Bactericidal Studies of Chlorine R. S. Ingols, H. A. Wyckoff, T. W. Kethley,H. W. Hodgden,
E. L. Fincher, J. C. Hildebrand, and J. E. Mandel ENGINEERING EXPERIMENT STATION, GEORGIA INSTITUTE OF TECHNOLOGY, ATLANTA, GA.
T
,
H E use of chlorine in water treatment for killing pathogenic bacteria has been recognized as a great factor in reducing diseases transmitted by public water supplies. Its wide acceptance has been favored by the small quantities of chlorine needed and by the low cost of the chlorine. The mechanism of its bactericidal action has not been thoroughly understood despite its wide acceptance for practical applications (10). Difficulties, both in measuring the amounts of chlorine that are left after contact with a bacterial suspension and in identifying the nature of active chlorine compounds present, have led to much confusion in interpreting many of the early bacteriological studies. The small amounts of chlorine needed for greatly reducing the number of organisms in suspension led one early research group (6) to the conclusion that the reaction could not be chemical, and the same fact recently caused a research group (11) to conclude that the reaction must attack a specific, easily oxidizable radical of an essential enzyme. I n spite of the small concentration of chlorine required to kill E. coli, the ratio of the amount of chlorine to the amount of cellular material is still very large. Assuming that E. coli is a sphere which has a diameter of 2 microns, each cell would be approximately 4 cubic microns in volume. This would represent about 4 X 1 0 - g mg. per cell (assuming a specific gravity of one) or 280,000 organisms would be required for 1 microgram, or the amount needed for 1p.p.m. in 1ml. Nost of the suspensions studied in the work reported here had about 1000 organisms or (4 X 10-6 me.) per ml. Thus, a concentration of 1.0 p.p.m. chlorine would represent about two hundred times the amount of chlorine to bacteria on a weight basis. Because of this high ratio there are bacteriologists (1) today who feel that it is the general chemical action of the chlorine on the bacterial protoplasm that is responsible for the death of the bacteria. Green and Stumpf (If) have presented evidence which they interpret as proving that the chlorine attacks the sulfhydryl radical of one of the enzymes of the glucose oxidation series. Their article does not indicate whether “free” chlorine or chloramines were used in attacking the bacteria, but they state that the reaction is irreversible. I n 1938, Douglas and Johnson (8) showed that sulfhydryl radicals were readily oxidized to sulfonyl compounds by high chlorine concentrations a t low p H values. I n a very recent paper, Black and Goodson ( 5 ) have shown that hypochlorous acid oxidizes sulfide to sulfate a t concentrations and pH values normal to potable waters. The purpose of the research reported in this paper was to determine the nature of the oxidation product of cysteine and other common amino acids with hypochlorous acid, monochloramine, and chlorine dioxide in order to evaluate the possible importance of the various mechanisms that may be involved in bacterial death. Numerous workers have shown that the hypochlorous acid is a much more effective bactericide than monochloramine. I n a recent review, the senior author (15) concluded that chlorine dioxide is an excellent bactericide, but i t has also been shown (16) that i t reacts with only a relatively few organic compounds. It is hoped to reconcile these data and concepts. Bacterial studies were made paralleling the chemical investigations, I n this work, standard chlorine techniques for testing
the efficiency of various chlorine compounds were used except that different dechlorinating agents were checked against each other. Because hypochlorous acid, monochloramine, and chlorine dioxide have high oxidation-reduction potentials these chemicals can readily be reduced by sodium thiosulfate or sodium sulfite. These highly oxidized compounds of sulfur would be unable, however, to restore or aid the “chlorinated” organism in restoring the unoxidized form of the sulfhydryl radical of its enzymes. A molecule of cysteine would not be able to reduce the sulfonyl Oxidation state, but i t is conceivable that it could aid a bacterium which was not otherwise too disorganized to use the cysteine t o reduce a disulfide state of oxidation. Therefore, the relative effectiveness of cysteine in comparison with sulfite and thiosulfate was tested with monochloramine, hypochlorous acid, and chlorine dioxide. I n an extensive study of the effect of freezing on bacteria ( l 7 ) , it was found that the state or “well being” of the organism has a tremendous effect on the number of survivors. Thus, the resisb ance of organisms to freezing death can be lowcred by suspending them in water for 2 hours prior to freezing. It was decided to investigate the effect of storing bacteria at room temperature in distilled water for 2 hours on their resistance t o chlorine. Only preliminary results are given (Figure I ) .
