臭氧对水中微生物的杀灭效果及其影响因素
作者：谈 智 吉钟山 徐 燕
单位：江苏省卫生防疫站，南京 210009
关键词：臭氧；水消毒；杀芽胞作用；枯草杆菌黑色变种；大肠杆菌；金黄色葡萄球菌；HBsAg；影响因素
中国消毒学杂志990307 提要 经插入水底的带多孔喷头导管，将臭氧通入水中。向12cm内径玻璃瓶装3L含菌蒸馏水连续加臭氧10min(10.56mg)，对大肠杆菌和金黄色葡萄球菌杀灭率均为100%，连续加臭氧120min对枯草杆菌黑色变种芽胞杀灭率亦达100%。向30mlHBsAg悬液中连续加臭氧15min(11.88mg)，可将HBsAg抗原性完全破坏。其杀菌效果受有机物影响较大。
EFFICACY OF OZONE IN KILLING MICROORGANISMS
IN WATER AND ITS INFLUENCING FACTORS
, 百拇医药
Tan Zhi Ji Zhongshan Xu Yan
Jiangsu Provincial Sanitary and Anti-epidemic Station, Nanjing 210009
Abstract
When ozone was introduced continuously for 10 min (10.56 mg) into a glass bottle with an inside diameter of 12 cm and containing 3 L of bacteria-containing distilled water through insertion of a tube with multihole nozzle to bottom of water, the killing rate of both Escherichia coli and Staphylococcus aureus in water was 100%. The killing rate of spores of Bacillus subtilis var. niger was also 100% when ozone was introduced continuously for 120 min. Continuous introduction of ozone to 30 ml of HBsAg suspension for 15 min (11.88 mg) could destroy HBsAg antigenicity completely. Its bactericidal efficacy was influenced significantly by organic substance.
, http://www.100md.com
Key words ozone water disinfection sporicidal effect Bacillus subtilis var. niger Escherichia coli Staphylococcus aureus HBsAg influencing factor
用宝生牌臭氧多功能消毒机(下简称消毒机，香港利华企业有限公司产)进行了臭氧对水中微生物的杀灭效果及其影响因素的试验观察。现报告结果如下。
1 方法
1.1 定量杀菌试验
试验菌为大肠杆菌(8099)、金黄色葡萄球菌(ATCC 6538)、枯草杆菌黑色变种(ATCC 9372)芽胞。试验时，将菌悬液加入盛于玻璃瓶的水中，含菌量达5×105～5×106cfu/ml。将消毒机臭氧导管喷头插入水底。加臭氧(对照组不加)至预定时间，取样液0.5ml，加入4.5ml中和剂(经预试验选为含0.5%硫代硫酸钠的0.03mol/L磷酸盐缓冲液)中。中和作用10min，作活菌计数，计算杀灭率。观察有机物影响时，向大肠杆菌悬液中加1%小牛血清。
, http://www.100md.com
1.2 破坏乙型肝炎表面抗原试验
将乙型肝炎表面抗原(HBsAg)悬液30ml加入大试管中。加臭氧至预定时间，加入等量中和剂(预试验选为含1.0%硫代硫酸钠的0.03mol/L磷酸盐缓冲液)。中和作用10～30min，用DG5031型酶联免疫检测仪(华东电子管厂产)与上海科华公司提供的试剂盒(经测试剂灵敏度为1ng/ml)测定OD值。将臭氧通入不含HBsAg的30ml磷酸盐缓冲液中，加入等量中和剂，制成中和产物作阴性对照，以中和产物加HBsAg为阳性对照。测定结果，以试验组OD值(S)与阴性对照OD值(N) 的比值(S/N)<2.1为HBsAg阴性。
2 结果
2.1 杀菌效果
经用DCS-1型臭氧检测仪(上海物理光学仪器厂产)测定，该消毒机20min可使150L密闭试验箱内臭氧浓度达82.20mg/m3，相当于臭氧12.33mg(表1)。
, http://www.100md.com
表1 在150L箱内消毒机工作不同时间形成的臭氧浓度
Table 1 Concentration of ozone produced by opera-tion of the disinfection machine for different periods of time in 150 L chamber 时间
Time(min)
臭氧浓度
Ozone concentration(mg/m3)
臭氧量
Ozone amount(mg)
5.0
, http://www.100md.com
28.00
4.20
10.0
70.40
10.56
12.5
76.20
11.43
15.0
79.20
11.88
20.0
82.20
, http://www.100md.com
12.33
