过氧化氢与戊二醛对细菌芽胞协同杀灭作用的研究
作者：李少岚 冯伶
单位：李少岚 冯伶(太原铁路中心卫生防疫站，太原 030013)
关键词：协同杀菌作用；过氧化氢；戊二醛；杀芽胞剂；枯草杆菌黑色变种；pH值的影响
中国消毒学杂志980403
提 要 试验表明，5.0%过氧化氢与0.5%戊二醛协同作用10～30 min，对枯草杆菌黑色变种芽胞的杀灭率远高于单以过氧化氢或戊二醛作用者。戊二醛浓度对复方杀菌效果影响较大，过氧化氢浓度者次之，溶液pH影响较小。
STUDY ON SYNERGETIC SPORICIDAL EFFECT OF HYDROGEN#C
PEROXIDE AND GLUTARALDEHYDE
, http://www.100md.com
Li Shaolan Feng Ling
( Taiyuan Railway Central Sanitary and Anti-epidemic Station, Taiyuan 030013)
Abstract The germicidal efficacies of hydrogen peroxide alone, glutaraldehyde alone and their combination were compared experimentally using spores of Bacillus subtilis var. niger and the influences of their concentration and pH of their solution on germicidal efficacy were observed in laboratory.
, 百拇医药 The results indicated that the killing rates of B. subtilis var. niger spores exposed to 5.0% hydrogen peroxide, 0.5% glutaraldehyde and their combination for 30 min were 60.12%, 88.18% and 99.99% respectively. The range obtained from orthogonal test indicated that the concentration of glutaraldehyde influenced considerably the synergetic germicidal efficacy, the influence of concentration of hydrogen peroxide was the next and pH change of the solution ranging from 3.0～9.0 exerted little influence.
, http://www.100md.com
Key words synergetic germicidal effect hydrogen peroxide glutaraldehydesporicide Bacillus subtilis var. niger influence of pH
过氧化氢与戊二醛混合可产生醛过氧化物，有协同杀菌作用〔1〕。对其协同杀灭细菌芽胞效果，以及其成分浓度与溶液pH值对杀菌效果的影响进行了试验观察。现报道结果于下。
1 方法
1.1 试验菌片的制备
将枯草杆菌黑色变种(ATCC 9372)芽胞悬液0.1 ml染于无菌玻璃片(1.5 cm×1.5 cm)上，置37℃温箱中干燥30 min，备用。
1.2 中和剂选择试验
, 百拇医药
试验用上述菌片进行。分组为：① (消毒剂+菌片)+中和剂；② 消毒剂+菌片；③ 中和剂+菌片；④ (消毒剂+中和剂)+菌片；⑤ 磷酸盐缓冲液+菌片(正常菌对照)；⑥ 未接种菌的培养基对照〔2〕。试验结果，当第6组不长菌，第3、4、5组组间菌数差不超过10%，第1组长菌量超过50 cfu/片，第2组不长菌或长菌量远少于第1组时，表明所选中和剂及其浓度适宜。
1.3 杀菌试验
采用载体定量试验法。试验时，将菌片浸泡于10 ml消毒液(对照为无菌蒸馏水)中，静置。作用到规定时间，将菌片移入盛有5 ml中和剂溶液的试管中。振打试管80～100次，以洗 下菌片上的菌。取洗脱液0.1 ml接种平皿，倾注普通营养琼脂并混匀。然后，置于37℃培养48 h，计数菌落数，计算杀灭率。
2 结果
2.1 #FK中和剂试验结果
, 百拇医药
试验表明，含0.5%甘氨酸、0.5%硫代硫酸钠的磷酸盐缓冲液，对10.0%过氧化氢与1.0%戊二醛复方溶液的中和作用符合要求(表1)。
表1 过氧化氢与戊二醛复方消毒剂中和试验结果
Table1 Results of neutralization test for compound disinfectant of hydrogen peroxide and glutaraldehyde 组别
Group
平均菌数
(cfu/片)
Average bacterial count
(cfu/carrier)
, http://www.100md.com
(消毒剂+菌片)+中和剂
(Disinfectant+bacterial carrier)+neutralizer
91
消毒剂+菌片
Disinfectant+bacterial carrier
0
中和剂+菌片
Neutraliaer+bacterial carrier
1 130 000
(消毒剂+中和剂)+菌片
, http://www.100md.com
(Disinfectant+neutralizer)+bacterial carrier
1 080 000
正常菌对照
Normal bacteria control
1 230 000
未接种菌的培养基对照
Uninoculated culture medium control
0
注：试验菌为枯草杆菌黑色变种芽胞，以玻璃片为载体制成菌片。
试验温度为18～22℃。消毒剂为含10.0%过氧化氢、1.0%戊二醛溶液，加菌后作用30 min。加中和剂后作用10 min。结果为5次试验平均值。
, http://www.100md.com
Note: Test bacteria was B. subtilis var. niger spore. Glass
strip was used as carrier to prepare bacterial carrier.
