The Chemistry of Disinfection

T H E CHEMISTRY O F DISINFECTION* BY WILDER D. BANCROFT AND G. HOLMES RICHTER**

The development of the problem of disinfection and antisepsis has undergone the usual series of events that one finds in the evolution of many problems of biology and medicine. I n the race to gain new knowledge of this subject the empirical data have become quite voluminous, while the theoretical interpretations are very meager. Few, if any, of the theories of disinfection attempt to cover all phases of the subject, and up to the present time, these attempts have not been in keeping with the development of other branches of knowledge that have a direct bearing on this problem. After the discovery of Lister, that phenol is an antiseptic, countless numbers of organic compounds have been tested, by empirical methods, for this property and many good disinfectants and antiseptics have been discovered. Since the demands of the professional men have been satisfied by the provision of fairly good drugs and our patience has been taxed by the trial and error methods, the time is now ripe to turn our thoughts to the theory of the action of toxic agents. At the outset we realize that bacteriology is a descriptive science and can offer no basis for a theoretical interpretation. I n dealing with the effect of drugs on living tissue we must realize the colloid nature of all living material and express our results in terms of colloid chemistry rather than clinical symptoms. The most outstanding and universal physical property of protoplasm is its colloidal structure. Theoretically it is possible to alter colloidal systems by chemical or physical means, the result being: coagulation or alteration of surface conditions such as swelling or contraction, displacement of adsorbed material, formation of emulsions, the increasing or decreasing of the charge on the particles, or reversal of the sign of the charge, peptization, formation of a jelly, stabilizing the sol, etc. The normal state of the colloids of any tissue varies with the age and the individual. Some of the variations, mentioned above, of the cellular colloids will bring about rather startling reactions in the tissue. For example it was found that a reversible coagulation was responsible for the action of anesthetics or narcotics.' Substances with a narcotic action when placed in contact with living protoplasm either directly or indirectly caused the colloids to coagulate. Upon the removal of the narcotic the colloids again became peptized to the normal state. I n many cases these changes are easily observed with the aid of the ultra-microscope. The case is just this, a direct narcotic enters the cell and is adsorbed upon the colloids to which it is most attracted. The first effect is the displacement * Ths work 18 art of the rogramme now being carried out at Cornell University under a grant from the Geckscher eoundation for the Advancement of Research established by August Xeckscher at Cornell Universlty. * * National Research Fellow in Chemistry. Bancroft and Richter: J. Phys. Chem., 35, 2 1 5 (1930).

512

WILDER D. BANCROFT AND G. HOLMES RICHTER

of material that’ was already adsorbed upon the surface. The displacement of this normal material increases its “effective concentration” in the water phase. This increase in concentration, in accordance with the mags law, will speed up the chemical reactions which the substance is undergoing, This is nothing more than the stimulating effects on the chemical reactions of a cell by narcotics and toxic agents that have been observed by all workers. The greatest stimulating effect will be on those reactions that show a small tendency to be reversible, such as oxidations. The accumulation of the narcotic upon the colloids of the cell eventually reaches such a point that t,he combined effec,tof the narcotic and the electrolytes of the cell will result, in the coagulation. The slowing down of the diffusion of the material in the cell from the high viscosity of the coagulated protoplasm, and the blocking of the surface of the enzymes by the narcotic will result in narcosis. An indirect narcotic does practically the same thing but in a different manner. Here the coagulating action is due to the products that accumulate through the interference by the narcotic of some normal function. Thus a substance that interferes with the adsorption of oxygen by the respiratory enzymes can behave as an indirect narcotic; the incompletely oxidized products such as acids will cause the coagulation. Upon removal of the agent that is responsible for the coagulation, the colloids may be peptized again. If this happens there is a return to normal without any symptoms of toxic action. However if the surfaces of the colloid micellae have become so drastically altered that peptization does not take place upon removal of the agents that caused the coagulation, or if the agent is irreversibly adsorbed, then clearly, this effect can cause considerable damage to the affected tissue or cell. Kow a moment’s reflection will show that always two effects, narcosis and toxic action, and nearly always a third, stimulation, can be obtained from one and the same substance, I t is probable that the initial stimulation is always present, but if the increase in the effective concentration by displacement is negligible the stimulation may be overlooked. These effects are controlled by the concentration of the drug or more properly by the degree of adsorption, There is nothing astounding in this conclusion, it has been known from empirical data for a long time. Some prefer t o regard it as a law and have named it the “Schulz-hrndt lam”. The only new thing about it is the correct explanation. The organisms that are most frequently exposed, or are involved in man’s attempt to expose them, to toxic agents are tho pathogenic bacteria and protozoa. The three stages or effect mentioned above are frequently observed.’ Very low concentrations sometimes stimulate the organisms which we wish to kill, by increasing the concentration of the drug we can inhibit the growth, but not produce death until still higher concentrations are used. The in-

’ Archiv Hyg., 91, 231 (1922).