以该消毒机向3L带菌蒸馏水中连续加臭氧5min，对其中大肠杆菌和金黄色葡萄崐球菌的杀灭率均达99.9%，10min的杀灭率皆为100%(表2)。对枯草杆菌黑色变种芽胞作用60min的杀灭率为99.98%，120min的杀灭率为100%(表3)。表2 臭氧对水中细菌繁殖体的杀灭效果
Table 2 Efficacy of ozone in killing vegetative bacteria in water 细菌
Bacteria
对照组平均菌数
Average bacterial count of control group(cfu/ml)
连续加臭氧不同时间(min)的杀灭率(%)
, 百拇医药
Killing rate(%)after introducing ozone continuously for different periods of time(min)
2
5
7
10
15
大肠杆菌
E.coli
2000000
99.78
99.98
, 百拇医药
99.99
100.00
100.00
金黄色葡萄球菌
S.aureus
1445000
99.44
99.96
99.99
100.00
100.00
注:盛水玻璃瓶内径为12cm，将臭氧导管多孔喷头插入水底。试验温度为20±2℃。结果为5次试验平均值。
, 百拇医药
Note: The inside diameter of glass bottle containing water was 12 cm. The multihole nozzle of ozone tube was inserted to the bottom of water. The temperature was 20±2℃. The results were means of 5 tests.表3 臭氧对水中枯草杆菌黑色变种芽胞的杀灭效果
Table 3 Effcacy of ozone in killing spores of Bacilllus subtilis var. niger in water 连续加臭氧时间
Time of continuous introduction of ozone(min)
杀灭率
, 百拇医药
Killing rate(%)
15
29.45
30
96.80
60
99.98
90
99.99
120
100.00
注:盛水玻璃瓶内径为12cm，将臭氧导管多孔喷头插入水底。试验温度为20±2℃。对照组平均菌数为2690000cfu/ml。结果为5次试验平均值。
, 百拇医药
Note: The inside diameter of the glass bottle containing water was 12 cm. The multihole nozzle of ozone tube was inserted to the bottom of water. The tempera-ture was 20±2℃.The average bacterial count of control group was 2690000cfu/ml.The results were means of 5 tests.
2.2 对HBsAg抗原性破坏作用
以该消毒机向30mlHBsAg悬液中连续加臭氧15min，可将其中HBsAg抗原性完全破坏(表4)。
表4 臭氧对悬液中HBsAg抗原性的破坏作用
, 百拇医药 Table 4 Destroying effect of ozone on HBsAg antigenicity in suspension 连续加臭氧时间
Time of continuous introduction of ozone(min)
S/N值
S/N value
5
6.70
15
1.73
30
1.40
, http://www.100md.com
60
1.07
90
0.99
注: 试验温度为20±2℃。阳性对照S/N值为71.69。结果为5次试验平均值。
Note: The temperature was 20±2℃. S/N value of posi-tive control group was 71.69. The results were means of 5 tests.
2.3 影响因素
2.3.1 有机物 经检测，当大肠杆菌悬液中含1%小牛血清时，连续加臭氧5min的杀灭率由不含小牛血清时的99.98%降至44.38%，加臭氧15min亦仅杀灭55.18%(表5)。
, http://www.100md.com
表5 有机物对臭氧杀灭水中大肠杆菌效果的影响
Table 5 Influence of organic substance on efficacy of ozone in killing Escherichia coli in water 小牛血清浓度
Concentration of calf serum(%)
对照组平均菌数
Average bacterial count of control group(cfu/ml)
连续加臭氧不同时间(min)的杀灭率(%)
Killing rate(%)after continuous introduction of ozone for different periods of time(min)
, http://www.100md.com
5
10
15
0
2000000
99.98
100.00
100.00
1
2835000
44.38
52.42
55.18
, 百拇医药
注: 试验温度为20±2℃。结果为5次试验平均值。
Note: The temperature was 20±2℃. The results were means of 5 tests.