The temperature was 18～22℃. The disinfectants
were solution containing 10.0% hydrogen peroxide
and 1.0% glutaraldehyde. The contact time after
adding bacteria was 3 min and that after adding
, http://www.100md.com
neutralize was 10 min. The results were means of 5 tests.
2.2 #FK协同杀菌作用
对过氧化氢或戊二醛单独作用与两者合用杀灭玻璃片上枯草杆菌黑色变种芽胞的效果进行比较。结果，单以5.0%过氧化氢或0.5%戊二醛作用30 min，对枯草杆菌黑色变种芽胞的杀灭率分别为60.12%与88.18%，但两者协同作用30 min，则可将其杀灭99.99%(表2)。
表2 过氧化氢与戊二醛对枯草杆菌黑色变种芽胞的协同杀灭效果
Table2 The synergetic killing effect of hydrogen
peroxide and glutaraldehyde on B. subtilis var. niger spores 消毒剂及其浓度
, http://www.100md.com
Disinfectant and concentration
(%)
作用不同时间(min)
的平均杀灭率(%)
Average killing rate(%)
after exposure for different
periods of tine(min)
10
20
30
5.0%过氧化氢
, 百拇医药
5.0%hydrogen peroxide
11.18
39.25
60.12
0.5%戊二醛
0.5%glutaraldehyde
32.12
78.56
88.18
5.0%过氧化氢+0.5%戊二醛
5.0%hydrogen peroxide+0.5%glutaraldehyde
, 百拇医药
80.56
99.17
99.99
注：试验温度为18～22℃。对照组平均菌数为1 100 000
cfu/片。杀灭率≥99.9%为5次重复试验平均值， 其余为3次重复试验平均值。
Note: The temperature was 18～22℃. The average
bacterial count of control group was 1 100 000
cfu/carrier. The killing rate ≥99.9% is mean of
, http://www.100md.com
5 repeated tests and other figures are means of 3 repeated tests.
2.3 成分浓度与溶液pH#FK对杀菌效果的影响
试验时，对过氧化氢浓度、戊二醛浓度与溶液pH值(用盐酸或氢氧化钠溶液调节)3因素各取3个水平(表3)，按L9(34)正交表安排试验。试验条件下的结果表明，两成分浓度较高与溶度碱性时杀菌效果较好.极差表明,戊二醛浓度的影响较大，过氧化氢浓度次之，溶液pH影响较小(表4)。
表3 试验因素与水平
Table3 Test factors and levels 因素
Factor
, 百拇医药
水平
Level
1
2
3
A戊二醛浓度
Glutaraldehyde concentration(%)
0.3
1.0
0.5
B过氧化氢浓度
Hydrogen peroxide concentration(%)
, 百拇医药
10.0
3.0
5.0
C溶液pH值
pH of the solution
3.0
9.0
6.0
表4 过氧化氢与戊二醛浓度及溶液pH对该复方杀灭枯草杆菌黑色变种芽胞
效果影响试验编组与检测结果
Table4 Grouping and results of test for influences of concentrations
, 百拇医药
of hydrogen peroxide and glutaraldehyde and pH of the solution on efficacy
of the compound disinfectant in killing B. subtilis var. niger spores 试验序号
Test No.