THE CHEMISTRY OF DISINFECTION

513

hibition of the organism by the drug is due to narcosis and the toxic effect of high concentrations is undoubtedly due to an irreversible coagulation. X closer analysis of this last phase of drug action is the object of this paper. The first important question is whether the effects of drugs onanorganism are physical or chemical. Colloid chemists for a number of years have been submitting evidence that dyes, electrolytes, drugs, and most organic compounds are only adsorbed upon bio-colloids. The work of Traube has been of great value because he has clearly emphasized the role of adsorption in drug action.’ Bechhold* also has presented a vast amount of data supporting this view. The quantitative experiments of Herzog und Betael’ show clearly that in the average case we are dealing with an adsorption of the drug upon the colloids of the organism. An application of the phase rule to such a system as organism-disinfectant showed that there was no definite chemical compound formed with a number of the common disinfectants. Thus the amount of disinfectant taken up by the organism is proportional to the concentration if the temperature is constant; and there is no possibility of a chemical reaction because there is no stoichiometrical relation between the two substances. The disinfectants that behaved in this manner were: silver nitrate, mercuric chloride, chloroform, and phenol. The only compound that showed evidence of a chemical reaction was formaldehyde, that is, the amount taken up was independent of the concentration of the formaldehyde. One ca:n readily understand why it is active over a wide range of dilutions whereas other substances like phenol become inactive on dilution. Perhaps the easiest approach to the problem is by first examining the effect of toxic elect,rolytes on lower forms of life. Wolfgang Ostwald4 in his study of the toxic effect of electrolytes on living tissue recognized clearly that adsorption of the ion or molecule was necessary before any effect was produced and furthermore that the toxic effect was proportional to the adsorpt,ion. What happens after the adsorption of the toxic ion takes place? Either one of two things can take place, there will be a tendency to increase or decrease the degree of dispersion, that is, peptize or coagulate the colloids of the cell. The colloid theory of narcosis has already indicated that irreversible coagulation is the most probable. Most workers prefer to regard disinfection as occurring in a t least two stages, the first being the true adsorption of the agent by the bacteria and the second step being the further action of the agent on the protoplasm. The first condition, that adsorption occurs, is generally accepted; t,he nature of the action after adsorption is subject to great differences in opinion, in spite of the logical conclusion that it is either a peptization or coagulation. There is a natural reluctance on the part of some workers to believe that, dilute Biochern. Z., 19, 197 (1919);120,90 (1921). in Biologie und Mediain” (1929). 2. physiol. Chem., 67, 309 (1910); 74,221 (1911). Archiv. ges. Physiol., 120, 19 (1908).

* “Die Kolloide

514


solutions of disinfectants can coagulate the cell colloids. Nevertheless, the work of Heilbrunn gives undisputed evidence that this is the case. Heilbrunnl in his study of the effect of mercuric chloride on the colloids of protoplasm gives us a clear picture of this coagulative effect. He says: “By actual viscosity tests it can be readily shown that low concentrations of mercuric chloride produce a coagulation, or at least a great increase in viscosity, in various types of protoplasm. Arbacza eggs treated with m / 10000 HgCh in sea-water very soon become altered so that the granules of the cell can no longer be moved by the centrifugal force. The same experiment can also be performed on protozoan cells. When the flagellate Euglena is centrifuged, it loses its spindle-shaped contour and becomes spherical, the granular inclusions massing a t one end. But when a small quantity of mercuric chloride solution is added to the culture medium in which the Euglena lives, no shifting of the granules occurs when the protozoa are centrifuged. The protoplasm ceases to behave as a fluid and is a gel or coagulum. The coagulative action of mercuric chloride, a t any rate in higher concentrations, apparently involves a precipitation of proteins from the hyaline ground-substance, that is to say the intergranular material, of the protoplasm. Xot only is this a logical assumption, but there is also actual experimental evidence in its favor. When Arbacia are first centrifuged, and then treated with dilute solutions of mercuric chloride in sea-water, new granules can be seen to appear in the hyaline region previously free from granules. “Dilute solutions of copper chloride which have little or no acidity may have a very pronounced action on protoplasm. Thus in one series of unpublished experiments, the following five solutions of copper chloride were prepared: m/Iooo, m/gooo, m/Ioooo, m/5oooo1 and m/Ioo,ooo. Even the m/5,ooo was dilute enough so that there was practically no effect of the hydrogen ion concentration (the pH of this solution was 7.8). All the solutions produced a coagulative effect on sea-urchin eggs, as was clearly shown by centrifuge tests. This coagulation did not occur very rapidly. Thus a test of the eggs in the m/5ooo solution showed no coagulation and no increase in viscosity after a 2 0 minute exposure, whereas a test after 54 minutes did show coagulation.” I n these experiments the solutions are so dilute that there can be no question of an osmotic effect on the organisms, furthermore the pH of the solutions was normal so the effect can only be ascribed to the colloidal changes, that is, coagulation. Research workers in Naegeli’s time were so completely mystified by this simple coagulation that they immediately named it “oligodynamic action”. Naturally, it is desirable to extend and confirm this work by using other disinfectants and testing for coagulation by a different method. Some experiments along this line have been carried out in this laboratory. A direct observation of some of the colloids in the living cell of an organism is possible by using an ultra-microscope. If we work within the natural 1

“The Colloid Chemistry of Protoplssm” (1928).

5'5

'TEE CHDMISTHY O F UISlN

limitations of this iustrument, then it. is possible to satisfy our curiosity concerning coagulation by disinfeciuntr and antiseptics. The yeast, cell is ~n excellent abjret to stari with and it is not too difficult to observc colloidal rnat,crial within thr ceil. In addition, the work of Hrrzog and Betzel has already shown that yeast oells adsorb the disinfectants and that there was no chemica,l reaction except with forriraldehycir. Tbus thrse organisms are an ideal starting point. Ordinary baker's ye& inoculated in Laurent's rnediurn with I .sy0gluease was the stock culture. When fresh material irom this culiure was examined undsr tho ultra-microscope one could see fxintly somc colloid material in Brownian movernent. A photograph of the ltpprararice of the normal ccll

VI". I

Fza.

