Vol. 30, No. 4 Printed in U.SA.
APPLIED MICROBIOLOGY, Oct. 1975, p. 710-711
Copyright ©) 1975 American Society for Microbiology
Bactericidal Effect of Hydrogen Peroxide Spacecraft Isolates
on
M. D. WARDLE* AND G. M. RENNINGER'
Jet Propulsion Laboratory, California Institute of Technology, Pasadena, California 91103* and Bionetics Corporation, Hampton, Virginia 23665 Received for publication 28 May 1975
Solutions of 3, 10, and 15% hydrogen peroxide were found to have pronounced bactericidal effects, as a function of time of exposure, on sporeformers and nonsporeformers isolated from spacecraft. the effect of hydrogen peroxide on both sporeforming and nonsporeforming spacecraft bacterial isolates as an initial step in determining its appropriateness for the microbiological decontamination of certain United States spacecraft.
The objective of this study was to evaluate hydrogen peroxide as a candidate bactericidal agent for use on spacecraft hardware. Relevant work in the USSR (6), and elsewhere (1, 2, 4, 5), has indicated the practicality ofsuch an application of hydrogen peroxide. To the present time,
assess
TABLz 1. Percentage of survivla of spores exposed to hydrogen peroxide H202
Exposure time (min)
Spacecraft isolate
(%)
2
30
60
150
21.8 57.9 25.8 129 0.0967 11.9 4.66
2.10 0.712 5.90 44.3 0.00224 0.950 0.00644
0 0 0 0.302 0 0 0
3
Bacillus subtilis B. subtilis B. licheniformis B. subtilis Atypical bacillus B. polymyxa B. subtilis var. nigerb
84.8 97.5 114 122 21.4 43.5 112
10
B. subtilis B. subtilis B. licheniformis B. subtilis Atypical bacillus B. polymyxa B. subtilis var. nigerb
34.9 81.7 111 149 2.00 3.22 102
0.00247 0 0.112 3.60 0 0 0.0125
0 0 0 0 0 0 0
0 0 0 0 0 0 0
15
B. subtilis B. subtilis B. licheniformis B. subtilis Atypical bacillus B. polymyxa B. subtilis var. nigerb
22.0 67.1 101 139
0.00220 0 0 0 0 0 0.0125
0 0 0 0 0 0 0
0 0 0 0
0.654 0.631 69.5
0 0 0
Mean of three tests; significant to two figures. b Not a spacecraft isolate.
a
the accepted agent for reducing surface bacterial populations on the United States Mariner spacecraft has been isopropyl alcohol (95%), which is generally regarded as being nonsporicidal. It was the purpose of the present study to I Present address: American Sterilizer Company, Erie,
Pa.
Survivor data were obtained for eight bacterial isolates (six sporeformers and two nonsporeformers) recovered before launch from the Mariner Mars 1971 flyby spacecraft and exposed to concentrations of 3, 10, and 15% hydrogen peroxide. For comparative purposes, Staphylococcus epidermidis and spores of Bacillus subtilis var. 710
VOL. 30, 1975
niger were also studied. Challenge cultures of spores, sporulated in a liquid synthetic medium (7), were suspended in 95% ethyl alcohol, whereas those of nonsporeformers were water suspensions harvested from 48-h Trypticase soy agar (BBL) culture plates. Challenge suspensions had approximately 108 colony-forming units per ml. The test procedure involved the inoculation of 1 ml of challenge suspension into a flask containing 100 ml of 3, 10, or 15% H202 (prepared by dilution of a 30% [by weight] stock solution) held in a water bath at 25 C. (A KMnO4 titration of test solutions was performed at the beginning and end of a test to verify H202 concentrations.) After a prescribed exposure time, a 1-ml aliquot was removed from the flask and placed in 9 ml of sterile catalase to decompose H202. After all of the H202 was decomposed (determination of which was made by enzyme activity and a KI/FeSO4 colorimetric test) 10-fold serial dilutions were made in 0.1% peptone water and from these triplicate Trypticase soy agar pour plates formed. All tests were performed in triplicate. An identical scheme, without hydrogen peroxide, was followed for the control samples which on an average yielded approximately 106 colony-forming units per ml. The percentage of survival was calculated from the ratio of the test and control sample geometric means. Table 1 shows the effects of 3, 10, and 15% H202 on spores. Isolate variability and possibly some spore activation (see reference 3) was observed; however, with moderate exposure times the peroxide showed pronounced sporicidal effects. The percentage of survival of nonsporeformers exposed to hydrogen peroxide is shown in Table 2. No viable vegetative cells were recovered after a 10-min exposure time to any of the three concentrations of hydrogen peroxide used. Although these data point to the efficacy of hydrogen peroxide as an agent for reducing spacecraft bacterial populations, further evaluation is required in terms of method of applica-
NOTES
711
TABLz 2. Percentage ofsurvival" of nonsporeformers exposed to hydrogen peroxide Hydrogen peroxide (%) Spacecraft
3
10
15
2 10 min min
2 10 min min
isolate
2
10
minj min Micrococcus p. 28 Micrococcus op. 2 Staphylooccus 63 edrmidisc
0 0
0 0
0
0
0 0 0
0 0 0
0 0
0
* Mean of three tests. ' Exposure time. ' Not a spacecraft isolate.