1
A
2
B
3
A+B
4
, http://www.100md.com
D
消毒后平均菌数的对数值
Log of average bacterial count after disinfection
1
1
1
1
1
3.68
2
1
2
, 百拇医药
2
2
5.04
3
1
3
3
3
4.80
4
2
1
2
3
, 百拇医药
1.08
5
2
2
3
1
2.94
6
2
3
1
2
2.25
7
, http://www.100md.com
3
1
3
2
2.21
8
3
2
1
3
3.67
9
3
3
, 百拇医药
2
1
3.86
Ⅰ
13.52
6.97
9.60
10.48
Ⅱ
6.27
11.65
9.98
9.50
, http://www.100md.com
Ⅲ
9.74
10.91
9.95
9.55
极差
Range
7.25
4.68
0.38
0.98
注: 试验温度为18～22℃。消毒剂加菌后作用20 min。未消毒对照组平均菌数为1 000 000 cfu/片。消毒后平均菌数的对数值> 3者为3次重复试验平均值，其余为5次重复试验平均值。
, http://www.100md.com
Note: The temperature was 18～22℃. The contact time of disinfectant after adding bacteria was 20 min. The average bacterial count of the control group was 1 000 000 cfu/carrier. The figures of log of average bacterial count >3 after disinfection are means of triplicate tests and others are means of 5 repeated tests.
3 结语
经比较,5.0%过氧化氢与0.5%戊二醛协同作用30min,对玻璃片上枯草杆菌黑色变种芽胞的杀灭率(99.99%)明显高于单以过氧化氢作用者(60.12%)与戊二醛作用者(88.18%)。正交试验所得极差表明，戊二醛浓度对复方协同杀菌效果影响较大，过氧化氢浓度者次之，溶液的pH影响较小。
参考文献
〔1〕 张文福. 过氧化氢消毒研究进展. 中国消毒学杂志 1992; 9 (3) :179.
〔2〕 王云峰，王有森，白竟玉，等. 消毒技术规范. 北京：卫生部， 1991: 84～85.
(1998-02-27收稿 1998-05-10修回), 百拇医药
单位：李少岚 冯伶(太原铁路中心卫生防疫站，太原 030013)
关键词：协同杀菌作用；过氧化氢；戊二醛；杀芽胞剂；枯草杆菌黑色变种；pH值的影响
中国消毒学杂志980403
提 要 试验表明，5.0%过氧化氢与0.5%戊二醛协同作用10～30 min，对枯草杆菌黑色变种芽胞的杀灭率远高于单以过氧化氢或戊二醛作用者。戊二醛浓度对复方杀菌效果影响较大，过氧化氢浓度者次之，溶液pH影响较小。
STUDY ON SYNERGETIC SPORICIDAL EFFECT OF HYDROGEN#C
PEROXIDE AND GLUTARALDEHYDE
, http://www.100md.com
Li Shaolan Feng Ling
( Taiyuan Railway Central Sanitary and Anti-epidemic Station, Taiyuan 030013)
Abstract The germicidal efficacies of hydrogen peroxide alone, glutaraldehyde alone and their combination were compared experimentally using spores of Bacillus subtilis var. niger and the influences of their concentration and pH of their solution on germicidal efficacy were observed in laboratory.
, 百拇医药 The results indicated that the killing rates of B. subtilis var. niger spores exposed to 5.0% hydrogen peroxide, 0.5% glutaraldehyde and their combination for 30 min were 60.12%, 88.18% and 99.99% respectively. The range obtained from orthogonal test indicated that the concentration of glutaraldehyde influenced considerably the synergetic germicidal efficacy, the influence of concentration of hydrogen peroxide was the next and pH change of the solution ranging from 3.0～9.0 exerted little influence.
