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
2571
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"
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. 4
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
DILUTIONPROCEDURE 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
INDUSTRIAL AND ENGINEERING CHEMISTRY
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Vol. 45, No. 11
conducted betaeen 4 ' and 39" C., averaged 1.77 for each 10' 0. over this range. I n keeping with the usual procedure in this laboratory, the reciprocal of the k values are recorded rather than the death rates themselves, since the reciprocals are numerically equivalent to the time required for 90% of the organisms to be killed, and this value has a more readily comprehended meaning than the usual death rate constant or k value. All death rate values given in this paper were calculated from the secondary death rates. Hours 4'C. l / k = 7 . 6 27" C. l / k = 3 . 4 39" C. l / k = 1 . 3
IOC
IC
25 K
3
v)
EFFECT OF CONCENTRATION
S I w
The effect of various concentrations of hlovidyn against E'. coli is shown in Figure 3. Again, it will be noted that, where measurable, two death rates were present for each dilution. As would be expected, the higher the concentration of Movidyn, the greater the death rate as exemplified by the steeper the line. I l k values I\-ere: M ovidyn Hours 1/100,000,000 = 13.4 1/20,000,000 = 3 . 4 1/10,000,000 = 2 . 1 1/1,000,000 = 0.7
0
6 a 0.I
COMPARATIVE RESULTS WITH SILVER NITRATE
0.01 TIME (HOURS) Figure 1. Bactericidal Action of Movidyn (1/20,000,000) against Various Test Organisms at 27"
c.
B. Globigii spores 1/1000
listed in order to determine the death rates versus dilutions of Movidyn for a variety of test organisms. The suspensions of Movidyn and test organisms were prepared according to the diagrammatic scheme and allowed to stand a t room temperature (27" C.). At various time intervals, aliquots were withdrawn for viable plate counts. One ml. portions of these suspensions, undiluted and a t 1/100 dilution were plated in duplicate in enriched nutrient agar. Plates were incubated a t 37' C. for 48 hours before counting. The points on the graphs represent average per cent kills, for the specified time, of three t o eight experiments. Although some of the curves appear to be determined by only two or three points, other points were obtained, but they showed a survival of less than O.OlojC, the lowest percentage indicated on the graphs.
A series of tests was conducted for the purpose of comparing the activity of Movidyn with that of silver nitrate and the effect of any residual silver deposited on the container walls as a result of having Movidyn or silver nitrate in the container. Figure 4 shows the time survival curves for E. coZi exposed to concentrations of 0.1 p.p.m. Rlovidyn (approx. 0.005 p.p.m. silver nitrate) and 0.005 p.p.m. silver nitrate. The death rates indicate that i t required about seven and one-half times
3.C
I/k ' 2 6 HOURS
EFFECT O F MOVIDYN ON VARIOUS ORGANISMS
Figure 1 shows the time-survival curves for the five test organisms when exposed to a 1/20,000,000 dilution of Movidyn, except in the case of B. globigii spores where a 1/1000 dilution was used. As is typical of many disinfectants, two death rates were noted with some of the organisms, an initial rapid death rate and a secondary slower death rate. When a 1/20,000,000 dilution of Movidyn was used, the time for 99.9970 kill of the test organisms was 1.5 hours for S. marcescens; 3.5 hours for S. typhosa; 11.5 hours for M . pyogenes var. aureus; and 12 hours for E. coli. When B. globigii spores and a 1/1000 dilution of Movidyn were used, almost 24 hours were required.