tion and material compatibility before the qualification of hydrogen peroxide for use on space-
craft hardware. This work was supported by contract NAS 7-100 from the National Aeronautics and Space Administration. LITERATURE CITED 1. Campbell, J. E., and R. L. Dimmick. 1966. Effect of 3% hydrogen peroxide on the viability of Serratia marcescens. J. Bacteriol. 91:.25-929. 2. Curran, H. R., F. R. Evans, and A. Leviton. 1940. The sporicidal action of hydrogen peroxide and the use of crystalline catalase to dissipate residual peroxide. J. Bacteriol. 40:423-434. 3. Falcone, G., G. Salvatore, and I. Covelli. 1959. Mechanism of induction of spore germination in Bacillus subtilis by L-alanine and hydrogen peroxide. Biochim. Biophys. Acta 36:390-397. 4. Swartling, P., and B. Lindgren. 1968. The sterilizing effect against Bacilus aubtilis spores of hydrogen peroxide at different temperatures and concentrations. J. Dairy Res. 35:423428. 5. Toledo, R. T., F. E. Escher, and J. C. Ayres. 1973. Sporicidal properties of hydrogen peroxide against food spoilage organisms. Appl. Microbiol. 26:592-597. 6. Vashkov, V. I., N. V. Rashkova, and G. V. Shcheglova. 1970. Principles, methods and problems (methods and means of sterilization of spacecraft), NASA technical translation F-13,769. National Aeronautics and Space Administration, Washington, D.C. 7. Wardle, M. D., W. A. Brewer, and M. L. Peterson. 1971. Dry-heat resistance of bacterial spores recovered from Mariner-Mars 1969 spacecraft. Appl. Microbiol. 21:827-831.
APPLIED MICROBIOLOGY, Oct. 1975, p. 710-711
Copyright ©) 1975 American Society for Microbiology
Bactericidal Effect of Hydrogen Peroxide Spacecraft Isolates
on
M. D. WARDLE* AND G. M. RENNINGER'
Jet Propulsion Laboratory, California Institute of Technology, Pasadena, California 91103* and Bionetics Corporation, Hampton, Virginia 23665 Received for publication 28 May 1975
Solutions of 3, 10, and 15% hydrogen peroxide were found to have pronounced bactericidal effects, as a function of time of exposure, on sporeformers and nonsporeformers isolated from spacecraft. the effect of hydrogen peroxide on both sporeforming and nonsporeforming spacecraft bacterial isolates as an initial step in determining its appropriateness for the microbiological decontamination of certain United States spacecraft.
The objective of this study was to evaluate hydrogen peroxide as a candidate bactericidal agent for use on spacecraft hardware. Relevant work in the USSR (6), and elsewhere (1, 2, 4, 5), has indicated the practicality ofsuch an application of hydrogen peroxide. To the present time,
assess
TABLz 1. Percentage of survivla of spores exposed to hydrogen peroxide H202
Exposure time (min)
Spacecraft isolate
(%)
2
30
60
150
21.8 57.9 25.8 129 0.0967 11.9 4.66
2.10 0.712 5.90 44.3 0.00224 0.950 0.00644
0 0 0 0.302 0 0 0
3
Bacillus subtilis B. subtilis B. licheniformis B. subtilis Atypical bacillus B. polymyxa B. subtilis var. nigerb
84.8 97.5 114 122 21.4 43.5 112
10
B. subtilis B. subtilis B. licheniformis B. subtilis Atypical bacillus B. polymyxa B. subtilis var. nigerb
34.9 81.7 111 149 2.00 3.22 102
0.00247 0 0.112 3.60 0 0 0.0125
0 0 0 0 0 0 0
0 0 0 0 0 0 0
15
B. subtilis B. subtilis B. licheniformis B. subtilis Atypical bacillus B. polymyxa B. subtilis var. nigerb
22.0 67.1 101 139
0.00220 0 0 0 0 0 0.0125
0 0 0 0 0 0 0
0 0 0 0
0.654 0.631 69.5
0 0 0
Mean of three tests; significant to two figures. b Not a spacecraft isolate.
a
the accepted agent for reducing surface bacterial populations on the United States Mariner spacecraft has been isopropyl alcohol (95%), which is generally regarded as being nonsporicidal. It was the purpose of the present study to I Present address: American Sterilizer Company, Erie,
Pa.