*

Cu*r"IatRd Y C h R t cell5 is giveii in Fig. 1, thc cells appear optically eriipty due to thr fact t,hiit tlici light froin the micellae is too fa,int t o make an impression on thr platr la Croix' showed that, generally, concentrations of 1-roo would inhibit the growth of bacteria but did not kill ihein until much greater concentrations were used. Koch2 showed that this inhibition did not kill certain bacteria even if ii. were maintained over long periods of time. Rnllner's3 study of this phenomenon led him to thP view that it was closely related to narcosis. ' Arehiv. O X ~ Path. . Phsrmakol., 13, 175 (b88c). Mitt. Kaiserl. Gesundsheitsrnt, 1, 234 (rR8t). Svrrnal yeast rellr

~~~~

8

Z. Bpkt.,

(2)

19,572 (1907).

j1 0

I(,,.,i*:,t

u.

R A X i ( ’ l t 0 F - 1 .,VI) MES It1CWPI:K

‘This ciffmt, ii m~rco8iiior aseptic stale can also be demonstr;tted. Vig. ,3 reproduction of a photograph of thc same culturr trcated with a small arriount of chloroform. Tbere is a striking difference betwwn this and this pffret of lhiz mercuric ebloridc. It, is quite easy to re by mrrely washing the orK;aniiims with fresh rnedi:%. relation bet ~ ( I P D these iwo t y p ~ sof action is that of the mversihility of the coagulnt,ion. In most CBSCS in which irreversible cungulntion is the ultimate result thm? is a ibilily is possiblr. On the other hard, preliorin;iry stage in which re subitairci*s t h a t generally produce a reversible co:rgolation aln) produce an irrcvcrsihlc coagulation if nilowed to act. in higher concentmtions. Thus it is ffcifcr of phtmol on yeast w:is studicii io t,he same manner. Herzog and Betael In~veshown t,hat there is no chemical reaction between phmol ttnd yeast. The ye& only takes up the phenol in the senee of a physical adsorption. 1)ilute solutions of phenol are toxic to yeast, Herzog and B ~ t z e l state that .i% solutions will kill tho cells after exposures of 5 hours. Phenol solutions of twier 1,hiscancentration ii’ere prepared and diluted with an equal volumr of the culture, the material was ihcn eaamined under the ult,ramicroseapr. As to be expected, coagulation occurred. The coagulation is quitr marked after five hours. However, phenol and other agents, to he described later, did not show the same density of coagiilation that, Hg(& produces; tlius there is a greater tendency towards antiseptic action than disinfection, at least in solutions of this concentration. a n antiseptic it would he vf interest Since phenol is so generally used to examine this effect on some other form of organism than yeast, i.e., bacterin. It, must be kept in mind that rather concentrated solutions are requirrd to kill many bacteria. Thus in order to kill the following organisms in 1 j minutes the concentrations of phenol required me:‘ Ftc;. j

’ Bioehem. J., 6 , 362 (19x2).

T H E CHEMISTRY O F DISINFECTION

. . . . . . . . . . . . . . . 7% Pest and Typhus Diphtheria.. . . . . . . . . . . . . . . . . . . . . . . . . . . . 5% ............................... 8% Staphylococcus. . . . . . . . . . . . . . . . . . . . . . . . .IO%

If weaker solutions are used a greater length of time is necessary before the organisms are killed ; in still lower concentrations only inhibition is produced. The concentration of phenol in the two following experiments was 0.5%. A pure strain of B. Megatherium was grown in the ordinary manner and a loop-full of the material was placed in 3cc of normal saline, an equal volume of 1 7phenol ~ was added; the concentration of phenol being 0.5 %. This material was then examined periodically in the ultra-microscope. At the end of two or three hours there was evidence of coagulation in a large number of the cells; another examination was made 48 hours later and the colloids of all the organisms were coagulated. The same type of experiment was also tried on B. Aeroyenes. I n the same concentration of phenol as above there was no visible evidence of coagulation within the first two or three hours, at the end of 48 hours coagulation had taken place in all cells. I n neither of the above experiments was the reversibility of the coagulation tested. a The observation of colloid material in these two bacterial cells is a little difficult, especially in B. Serogenes. This is due to the smallness of the cells and also to their shape, The light reflected from B. Aerogenes when the substage mirror is at certain angles makes these organisms appear as coagulated diplococci. However, when all adjustments are carefully made there is no doubt that the effect of the phenol on these organisms is no different from that on yeast cells. There are several different types of disinfectants and antiseptics: heavy metals such as mercury compounds, aliphatic compounds such as chloroform and carbon tetrachloride, aromatic phenols, oxidizing agents, and acid or basic dyes, or analogous substances. The evidence up to this point, based on the study of a representative member of the first three groups, shows that they all act in the same manner, i,e., by coagulation. I t is desirable to see if members of the other groups behave in this same manner. .Is a representative of the oxidizing class of compounds hydrogen peroxide was chosen, as it is a mild agent and no one would be inclined t o believe that it was a coagulating agent. The experiment was performed on the same type of yeast culture as was used above. By means of a graduated pipette a given volume of the culture was treated with a three percent solution of hydrogen peroxide This treated culture was permitted to until a final concentration was 1%. stand for four hours and then examined in the ultra-microscope. The changes were quite marked, a very extensive coagulation had taken place. The same type of experiment was next tried with Yatren,’ an acidic substance. A solution whose concentration was 1-500 of the sodium salt of The authors are indebted t o E. H. Volweiier of the Abbott Laboratories for the samples of Yatren and Acriflavine.

5’8

WILDER D. BANCROFT AND G . HOLMES RICHTER

Yatren was prepared and diluted with an equal volume of the yeast culture. The treated culture was allowed to stand for four hours and then examined as above. The culture used in this experiment was not pure, it was contaminakd with some unidentified bacteria. The examination revealed that all the organisms were affected in the same manner, the colloids within the cells were flocculated. The effect of Acriflavine on the organisms was next investigated. The culture in this case also was contaminated with foreign bacteria. The hcriflavine was made up to a concentration of 1-500and diluted with an equal volume of the culture. After standing three or four hours the material was examined in the usual manner. In this case the yeast cells were slightly coagulated while the other organisms were not so affected, thus showing a selective action. The coagulation of the yeast cells is not marked as in the other cases and is reversible at this stage. Since the culture is acidic and Acriflavine is most active in alkaline media this effect was to be expected. Thus from this superficial survey of the action of representative members of various groups of antiseptics and disinfectants, we are drawn to the conclusion that they all affect the organismtiin the same manner, i.e., by coagulating the colloids of the cell. This coagulation may be of two types, reversible or irreversible bringing about the conditions known as antisepsis or disinfect ion. The kinetics of the coagulation of biological colloids offers an interesting study. The fact that the popular belief that there is a “relation between chemical constitution and physiological action” is evidence enough that many think that the effect of drugs on living tissues is, in the ultimate analysis, chemica1. The justification of this view is based upon the