Methods of Study Recently, Palin (26) published a technique for differentiating hypochlorous acid from mono- , di- , and trichloramine. This technique uses orthotolidine (OT) as the indicator a t p H 6.5 for titrating directly the hypochlorous acid with ferrous ammonium sulfate; hypochlorous acid oxidizes OT to a blue color which is reduced to a colorless end point with the ferrous ion. Then the monochloramine is determined by adding a small amount of iodide and again titrating the blue color M ith the ferrous ammonium sulfate. The dichloramine is determined by lon wing the
Y
Buda Industrial Engine with Vapor Phase Cooling Uses Gas from Sludge Digestion for Fuel
996
Water Treatment pH to liberate iodine from the iodide and then raising.the pH to form the blue color with the orthotolidine a t H 6.5. The blue color is again titrated. A portion of the trichEramine is titrated with the hypochlorous acid in the first step of the series, but it can be differentiated from the hypochlorous acid by extraction of the trichloramine with carbon tetrachloride and then titration of the hypochlorous acid in the aqueous phase. This technique for the identification of the various fractions has worked very well in this laboratory with the materials for which it was designed, but it does give trouble with certain nitrogenous organic compounds such as proline. A more recent method for differentiating the same compounds has been published by Marks, Williams, and Glasgow (go), using similar chemical steps but employing the amperometric device of Wallace and Tiernan Co. as the indicator instead of orthotolidine. This method has more flexibility of operation than the Palin technique but is somewhat slower for titrating many samples that are ready a t one time. It gives easier end points with such compounds as proline and tyrosine. Although each of the techniques gives a monochloramine titration value, neither is capable of indicating whether this value represents the inorganic material added or the organic derivative of the amine treated with monochloramine. Thus, all the organic reaction products under study must be identified by a third technique. Weil and Morris (28) have shown that the chloramine compounds can be identified and measured by using the ultraviolet spectrum. A survey of the amino acids indicates that these acids can also be studied with ultraviolet and that many of the reaction products can be identified tentatively by correlating the chemical and physical data. Thus, an amino acid can be treated with an amount of chlorine to yield a monochloramine which can be determined chemically. The ultraviolet spectrum will indicate whether the monochloramine is a derivative of ammonia or the organic compounds, or a mixture. Further, if the inorganic monochloramine is added to the organic compound, a mixture of the organic and inorganic chlorine compounds is formed, and it is sometimes possible that the relative amount of each can be evaluated with the ultraviolet spectrum data. The chlorination of the amino acids was studied a t concentrations of 10-4 M amino acid in order to get good curves in the ultraviolet spectrum. The pH was maintained a t 8.0. The distilled water was first buffered, and then an excess of chlorine was added and allowed to stand for 24 hours or longer. The excess chlorine was removed with a large ultraviolet lamp.
Results
74
+ HOC1 + CHaCHNHClCOOH
+
~ C H ~ C H N H L C O O H5HOC1+ 2CHsCOCOOH
+ H20
+ 5HC1 + Nz + 3He0
(1) (2)
When a large excess of hypochlorous acid is added rapidly to alanine the deamination may yield some trichloramine, but .it appears as only a very small fraction of the total nitrogen. When cysteine is reacted with six equivalents of hypochlorous
May 1953
+ 3HOCIHSOaCHzCHNHzCOOH + 3HC1 (3) 2HSCH2CHNHnCOOH + l l H O C l + 2HSOsCH~COCOOH+ l l H C l + N2 + 3Hz0 (4) HSCHzCHNHzCOOH
p.p.rn. dose 04 Monoohloramine
Figure 1. Effectiveness of Like Amounts of Monochloramine on Portions of Same Dilute Bacterial Sus ension, Dosed Immediately after Dilution and 2 Hours ager Dilution
When cysteine is reacted with chlorine dioxide, the same SUIfonyl amino acid is formed as in Equation 3, but there is no further demand or reaction with the amino group as in Equation 4. When cysteine is reacted with monochloramine, the oxidation of the sulfhydryl is carried only to the disulfide state, and no further reactions occur. This reaction is shown in Equation 5. NH&l
Chemical. Alanine is used as a simple amino acid to illustrate the various reactions with the different oxidizing compounds. Chlorine dioxide does not react with alanine. Monochloramine forms a mixture of the organic and inorganic monochloramine in a 1:l mixture of ammonia and alanine with two equivalents of chlorine added. Hypochlorous acid forms organic monochloramine as shown in Equation 1. With time the organic chloramine decomposes, or when more chlorine is used (a higher chlorinealanine ratio), the chlorine oxidatively deaminates the alanine to form pyruvic acid or acetaldehyde with decarboxylation of the acid as shown in Equation 2. The pyruvic acid gives a burned taste, which can easily be detected and identified, as well as a characteristic ultravioIet spectrum.
