obtained are what would normally be expected judging from the molecular weight of this compound. The os­ motic pressure exerted by the compound as determined by a purely physical test, would not seem to exert an appreciable effect* However, this does not reveal the entire condition. It should be remembered that in very dilute solutions of a cationic com^ pound enough is adsorbed on the surface of the organism to change its charge from a negative one to a positive one. Further­ more the cationics are surface tension depressors which means that they will collect at the interface between the solid and the liquid phase. This, then, would indicate that the concen­ tration of the compound upon the surface of the organism is much greater than the concentration of the compound in the solution. The author believes that this adsorbed material exerts an osmotic pressure comparable to its concentration on the bacterial cell. This means that an organism in a 1:1,000 dilution of a cationic compound would not be exposed to an osmotic pressure of only a 1:1,000 dilution but would be sub­ jected to an osmotic pressure greatly in excess of that owing to the high concentration of the compound upon the organism. 31. This osmotic pressure would tend to draw the cell fluids through the disrupted cell wall into the surrounding fluids. This be­ lief was further strengthened by Dyar (1947), who observed, while employing a 1:300 dilution of cetyl pyridinium chloride for a cell wall stain, that the examination of the bacterial cells in water under a cover slip showed the cytoplasm to be shrunk away from the cell wall. It is quite probable that the surface-active agents are adsorbed on the surface of the bacterial cells in sufficient concentration to interfere with the osmotic balance of the organism and its surrounding menstruum, and in this manner may prevent the intake of nutrients. This would help to explain why these compounds are so highly bacteriostatic in high dilu­ tions, and why the bacteriostatic effect can be removed by dilution and vigorous shaking or by the use of neutralizing agents. Further, it would explain partially why reversal can be obtained using spores since they have a very thick cell wall which is resistant to both denaturation and osmotic effects, and the moisture content is much lower causing the internal os­ motic pressure of the spore to be much higher than that of vegetative cells. The reason for the high bacteriostasis on bacterial spores when the cationic is not removed may be caused by the bacteriostatic effect of the cationic oh the vegetative cell as it emerges from the bacterial spore, for in the vege­ tative state a spore forming organism is as susceptible to chemical and physical factors as any other organism. o Quaternary in grams per liter FIGURE 1 - The Osmotic Pressure of a Surface-Active Cation (Roccal) as Determined by Freezing Point Depression Methods 32. o to rH o O O ° C (S 8Uoqdsoinq.v) aunssovij oxq.omso SUMMARY Bacteriostatic titres of some surface—active cationic com­ pounds were run on Micrococcus pyogenes. Bacteriostasis was noted in high dilutions. Tolerance and resistance data were collected on the com­ pounds using Escherichia coli as the test organism. The tolerance can be increased, but the resistance can not. Reversal of the action of the cationics was attempted on Bacillus subtilis spores using several methods including neutralisation with an anionic detergent, centrifuging and washing, and washing by shaking and dilution. The last two methods gave reversal with good recovery of the spores. Electrophoretic mobilities were determined on untreated spores of B. subtilis. spores treated with a surface-active cationic, and spores subsequently washed by centrifugation after treatment with a cationic. The cationic changed the charge on the organism to positive but subsequent washing re­ moved the positive charge and the negative charge was restored. Clumping of organisms caused by cationics wa3 determined. Some organisms clumped, and some did not. It is assumed that those which do clump are still under the influence of the com^ pound so that clumping does not interfere with the destruction of the organisms. 34. Osmotic pressure of a typical cationic was determined. Results were as expected judging from the molecular weight of the compound* However, the theory is advanced that due to adsorption, the concentration and thus the osmotic pressure on the surface of the organism is greater than a physical test will demonstrate. Thus the compounds may act by upsetting the osmotic balance between the organism and its surroundings. 35 LITERATURE CITED Ackley, Robert R. (1947) Properties and uses of 3urface-active cations. Soap & San, Chem., 22:39-42. Adrien, Albert (1942) Chemistry and physics of antiseptics in relation to mode of action. Lancet, 243 (6222):633- 636. Anson, M. L. (1939) The denaturation of proteins by synthetic detergents and bile salts. J. Gen. Physiol., 23: 239-246. Baker, Zelma, Harrison, R. W #, and Miller, B. J. (1941) Action of synthetic detergents on the metabolism of bacteria. J. Exptl. Med., 22s249-271. Baker, Z., Harrison, R. W., and Miller, B. F. (1941) Inhibition by phospholipids of the action of synthetic detergents on bacteria. J. Exptl. Med., 24,:621-637. DuBois, A. S. (1947) Bacteriological evaluation of cationic germicides. Soap & San. Chem., 23:139-143. Dyar, M. T., and Ordal, E. J. (1946) The effects of surfaceactive agents on theelectrophoretic mobilities of bacteria. J.Bact., 51:149-167. Dyar, M. T. (1947) A cellwall stain employing a cationic surface-active agent as a mordant. J* Bact., 52*496. Findlay, Alexander. (1919) Osmotic Pressure, second edition, Longmans, Green & Co., London, England, pp. 77-93. Frampton, V. L., and Hildebrand, E. M. (1944) Electrokinetic studies on Erwinia amylovora and Phytomonas stewartii in relation to virulence, J. Bact., 4§*537-545. Hotchkiss, R. D. (1946) The nature of the bactericidal action of surface-active agents. Ann. N. Y. Acad. Sci., 46:479-493. Hucker, G. J., Brooks, R. F., Metcalf, Dorothea, and VanEseltine, Wm. (1947) The activity of certain cationic germicides. 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(1947) The real and apparent bacteri­ cidal efficiencies of quaternary ammonium compounds. J. Bact., 22:370. McCulloch, Ernest C. (May 2, 1947) False disinfection velocity curves produced by quaternary ammonium compounds. Science, 105:(2731) 480. McCulloch, E. C., Steinar Hauge, and Hoyo Migaki. (1948) The quaternary ammonium compounds in sanitization. Am. J. Pub. Health, 38:493-503* Mallmann, W. L., Kivela, E. W., and Turney, Gray. (1946) Sanitizing dishes* Soap & San. Chem., 22:130-134* Moyer, L. S. (1936a) A suggested standard method for the investigation of electrophoresis. J. Bact., 31: 531-546* Moyer, L. S* (1936b) Ganges in electrokinetic potential of bacteria at various phases of the culture cycle. J. Bact., 22:433-464. 37 Ordal, E. J., and Borg, A. F. (1942) Effect of surfaceactive agents on oxidations of lactate by bacteria* Proc. Soc. Exptl* Biol. Med., 20:332-336. Powney, J*, and Wood, L» J. (1940) The properties of detergent solutions* Part IX. 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