2.3.2 喷头与容器内径 试验表明，在气体导管末端拆去多孔喷头时，对12cm内径玻璃瓶装3L水中连续加臭氧7min与10min，对水中金黄色葡萄球菌的杀灭率，由有多孔喷头时的99.99%与100%分别降至99.39%与99.54%，加臭氧15min亦仅 杀灭99.79%。对22cm内径玻璃瓶装3L水，以多孔喷头连续加臭氧10min，对金黄色葡萄球菌的杀灭率亦降至99.57%，加臭氧15min亦仅杀灭99.96%。
2.3.3 水质 经检测，以多孔喷头向12cm内径玻璃瓶装3L水箱所存水中连续加臭氧60min，对水中枯草杆菌黑色变种芽胞的杀灭率仅为99.01%(蒸馏水中者可达99.98%)，加臭氧120min才杀灭99.96%。
, http://www.100md.com
3 讨论
3.1 将消毒机产生的臭氧经带多孔喷头导管通入水中。向12cm内径玻璃瓶装3L带菌蒸馏水连续加臭氧10min，对大肠杆菌和金黄色葡萄球菌的杀灭率均达100%；连续加臭氧120min，对枯草杆菌黑色变种芽胞的杀灭率亦达100%。向30mlHBsAg悬液连续加臭氧15min，可将HBsAg抗原性完全破坏。菌悬液中含1%小牛血清时，对臭氧杀菌效果有明显影响。对水箱贮存水中细菌的杀灭效果较对蒸馏水中者为低，可能与前者含杂质较多有关。
3.2 臭氧气体经多孔喷头通入水中，臭氧杀灭水中细菌效果较好。可能因多孔喷头增加了臭氧与水的接触面，使臭氧溶于水中较多所致。同样水量装于内崐径较小容器时，臭氧对其中细菌杀灭效果较好。可能是因为内径小的容器中同量水较深，由置于水底喷头释放的臭氧，在溢至水面过程中与水接触较多，致水中溶解臭氧量增加。
收稿日期：1998-11-28 修回日期：1999-04-23, http://www.100md.com
单位：江苏省卫生防疫站，南京 210009
关键词：臭氧；水消毒；杀芽胞作用；枯草杆菌黑色变种；大肠杆菌；金黄色葡萄球菌；HBsAg；影响因素
中国消毒学杂志990307 提要 经插入水底的带多孔喷头导管，将臭氧通入水中。向12cm内径玻璃瓶装3L含菌蒸馏水连续加臭氧10min(10.56mg)，对大肠杆菌和金黄色葡萄球菌杀灭率均为100%，连续加臭氧120min对枯草杆菌黑色变种芽胞杀灭率亦达100%。向30mlHBsAg悬液中连续加臭氧15min(11.88mg)，可将HBsAg抗原性完全破坏。其杀菌效果受有机物影响较大。
EFFICACY OF OZONE IN KILLING MICROORGANISMS
IN WATER AND ITS INFLUENCING FACTORS
, 百拇医药
Tan Zhi Ji Zhongshan Xu Yan
Jiangsu Provincial Sanitary and Anti-epidemic Station, Nanjing 210009
Abstract
When ozone was introduced continuously for 10 min (10.56 mg) into a glass bottle with an inside diameter of 12 cm and containing 3 L of bacteria-containing distilled water through insertion of a tube with multihole nozzle to bottom of water, the killing rate of both Escherichia coli and Staphylococcus aureus in water was 100%. The killing rate of spores of Bacillus subtilis var. niger was also 100% when ozone was introduced continuously for 120 min. Continuous introduction of ozone to 30 ml of HBsAg suspension for 15 min (11.88 mg) could destroy HBsAg antigenicity completely. Its bactericidal efficacy was influenced significantly by organic substance.
