APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 1978, p. 1116-1120 0099-2240/78/0035-11 16$2.00/0 Copyright © 1978 American Society for Microbiology
Vol. 35, No. 6 Printed in U.S.A.
Reversal of the Silver Inhibition of Microorganisms by Agar RICHARD C. TILTON'* AND BERNARD ROSENBERG2 University of Connecticut Health Center, Farmington, Connecticut 06032,' and Electrolux Corporation, Old
Greenwich, Connecticut 068702 Received for publication 25 October 1977
Increasing use of silver in the treatment of water has necessitated an examination of microbiological methods for the measurement of silver inactivation of microorganisms. Three common agar media were tested for their ability to neutralize the bacteriostatic effects of silver. Results suggested that growth media differed in their neutralizing capacity; that is, the non-inhibitory media tryptone glucose agar and Trypticase soy agar showed more neutralizing capacity than eosin methylene blue agar. Furthermore, the neutralizing effect appeared to be a function of the soluble component of the media and not of the agar itself. In the latter part of the 19th century, Carl von Nageli observed the disappearance of algae exposed to very small amounts of copper or silver. Subsequently, he proposed that silver was germicidal in very small amounts (6). There are several possible mechanisms of silver inhibition of bacteria that have been proposed, namely, interference with electron transport, binding of silver to deoxyribonucleic acid, and cell membrane interaction. Despite the lack of information on the mechanism of silver inhibition, silver has been used for years as a disinfectant. Numerous processes and products using silver in water treatment have been proposed. Most recently, filters containing a carbon-silver bed have shown promise as water purifiers. Development of such filtration technology justified attempts to determine the bacteriological efficacy of such products. It has been assumed that accurate testing of a disinfectant includes the use of a substance to neutralize the effects of the disinfectant before plating on agar. Chambers et al. (1) proposed the use of a thioglycolate-thiosulfate mixture to neutralize the effects of Ag+ during microbiological analysis of Ag+-treated water. The use of such a neutralizer has been widely accepted by regulatory agencies. Evidence now exists in a number of laboratories that other factors are involved in the neutralization of Ag+ subsequent to bacterial exposure. They include: presence of amino acids (2), hardness of water, phosphate and chloride content, temperature of incubation (7), type of buffer, light, bacterial density, and salt content of the growth medium (1). The intent of the present study was to determine the neutralizing effect of agar growth media on silver disinfection of bacteria.
MATERIALS AND METHODS Reagents, chemicals, and media. Thioglycolatethiosulfate neutralizer stock solution was prepared by adding 1.0 g of sodium thioglycolate and 1.46 g of sodium thiosulfate to 1,000 ml of distilled deionized water (1). This reagent was prepared daily and diluted as necessary.
A silver nitrate stock solution (5.0 mg of Ag+ per liter) was prepared in distilled deionized water and stored in the dark in a glass flask before use. The silver solution was routinely analyzed with a Perkin-Elmer 403 atomic absorption spectrophotometer. Bacteriological agars (eosin methylene blue [EMB] Trypticase soy agar [TSA], and tryptone glucose extract agar [TGE]) were purchased from Baltimore Biological Laboratory, Cockeysville, Md., and prepared weekly according to the manufacturer's instructions. Cultures. Escherichia coli ATCC 25822 was maintained on TSA slants. Before use, it was suspended in Trypticase soy broth, and incubated for 4 to 6 h at 35°C, and the number of organisms was standardized to 3 x 107 colony-forming units (CFU) per ml with an Autobac (Pfizer Diagnostics, Groton, Conn.) nephelometer (5). Experimental procedure. The standardized suspension of E. coli was further diluted in distilled deionized water to 3 x 103 CFU/ml. A 5-ml portion of this suspension was added to 45 ml of distilled deionized water containing 50 ,ug of Ag+ per liter (sample A) and similarly to 45 ml of distilled deionized water without Ag+ (sample B). The final working concentration of E. coli was 3 x 102 CFU/ml (Fig. 1). From each of these two suspensions of E. coli, five 5-ml test samples were prepared as follows: (i) test sample with no additions; (ii) test sample plus an equal volume of undiluted thioneutralizer stock solution; (iii) test sample plus an equal volume of a 1:100 dilution of thioneutralizer stock solution; (iv) test sample plus an equal volume of a 1:1,000 dilution of thioneutralizer stock solution; (v) test sample plus an equal volume of thioneutralizer stock solution (plating of the treated E. coli inoculum on agar plates was delayed an addi-
1116
REVERSAL OF SILVER INHIBITION BY AGAR
VOL. 35, 1978
Sample B 103
Sample A
103E. coli |+
1117
50ug of Ag+
|E. coli| B
Samples i, ii, iii, and iv plated on Ag+-pretreated TSA, TGE, and EMB. Sample v plating delayed 5 min and similarly processed.