fact that such phenomena can be expressed by the equations that are used to represent the velocity of chemical reactions. The literature is filled with studies showing that disinfection is a “monomolecular reaction”. This is indeed unfortunate; these equations that express velocities of chemical reactions are empirical and consequentIy are not specific for chemical reactions. They can express the velocities of physical processes with equal readiness, thus if we have a beaker full of a solution of a dye and turn a stream of water into the solution, the rate of disappearance of the dye follows the monomolecular equation. Or if a given volume is filled with a certain gas and another gas is blown in, the rate of change in the concentration of the gases can be represented by these equations. The rate of the dissolving of a crystal or the velocity of the swelling of gelatin in water can be expressed by the monomolecular equation, etc. The velocity of coagulation of many sols, in certain regions, can also be expressed by these equations. Taking the data of Westgren and Reitstotter on the coagulation of a gold sol, we can express the data by the “bimolecular equation”.


Time in min.

Rel. No. particles per cc

519

“K” -

0

5.22

I

4.35

. 0 380

2

3.63

,0419

3

3.38

,0347

5

2.75

,0344

7

2

.31

,0344

In spite of this good agreement, there can be no question of a chemical reaction, the process is purely physical. I t is not surprising that many coagutions resemble chemical reactions, in the one case the velocity depends upon the collision of particles and in the other, collision of molecules. I t is this same type of confusion that leads many to believe that the coagulation of proteins is a chemical process, or that disinfection is chemical in nature. There is a characteristic difference however, between chemical and physical changes. This difference is in the acceleration of the velocity when the temperature is increased, for an increase of 10’ the acceleration of the velocity of chemical changes is from 1.8 to 4, the average for a great number of cases is approximately 2.l Cooper2 has studied the effect of temperature on disinfection and has obtained some interesting results. The temperature range of his experiments was I j o (from zoo to 3 7’) and if purely chemical changes were taking place one should expect the effect of the disinfectant to be about four times greater at the higher temperature. Hydroxylamine hydrochloride, pyrogallol, mcresol, and p-bromophenol acting on B. Coli were just as effective at the lower as at the higher temperature, the acceleration being zero. Phenol, hydrogen peroxide, ethyl alcohol, acetone, and quinol acting on B. Colz had a coefficient of 2-3 over this range. Picric acid, benzoquinone, toluquinone, quinhydrone, 2,6-dichloroquinone, and potassium permanganate acting on the same organisms had coefficients ranging from I O to 20. The coefficient of 2-3 for the phenol group indicates that a chemical reaction might be responsible for the effect. However, if the same series of disinfectants are tried on other organisms the above classification no longer holds, the individual members jump from one group to another depending upon the organism. One gathers the impression then that the effect of disinfectants, in the general case, is physical rather than chemical, this physical change being coagulation of the cell colloids. There are other difficulties with a purely chemical concept of toxic action. In a typical chemical reaction one can predict accurately from a knowledge of the mass law the effect of varying the amounts of the reacting materials. Thus in the case of the reaction: alcohol

+ acid

Taylor, “Treatise on Physical Chemistry”.

* J. Hyg., 28,

163 (1928).

ester

+ water

520


an increase in the amount of the acid or alcohol will increase the velocity of the reaction toward the right side of the equation and increase the yield of ester and water. Or if either the ester or water is added, the reaction is displaced towards the left side of the equation with the corresponding increase in the amount of acid and alcohol. h’ow assume a reaction between the bacteria and disinfectant : bacteria

+ disinfectant

dead bacteria

I t is common knowledge that increasing the concentration of the disinfectant will increase the velocity of disinfection and the number of dead bacteria at the new equilibrium will be increased. An experiment of McClintic will illustrate this point.’ With a constant amount of bacteria a dilution of disinfectant “B“ 1-1600 had no effect on B. Typhosus in 15 minutes, a dilution of 1-1500 killed the entire culture in I O minutes, a dilution of 1-1400 would do the same thing in j minutes, and a dilution of 1-1300 killed all the bacteria in 24 minutes. This is in qualitative agreement with the mass action law. The experiment can be carried out in yet another way. If dead bacteria be added to the disinfection experiment the effect of the disinfectant should be decreased, if the,reaction is reversible. Lange2 did this experiment and indeed the effect of the disinfectant was decreased. Up to this point there is an excellent qualitative agreement with the analogy of a chemical reaction. There is left only one other experiment to make the analogy complete, the investigation of the behavior of the reaction when the amount of Zizie bacteria are varied. If larger amounts of live bacteria be used then on the analogy of a chemical reaction the velocity of disinfection should be much greater. Experiments with constant amounts of disinfectant, and increasing amounts of bacteria reveal a hideous disagreement3 with the idea of a chemical reaction. The increasing mass of the bacteria cut down the effect of the disinfectant to a marked degree. RlcClintic found that when the bacterial mass was increased five-fold the phenol coefficient of disinfectant “B” dropped from 15.6 to 11.6. In any experiment if the number of bacteria be made very great then there will be no effect produced by the disinfectant. The analogy therefore breaks down beyond repair and we are driven to the conclusion. again that disinfection, as a general rule, does not depend upon chemical reactions for its basis. Some workers prefer to abandon chemical concepts altogether and explain these results on a biological basis, with the aid of mathematics. This is not necessary, for the phenomena are colloidal and can be explained by colloid chemist,ry. I t has already been shown that the biological effects are produced by coagulation. The coagulation in turn depends upon the adsorption of sufficient ions or particles of opposite charge to lower the stabilizing charge to the point of coagulation. The stabilizing charge of the cell colloids can Hygenic Lab. Bull., No. 82 (1912). Z. Hyg. Infektionskrankh., 96, 108 (1922). 3 Lange gives a summary of the literature up to 1922 in Z. Hyg. Infektionskrankh., 96, 117 (1922). 2


j2I