Survivor data were obtained for eight bacterial isolates (six sporeformers and two nonsporeformers) recovered before launch from the Mariner Mars 1971 flyby spacecraft and exposed to concentrations of 3, 10, and 15% hydrogen peroxide. For comparative purposes, Staphylococcus epidermidis and spores of Bacillus subtilis var. 710
VOL. 30, 1975
niger were also studied. Challenge cultures of spores, sporulated in a liquid synthetic medium (7), were suspended in 95% ethyl alcohol, whereas those of nonsporeformers were water suspensions harvested from 48-h Trypticase soy agar (BBL) culture plates. Challenge suspensions had approximately 108 colony-forming units per ml. The test procedure involved the inoculation of 1 ml of challenge suspension into a flask containing 100 ml of 3, 10, or 15% H202 (prepared by dilution of a 30% [by weight] stock solution) held in a water bath at 25 C. (A KMnO4 titration of test solutions was performed at the beginning and end of a test to verify H202 concentrations.) After a prescribed exposure time, a 1-ml aliquot was removed from the flask and placed in 9 ml of sterile catalase to decompose H202. After all of the H202 was decomposed (determination of which was made by enzyme activity and a KI/FeSO4 colorimetric test) 10-fold serial dilutions were made in 0.1% peptone water and from these triplicate Trypticase soy agar pour plates formed. All tests were performed in triplicate. An identical scheme, without hydrogen peroxide, was followed for the control samples which on an average yielded approximately 106 colony-forming units per ml. The percentage of survival was calculated from the ratio of the test and control sample geometric means. Table 1 shows the effects of 3, 10, and 15% H202 on spores. Isolate variability and possibly some spore activation (see reference 3) was observed; however, with moderate exposure times the peroxide showed pronounced sporicidal effects. The percentage of survival of nonsporeformers exposed to hydrogen peroxide is shown in Table 2. No viable vegetative cells were recovered after a 10-min exposure time to any of the three concentrations of hydrogen peroxide used. Although these data point to the efficacy of hydrogen peroxide as an agent for reducing spacecraft bacterial populations, further evaluation is required in terms of method of applica-
NOTES
711
TABLz 2. Percentage ofsurvival" of nonsporeformers exposed to hydrogen peroxide Hydrogen peroxide (%) Spacecraft
3
10
15
2 10 min min
2 10 min min
isolate
2
10
minj min Micrococcus p. 28 Micrococcus op. 2 Staphylooccus 63 edrmidisc
0 0
0 0
0
0
0 0 0
0 0 0
0 0
0
* Mean of three tests. ' Exposure time. ' Not a spacecraft isolate.
tion and material compatibility before the qualification of hydrogen peroxide for use on space-
craft hardware. This work was supported by contract NAS 7-100 from the National Aeronautics and Space Administration. LITERATURE CITED 1. Campbell, J. E., and R. L. Dimmick. 1966. Effect of 3% hydrogen peroxide on the viability of Serratia marcescens. J. Bacteriol. 91:.25-929. 2. Curran, H. R., F. R. Evans, and A. Leviton. 1940. The sporicidal action of hydrogen peroxide and the use of crystalline catalase to dissipate residual peroxide. J. Bacteriol. 40:423-434. 3. Falcone, G., G. Salvatore, and I. Covelli. 1959. Mechanism of induction of spore germination in Bacillus subtilis by L-alanine and hydrogen peroxide. Biochim. Biophys. Acta 36:390-397. 4. Swartling, P., and B. Lindgren. 1968. The sterilizing effect against Bacilus aubtilis spores of hydrogen peroxide at different temperatures and concentrations. J. Dairy Res. 35:423428. 5. Toledo, R. T., F. E. Escher, and J. C. Ayres. 1973. Sporicidal properties of hydrogen peroxide against food spoilage organisms. Appl. Microbiol. 26:592-597. 6. Vashkov, V. I., N. V. Rashkova, and G. V. Shcheglova. 1970. Principles, methods and problems (methods and means of sterilization of spacecraft), NASA technical translation F-13,769. National Aeronautics and Space Administration, Washington, D.C. 7. Wardle, M. D., W. A. Brewer, and M. L. Peterson. 1971. Dry-heat resistance of bacterial spores recovered from Mariner-Mars 1969 spacecraft. Appl. Microbiol. 21:827-831.