B
(i) Untreated test sample (TA) (ii) TA + 5 ml of undiluted thioneutralizer (TN) (iii) TA + 5 ml of 1:100 TN (iv) TA + 5 ml of 1:1,000 TN (v) TA + 5 ml of undiluted TN (plating delayed 5 min)
Al
B
Samples i, ii, iii, and iv plated (TSA, TGE, and EMB) at 0, 10, 20, and 30 min. Sample v plated, after 5-min hold, at recalculated intervals of 0, 10, 20, and 30 min.
FIG. 1. Laboratory procedure for the silver neutralization experiment.
tional 5 min). Each of the five test samples of the two suspensions of E. coli (A, containing 50 jig of Ag+ per liter; B, containing no Ag+) were further treated as follows. (i) A 1-ml volume of each of the five samples of Ag+treated and untreated E. coli suspension was surface plated in triplicate on TSA, TGE, and EMB at intervals of 0, 10, 20, and 30 min of exposure. (ii) E. coli suspension B (no Ag+) was also divided into five samples and plated at 0, 10, 20, and 30 min in triplicate on TSA, TGE, and EMB. These plates were surface treated with 0.1 ml of a solution of 50 ug of Ag+ per liter and dried for 30 min before the plating of the five test samples. All plates were incubated in air at 35°C. Agar plates were counted daily for 2 days. Results were plotted as numbers of microbial survivors of a particular treatment protocol. To demonstrate that the substances in the agar growth medium, rather than the agar itself, were responsible for the neutralization of the silver toxicity, TGE plates and TGE broth tubes were prepared containing serial twofold concentrations of Ag+ ranging from 50 to 1,600 ug/liter. Each dilution of both Ag+treated agar and broth was inoculated with 103 CFU of E. coli per ml and incubated at 35°C overnight. Results were recorded daily for 2 days.
was added, the initial colony counts were approximately 30% higher than in the non-thioneutralized sample. After a 10-min exposure to silver, half of the original inoculum survived after thioneutralization, as compared with no survivors in the non-thioneutralized cultures. After a 20-min exposure to Ag+, no survivors were detected, whether or not the thioneutralizer was used. In this experiment, where such rapid microbial kill was observed, no differences could be seen in the ability of the three agars (TGE, TSA, EMB) to neutralize the effects of silver. Figure 3 presents data on control cultures of E. coli which were not exposed to Ag+. Once again, there was no difference in the rate of survival between thioglycolate-thiosulfate-neutralized cultures and those not so treated. However, there appeared to be a marked difference in the ability of the three agars to support the growth of non-Ag+-treated survivors. Figure 4 supports the contention that the agar growth medium rapidly neutralizes the bacteriostatic effects of Ag+. Non-Ag+-treated E. coli showed no marked reduction in numbers when plated on three agars which had been previously RESULTS spread with a solution containing 50 jig of Ag+ Figure 2 shows the survival of E. coli in the per liter. It is apparent, however, that TGE and presence of 50 ,ug of Ag+ per liter as a function of TSA neutralized the activity of Ag+ more effectime. Silver rapidly inactivated E. coli, resulting tively than did EMB. Data not presented graphically indicate that, in 50% kill in the initial 10-min exposure. Thioglycolate-thiosulfate reagent was added to sam- if the thioneutralizer was diluted 1:100 or 1:1,000 ples of E. coli to neutralize residual silver before and added before plating, survival of E. coli was plating on one of three agar media. The thioneu- similar to those instances in which no thioneutralizer was equivalent to the agar media in its tralizer was added. If thioneutralized samples of ability to promote the growth of survivors. How- culture fluid were held 5 min before plating, no ever, in those samples to which thioneutralizer salutary effect on the survival of E. coli was
6.0
2 Q
-4.0 x
\\ 0 NO
3
ADDED
THIONEUTRALIZER
A THIONEUTRALIZER ADDED EMS -.- TGE -
!