also be lowered by the adsorption of organic non-electrolytes as Freundlich and Rona’ have demonstrated and explained. I n this case the coagulation is due to the combined effect of the organic compound and the electrolytes of the cell. The colloids of the cell behave as the substrate upon which the disinfectant is adsorbed. The coagulation of these bio-colloids proceeds in the same manner as that of other colloids, there must be the adsorption of some minimal amount of the coagulating agent before flocculation begins. Increasing concentrations of the flocculating agent , within certain limits, will hasten the coagulation. Thus it is not surprising that increasing the concentration of disinfectant will hasten the process of disinfection. Suppose that dead cells are added to the experiment (cells killed by heat), these cells will also adsorb the disinfectant and lower its effective concentration and thus slow the process down. The addition of living cells does the same thing and they are not materially damaged if they are numerous enough because the amount of disinfectant per cell is less than that required t o produce irreversible coagulation. The whole process is clearly colloidal in nature and depends upon adsorption and coagulation. If this conclusion is correct then the methods employed in disinfection and antisepsis must all be methods of coagulating bio-colloids. The nvailable data seem to indicate that this is indeed the case. One can further postulate that any method of producing narcosis in higher animals is likely to inhibit the growth and activity of the bacteria and if pushed far enough will behave as disinfectants. Consider some of the less common methods of narcosis and see if the conditions outlined above are not fulfilled. X physical blow will cause narcosis. K e are familiar with the effects of a sharp blow on the head or striking our “funny-bone”. In the discussion of narcosis we have presented evidence that this is due to the mechanical coagulation of the cell colloids.? The effect of mechanical agitation on bacteria has been investigated by several workers and the data are very interesting. Two effects are at once apparent, at first there is increased growth and activity and later inhibition but not death.3 The experiments are usually carried out by shaking bacterial suspensions in a shaking machine for several hours. The first effect on the cultures was to increase the growth activity above that of the unshaken controls. On shaking for longer periods of time it was found that the growth was greatly retarded, or completely inhibited. This is not due t o any mechanical damage to the cells for the culture would grow in the normal way when removed from the shaker. Thus there is a true stimulation followed by narcosis, the effect being due to mechanical coagulation. Heilbrunn4 discusses many cases of coagulation of cell colloids by mechanical means and has measured the viscosity changes produced by the coagu?

Biochem. Z., 81, 87 (191;). Bancroft and Richter: J. Phys. Chem., 35, 2 1 5 (1931) Brchiv. ges. Physiol., 17, 125 (1878). “Colloid Chemistry of Protoplasm” (1928).

522


lation. The mechanical coagulation of other bio-colloids is by no means rare, the coagulation of blood fibrin, the inactivation of enzymes, and of immune serum by shaking are commonplace examples of this phenomenon. The practical importance of the antisepsis produced by shaking is nil, although from the theoretical point of view it is valuable because it shows clearly that the process of disinfection is colloidal in nature. It has been known for quite a time that the injection of distilled water around a nerve will cause narcosis. The effect is explained by the coagulation produced. Let us see if this type of coagulation plays any role in disinfection. The effects of distilled water on bacteria are very interesting. Fisher’ would have us believe that the deleterious action of distilled water on bacteria was due to “plasmopsis” of the cell i.e., a bursting of the cell due to the high internal osmotic pressure. He was able to observe the degeneration of the exposed organisms. Leuch* carefully repeated these observations and found that the degenerated fragments could be seen even if the bacteria were originally absent; or if the glass slides were well cleaned there was no plasmolysis. One gathers the impression then that the osmotic effects are of only secondary importance and that the distilled water exerts its effect by some other means. Concerning the colloidal mechanism of this action it is not unlikely that the bio-colloids of the cell are peptized by the electrolytes of the medium. There must be a minimum concentration below which the adsorption of the ions is not sufficient to peptize the material to the proper degree. When the addition of the distilled water has been sufficient to dilute the ions to this value the bio-colloids merely flocculate. The whole process is similar to the flocculation of sols that have been dialyzed too long, or the coagulation of globulins when they are diluted with distilled water. The flocculation, as in most cases, is more easily reversible in the earlier stages than a t a later period. I n keeping with this it is found that bacteria exposed to the action of distilled water for short periods of time are not damaged as much as when the exposure is longer. The practical importance of disinfection by this means is not worth mentioning. However, the theoretical value of this phenomenon must not be underestimated. It is a well-established phenomenon and is not dependent upon osmotic effects as Leuch has shown. Still no theory of disinfection except the colloid theory is able to account for the action on the same basis as other types of disinfection. There could be hardly a question concerning distribution coefficients, surface tension changes, or chemical reactions; yet there are prominent theories that rest upon these effects as a basis. This is just another example of the frequent deviation of fact from the theories that do not consider the colloidal nature of living matter. The effect of heat on bio-colloids is well known and little persuasion is needed to convince one that it is coagulation. Disinfection by means of heat is quite common, the colloid aspects of this method are however, not often “Vorleaungen uber Bakterien” (1903). Archiv. Hyg., 54, 396 (1905).

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523

clearly recognized. Most workers agree that coagulation occurs. The disagreement arises in the different concepts of coagulation. Biological workers in general support the view that the process of coagulation by heat is chemical in nature. The principal basis of this view is that the velocity of coagulation can be expressed by an equation that is “characteristic” for the occurrence of a chemical reaction.’ The mistake in this view lies in the assumption that the equation is specific for a chemical reaction. That such an assumption is wrong has already been illustrated in the case of the coagulation of a gold sol, which can also be fitted to the equation. The temperature coefficients of heat disinfection are high and show no tendency to remain constant over equal ranges of temperature. This is exactly what one would suspect if the phenomenon were physical or colloidal and by no reasonable twist of the inagination could such evidence be used to support a chemical view. Bancroft and Rutzler2 have investigated the effects of heat on protein bio-colloids and their conclusions are that the coagulation is colloidal throughout. Their paper should be consulted for the theory and many interesting details of the phenomena of heat coagulation. Turning to a less well known method of coagulation, that produced by light, let us examine the role of light in disinfection. Concerning the coagulation of proteins by light of short wave-length, &fonda has clearly demonstrated that the colloidal condition of many bio-colloids can be altered. The investigations of Clark4 support the theory that the action of ultraviolet light on organic substances consists in the emission of electrons from the material. I n sols that had a negative charge, the loss of electrons by light action would leave neutral or positive particles, the mutual action of the positive and negative colloids would tend to coagulate the sol. On the other hand, a positive sol when exposed to ultraviolet light, loses electrons and becomes more positive, hence it peptized to a greater degree. Seutral sols become peptized through the positive charge gained by the loss of an electron. Clark was able to demonstrate these effects with egg albumin exposed to ultraviolet light. The coagulation by light is also produced zn U Z I J O . Gibbs5 exposed Spzrog y m to the rays from a mercury vapor arc, and followed the colloidal changes by viscosity measurements. The variations of viscosity that are characteristic for coagulating protoplasm were found to take place. .4ddoms6 was able to see the coagulation, by means of the ultramicroscope, in wheat seedlings root hairs exposed to ultraviolet light. Here, as in other types of coagulation, the process is easily reversible in the initial stages; the toxic effects arc associated with the irreversible stage of coagulation.