a
39'C
Y
= 3.4
EFFECT O F TEMPERATURE ON DEATH RATES
Figure 2 shows the effect of temperature on the death rate of E. coli exposed to a 1/20,000,000 dilution of Movidyn. Death rate constants (IC values) were calculated for each curve This with the temperature coefficient per 10" C. rise (&). temperature quotient, calculated on the basis of experiments
TIME (HOURS)
Figure 2. Bactericidal Action of Movidyn (1/20,000,000) against E . Coli at Different Temperatures
INDUSTRIAL AND ENGINEERING CHEMISTRY
November 1953
2513
+
TABLE I. SODIUMTHIOGLYCOLLATE AS NEUTRALIZING AGENT FOR MOVIDYN Time, Hr. 1/2
1 2 5 24
*
(l/20,000,000 against E . Coli at 27' C.) Percentage Survival Agar Agar 0.1% sodium thioglycollate 23.2 19.5 11.5 12.1 5.7 6.5 3.1 5.8 0.0 0.0
+
longer to kill E. coli with pure silver nitrate, compared with the same concentration of silver in the form of Movidyn. Likewise, the death rate of E. coli was much higher as a result of the residual Movidyn, compared to silver nitrate. I n fact, the residual Movidyn appeared more effective as a bactericidal agent than silver nitrate dissolved in the water. It was evident that the glassware in which Movidyn was placed had adsorbed some of the silver that remained active even though this glassware had been chemically cleaned with potassium dichromate-sulfuric acid cleaning solution. However, this residual activity was removed from the walls of the glassware by rinsing with sodium thiosulfate. CHEMICAL'NEUTRALIZATION
The percentage survivals for E. coli exposed for various lengths of time to a 1/20,000,000dilution of Movidyn plated in agar with
and without sodium thioglycollate are shown on Table I. There was no significant difference in percentage survivals regardless of the media used. Thus, under the conditions employed, 0.1% sodium thioglycollate did not reverse the action of Movidyn on the test organism nor was the action reversed when agar containing 0.1% sodium thiosulfate, 0.1% sodium chloride, or 1% skim milk was employed as the plating media. Inhibition of growth in agar due to sufficient quantities of Movidyn being carried over in the plating proce100 dure was observed only when the dilution of Movidyn in the agar was less than 1/10,000. In
!5d 3
P
2
4
0 = 1/10,000,000 Movidyn
(1 200 000 OOO silver nitrate) l/k ='2.1'hours .O= 1/200,000,000 silver nitrate l / k = 15.8 hours
X
-
Residual Movidyn l / k = 12.4 hours
+ = Residual silver nitrate l / k = 60 hours
DISCUSSION
a
f;
6 8 10 12 TIME (HOURS) Figure 4. Comparative and Residual Bactericidal Action of Movidyn and Silver Nitrate against E. Coli a t 27" c.
0
such cases, evidence that sodium thioglycollate or sodium thiosulfate in the agar neutralized the inhibitory effect of the Movidyn was obtained. A dilution of 1/1000 (1000 p.p.m.) of Movidyn in agar prevented the growth of B. globigii. However, the addition of 1000 to 1500 p.p.m. of sodium thiosulfate to agar containing 1000 p.p.m. of Movidyn neutralized the effect of the Movidyn and permitted growth of the organism,
10
v)
0.01
'*
W 0
0 .I
0.01 0
6 8 IO 12 TIME (HOURS) Figure 3. Bactericidal Action of Movidyn of Different Dilutions against E. Coli at 27' C . 0 = 1/100 OOO 000 x = l/lOOOOOOO 0 = 1/20,d00,dOo 0 l/l,doo,bll
2
4
-
The product thus appeared to have many interesting properties, a few of which may be described as follows: It was effective a t extremely high dilutions, and this effect was considerably greater than that of the same concentration of silver in ionic form. At these high dilutions the rate of kill was slow but steady. A most interesting property was that of its residual effect. As a solid it does not disappear from water as a gaseous constituent would do, so its residual effect does not rapidly disappear. Moreover, what losses do occur in suspension appear to result from its adsorption on the walls of the container imparting a bactericidal residual that persists when fresh untreated water is added, to the container, even though the container is well washed and rinsed between tests. Finally, there was evidence that its effect was bactericidal, rather than the bacteriostatic effect often evidenced by certain heavy metal disinfectants, as shown by the inability of sulfhydryl compounds, thiosulfate, chloride, or organic matter to neutralize the product and allow treated organisms to multiply. RECIOIVED for review January 10, 1953. ACCEPTBID June 6, 1953. Publication of this paper does not constitute endorsement of this product by the United States Army. Presented before the Division of Water, Sewage, CHEMICAL and Sanitation Chemistry a t the 122nd Meeting, AMERICAN SOCIETY, Atlantic City, N. J., 1952.