0
TSA
0 2.0
1.0,
TIME
20 10 OF INCUBATION PRIOR TO PLATING (ml..)
30
FIG. 2. Effects ofplating on the survival of E. coli incubated in the presence of 50 jig of Ag+ per liter. to
-
5.0
0 x
o
_
otO
0 o
0 20
a\ z\ o NO THIONEUTRALIZER ADDED 1.0
A
A THIONEUTRALIZER ADDED
EEMS --TE
10
TIME OF INCUBATION PRIOR
-- TSA
20 TO PLATING (min.)
30
FIG. 3. Effects ofplating on control cultures of E. coli (no Ag' added). 1118
REVERSAL OF SILVER INHIBITION BY AGAR
VOL. 35, 1978 6.0
5.0
1119
+
t
0
x 4.0
'
E
0
-0
I 0
----
so 2a.0
A
0-
-0
£~~~~~~~ 0 z
O
NO THIONEUTRALIZER ADDED
A
THIONEUTRALIZER ADDED
1.0
EMS
TIME
-.-
TOE .-
TSA
10 20 OF INCUBATION PRIOR TO PLATING (min.)
30
FIG. 4. Effects of Ag+-pretreated agar plates on the survival of E. coli. observed.
Table 1 supports the contention that the nutrient substances in the agar medium, and not the agar itself, neutralize the toxic effects of Ag+. The media, both broth and agar, absorbed Ag+ to an approximate concentration of 400 ,ug/ml, at which point free Ag+ was available to inhibit the challenge dose of E. coli.
DISCUSSION Unpublished observations on the ability of various agar media to neutralize the inhibitory effect of silver on bacteria have concerned those investigators who attempt to measure the kinetics of silver inactivation. At present, the standard plate count is the most practical method for the determination of viable count. Although it is essential to neutralize the effects of unbound silver before plating, such a procedure may strip silver ions from the bacterial cell envelope with subsequent "rescue" of these microorganisms. This rescue effect appears to be a function, in part, of the bacteriological plating medium. The data in Table 1 indicate that the agar itself does not neutralize silver. Greun (2) has reported that lysine, arginine, methionine, and cystine react
TABLE 1. Effects of TGE agar versus TGE broth on the survival of E. coli in serial twofold concentrations of Ag+ Ag+
Growtha
(Jg/ml)
TGE agar TGE broth
0 (control) 50
+ +
+ +
+ + 100 + + 200 + + 400 800 1,600 Growth equal to or equivalent to control; -, no
a +, growth.
with silver. There are amino acid-containing organic substances, such as peptones, which act as chelating agents of metal ions in media (3). The data in this study demonstrate that the agar constituent of the medium does not neutralize silver and that the soluble components of media effectively neutralize 400 to 800 ug of Ag+ per ml. The data also confirm the time-dependent nature of silver inactivation of bacteria. At the
1120 TILTON AND ROSENBERG end of 20 min of exposure to 50 ,ug of Ag+ per liter, there was virtually 100% kill with or without thioneutralizer and independent of the plating medium. Only during the initial stages of Ag+ exposure (0 to 20 min) could differences be observed. The presence of thioneutralizer promoted survival of E. coli at least during the initial 10 min of exposure. The slopes of the death curves for non-thioneutralized cultures were consistently steeper than the slopes of those treated with thioneutralizer. Virtually no survivors were observed after a 10-min exposure to Ag+. The data in Fig. 4 demonstrate the neutralizing capacity of agar media. As compared with the rapid death rate of the Ag+-exposed organisms seen in Fig. 2, E. coli survived plating on agar which had been previously spread with an AgNO3 solution. In this experiment, thioneutralizer added to the inoculum showed little effect. Those organisms plated on EMB revealed the lowest initial plate counts and the most rapid die-off. Such an observation was expected because, although EMB is a selective medium designed for the isolation of coliform bacteria, it contains methylene blue, which may be inhibitory for bacteria whose cell envelopes have been damaged by silver (4). Rescue is increased in the richer media such as TSA and TGE agar. As a result of this study, it appears that the
APPL. ENVIRON. MICROBIOL.
