New Colloidal Silver Disinfectant Effect of Environmental Factors on Bactericidal Action CECIL W. CHAMBERS, LESLIE A. CHAMBERS, AND PAUL KABLER Environmental Health Center, 1014 Broadway, Cincinnati 2, O h b
T
HIS oligodynamic germicide was provided to the Public Health Service, under a lot number, by the Chemical Corps, Biological Laboratories of the United States Army. It is composed of approximately 30% metallic silver, the average particle size of which is about 75 A. units, and is stabilized by gelatin. The procedure for obtaining the extraordinary degree of dispersion of the silver is not known to the reporting laboratory. This investigation was undertaken to determine the effect of environmental variants such as pH, temperature, and composition of different waters on the efficiency of the disinfecting action of the material in question, TEST METHODS AND MATERIALS
The test or anism used throughout the study was a laboratory strain of %scherichia coli C-46. Test suspensions were prepared by introducing the growth from an agar slant incubated 18 to 24 hours at 37' C. into 99 ml. of buffered dilution water. The suspension was violently shaken and allowed to settle 5 minutes. Transfer of 0.25 ml. from the suspension to another 99 d. of dilution water resulted in a final bacterial concentration of about 3500 per ml. in the test. Duplicate counts for each determination were made by use of the nutrient agar our plate method. Identity of surviving organisms was estabgshed by transfer of the colony to lactose broth. Production of gas was considered to constitute identification of organisms as E. coli. Hydrogen ion concentrations were determined with a line operated pH meter. Germicide concentrations were in terms of gross aqueous stock material used, of the com osition previously indicated, without regard for the amount of sif)verpresent. PRELIMINARY INVESTIGATIONS
TOXIC RESIDUAL ON GLASSWABE.It became apparent early in the study that active bactericidal material remained on glassware that had been in contact with this substance. Attempts t o remove this residual with soap or detergent water wash, concentrated potassium dichromate cleaning solution, or by boiling in aqueous 5 70cysteine hydrochloride were unsuccessful. Cysteine hydrochloride seemed to inactivate the residual silver initially but glassware so treated, subsequently exhibited bactericidal action when cleaned in acid solution. Treatment of glassware a t 121' C. for 15 minutes submerged in saturated sodium chloride solution containing 1 gram of copper sulfate and 2 ml. of concentrated hydrochloric acid per liter effectively removed the bactericidal material. Effective removal from bottles was demonstrated by plating in parallel E. coli suspensions that had been stored in germicide contaminated bottles cleaned by the sodium chloride method, and from bottles that had never contained any of the active material. The bacterial counts of the suspensions showed no significant differences. Bacterial suspensions stored in bottles contaminated by material tested, and washed by several routine methods, showed marked reductions in survival. Fifty-one Petri plates in which bacterial suspensions containing as much as 200 p.p.m. of this formulation (cystine neutralized) had been enumerated were tested for residual activity simultaneously with 18 plates that had not been in contact with the disinfectant. The mean, maximum, and minimum counts for the two series were almost identical. This apparently anomalous result was due to the practice of introducing the disinfectant containing material into Petri dishes having a thin layer of solidified
agar in the bottom. The thin agar underlay effectively reduced "spreader" bacterial growths and also prevented the fixation of active material to the plates. NEUTRALIZERS.I n order to determine accurately the disinfecting action of a material, it is believed that an efficient neutralizer should be available to stop the reaction when desired. Lcystine had been suggested for use as a neutralizer of silver disinfectants. For best efficiency the cystine must be finely ground to facilitate pipeting. The desired concentrations were prepared with- 1 N sodium hydroxide before sterilization to a level which resulted in a pH of 6.0 to 6.5 after sterilization a t 121' C. for 15 minutes. The neutralizing efficiency of cystine was determined by adding 1 ml. of various germicide concentrations to 9 ml. of cystine suspension. The resultant mixture was violently shaken and the proper amount of bacterial suspension immediately added and mixed. After various exposure times, from 10 minutes to 4 hours, portions were removed and the bacterial populations determined by agar plate counts. Table I shows that under the conditions of test, 1% cystine effectively neutralized up to 20 p.p.m. and 10% cystine neutralized u p to 500 p.p.m. of the germicide tested.
TABLE I. NEUTRALIZING EFFICIENCY OF I~CYSTINE Germioide, Teat 50 6'3
.
Control*
7b
>Cystine, %
10.0 10.0 10.0 1.0
Control8 1.0 0 Single test. b Average of 10 tests.
