软式内镜终末漂洗纯化水微生物监测方法溯源
搜了下百度，发现 蓝色星空0r7agg和penazy的两篇文章，刘运喜教授在2017年5月19日在中华预防医学会第26次全国医院感染学术年会的疑惑解答，和2017年8月28-30日在黑龙江全国消毒与感染控制学术年会的35个问题全面解答，内镜器械冲（清）洗用水细菌菌落总数标准来源于GB 30689—2014《 内镜自动清洗消毒机》和ISO15883.4-2018。GB 30689—2014中只有菌落数标准，没有检测方法。ISO15883.4-2018附录E、D介绍了微生物的检测方法，可全英文，实在看不懂。垦请老师百忙之中对ISO15883.4-2018作个介绍，并讲解下与药典纯化水微生物检测方法的异同本帖最后由 jerkran 于 2023-11-16 17:33 编辑
英文：Annex E(normative)Tests for microbial contamination of post-disinfection rinse water
E.1 Water samples
l Take samples from draw-off points adjacent to the WD and from the point of discharge into the WD chamber or load, and label “Supply Sample” and “WD Sample” respectively.l Sampling containers shall be 250 ml, or larger, and shall be sterile.l Test the samples within 4 h of collection or store at 2 °C to 5 °C and test within 48 h of collection.l Swab the discharge surfaces of the sampling points thoroughly with 0,2 µm filtered 70 % iso-propanol and allow to dry by evaporation immediately before the sample is taken.l Collect a sample of not less than 200 ml, or as specified, from each sampling point for each test to be carried out.
E.2 Test for aerobic mesophilic bacteriaTest post-disinfection rinse water for aerobic mesophilic bacteria in accordance with ISO 15883-1:2006,6.4.2.4 and Annex C.
E.3 Test for environmental mycobacterial Filter a 100 ml aliquot of the sample under vacuum through a 0,45 µm filter that is of appropriate size to allow its transfer and incubation as described below (e.g. 47 mm diameter).l Transfer the filter aseptically to the surface of a Middlebrook 2) 7H10 Agar plate and incubate at (30 ± 2) °C.The plates should be read regularly. Incubation should be continued for 28 d before it is concluded that no growth has occurred.l The petri dish should be sealed to prevent dehydration of the growth medium.l Carry out the test in duplicate. Examine the filters weekly and count and record the number of colonies of bacterial growth.l Report the mean number of colony forming units per sample.
NOTEIf plates are overgrown by relatively faster growing contaminants within 48 h to 72 h, it may be necessary to resample and perform a preliminary partial decontamination of the sample with one or more chemicals to which mycobacteria are more resistant than the other organisms.If growth of mycobacteria is observed consideration should be given to having the cultures transferred to a specialist laboratory for identification of the mycobacterial strains isolated.
E.4 Test for legionellaeTest post-disinfection rinse water for legionelle in accordance with ISO 11731-2.2)Middlebrook is a trade name. This information is given for the convenience of users of this part of ISO 15583 and does not constitute an endorsement by ISO of the product named. Equivalent products may be used if they can be shown to lead to the same results.中文（机翻）1：
附件E
（规范性）
消毒后冲洗水微生物污染检测
E.1水样
从WD附近的抽取点和从排放点到WD室或负载中取样，并分别标记“供应样品”和“WD样品”。
3.取样容器应为250 ml或更大，并应无菌。
在采集后4小时内检测样品，或在2 °C至5 °C下储存，并在采集后48小时内检测。
用0.2 µm过滤的70%异丙醇彻底擦拭取样点的排放表面，并在取样前立即蒸发干燥。
从每个取样点采集不少于200 ml的样品，或按照规定采集，用于进行每个试验。
E.2好氧嗜温菌试验
根据ISO 15883-1：2006第6.4.2.4节和附录C，对消毒后冲洗水进行嗜温好氧菌检测。
E.3环境分枝杆菌检测
将100 ml等份样品在真空下通过0.45 µm过滤器过滤，该过滤器具有适当的尺寸，以允许其转移和孵育，如下所述（例如47 mm直径）。
将滤膜无菌转移至Middlebrook 2）7 H10琼脂平板表面，并在（30 ± 2）°C下孵育。应定期读取平板读数。 应继续培养28天，然后得出结论，没有发生生长。
培养皿应密封，以防止生长培养基脱水。
重复进行两次试验。 每周检查过滤器，计数并记录细菌生长菌落数。
报告每个样品的平均菌落形成单位数。
注：如果平板在48 h至72 h内被相对较快生长的污染物过度生长，则可能需要重新取样，并使用分枝杆菌比其他微生物更耐的一种或多种化学品对样品进行初步部分去污。
如果观察到分枝杆菌生长，应考虑将培养物转移到专业实验室，以鉴定分离的分枝杆菌菌株。
E.4军团菌试验
根据ISO 11731-2检测消毒后冲洗水的军团菌。
2)Middlebrook是个商标名 本信息是为了方便ISO 15583本部分的用户而提供的，并不构成ISO对所述产品的认可。 如果可以证明等同产品可导致相同的结果，则可使用等同产品。
翻译2:
Annex E(normative) （规范性）Tests for microbial contamination of post-disinfection rinse water
消毒后冲洗水的微生物污染测试
E.1 Water samples E.1 水样
l Take samples from draw-off points adjacent to the WD and from the point of discharge into the WD chamber or load, and label “Supply Sample” and “WD Sample” respectively.