neutralization of Ag+ activity is a function both of the chelating substances in the agar medium and of the thioneutralizer added to the inoculum before plating. Claims of silver resistance in microorganisms should now be examined in light of the effects of nutrient media on the ability of silver to inactivate bacteria. ACKNOWLEDGMENTS This study was supported by a grant from the Electrolux Division of Consolidated Foods Corp., Chicago, Ill.
LITERATURE CITED 1. Chambers, C. W., C. M. Procter and P. W. Kabler. 1962. Bactericidal effect of low concentrations of silver. J. Am. Water Works Assoc. 54:208-216. 2. Greun, L. C. 1975. Interaction of amino acids with silver ions. Biochim. Biophys. Acta 386:270-274. 3. Lennette, E. H., E. H. Spaulding, and J. P. Truant (ed.). 1974. Manual of clinical microbiology, 2nd ed. American Society for Microbiology, Washington, D.C. 4. Rosencranz, H. S., and H. S. Carr. 1972. Silver sulfadiazine: effect on the growth and metabolism of bacteria. Antimicrob. Agents Chemother. 2:367-372. 5. Tilton, R. C., L. Lieberman and E. H. Gerlach. 1973. Microdilution antibiotic susceptibility test: examination of certain variables. Appl. Microbiol. 26:658-665. 6. von Nageli, C. 1893. Leben die oligodynamischen Erscheinungen an lebenden Zellen. Denkschr. Schweiz. Naturforsch. Ges. 33:174. 7. Woodward, R. L. 1963. Review of the effectiveness of silver. J. Am. Water Works Assoc. 55:881-886.
Vol. 35, No. 6 Printed in U.S.A.
Reversal of the Silver Inhibition of Microorganisms by Agar RICHARD C. TILTON'* AND BERNARD ROSENBERG2 University of Connecticut Health Center, Farmington, Connecticut 06032,' and Electrolux Corporation, Old
Greenwich, Connecticut 068702 Received for publication 25 October 1977
Increasing use of silver in the treatment of water has necessitated an examination of microbiological methods for the measurement of silver inactivation of microorganisms. Three common agar media were tested for their ability to neutralize the bacteriostatic effects of silver. Results suggested that growth media differed in their neutralizing capacity; that is, the non-inhibitory media tryptone glucose agar and Trypticase soy agar showed more neutralizing capacity than eosin methylene blue agar. Furthermore, the neutralizing effect appeared to be a function of the soluble component of the media and not of the agar itself. In the latter part of the 19th century, Carl von Nageli observed the disappearance of algae exposed to very small amounts of copper or silver. Subsequently, he proposed that silver was germicidal in very small amounts (6). There are several possible mechanisms of silver inhibition of bacteria that have been proposed, namely, interference with electron transport, binding of silver to deoxyribonucleic acid, and cell membrane interaction. Despite the lack of information on the mechanism of silver inhibition, silver has been used for years as a disinfectant. Numerous processes and products using silver in water treatment have been proposed. Most recently, filters containing a carbon-silver bed have shown promise as water purifiers. Development of such filtration technology justified attempts to determine the bacteriological efficacy of such products. It has been assumed that accurate testing of a disinfectant includes the use of a substance to neutralize the effects of the disinfectant before plating on agar. Chambers et al. (1) proposed the use of a thioglycolate-thiosulfate mixture to neutralize the effects of Ag+ during microbiological analysis of Ag+-treated water. The use of such a neutralizer has been widely accepted by regulatory agencies. Evidence now exists in a number of laboratories that other factors are involved in the neutralization of Ag+ subsequent to bacterial exposure. They include: presence of amino acids (2), hardness of water, phosphate and chloride content, temperature of incubation (7), type of buffer, light, bacterial density, and salt content of the growth medium (1). The intent of the present study was to determine the neutralizing effect of agar growth media on silver disinfection of bacteria.