CHaCHNHzCOOH
acid (3 moles), the chlorine disappears entirely before a chemical analysis can be made when p H 8.0 is maintained as shown in Equation 3. When eight equivalents were added, monochloramine was found and no free chlorine was left in 5 minutes. When eight equivalents of chlorine are added to cysteine, monochloramine is found for a period of 45 minutes to 1 hour, and then it is gone. With more chlorine, little free chlorine is found for a few minutes, but it disappears as shown in Equation 4. These results indicate the following reactions which are in agreement with Douglas and Johnson (8):
+ 2HSCH&HNH&OOH +NHs + HCl +
HOOCCHNH&HZS-SCH~CHNH&OOH (5)
The disulfide formed has an ultraviolet spectrum identical with that for cysteine, and two equivalents of monochloramine are used in the process. Three equivalents of monochloramine give a residual of one equivalent which is stable for several hours. With both glycylglycine and glycylglycylglycine, hypochlorous acid reacts to deaminate the free amino group rapidly (complete within 20 minutes a t 25' C. and p H 8.0) and then continues the reaction to give an oxidative hydrolysis within 2 hours. By maintaining a constant amount of chlorine and a constant chlorine-nitrogen ratio it can be shown that the hydrolytic reaction is much slower than the deamination. With tyrosine, hypochlorous acid is rapidly reduced in amounts similar to that for alanine, indicating deamination and ketone formation. With monochloramine, there is no obvious reaction with tyrosine except the formation of organic monochloramine which is slowly (over several hours) broken down. With chlorine dioxide there is a reaction with the phenol ring to give color. This reaction can be used to determine either the chlorine dioxide or the tyrosine, although the details for optimum conditions for these analytical procedures have not been developed. This same color is developed in certain proteins when
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997
.
they are mixed with chlorine dioxide so that care must be used with data obtained with photocolorimetric procedures using chlorine dioxide and protein. The complexity of the hemin molecule is such that no specific reactions of the various chlorinating compounds are suggested. Using ultraviolet light, changes in the adsorption spectrum can be noted for hypochlorous acid, monochloramine, and chlorine dioxide. The shift in the spectrum for hypochlorous acid with
c
s 40 Dechlorindted with -Sulfite Cysteine
_--
01
I
I
1
I
I
I
0.5
1.0
1.5
2.0
2.5
3.0
p.p.m.
Chlorine
I
added
Figure 2. Effect of Different Dechlorinating Agents on a Single Bacterial Suspension Containing Ammonia and Dosed with Free Chlorine
centrifuging of the cells was used between the culture medium and the final dilution water because the final organic matter concentration from the nutrients must be less than 0.01 of 1 p.p.m. a t the one million dilution used. (The original nutrient broth count was 300,000,000 per ml.) Short chlorine contact periods of 5 minutes were used and the suspensions were dechlorinated with cysteine. Because monochloramine oxidizes the sulfhydryl radical to a compound that can be reduced biologically it was believed that this possible reaction should be studied biologically. It is realized that counting bacteria is not very accurate and that as chlorine contact time is shortened it becomes more difficult to control intervals of time accurately. However, many runs have been made and all results wereso consistent that the representative data of two runs are given in Figures 2 and 3. I n Figure 2 the ammonia was present in the dilution water of the suspension, and a chlorine solution was added. Very little difference was obtained, but four out of five points indicate that cysteine gives a greater number of survivors than sodium sulfite as a dechlorinating agent. Holwerda (IS), Hoather ( I d ) , and Houghton ( I 4 have shown that when chlorine is added to a bacterial suspension containing ammonia, it is much more effective than when preformed monochloramine is added to a suspension. The data of Figure 3 were obtained by adding preformed monochloramine to a bacterial suspension and then comparing the effectiveness of cysteine and sulfite for dechlorinating. These differences which occur only with short contact times of 5 minutes or less indicate that more bacteria live after chlorine contact by dechlorinating with cysteine than with sulfite. 100
a dose of less than 1.0 p.p.m. is large and irreversible to any amount of added cysteine. With monochloramine there is a shift that corresponds to that which can also be produced with carbon monoxide. Because it is also irreversible with cysteine i t is not certain whether i t is an addition product or oxidation product. Chlorine dioxide also produces a marked change in the spectrum of the hemin. A summary of the chemical results is given in Table I.