, http://www.100md.com
Key words ozone water disinfection sporicidal effect Bacillus subtilis var. niger Escherichia coli Staphylococcus aureus HBsAg influencing factor
用宝生牌臭氧多功能消毒机(下简称消毒机，香港利华企业有限公司产)进行了臭氧对水中微生物的杀灭效果及其影响因素的试验观察。现报告结果如下。
1 方法
1.1 定量杀菌试验
试验菌为大肠杆菌(8099)、金黄色葡萄球菌(ATCC 6538)、枯草杆菌黑色变种(ATCC 9372)芽胞。试验时，将菌悬液加入盛于玻璃瓶的水中，含菌量达5×105～5×106cfu/ml。将消毒机臭氧导管喷头插入水底。加臭氧(对照组不加)至预定时间，取样液0.5ml，加入4.5ml中和剂(经预试验选为含0.5%硫代硫酸钠的0.03mol/L磷酸盐缓冲液)中。中和作用10min，作活菌计数，计算杀灭率。观察有机物影响时，向大肠杆菌悬液中加1%小牛血清。
, http://www.100md.com
1.2 破坏乙型肝炎表面抗原试验
将乙型肝炎表面抗原(HBsAg)悬液30ml加入大试管中。加臭氧至预定时间，加入等量中和剂(预试验选为含1.0%硫代硫酸钠的0.03mol/L磷酸盐缓冲液)。中和作用10～30min，用DG5031型酶联免疫检测仪(华东电子管厂产)与上海科华公司提供的试剂盒(经测试剂灵敏度为1ng/ml)测定OD值。将臭氧通入不含HBsAg的30ml磷酸盐缓冲液中，加入等量中和剂，制成中和产物作阴性对照，以中和产物加HBsAg为阳性对照。测定结果，以试验组OD值(S)与阴性对照OD值(N) 的比值(S/N)<2.1为HBsAg阴性。
2 结果
2.1 杀菌效果
经用DCS-1型臭氧检测仪(上海物理光学仪器厂产)测定，该消毒机20min可使150L密闭试验箱内臭氧浓度达82.20mg/m3，相当于臭氧12.33mg(表1)。
, http://www.100md.com
表1 在150L箱内消毒机工作不同时间形成的臭氧浓度
Table 1 Concentration of ozone produced by opera-tion of the disinfection machine for different periods of time in 150 L chamber 时间
Time(min)
臭氧浓度
Ozone concentration(mg/m3)
臭氧量
Ozone amount(mg)
5.0
, http://www.100md.com
28.00
4.20
10.0
70.40
10.56
12.5
76.20
11.43
15.0
79.20
11.88
20.0
82.20
, http://www.100md.com
12.33
以该消毒机向3L带菌蒸馏水中连续加臭氧5min，对其中大肠杆菌和金黄色葡萄崐球菌的杀灭率均达99.9%，10min的杀灭率皆为100%(表2)。对枯草杆菌黑色变种芽胞作用60min的杀灭率为99.98%，120min的杀灭率为100%(表3)。表2 臭氧对水中细菌繁殖体的杀灭效果
Table 2 Efficacy of ozone in killing vegetative bacteria in water 细菌
Bacteria
对照组平均菌数
Average bacterial count of control group(cfu/ml)
连续加臭氧不同时间(min)的杀灭率(%)
, 百拇医药
Killing rate(%)after introducing ozone continuously for different periods of time(min)
2
5
7
10
15
大肠杆菌
E.coli
2000000
99.78
99.98
, 百拇医药
99.99
100.00
100.00
金黄色葡萄球菌
S.aureus
1445000
99.44
99.96
99.99
100.00
100.00
注:盛水玻璃瓶内径为12cm，将臭氧导管多孔喷头插入水底。试验温度为20±2℃。结果为5次试验平均值。
, 百拇医药
Note: The inside diameter of glass bottle containing water was 12 cm. The multihole nozzle of ozone tube was inserted to the bottom of water. The temperature was 20±2℃. The results were means of 5 tests.表3 臭氧对水中枯草杆菌黑色变种芽胞的杀灭效果
Table 3 Effcacy of ozone in killing spores of Bacilllus subtilis var. niger in water 连续加臭氧时间
Time of continuous introduction of ozone(min)
杀灭率
, 百拇医药
Killing rate(%)
15
29.45
30
96.80
60
99.98
90
99.99
120
100.00
注:盛水玻璃瓶内径为12cm，将臭氧导管多孔喷头插入水底。试验温度为20±2℃。对照组平均菌数为2690000cfu/ml。结果为5次试验平均值。
, 百拇医药
Note: The inside diameter of the glass bottle containing water was 12 cm. The multihole nozzle of ozone tube was inserted to the bottom of water. The tempera-ture was 20±2℃.The average bacterial count of control group was 2690000cfu/ml.The results were means of 5 tests.