MATERIALS AND METHODS Reagents, chemicals, and media. Thioglycolatethiosulfate neutralizer stock solution was prepared by adding 1.0 g of sodium thioglycolate and 1.46 g of sodium thiosulfate to 1,000 ml of distilled deionized water (1). This reagent was prepared daily and diluted as necessary.
A silver nitrate stock solution (5.0 mg of Ag+ per liter) was prepared in distilled deionized water and stored in the dark in a glass flask before use. The silver solution was routinely analyzed with a Perkin-Elmer 403 atomic absorption spectrophotometer. Bacteriological agars (eosin methylene blue [EMB] Trypticase soy agar [TSA], and tryptone glucose extract agar [TGE]) were purchased from Baltimore Biological Laboratory, Cockeysville, Md., and prepared weekly according to the manufacturer's instructions. Cultures. Escherichia coli ATCC 25822 was maintained on TSA slants. Before use, it was suspended in Trypticase soy broth, and incubated for 4 to 6 h at 35°C, and the number of organisms was standardized to 3 x 107 colony-forming units (CFU) per ml with an Autobac (Pfizer Diagnostics, Groton, Conn.) nephelometer (5). Experimental procedure. The standardized suspension of E. coli was further diluted in distilled deionized water to 3 x 103 CFU/ml. A 5-ml portion of this suspension was added to 45 ml of distilled deionized water containing 50 ,ug of Ag+ per liter (sample A) and similarly to 45 ml of distilled deionized water without Ag+ (sample B). The final working concentration of E. coli was 3 x 102 CFU/ml (Fig. 1). From each of these two suspensions of E. coli, five 5-ml test samples were prepared as follows: (i) test sample with no additions; (ii) test sample plus an equal volume of undiluted thioneutralizer stock solution; (iii) test sample plus an equal volume of a 1:100 dilution of thioneutralizer stock solution; (iv) test sample plus an equal volume of a 1:1,000 dilution of thioneutralizer stock solution; (v) test sample plus an equal volume of thioneutralizer stock solution (plating of the treated E. coli inoculum on agar plates was delayed an addi-
1116
REVERSAL OF SILVER INHIBITION BY AGAR
VOL. 35, 1978
Sample B 103
Sample A
103E. coli |+
1117
50ug of Ag+
|E. coli| B
Samples i, ii, iii, and iv plated on Ag+-pretreated TSA, TGE, and EMB. Sample v plating delayed 5 min and similarly processed.
B
(i) Untreated test sample (TA) (ii) TA + 5 ml of undiluted thioneutralizer (TN) (iii) TA + 5 ml of 1:100 TN (iv) TA + 5 ml of 1:1,000 TN (v) TA + 5 ml of undiluted TN (plating delayed 5 min)
Al
B
Samples i, ii, iii, and iv plated (TSA, TGE, and EMB) at 0, 10, 20, and 30 min. Sample v plated, after 5-min hold, at recalculated intervals of 0, 10, 20, and 30 min.
FIG. 1. Laboratory procedure for the silver neutralization experiment.
tional 5 min). Each of the five test samples of the two suspensions of E. coli (A, containing 50 jig of Ag+ per liter; B, containing no Ag+) were further treated as follows. (i) A 1-ml volume of each of the five samples of Ag+treated and untreated E. coli suspension was surface plated in triplicate on TSA, TGE, and EMB at intervals of 0, 10, 20, and 30 min of exposure. (ii) E. coli suspension B (no Ag+) was also divided into five samples and plated at 0, 10, 20, and 30 min in triplicate on TSA, TGE, and EMB. These plates were surface treated with 0.1 ml of a solution of 50 ug of Ag+ per liter and dried for 30 min before the plating of the five test samples. All plates were incubated in air at 35°C. Agar plates were counted daily for 2 days. Results were plotted as numbers of microbial survivors of a particular treatment protocol. To demonstrate that the substances in the agar growth medium, rather than the agar itself, were responsible for the neutralization of the silver toxicity, TGE plates and TGE broth tubes were prepared containing serial twofold concentrations of Ag+ ranging from 50 to 1,600 ug/liter. Each dilution of both Ag+treated agar and broth was inoculated with 103 CFU of E. coli per ml and incubated at 35°C overnight. Results were recorded daily for 2 days.