80
f
'260 w
L
0
2 40
B Table I.
'
Summary of Reactions of Chlorine with Pertinent Organic Compounds
Organic Substrate Alanine
Hypochlorous Acid Pyruvic acid
Cysteine Glycylglycine Qlycylglycylglycine
RSOsH Oxidative Hydrolysis and deamination
Tyrosine
Ketone
Hemin
Violent change
iUonoqhloramine
Organic monochloramine
RSSR .....
Termi,nal organic monochloramine Organic monochloramine Irreversible addition or oxidation
Chlorine Dioxide l i o reaction
RS03H
....
No reaction Color
A shift in spectrum
Biological. The authors have always had a certain amount of trouble predicting the proper range in a series of chlorine doses to get a good spread in numbers of bacteria remaining. Some studies are in progress in our laboratories to determine what makes organisms sensitive t o low temperatures (0' to - 2 5 O C.). It was found that sensitivity to freezing was affected by age (rate of growth), salt concentration, concentration of colloids (gelatin) in the environment, and the manner in which the culture was handled. It was found that a culture was much more resistant t o freezing if i t was frozen immediately after a million t o one dilution in distilled, deionized water than i t was after standing at room temperature in the million to one dilution. The results shown in Figure 1 indicate that a dilute suspension of bacteria (300 per ml. in distilled water) becomes more sensitive to monochloramine after 2 hours' standing in the distilled water. No
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20
0
0.5
1.0
1.5 p.p.m.
2.0 2.5 3.0 Monochloramine dose
Figure 3. Effect of Different Dechlorinating Agents on Survival of 5 Minutes of Contact with Preformed Monochloramine
During the early part of this work thiosulfate was compared with cysteine, and then it was suggested by Marks in private communication, that sulfite would be a better dechlorinating reagent than thiosulfate. I n many runs using the three dechlorinating reagents in direct comparison the thiosulfate and sulfite have given the same results, while cysteine has always given better results than either sulfite or thiosulfate.
Discussion The importance of the condition or well being of the organism a t the time that i t is suhjected to chlorine contact is demonstrated by the results given in Figure 1. Consideration was given to the inclusion of data comparing the effectiveness of chlorine against the same organism grown overnight on the surface of an agar dish (aerobic) in comparison with those grown in broth (anaerobic). However, the preparation of the suspensions and the period of leaving the organism in the final suspension were not recorded.