, http://www.100md.com
Key words synergetic germicidal effect hydrogen peroxide glutaraldehydesporicide Bacillus subtilis var. niger influence of pH
过氧化氢与戊二醛混合可产生醛过氧化物，有协同杀菌作用〔1〕。对其协同杀灭细菌芽胞效果，以及其成分浓度与溶液pH值对杀菌效果的影响进行了试验观察。现报道结果于下。
1 方法
1.1 试验菌片的制备
将枯草杆菌黑色变种(ATCC 9372)芽胞悬液0.1 ml染于无菌玻璃片(1.5 cm×1.5 cm)上，置37℃温箱中干燥30 min，备用。
1.2 中和剂选择试验
, 百拇医药
试验用上述菌片进行。分组为：① (消毒剂+菌片)+中和剂；② 消毒剂+菌片；③ 中和剂+菌片；④ (消毒剂+中和剂)+菌片；⑤ 磷酸盐缓冲液+菌片(正常菌对照)；⑥ 未接种菌的培养基对照〔2〕。试验结果，当第6组不长菌，第3、4、5组组间菌数差不超过10%，第1组长菌量超过50 cfu/片，第2组不长菌或长菌量远少于第1组时，表明所选中和剂及其浓度适宜。
1.3 杀菌试验
采用载体定量试验法。试验时，将菌片浸泡于10 ml消毒液(对照为无菌蒸馏水)中，静置。作用到规定时间，将菌片移入盛有5 ml中和剂溶液的试管中。振打试管80～100次，以洗 下菌片上的菌。取洗脱液0.1 ml接种平皿，倾注普通营养琼脂并混匀。然后，置于37℃培养48 h，计数菌落数，计算杀灭率。
2 结果
2.1 #FK中和剂试验结果
, 百拇医药
试验表明，含0.5%甘氨酸、0.5%硫代硫酸钠的磷酸盐缓冲液，对10.0%过氧化氢与1.0%戊二醛复方溶液的中和作用符合要求(表1)。
表1 过氧化氢与戊二醛复方消毒剂中和试验结果
Table1 Results of neutralization test for compound disinfectant of hydrogen peroxide and glutaraldehyde 组别
Group
平均菌数
(cfu/片)
Average bacterial count
(cfu/carrier)
, http://www.100md.com
(消毒剂+菌片)+中和剂
(Disinfectant+bacterial carrier)+neutralizer
91
消毒剂+菌片
Disinfectant+bacterial carrier
0
中和剂+菌片
Neutraliaer+bacterial carrier
1 130 000
(消毒剂+中和剂)+菌片
, http://www.100md.com
(Disinfectant+neutralizer)+bacterial carrier
1 080 000
正常菌对照
Normal bacteria control
1 230 000
未接种菌的培养基对照
Uninoculated culture medium control
0
注：试验菌为枯草杆菌黑色变种芽胞，以玻璃片为载体制成菌片。
试验温度为18～22℃。消毒剂为含10.0%过氧化氢、1.0%戊二醛溶液，加菌后作用30 min。加中和剂后作用10 min。结果为5次试验平均值。
, http://www.100md.com
Note: Test bacteria was B. subtilis var. niger spore. Glass
strip was used as carrier to prepare bacterial carrier.
The temperature was 18～22℃. The disinfectants
were solution containing 10.0% hydrogen peroxide
and 1.0% glutaraldehyde. The contact time after
adding bacteria was 3 min and that after adding
, http://www.100md.com
neutralize was 10 min. The results were means of 5 tests.
2.2 #FK协同杀菌作用
对过氧化氢或戊二醛单独作用与两者合用杀灭玻璃片上枯草杆菌黑色变种芽胞的效果进行比较。结果，单以5.0%过氧化氢或0.5%戊二醛作用30 min，对枯草杆菌黑色变种芽胞的杀灭率分别为60.12%与88.18%，但两者协同作用30 min，则可将其杀灭99.99%(表2)。
表2 过氧化氢与戊二醛对枯草杆菌黑色变种芽胞的协同杀灭效果
Table2 The synergetic killing effect of hydrogen
peroxide and glutaraldehyde on B. subtilis var. niger spores 消毒剂及其浓度
, http://www.100md.com
Disinfectant and concentration
(%)
作用不同时间(min)
的平均杀灭率(%)
Average killing rate(%)
after exposure for different
periods of tine(min)
10
20
30
5.0%过氧化氢
, 百拇医药
5.0%hydrogen peroxide
11.18
39.25
60.12
0.5%戊二醛
0.5%glutaraldehyde
32.12
78.56
88.18
5.0%过氧化氢+0.5%戊二醛
5.0%hydrogen peroxide+0.5%glutaraldehyde
, 百拇医药
80.56
99.17
99.99
注：试验温度为18～22℃。对照组平均菌数为1 100 000
cfu/片。杀灭率≥99.9%为5次重复试验平均值， 其余为3次重复试验平均值。
Note: The temperature was 18～22℃. The average
bacterial count of control group was 1 100 000
cfu/carrier. The killing rate ≥99.9% is mean of
, http://www.100md.com
5 repeated tests and other figures are means of 3 repeated tests.