P.P.M. 600 100 0
20 0
Baoteris Surviving, % Hours 1/a 1 2 4 96.8 90.6 91.7 109.1 86.1 91.1 89.1 96.0 97.4 91.8 87.2 100.8 91.9 *. 99.0 96.1 .. 84.2
..
It was technically not feasible to use a suspension of cystine containing more than lo%, because it settled out in the pipets and because it produced an opaque agar plate. In similar tests it was determined that the following were unsatisfactory for the reasons indicated: 1. Cysteine hydrochloride solutions were sufficiently acid to be toxic to the test bacteria. Adjustment of the p H to a compatible level resulted in precipitation of cystine. 2. Two per cent trypticase plus 0.6% yeast extract showed no evidence of even partial neutralization when tested with 100 p.p.m. of germicide. 3. Results with sodium sulfite indicated probable neutralizing capacity, but it was discarded because of extreme susceptibility to oxidation.
TESTWATERS. The survival of bacteria is dependent upon the composition and character of the suspending medium. Thus to estimate accurately the bactericidal action of the material in question i t was necessary to determine the survival rate of the test organisms in the dilution waters with no bactericide present. The initial number of test organisms, when stored for varying periods of time, changed less in 1.5-strength Formula C water ( I ) than in other test waters studied. No significant ahanges in population density were observed during 24-hour storage a t 2" to 5" C. in 1.5 strength Formula C, 2569
INDUSTRIAL A N D E N G I N E E R I N G C H E M I S T R Y
25'20
Norwood or Cincinnati tap waters. At 22' to 25" C. the survival rate was a t least 84% in Norwood and Formula C water after 6 hours' storage, and approximately 50% survived after 24 hours. I n Cincinnati tap water a t 22" to 25' C. the survivals were 38'% at 6 hours, 23% at 12 hours, and 9% at 24 hours, Cincinnati and Norwood tap waters were dechlorinated with sodium sulfite but weie not sterilized before use. Control tests showed that neither water contained coliform bacteria initially.
Vol. 45, No. 11
At reduced temperature (2" to 5" C.) the bactericidal act,ion was adversely affected. As shown in Table 111, at 2' t o 5 " C. with other conditions unchanged, more than 100 times the amount of germicide was required to kill bacteria than was required at 22" to 25" C. When the unfavorable conditions of low temperature (2" to 5" C.) and low pH (6.0 t o 6.5) were compounded, high concentrations and long exposure times were required to produce satisfactory disinfection.
EXPERIMENTAL PROCEDURE
Essentially the same procedure was used throughout the study except for adjustments in time intervals of exposure and concentrations of germicide tested. A series of 175-ml. French square bottles (borosilicate glass) having Escher-type stoppers and containing 99 ml. of test water were numbered consecutively. Beginning with bottle No. 2 increasing amounts of the substance tested were placed in succeeding bottles. One ml. of test organism suspension was added to each bottle. Bottle No, 1 contained only test water and test organisms and was therefore a survival control of the water. After exposure intervals of from 0.5 to 24 hours, portions were transferred from the control bottle and each test bottle to cystine neutralizer, from which agar plate count determinations were made. Percentage survivals were computed from the number of organisms present in a test bottle as related to the number remaining in the control a t the corresponding exposure interval. A control test of cystine neutralizing efficiency was also included. RESULTS
This study included 88 sets of tests in each of which the survival of E . coli was determined in multiple concentrations of disinfectant with various combinations of pH, temperature, and test waters. Each of the variants affected the bactericidal action. Because the available data are too voluminous to present in their entirety, representative segments are presented in Tables I1 to V, inclusive. I n adjusting the p H of natural waters it was desired to preserve the natural state except for the addition of hydroxyl ions, This made it necessary to avoid the addition of otherwise desirable buffer salts. I n the synthetic water the buffer concentration was governed by using the amount of buffer resulting in the least variation in bacterial numbers in the control, throughout the test period, rather than using an amount that had high buffering capacity. Therefore, in experiments covering a 24-hour period, the buffer capacity of the test waters was insufficient to prevent some shift in p H during the course of an experiment. Accordingly, p H conditions were reported in ranges rather than at fixed levels. This method of reporting could be used because pH changes of 0.5 of one unit did not result in discernible variations in bactericida1 efficiency. Under the normal summer temperatures (22' to 25' C.) of most of the United States and in alkaline water (pH 8.5), this material in concentrations of as little as 0.1 p.p.m., effectively disinfected water in 6 to 8 hours. The exposure time could be reduced to 2 hours when 0.4 p.p.m. was used. In waters having a p H of 6.0 to 6.5 (Table 11), 0.4 p.p.m. killed the test organism in 24 hours, and approximately 99.9% were killed in 24 hours by 0.1 p.p.m.