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Water Treatment More careful techniques will be needed in preparing the two suspensions of bacteria for comparison of the effect of aerobic and anaerobic conditions in relation to chlorine efficiency in order to develop data with any meaning. Careful consideration must be given to the preparation of bacterial suspensions where the results are to be used for theoretical interpretation. Allen et al. ( 2 ) indicate that the number of organisms which can grow on differential media is dependent on the time they are maintained in “pure” water. Thus in spite of the large amount of chemical data presented, the great importance of the bacterium as a biological organism must be stressed in understanding this problem. The bacteriological results on the relative effect of cysteine in comparison with sulfite or thiosulfate is another effort to focus attention on the importance of the condition of the chlorinated organism. Cysteine can reduce the chlorine compounds more rapidly than sulfite, but if this were the only factor of importance then it would seem that the more effective bactericide should show the greater difference between cysteine and sulfite in the number of survivors. The data presented here indicate that generally the preformed monochloramine, which is the least effective bactericide that has been studied, gives the greatest difference in the number of survivors between cysteine and sulfite when used to dechlorinate the suspension, It is hoped that the biological material discussed and the chemical results presented will clarify the mechanism of the bactericidal action of chlorine by comparing the action of the various oxidizing forms in which this element is commonly used in water treatment. Hypochlorous Acid. The bactericidal studies indicate that once the bacteria have been treated with hypochlorous acid, the cysteine does not allow any more organisms t o live after contact by this form of chlorine than sulfite does. Because the hypochlorous acid changes the sulfhydryl groups to the completely oxidized sulfur state, the cysteine cannot help the bacterium restore the sulfonyl radical to the functional sulfhydryl form. Thus, this one irreversible and complete change in the state of oxidation of one essential enzyme would be adequate to explain the irreversible bactericidal action of hypochlorous acid. However, this form of chlorine also rapidly removes the free amino radicals from organic amino acids in high chlorine-nitrogen ratios and thus can cause changes in the fundamental structure of the proteins of the general protoplasm which would require intricate chemical change to restore. At this point, it is not known how significant this irreversible change in the protoplasm of bacteria is, but it seems probable that the change would not help in the recovery of the organisms on dechlorination. Further, the hypochlorous acid in low residuals causes extreme changes in the iron-bearing components of enzyme systems (hemin and hemoglobin) which are not restored t o the original state by adding cysteine. This again may be an important factor in causing cessation of vital functions which cannot be restored by adding cysteine as the dechlorinating agent. Thus, any one of these chemical changes might cause death of the bacteria, and the major extent of these changes is one strong argument in favor of hypochlorous acid as the bactericidal agent in sterilization. Monochloramine. In comparison with hypochlorous acid, monochloramine requires much more time and much larger concentrations of oxidation capacity to bring bacterial death. The chemical information indicates that the sulfhydryl radical is first changed only to the disulfide state typical of cystine. From the studies (4) of the action of antibiotics on bacteria, it is known that disulfide linkages can kill bacteria, but it is also known that cysteine added to the medium can restore the life of bacteria dosed with such antibiotics as gliotoxin, though not with penicillin. Because of the lack of restoration, it is assumed by Bailey and Cavallito ( 4 ) that penicillin attacks more than the sulfhydryl radical alone. Thus, if the sulfhydryl radical oxidation is the May 1953
only important change that is brought about by the monochloramine, the cysteine should be able to restore a significant number of bacteria after the organisms have had only a short period of contact with monochloramine-i.e., if only the disulfide radical is formed. However, the bacterial data indicate that a possible restoration of the sulfhydryl radical revives a t best only a small number of the bacteria and that this restoration occurs only in moderate concentrations of monochloramine. It is not known what the extra chemical reaction is that explains why the cysteine does not restore more of the bacteria. The monochloramine reacts with an alpha amino group of certain amino acids to form a mixture of organic and inorganic monochloramine compounds. These can be reduced to the ammonia or organic amine group with cysteine, sulfite, or thisulfate. Thus, it does not seem likely that this reversible chemical change can either cause death or explain the failure of cysteine or sulfite to restore the bacteria treated with monochloramine. When hemin is treated with monochloramine there is an extensive and irreversible change in the structure of the molecule which appears similar to the change brought about by carbon monoxide. This latter change may not be extensive chemically, but it may be able to kill organisms because the iron enzyme systems cannot function even though the valence of the iron remains unchanged. The spectral changes of the hemin or hemoglobin with monochloramine are not reversible with cysteine. The authors believe that