was added, the initial colony counts were approximately 30% higher than in the non-thioneutralized sample. After a 10-min exposure to silver, half of the original inoculum survived after thioneutralization, as compared with no survivors in the non-thioneutralized cultures. After a 20-min exposure to Ag+, no survivors were detected, whether or not the thioneutralizer was used. In this experiment, where such rapid microbial kill was observed, no differences could be seen in the ability of the three agars (TGE, TSA, EMB) to neutralize the effects of silver. Figure 3 presents data on control cultures of E. coli which were not exposed to Ag+. Once again, there was no difference in the rate of survival between thioglycolate-thiosulfate-neutralized cultures and those not so treated. However, there appeared to be a marked difference in the ability of the three agars to support the growth of non-Ag+-treated survivors. Figure 4 supports the contention that the agar growth medium rapidly neutralizes the bacteriostatic effects of Ag+. Non-Ag+-treated E. coli showed no marked reduction in numbers when plated on three agars which had been previously RESULTS spread with a solution containing 50 jig of Ag+ Figure 2 shows the survival of E. coli in the per liter. It is apparent, however, that TGE and presence of 50 ,ug of Ag+ per liter as a function of TSA neutralized the activity of Ag+ more effectime. Silver rapidly inactivated E. coli, resulting tively than did EMB. Data not presented graphically indicate that, in 50% kill in the initial 10-min exposure. Thioglycolate-thiosulfate reagent was added to sam- if the thioneutralizer was diluted 1:100 or 1:1,000 ples of E. coli to neutralize residual silver before and added before plating, survival of E. coli was plating on one of three agar media. The thioneu- similar to those instances in which no thioneutralizer was equivalent to the agar media in its tralizer was added. If thioneutralized samples of ability to promote the growth of survivors. How- culture fluid were held 5 min before plating, no ever, in those samples to which thioneutralizer salutary effect on the survival of E. coli was
6.0
2 Q
-4.0 x
\\ 0 NO
3
ADDED
THIONEUTRALIZER
A THIONEUTRALIZER ADDED EMS -.- TGE -
!
0
TSA
0 2.0
1.0,
TIME
20 10 OF INCUBATION PRIOR TO PLATING (ml..)
30
FIG. 2. Effects ofplating on the survival of E. coli incubated in the presence of 50 jig of Ag+ per liter. to
-
5.0
0 x
o
_
otO
0 o
0 20
a\ z\ o NO THIONEUTRALIZER ADDED 1.0
A
A THIONEUTRALIZER ADDED
EEMS --TE
10
TIME OF INCUBATION PRIOR
-- TSA
20 TO PLATING (min.)
30
FIG. 3. Effects ofplating on control cultures of E. coli (no Ag' added). 1118
REVERSAL OF SILVER INHIBITION BY AGAR
VOL. 35, 1978 6.0
5.0
1119
+
t
0
x 4.0
'
E
0
-0
I 0
----
so 2a.0
A
0-
-0
£~~~~~~~ 0 z
O
NO THIONEUTRALIZER ADDED
A
THIONEUTRALIZER ADDED
1.0
EMS
TIME
-.-
TOE .-
TSA
10 20 OF INCUBATION PRIOR TO PLATING (min.)
30
FIG. 4. Effects of Ag+-pretreated agar plates on the survival of E. coli. observed.
Table 1 supports the contention that the nutrient substances in the agar medium, and not the agar itself, neutralize the toxic effects of Ag+. The media, both broth and agar, absorbed Ag+ to an approximate concentration of 400 ,ug/ml, at which point free Ag+ was available to inhibit the challenge dose of E. coli.