*

T . B. Robertson: “Physical Chemistry of Proteins.” J. Phys. Chem., 35, 144 (1931). Archiv ges. Physiol., 196, 540 (1922). Am. J. Physiol., 61, 72 (1922). Trans. Roy. SOC.Canada, 20, 419 (1926). Am. J. Botany, 14, 147 (1927).

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Light of short wave-length has the same effect on bacteria. As early as The most effective region of t>hespectrum is t,he range from 2 9 7 mp to Z I O mp, that is the samc region that is most effective in coagulating proteim2 Furthermore the action is direct and does not involve the formation of bactericidal substances such as peroxides, etc., as the amount of these substances produced is far below that necessary to cause the large effects. Moreover, the action is evident only when the light is on; after the light is cut off there is very little action due to the effects of the smalI amount of peroxides formed during the illumination. We can conclude then, that the action is direct. When ultraviolet rays are passed through suspensions of bacteria only the rays that are adsorbed are effective in killing the organisms. As already mentioned, Henri showed that the region of the ultraviolet light possessing the strongest bactericidal power is that which is absorbed by bacterial proteins. Moreover, the degree of action is almost exactly proportional to the extinction coefficient of protoplasm for these rays. Hence, we can be drawn to only one reasonable conclusion, the toxic action of light, is due to the coagulation of the protein colloids of the cell. This is in complete harmony with other types of disinfection and strengthens our belief that disinfection and irreversible coagulation, by whatever means, are intimately related. Briefly recapitulating, we can observe by means of the ultramicroscope, that all types of chemical disinfectants bring about an irreversible coagulation of the bacterial cglloids. Reversible coagulations of the colloids are associated with antisepsis. The corollary that coagulation, by whatever means, will result in antisepsis or disinfection is verified by the types of COagulation produced by distilled water, mechanical agitation, heat, and light. Furthermore, the coagulation is colloidal in nature and not chemical. The fact that disinfection data can be fitted to chemical reaction velocity equations is without significance. The colloid theory is the only theory that can adequately explain the types of disinfection produced by physical means. Thus, from every phase of disinfection we are drawn to the conclusion that the process is colloidal in nature and consists merely in the irreversible coagulation of the bio-colloids of the bacteria. In the development of this thesis up to this point, it has been stated or otherwise assumed that the coagulation has been produced directly by the bactericidal agent, This is by no means necessary, for as we have already indicated there is a group of narcotics that act indirectly and the same poseibility exists that such a thing may occur in disinfection. The experimental researches of C. Voegtlin have led him to conclude that the action of arsenic upon trypanosomes was indirect, In his own words: “A long series of experiments carried out in recent years at the U. S. Hygenic Laboratory in Washington has furnished evidence to the effect that arsenic enters into combination with a sulphur compound (glutathione) which is widely distributed in animal 1877 Downes and Blunt’ discovered the bactericidal action of light.

’ Proc. Roy. Soc., 26, 488 ( 1 8 7 7 ) . Compt. rend. sac. biol., 73, 323.


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cells and even in such low forms of plants as yeast. This sulphur compound had recently been discovered by the English bio-chemist, Hopkins, who found it essential for the maintenance of normal cellular combustion. S o w it is significant that certain sulphur compounds have a great affinity for arsenic, as every chemist knows. It was therefore very important to find that the toxic action of arsenic on the trypanosomes could be completely checked in the test tube as well as in the living animal by simply furnishing the cells with an extra supply of glutathione. The effect of the latter substance is quite specific, as it proved to be the only substance of a great number of others occurring in the body which offered protection. Without going into further details we may therefore assume that arsenic kills by interfering with the normal cellular combustion or oxidation mechanism, and the cells die of “internal asphyxia”.” 15-e may question the conclusion that death is due to “internal asphyxia.” This lowering of the oxidation process of the cell will allow the toxic (coagulative) substances that are normally destroyed by oxidation to accumulate to the point where they coagulate the cell colloids and cause narcosis or death, depending upon the reversibility of the coagulation. Hence, if the mechanism outlined above is true, arsenic behaves as an indirect disinfectant. Heffter has suggested, and supported by several experiments the theory that free sulphur acts upon the glutathione and forms complex sulfides which stop the cellular oxidation. This would explain the toxic action of sulphur. It may be that there are numerous examples of this indirect class in the reducing type of drugs, but actual knowledge is meager at present. Since the death of the organisms is due to irreversible coagulation the question of reversibility and the conditions which favor it are important. Normally in testing disinfectants the organisms are exposed to the agent, then placed in some medium to observe whether growth takes place or not. Indeed, this is a convenient but not very accurate method, for the peptizing agents of different substrates are quite different. For example, Liesegang mentions a case where the bacteria of bird cholera were exposed to I:IOOO mercuric chloride solutions for seven minutes; these organisms were then unable to develop on ordinary media. One would ordinarily conclude that they were dead. However, this is not the case for if they are placed in their natural environment, birds, they begin to develop after three hours. The conditions favoring peptization are much better in the animal body than in the laboratory media. Supflel gives a striking example of peptization of Anthrax bio-colloids after treatment with mercuric chloride. The organisms after exposure are apparently dead when tested on ordinary media. Siipfle mixed these organisms with a good grade of blood charcoal and cooled the mixture, to aid the adsorption. I n this case the sublimate is adsorbed much stronger on the charcoal than on the bacteria so the material leaves the Anthmz cells and is deposited on the charcoal. Upon removal of the charcoal, 1

Archiv. Hyg., 89, 351 (1920);93, 252 (1923).

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the organisms are found to be again normal and grow well on the ordinary media. In this way it was found that reversibility could be effected in organisms exposed to : 5 % sublimate after I I days 3% ” ” 38 I’ 1%

”

”

40

”