TABLE 11. EFFECT OF DH ox SURVIVAL OF E.
COLI
(Germicide a t 2Z0 to 25" C.; 1.5 X formula C water) Bactericide, P.P.M. 0.10 0.40 0.10 0.40
PH 6.0-6.5 6.0-6.5 8.5-9.0 8.5-9.0
47.2 55.3 30.0 24.4
1 44.1 39.7 23.6 12.6
TABLE 111. EFFECTO F TEMPERATURE Bactericide, P.P.M. 0.10 10.0
I
The bactericidal action of this preparation did not appear to conform to the traditional curve exhibited by most chemical disinfectants, in that increased concentrations did not necessarily produce correspondingly increased kills. This nonconformity was more apparent when tests were conducted under one or more adverse environmental conditions. Table IV shows that at 2" to 5' C. and a pH of 8.5 to 9.0 all concentrations of this disinfectant from 3 to 10 p.p.m. killed about the same percentage (80%) of the test organisms in 2 hours and more than 95% in 12 hours, but a few remained after 24 hours' exposure. Similar but less prominent tendencies were observed under nearly all conditions of tests. At 22" to 25" C. and pH 8.5 to 9.0 this action was not marked.
TABLEIv. EFFECTO F GERhfICIDE COSCENTRATION SURVIVAL O B E. COLI
O N THE
temperature 2' to 5' C.; 1.5 X formula C water) Bacteria Surviving, 70 Bactericide, Hours P.P.M. 2 G 12 24 3.0 17.8 6.2 3.6 0.02 4.0 19.4 9.7 4.7 0.30 2.9 0 17.8 8.1 5.9 7.0 17.2 7.7 3.1 0.03 10.0 19.0 2.7 0.04 4.9
(pH 8.5-9.0;
TABLE V. EFFECTOF WATERCOMPOSITION ON OF E. COLI Bactericide, P.P.M. 0.10 0.10
0.10
THE
SURVIVAL
(Germicide p H 8.5 to 9.0; temperature 2' t o 5' C.) Bacteria Surviving, % Hours Test Water 1/z 1 2 4 6 8 12 0 1 . 5 X F o r m u l a C 30.0 23.8 1 0 . 8 2 . 7 0 . 0 7 0 CincinnatiTap 43.5 35.5 20.3 5 . 0 2.0 0 0 43.9 25.3 31.5 3 3 . 1 22.5 RorwoodTap 17.3 9 . 3 ~
The results of this study indicate that, in general, the bactericidal action of this germicide is slightly better in 1.5 strength Formula C water than in Cincinnati tap water and somewhat less in Norwood tap water than in the other two test waters. Table V shows that a t pH 8.5 to 9.0 and 22" to 25" C. only slight percentage differences occur in the surviving organisms in Formula C water and Cincinnati tap water. Complete kills were obtained with 0.1 p.p.m. of this formulation in both in 8 hours. I n Norwood tap water under the same conditions approximately 10 % E. coli survived after 12 hours exposure. The differences in surviva1 rates for the various waters appeared to be accentuated at low temperature.
Bacteria Surviving, 3' % Hours 4 6 8 12 20.9 30.2 13.6 10.9 27.8 17.6 13.3 0.85 2.7 0.07 0 0 0 0 0 0
2 35.9 33.3 10.8 0.48
O N SURVIVAL O F E. C O L I (Germicide a t pH 8.5 to 9.0; 1.5 X formula C water) Bacteria Surviving, 70 Temperature, Hour c. 1 2 4 '6 8 12 24 0 0 22-25 30.0 23.6 10.8 2.7 0 . 0 7 0 2-5 57.0 34.4 1 9 . 0 9 . 5 4 . 9 5.5 2.7 0.04
24 0.12 0 0 0
SUMMARY
The effects of some environmental variants on the bactericidal action of a new silver-bearing germicide have been studied, The results observed indicate:
November 1953
INDUSTRIAL AND ENGINEERING CHEMISTRY
1. The bactericidal action of this material was neutralized by appropriate amounts of >cystine. 2. A measurable active residual remained on glassware, but the residue was effectively removed by a simple and inexpensive method. 3. Under favorable conditions (pH 8.5 to 9.0 and 22" to 25' C . ) this preparation was an effective bactericide in concentrations as low as 0.1 p.p.m. At pH 6.0 to 6.5 distinctly higher concentrations of the formulation were required. 4. At reduced temperatures, 2' to 5" C., the disinfecting action was considerably reduced. 5. The relationship of the disinfectant concentration to bactericidal action appeared to deviate from the accepted pattern
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of phenolic and halogen disinfectants to a greater degree than could be explained by experimental errors of the technique used. 6. The bactericidal efficiency of this bactericide was less in certain test waters than in others. LITERATURE CITED
(1) American Public Health Association, "Standard Methods for Examination of Water and Sewage," 9th ed., p. 140, par. 2.1, New York, 1946. RECEIVBD for review September 6, 1953. ACCEPTBD June 24, 1953. Presented before the Division of Water, Sewage, and Sanitation Chemistry at the 122nd Meeting, AMERICAN CHEMICAL SOCIETY, Atlantic City, N. J., 1952.