2.3 成分浓度与溶液pH#FK对杀菌效果的影响
试验时，对过氧化氢浓度、戊二醛浓度与溶液pH值(用盐酸或氢氧化钠溶液调节)3因素各取3个水平(表3)，按L9(34)正交表安排试验。试验条件下的结果表明，两成分浓度较高与溶度碱性时杀菌效果较好.极差表明,戊二醛浓度的影响较大，过氧化氢浓度次之，溶液pH影响较小(表4)。
表3 试验因素与水平
Table3 Test factors and levels 因素
Factor
, 百拇医药
水平
Level
1
2
3
A戊二醛浓度
Glutaraldehyde concentration(%)
0.3
1.0
0.5
B过氧化氢浓度
Hydrogen peroxide concentration(%)
, 百拇医药
10.0
3.0
5.0
C溶液pH值
pH of the solution
3.0
9.0
6.0
表4 过氧化氢与戊二醛浓度及溶液pH对该复方杀灭枯草杆菌黑色变种芽胞
效果影响试验编组与检测结果
Table4 Grouping and results of test for influences of concentrations
, 百拇医药
of hydrogen peroxide and glutaraldehyde and pH of the solution on efficacy
of the compound disinfectant in killing B. subtilis var. niger spores 试验序号
Test No.
1
A
2
B
3
A+B
4
, http://www.100md.com
D
消毒后平均菌数的对数值
Log of average bacterial count after disinfection
1
1
1
1
1
3.68
2
1
2
, 百拇医药
2
2
5.04
3
1
3
3
3
4.80
4
2
1
2
3
, 百拇医药
1.08
5
2
2
3
1
2.94
6
2
3
1
2
2.25
7
, http://www.100md.com
3
1
3
2
2.21
8
3
2
1
3
3.67
9
3
3
, 百拇医药
2
1
3.86
Ⅰ
13.52
6.97
9.60
10.48
Ⅱ
6.27
11.65
9.98
9.50
, http://www.100md.com
Ⅲ
9.74
10.91
9.95
9.55
极差
Range
7.25
4.68
0.38
0.98
注: 试验温度为18～22℃。消毒剂加菌后作用20 min。未消毒对照组平均菌数为1 000 000 cfu/片。消毒后平均菌数的对数值> 3者为3次重复试验平均值，其余为5次重复试验平均值。
, http://www.100md.com
Note: The temperature was 18～22℃. The contact time of disinfectant after adding bacteria was 20 min. The average bacterial count of the control group was 1 000 000 cfu/carrier. The figures of log of average bacterial count >3 after disinfection are means of triplicate tests and others are means of 5 repeated tests.
3 结语
经比较,5.0%过氧化氢与0.5%戊二醛协同作用30min,对玻璃片上枯草杆菌黑色变种芽胞的杀灭率(99.99%)明显高于单以过氧化氢作用者(60.12%)与戊二醛作用者(88.18%)。正交试验所得极差表明，戊二醛浓度对复方协同杀菌效果影响较大，过氧化氢浓度者次之，溶液的pH影响较小。
参考文献
〔1〕 张文福. 过氧化氢消毒研究进展. 中国消毒学杂志 1992; 9 (3) :179.
〔2〕 王云峰，王有森，白竟玉，等. 消毒技术规范. 北京：卫生部， 1991: 84～85.
(1998-02-27收稿 1998-05-10修回), 百拇医药