One can understand why it is much more difficult to disinfect living tissues with their favorable peptizing conditions, than inert substrates such as glassware, clothes, instruments, etc. The favorable peptizing conditions in the former case are probably the colloids of the tissue behaving like the charcoal in Siipfle’s experiments. I n the light of colloid chemistry the testing of disinfectants should be studied from the point of view of the reversibility of the coagulation produced by the disinfectant rather than the determination of growth, on relatively inert media, after exposure. Some workers regard disinfection in a totally different light, thus McClendon’ discusses disinfection under the head of cytolysis. However, he clearly recognizes that cytolysis does not always accompany death. Many anesthetics and disinfectants do actualy favor the cytolysis; from our knowledge of the effects of these drugs on protoplasm we can construct a mechanism of cytolysis. For example if a tissue is cut off from the blood supply it will after a time digest itself (autolysis); this is accelerated or favored by the presence of etherI2etc. We know that shortly after the oxygen supply is cut off narcosis occurs or a reversible coagulation of the tissue is produced; in time this passes into an irreversible coagulation and death. Autolysis occurs after this stage of irreversible coagulation and is brought about by the enzymes present in the tissue. I t is known that the enzymes are not as easily affected as protoplasm by anesthetics or mild disinfectants. For example, when toluene is added to digestion experiments, it affects the protoplasm of bacteria and kills them while it is almost harmless to the enzyme. On the other hand, if the coagulation is extensive enough to damage the enzymes then autolysis cannot take place. Heilbrunn has studied the viscosity of protoplasm that is undergoing cytolysis and the evidence is that coagulation precedes digestion : “In spite of the fact that Loeb many times emphasized the importance of the so-called cytolysis of sea-urchin eggs, he made little effort to explain the mechanism of the process. In pages 188 to 190 of his book he expresses some vague general ideas regarding the possible nature of cytolysis. Saponin and benzine, he states, dissolve the chorion or jelly of mollusc and annelid eggs. He then cites von Knaffl-Lends views to the effect that cytolysis is due primarily to the liquefaction of lipoids, and he makes the following quotation from von Knaffl-Lena, “The mechanism of cytolysis consists in the liquefaction of the lipoids, and thereupon the lipoid-free protein swells or is dissolved by taking up water.” 1

2

“Physical Chemistry of Vital Phenomena.” Archiv. exp. Path. Pharmakol., 60, 256 (1909).

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“Loeb thus apparently believes that cytolysis in sea-urchineggs is primarily a liquefaction of the protoplasm. This it most certainly is not, for viscosity tests indicate beyond the question of a doubt that during cytolysis, the viscosity of the egg protoplasm increases enormously. “Our general conclusion is that in all types of protoplasm there is a definite type of response to such agents as distilled water, fat solvents, the electric current, mechanical injury, etc. This response is typically characterized by the appearance of numerous small vacuoles within the protoplasm. Frequently, though not always, it is accompanied by an increase in the volume of the cell. When pigment is present in the cell, this always escapes.” The phenomena exhibited by the bacteriophage are also of this type although it is referred to as a lysis. To begin with it has been noted by several workers that small quantities of the bacteriophage stimulate the bacteria.’ We have already indicated that in the coagulation of cellular bio-colloids the increasing instability of the system is associated with the phenomena of stimulation. This alone would indicate that displacement adsorption and coagulation are concerned in the action of the bacteriophage. The excellent researches of J. Bronfenbrenner present more direct evidence that the colloidal condition of the cell is changed before digestion and that the digestion is due to the normal endoferments. “When swollen bacteria are stained by the method of Gutstein, the cytoplasm may be differentiated from the ectoplasm. The latter always appears continuous, and even in extremely distended cells it shows no evidence of “holes” described by D’Herelle as resulting from the puncturing of the membrane by the entering parasites. The cytoplasm, on the contrary, shows marked changes during swelling. I t takes the stain less intensely and less evenly as the swelling progresses, so that in many instances it appears segmented or beaded. I n cells photographed a t this stage unstained, by means of ultra-violet light illumination, the cytoplasm appears to be of uneven density, quite unlike that of normal bacteria.” “The lysis of bacteria in a synthetic medium, devoid of all protein, gave unmistakable evidence of hydrolysis of bacterial protein. “In the light of these experiments, the clearing of bacterial cultures in the presence of bacteriophage seems to be due to the hydrolysis of the bacteria. The active agent (bacteriophage) plays no part in the actual solution. The solution is the result of intercellular digestion brought about by normal endoferments.” Bacteriologists do not know, a t present, how these enzymes become activated. Colloid chemistry is able to suggest a mechanism; it is common knowledge that enzymes, as all catalysts, must adsorb or be adsorbed by the substrate before they can act. Colloidal studies have indicated that the adsorption capacity of coagulated proteins is frequently greater than that of the uncoagulated sol; this is probably due to the jelly structure of the coagulum. Consequently, we should expect that coagulated proteins should be 1 “ T h e Bacteriophage and Ita Behavior”, 76 (1926);J. Infectious Disessea, 37,35 (1925); Schweiz. med. Wochenschr., 52, 761 (1922).

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more easily and rapidly digested; this has been found to be the case.’ In regard to the swelling of the protoplasm referred to above, bacteriologists explain this as due to the increase in osmotic pressure within the cells.* Another possible explanation is that the swelling is due to the change in the pH of the cell during digestion, the degree of imbibition of most bio-colloids being very sensitive to changes in the acidity of the medium. I t is interesting tonote that when bacteria are shaken with distilled water or treated with other coagulating agents the “bacteriophage is spontaneously generated.”3 The coagulating action of distilled water has already been discussed. The phenomena of drug resistance are well known and are important in bacteriology and medicine. Briefly, it has been found that if organisms are exposed to small amounts of toxic compounds for long periods of time then subjected to a full dose of the toxic agent they are not killed. Every phenomenon from the probability of the survival of the fittest to the loss of the parabasal body in trypanosomes has been used to explain this ~ o n d i t i o n . ~ Findlay in discussing the problem at the beginning of 1930 says: “It is obvious that at present no satisfactory expianation of drug resistance can be given. I t is however, inevitable that the drug resist,ance which can be required by trypanosomes, spirochaetes, and bacteria, either in vitro or in z’ivo, should be compared with the acquired tolerance towards drugs, such as alcohol, exhibited by man.” The reason that this problem has been so difficult for the biologists is because the condition is not dependent on biological phenomena. The whole thing is essentially colloidal in nature and is easily explained, including drug tolerance in man. The toxic action of drugs as we have already indicated, depends upon the coagulation