DISCUSSION Unpublished observations on the ability of various agar media to neutralize the inhibitory effect of silver on bacteria have concerned those investigators who attempt to measure the kinetics of silver inactivation. At present, the standard plate count is the most practical method for the determination of viable count. Although it is essential to neutralize the effects of unbound silver before plating, such a procedure may strip silver ions from the bacterial cell envelope with subsequent "rescue" of these microorganisms. This rescue effect appears to be a function, in part, of the bacteriological plating medium. The data in Table 1 indicate that the agar itself does not neutralize silver. Greun (2) has reported that lysine, arginine, methionine, and cystine react
TABLE 1. Effects of TGE agar versus TGE broth on the survival of E. coli in serial twofold concentrations of Ag+ Ag+
Growtha
(Jg/ml)
TGE agar TGE broth
0 (control) 50
+ +
+ +
+ + 100 + + 200 + + 400 800 1,600 Growth equal to or equivalent to control; -, no
a +, growth.
with silver. There are amino acid-containing organic substances, such as peptones, which act as chelating agents of metal ions in media (3). The data in this study demonstrate that the agar constituent of the medium does not neutralize silver and that the soluble components of media effectively neutralize 400 to 800 ug of Ag+ per ml. The data also confirm the time-dependent nature of silver inactivation of bacteria. At the
1120 TILTON AND ROSENBERG end of 20 min of exposure to 50 ,ug of Ag+ per liter, there was virtually 100% kill with or without thioneutralizer and independent of the plating medium. Only during the initial stages of Ag+ exposure (0 to 20 min) could differences be observed. The presence of thioneutralizer promoted survival of E. coli at least during the initial 10 min of exposure. The slopes of the death curves for non-thioneutralized cultures were consistently steeper than the slopes of those treated with thioneutralizer. Virtually no survivors were observed after a 10-min exposure to Ag+. The data in Fig. 4 demonstrate the neutralizing capacity of agar media. As compared with the rapid death rate of the Ag+-exposed organisms seen in Fig. 2, E. coli survived plating on agar which had been previously spread with an AgNO3 solution. In this experiment, thioneutralizer added to the inoculum showed little effect. Those organisms plated on EMB revealed the lowest initial plate counts and the most rapid die-off. Such an observation was expected because, although EMB is a selective medium designed for the isolation of coliform bacteria, it contains methylene blue, which may be inhibitory for bacteria whose cell envelopes have been damaged by silver (4). Rescue is increased in the richer media such as TSA and TGE agar. As a result of this study, it appears that the
APPL. ENVIRON. MICROBIOL.
neutralization of Ag+ activity is a function both of the chelating substances in the agar medium and of the thioneutralizer added to the inoculum before plating. Claims of silver resistance in microorganisms should now be examined in light of the effects of nutrient media on the ability of silver to inactivate bacteria. ACKNOWLEDGMENTS This study was supported by a grant from the Electrolux Division of Consolidated Foods Corp., Chicago, Ill.
LITERATURE CITED 1. Chambers, C. W., C. M. Procter and P. W. Kabler. 1962. Bactericidal effect of low concentrations of silver. J. Am. Water Works Assoc. 54:208-216. 2. Greun, L. C. 1975. Interaction of amino acids with silver ions. Biochim. Biophys. Acta 386:270-274. 3. Lennette, E. H., E. H. Spaulding, and J. P. Truant (ed.). 1974. Manual of clinical microbiology, 2nd ed. American Society for Microbiology, Washington, D.C. 4. Rosencranz, H. S., and H. S. Carr. 1972. Silver sulfadiazine: effect on the growth and metabolism of bacteria. Antimicrob. Agents Chemother. 2:367-372. 5. Tilton, R. C., L. Lieberman and E. H. Gerlach. 1973. Microdilution antibiotic susceptibility test: examination of certain variables. Appl. Microbiol. 26:658-665. 6. von Nageli, C. 1893. Leben die oligodynamischen Erscheinungen an lebenden Zellen. Denkschr. Schweiz. Naturforsch. Ges. 33:174. 7. Woodward, R. L. 1963. Review of the effectiveness of silver. J. Am. Water Works Assoc. 55:881-886.