Bactericidal Action of Movidyn ROBERT K. HOFFMAN, BERNARD F. SURKIEWICZ, L. A. CHAMBERS, AND CHARLES R. PHILLIPS Chemical Corps Biological Laboratories, Camp Detrick, Frederick M d .
I"
4 CEMBER 1949, samples of Movidyn, a colloidal silver preparation, became available in experimental lots for bactericidal evaluation in this laboratory. This product consisted of finely dispersed colloidal silver, stabilized by the addition of a protective agent such as gelatin t o prevent recoagulation. It has been prepared commercially in Czechoslovakia and is now commercially available in this country. The tests reported here were made on laboratory sample Lot No. 227-B prepared in this laboratory by the Czechoslovakian developer of this product, Zdenek Moudry. The laboratory lot contained in suspension about 5% by weight of silver (expressed as silver nitrate). The commercial product (marketed by United States Movidyn Corp., 863 North Orleans St., Chicago 10, Ill.) may differ somewhat from this laboratory preparation since it is t o be made in larger volume lots, and the exact nature of the colloidal particles may not be precisely the same.
MATERIALS AND METHODS
The test organisms employed for the work reported here were Escherichia coli strain B, Serratia marcescens, Salmonella typhosa, Micrococcus pyogenes vw. aureus 209 and Bacillus globigii spores. With the exception of B. globigii spores, these organisms were carried on nutrient agar slants. For each experiment, transfers were made to nutrient broth, and after an 18- to 24-hour culture Present address, Environmental Health Center, U. 8. Public Health Service, Cincinnati, Ohio. f
period a t 37' C., they were centrifuged, washed twice with distilled water, and resuspended in distilled water. The B. globigii spores used as the test organisms had been harvested, washed, and suspended in distilled water 3 months before use. The spore suspension had been heat shocked for 30 minutes a t 60" C. to destroy any vegetative cells before using. I n all cases, suspensions of the test organisms were prepared with 10,000 to 40,000 organisms per ml. All dilutions of Movidyn referred t o in this paper were the actual dilutions made from the liquid sample 227-B. Dilutions are reported rather than concentrations, as is customary with bacteriological data. This sample of Movidyn contained about 5% by weight of silver (expressed as silver nitrate) so the actual dilution of silver (as silver nitrate) in the samples was twenty times greater than the Movidyn dilutions reported. A diagrammatic scheme of the dilution procedure is shown below. Only three dilutions were made in order to obtain any one of the test concentrations of Movidyn. It was necessary t o limit the number of dilutions made to reach the desired concentrations since there was a loss of silver in each dilution prepared, because of deposition on the walls of the container. Autoclaved tap water with the pH adjusted toabout7.0wasusedasthemenstruum for suspending the test organism. This was found more suitable than distilled water since several of the test organisms survive longer in the presence of traces of inorganic salts in water than in distilled water. Time dilution studies were undertaken with the five organisms
DILUTION PROCEDURE FOR J ~ O V I D Y TESTING N
I
5 ml. 990 ml. HzO
l/lOOO
1/10,000
1 ml.
BACTERIAL SUSPENSION
1/1,000,000
1/100,000
995 ml.
999 ml.
Hz0
Hz0
1/200,000
1/1,000,000
1 ml. 99 ml. BACTERIAL
99 ml. BACTERIAL SUSPENSION
1/10,000,000
1/20,000,000
BACTERIAL SUSPENSION 1/100,000,000