of the bio-colloids of the tissue or organ in question; drug tolerance is based upon the well-known phenomena of fractional coagulation. I t is known that in many cases as in hydrated ferric oxide; and albumin sols6 much less of the coagulating agent is required when added all at once than when added in small amounts over a long period of time, particularly when the slow addition causes a partial coagulation of the sol. This fractional precipitate not only removes the coagulating agent by adsorption on the precipitate which in many cases is greater than that of the original sol, but also alters the stability of the sol by decreasing its concentration. Other conditions being equal, a dilute sol, in general, is more stable than a concentrated sol, because the chance that any two particles can come in contact is less. Thus in the treatment of an organism or tissue, with a coagulating drug it need not surprise us that after several small doses the normal toxic dose does not produce the same effect as in the untreated controls, indeed it is rather to be expected. Since there are many sols in a living cell it frequently Lloyd: “Chemistry of Proteins”. J. Gen. Physiol., 4, 245 (1922). Deutsch. med. Wochenschr., 48, 383 (1922). Compt. rend., 153, 226 (1911); Z. Immunitats., 43, 253 (1925). 2.physik. Chem., 44, 143 (1903). Beitr. Chem. Physiol. Path., 5 , 436 (1904).

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happens that with two drugs, that are adsorbed upon the same sol, the one drug will make the cell resistant to the other drug. This is due to the fact that the first drug starts the fractional coagulation. In other cases where the two drugs are adsorbed on different sols, the one drug will fail to make the cell resistant to the other drug because it cannot partially coagulate the other sol. The phenomena of drug tolerance which are so difficult to explain on other theories are quite simple when examined in the light of colloid chemistry; it is the natural outcome of the coagulation theory. The colloid theory is based upon the groundwork of logical interpretation of existing data and experimental observation of the colloidal changes with the ultramicroscope. The interpretation of the data in the light of colloidal chemistry must in time become increasingly evident to biological workers that it is not only logical, but flexible enough to cover the great mass of data without any unusual assumptions. I n the consideration of this problem nothing but the most elementary facts and assumptions of colloid chemistry have been employed, yet this theory is able to account for and unify the most diversified facts in disinfection and antisepsis. Turning to the experimental observations with the ultramicroscope it behooves us to state the limitations of this instrument. No one instrument or method will illustrate all types of biological coagulations. The study of the changes in viscosity is, perhaps, the ideal method, but in this particular case there are great experimental difficulties. The ultramicroscope affords the easiest method of study, but unfortunately it is not universal in application. The colloid particles of protein sols are practically invisible due to the fact that the index of refraction of the particles and the surrounding medium are nearly the same, and colloid particles in the ultramicroscope are recognized by the reflected light. The coagulated sols are frequently easy to observe because their optical properties are much different, coagulated egg white being a familiar example. However, there are cases where the transformation of sol to gel is not accompanied by any marked or drastic change in optical properties, ie. in gelatine. Heilbrunn, in fact, regards this as of very frequent occurrence and if it were not for his classical studies of the variations in the viscosity we would still be in the dark concerning some of the most important problems of biology. Naturally such changes as these are quite difficult to observe. The opposite extreme also exists, if the organisms are surrounded by a thick membrane that reflects a large amount of light we will not be able to see within the cell, although the outlines of the organism are plainly visible. Staphylococci apparently belong to this group; even when these organisms are exposed to saturated bichloride of mercury solutions, or steam, the coagulation is barely perceptible. We do not wish to give the impression, that the ultramicroscope is not a valuable instrument for this type of work. If the ultramicroscope were used as much in biology and bacteriology as the ordinary microscope, most workers would not have failed to observe phenomena that was already familiar t o the minds of bacteriologists and colloid chemists.

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WILDER D. BANCROFT A N D G . HOLMES RICHTER

The conclusions of this study of disinfection in the light of colloid chemistry indicate that antisepsis is merely a state of narcosis, which depends upon the reversible coagulation of the cell colloids; disinfection is brought about by the irreversible coagulation of the cell colloids. The corollary, that coagulation by whatever means will bring about antisepsis or disinfection, also holds and is exemplified by the action of heat, light, distilled water, and mechanical agitation on bacteria.

Summary (I) Antisepsis and disinfection in bacteria are similar to narcosis and toxic action in the higher organisms. The decreasing stability of the cell colloids in the initial stages of (2) coagulation is associated with the phenomena of stimulation; the stage of coagulation that is reversible is responsible for the inhibition of the activity or the organism but does not kill them; the state of irreversible coagulation is responsible for the death of the bacteria. (3) These colloidal changes were observed in living cells and bacteria by means of the ultramicroscope. (4) Antiseptics and disinfectants, like narcotics, can act in either of two ways, by directly coagulating the cell colloids, or by interfering with some normal function of the cell to such an extent that the accumulated toxic products will cause the coagulation. Phenol is an example of the first case and arsenic derivatives seem to be of the second type. ( 5 ) The mechanism of disinfection consists of two phases, first the adsorption of the drug and secondly, the coagulation of the cell colloids. ( 6 ) Most workers have confused the nature of the second phase of action with that of a chemical reaction because the velocity of disinfection can be expressed by equations that are used to express the velocity of chemical reactions. This confusion is cleared away by showing that these equations are not “specific for chemical reactions” but can also be used, in certain regions, to express the velocity of coagulation and other physical actions. (7) The difficulties of a chemical concept of disinfection are shown by the inapplicability of the mass action law, lack of stoichiometrical relations and the abnormal temperature coefficients. ( 8 ) The disinfection by lytic agents is discussed and evidence produced to show that coagulation is the initial phase, the digestion follows the coagulation. ( 9 ) The phenomenon of drug tolerance is explained upon the basis of fractional coagulation: the adsorption of the drug by the coagulum and the increased stability of the diluted sol. This does not preclude the possible formation of substances which counteract the action of the drug. ( I O ) The limitations of the ultramicroscope in the observations depends on the optical properties of the coagulum being such that it reflects the light and the absence of a thick reflecting membrane around the cells. Cornel2 Unzverszty

The Chemistry of Disinfection

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