the irreversibility explains the failure of the cysteine to restore the bacteria treated with monochloramine. There is nothing in the data published by Knox et al. (11) that refutes this concept except the statement that chlorine does not destroy catalase a t the low concentrations necessary for kill. However, these same authors stated that the reaction of chlorine with bacteria is irreversible while the data resulting from these studies may be interpreted to refute their statement, a t least when chlorine refers to monochloramine. Thus, the idea that monochloramine “destroys” catalase is offered by the authors as an hypothesis for further study. Chlorine Dioxide. The complete oxidation of the sulfhydryl radical of cysteine by chlorine dioxide can explain the bactericidal action of this compound. Further, the change would be irreversible. These studies indicate that chlorine dioxide does not attack the amino radicals in amino acids; this s e e m to support the concept that the amino radical is not a highly vulnerable point in the bacterial structures and is in agreement with that claimed previously by the senior author.(lb) from other data. Phillips (9.4) in a recent review on the resistance of bacterial spores to disinfectants develops the hypothesis that spores do not have the sulfhydryl radical in the labile position which is typical of the vegetative form. This would explain the very poor sporicidal activity of monochloramine, for, besides its oxidation of the sulfhydryl, monochloramine can alter only the primary amino group of protein side chains and then only at a very low rate. The low rate of sporicidal activity by monochloramine is well substantiated by Levine (19) who has shown that B. metiens spores may require 100 minutes a t 22 p.p.m. of monochloramine for a 99% kill. On the other hand, Ridenour, Ingols, and Armbruster (27) have shown that chlorine dioxide is capable of killing 99% B. subtilis spores in 30 minutes u ~ i t h 0.2 p.p.m. residual. Since chlorine dioxide does not attack the free amino or carboxylic radicals and the sulfhydryl radical is not exposed, it is considered possible that the phenol radical of tyrosine may be the vulnerable point of attack by the chlorine dioxide in spores. The data of both Levine (19)and Ridenour (27) indicate that hypochlorous acid is much faster than monochloramine, but chlorine dioxide is much faster than hypochlorous acid according to Ridenour ($7). Hypochlorous acid does not react with phenol radical of tyrosine but it does cause more rapid general deamination than monochloramine. These facts indi-
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cate that the organic phenol radical of tyrosine may be an important protein side chain. This hypothesis may be evaluated in further enzyme studies. Viruses are apparently nucleoproteins and not enzymes, but there are some points of similarity between enzymes and viruses, Knight, however (18), has shown that some viruses do not contain sulfhydryl radicals because they lack sulfur entirely; however, the viruses are not involved in energy-liberating reactions such as the vulnerable enzyme of Green and Stumpf (11). Although other viruses are known to have cysteine in their structure, Anson and Stanley (5) have reported that, where present, the sulfhydryl radicals of tobacco mosaic virus may be oxidized with iodine without destroying the infectivity of the virus. The literature is quite clear, however, in showing that chlorine does destroy viruses. Monochloramine, hypochlorous acid, and chlorine dioxide (26) were used successfully against poliomyelitis in studies of the Lansing strain, and the relative effectiveness was approximately the same as against a single strain of bacteria. Neefe, Baty, Reinhold, and Stokes (21) indicate that the virus Qf infectious hepatitis is destroyed by 1.1 p.p.m. of chlorine in 30 minutes. Recently, Parker and Elliker (25) reported that the phage of Streptococcus lactis is sensitive to 25 p.p.m. of chlorine at pH 9.5 with only 15 seconds’ contact. These data from the literature indicate that either cysteine and its sulfhydryl radical are more important in the poliomyelitis and infectious hepatitis viruses and S. lactis phage than they are in the tobacco mosaic virus or that the chlorine is effective through its action at other points in the molecule. Since the cysteine cont,ent of these viruses is unknown, i t is not possible to decide where the chlorine is most effective. Fair, Morris, and Chang (9) concluded that many of the variables of bacterial resistance to chlorine may be correlated with the resistance of the cell wall to diffusion. The emphasis in the studies reported here has been on the chemical aspects of chlorination, and no attempt has been made to evaluate the relative importance of the chemical and physical factors. Rahn (26) indicates that death of bacteria by chlorine can only be understood on the assumption that “several moIecules of the cell surface must be destroyed to produce an injury from which the cell cannot recover.” Fair et al. (10) indicated that an analysis of the extensive data of Butterfield et al. (7) with hypochlorous acid must be interpreted on the assumption that there must be three or four attacks on an organism before death results. This multiple attack concept agrees rather well with the idea that a number of molecules of hypochlorous acid or chlorine dioxide are required to oxidize a sulfhydryl group to a sulfonic acid group. Or, it can agree with the idea that the monochloramine must alter mor0 than the sulfhydryl group (to the disulfide state) to be lethal. These ideas would involve a change in concept of the bactericidal properties of chlorine as expressed by the senior author (15)and tend to agree with those of Allen ( I ) .