2.2 对HBsAg抗原性破坏作用
以该消毒机向30mlHBsAg悬液中连续加臭氧15min，可将其中HBsAg抗原性完全破坏(表4)。
表4 臭氧对悬液中HBsAg抗原性的破坏作用
, 百拇医药 Table 4 Destroying effect of ozone on HBsAg antigenicity in suspension 连续加臭氧时间
Time of continuous introduction of ozone(min)
S/N值
S/N value
5
6.70
15
1.73
30
1.40
, http://www.100md.com
60
1.07
90
0.99
注: 试验温度为20±2℃。阳性对照S/N值为71.69。结果为5次试验平均值。
Note: The temperature was 20±2℃. S/N value of posi-tive control group was 71.69. The results were means of 5 tests.
2.3 影响因素
2.3.1 有机物 经检测，当大肠杆菌悬液中含1%小牛血清时，连续加臭氧5min的杀灭率由不含小牛血清时的99.98%降至44.38%，加臭氧15min亦仅杀灭55.18%(表5)。
, http://www.100md.com
表5 有机物对臭氧杀灭水中大肠杆菌效果的影响
Table 5 Influence of organic substance on efficacy of ozone in killing Escherichia coli in water 小牛血清浓度
Concentration of calf serum(%)
对照组平均菌数
Average bacterial count of control group(cfu/ml)
连续加臭氧不同时间(min)的杀灭率(%)
Killing rate(%)after continuous introduction of ozone for different periods of time(min)
, http://www.100md.com
5
10
15
0
2000000
99.98
100.00
100.00
1
2835000
44.38
52.42
55.18
, 百拇医药
注: 试验温度为20±2℃。结果为5次试验平均值。
Note: The temperature was 20±2℃. The results were means of 5 tests.
2.3.2 喷头与容器内径 试验表明，在气体导管末端拆去多孔喷头时，对12cm内径玻璃瓶装3L水中连续加臭氧7min与10min，对水中金黄色葡萄球菌的杀灭率，由有多孔喷头时的99.99%与100%分别降至99.39%与99.54%，加臭氧15min亦仅 杀灭99.79%。对22cm内径玻璃瓶装3L水，以多孔喷头连续加臭氧10min，对金黄色葡萄球菌的杀灭率亦降至99.57%，加臭氧15min亦仅杀灭99.96%。
2.3.3 水质 经检测，以多孔喷头向12cm内径玻璃瓶装3L水箱所存水中连续加臭氧60min，对水中枯草杆菌黑色变种芽胞的杀灭率仅为99.01%(蒸馏水中者可达99.98%)，加臭氧120min才杀灭99.96%。
, http://www.100md.com
3 讨论
3.1 将消毒机产生的臭氧经带多孔喷头导管通入水中。向12cm内径玻璃瓶装3L带菌蒸馏水连续加臭氧10min，对大肠杆菌和金黄色葡萄球菌的杀灭率均达100%；连续加臭氧120min，对枯草杆菌黑色变种芽胞的杀灭率亦达100%。向30mlHBsAg悬液连续加臭氧15min，可将HBsAg抗原性完全破坏。菌悬液中含1%小牛血清时，对臭氧杀菌效果有明显影响。对水箱贮存水中细菌的杀灭效果较对蒸馏水中者为低，可能与前者含杂质较多有关。
3.2 臭氧气体经多孔喷头通入水中，臭氧杀灭水中细菌效果较好。可能因多孔喷头增加了臭氧与水的接触面，使臭氧溶于水中较多所致。同样水量装于内崐径较小容器时，臭氧对其中细菌杀灭效果较好。可能是因为内径小的容器中同量水较深，由置于水底喷头释放的臭氧，在溢至水面过程中与水接触较多，致水中溶解臭氧量增加。
收稿日期：1998-11-28 修回日期：1999-04-23, http://www.100md.com