Summary and Conclusions This paper suggests that hypochlorous acid and chlorine dioxide are effective rn bactericidal agents in water treatment because either oxidant forms an irreversible product from its reaction
with the sulfhydryl radical. It further indicates that monochloramine fails to oxidize immediately the labile sulfhydryl radicals to an irreversible form. This may explain in part the need for much higher monochloramine residuals and longer contact periods for complete bacterial kill. The hypothesis is advanced that the failure of cysteine to restore the bacteria even though it might restore the sulfhydryl radical, is caused by changes in other enzyme systems which are brought about by the monochloramine and which may not be necessary for death caused by hypochlorous acid. Thus, the sulfhydryl group may be the most vulnerable group t o a strong oxidant, but changes in other groups may be important in bringing about death.
Acknowledgment These investigations were supported in part b y a research grant from the Kational Institutes of Health, U. S. Public Health Service.
Literature Cited (1j Allen, L. A., J.Inst. Water Engrs., 4 , 502 (1950). (2) Allen, L. A., Pasley, S. M.,and Peirce, M.A. F., J . Gen. Microb i d , 7, 36 (1952). (3) Anson, M. L., and Stanley, IT. hl., J. Gen. Physiol., 24, 679 (1951 ). (4) Bailey, J. H., and Cavallito, C. J., J . Bacteriol., 55, 175 (1948). (5) Black. A. P.. and Goodson. J. B. Jr.. J . Am. Water Works Assoc.. 44, 309 (i952). Bunau-Varilla, P., and Techoneyres, E., Compt. rend., 180, 1615 (1 925). Butterfield, C. T., Wattie, E., Megregians, S., and Chambers, C. W.,Public HealthRepts., 58, 1837 (1943). Douglas, I., and Johnson, T. B., J . Am. Chem. Soc., 60, 1486 (1938). Fair, G. M., Morris, J. C., and Chang, S. L., J . New Engl. Water Works Assoc., 61,286 (1947). Fair, G. M,, Morris, J. C., Chnng, S.L., Meil, I., and Burden, R. T., J . Am. Water Works Assoc., 40, 1051 (1948). Green, D. E., and Stumpf, P. K., Ibid., 38, 1301 (1946); Knox. K, E., Stumpf, P. K., Green, D. E., and .4uerbaoh, V. H., J . Bacteriol., 55, 451 (1948). Hoather. R. C.. J . Inst. Water Enars.. 3. 507 11949;. Holwerda, K., ‘Mededeel. Dienst i.’olksgezondheid Ned. IndiS, 17, Pt. 11, 251 (1928). FIoughton, G. U., J . Inat. Water Engrs., 4 , 4 3 4 (1950). Ingols, R. S,, Ibid., 4, 581 (1950). Ingols, R. S..and Ridenour, G. M.,J . Am, Water Works Assoc., 40,1207 (1948). Kethley, T. TI’., and Finoher, E. L., unpublished data. Knight, C. A., J . Bbl. Chem., 171,297 (1947). Levine, M., Bacteriol. Rets., 16, 117 (1952). Marks, H. C., Williams, D. B., and Glasgow, G. U., J . Am. Water W o ~ k Assoc., s 43,201 (1951). S e e f e , J. R., Baty, J. B., Reinhold, J. G., and Stokes, J., Jr., Am. J . Public Health, 37,365 (1947). Palin, -4.T., J . Inst. Wder Engrs., 3, 100 (1949). Parker, R. 13., and Elliker, P. R., J . Milk and Food Technol., 14, 52 (1951). Phillips, C.’R., Bacterid Revs., 16, 135 (1952). Rahn, O., Biodvnamica, 5 , l (1945). Ridenour, G. M., and Ingols, R. S., Am. J . Public Health, 36, 639 (1946). Ridenour, G . M . , Ingols, R. S., and Armbruster, E. H., Water & Sewage Works, 96,279 (1949). Weil, I.,and Morris, J. C . , J . Am. Chem. Soc., 71, 1664 (1949). RECEIVED for review October 16, 1952.
INDUSTRIAL AND ENGINEERING CHEMISTRY
ACCEPTED January 12, 1953.
Vol. 45, No. 5