l 从WD附近的抽取点和从放电点到WD室或负载中取样，并分别标记“供应样品”和“WD样品”。l Sampling containers shall be 250 ml, or larger, and shall be sterile.
l 取样容器应为250毫升或更大，并应为无菌。l Test the samples within 4 h of collection or store at 2 °C to 5 °C and test within 48 h of collection.
l 在收集后4小时内测试样品或在2°C至5°C下储存，并在收集后48小时内测试。l Swab the discharge surfaces of the sampling points thoroughly with 0,2 µm filtered 70 % iso-propanol and allow to dry by evaporation immediately before the sample is taken.
l 用0.2μm过滤的70%异丙醇彻底擦拭采样点的放电表面，并在取样前立即蒸发干燥。l Collect a sample of not less than 200 ml, or as specified, from each sampling point for each test to be carried out.
l 从每个采样点收集不少于 200 毫升的样品，或按规定进行每次测试。
E.2 Test for aerobic mesophilic bacteria
E.2需氧嗜温细菌试验Test post-disinfection rinse water for aerobic mesophilic bacteria in accordance with ISO 15883-1:2006,6.4.2.4 and Annex C.
根据 ISO 15883-1：2006、6.4.2.4 和附录 C 测试消毒后冲洗水中是否有好氧嗜温细菌。
E.3 Test for environmental mycobacteria
E.3 环境分枝杆菌检测l Filter a 100 ml aliquot of the sample under vacuum through a 0,45 µm filter that is of appropriate size to allow its transfer and incubation as described below (e.g. 47 mm diameter).
l 在真空下通过适当尺寸的 0,45 μm 过滤器过滤 100 ml 等分试样，以允许其如下所述转移和孵育（例如直径 47 mm）。l Transfer the filter aseptically to the surface of a Middlebrook 2) 7H10 Agar plate and incubate at (30 ± 2) °C.The plates should be read regularly. Incubation should be continued for 28 d before it is concluded that no growth has occurred.
l 将过滤器无菌转移到Middlebrook 2）7H10琼脂平板的表面，并在（30±2）°C下孵育。孵育应持续28天，然后得出结论，没有发生生长。l The petri dish should be sealed to prevent dehydration of the growth medium.
l 培养皿应密封，防止生长培养基脱水。l Carry out the test in duplicate. Examine the filters weekly and count and record the number of colonies of bacterial growth.
l 一式两份进行测试。每周检查过滤器，计算并记录细菌生长的菌落数。l Report the mean number of colony forming units per sample.
l 报告每个样品的平均菌落形成单位数。
NOTEIf plates are overgrown by relatively faster growing contaminants within 48 h to 72 h, it may be necessary to resample and perform a preliminary partial decontamination of the sample with one or more chemicals to which mycobacteria are more resistant than the other organisms.
注意 如果在 48 小时至 72 小时内，板被生长相对较快的污染物过度生长，则可能需要重新取样并用一种或多种化学物质对样品进行初步部分去污，分枝杆菌比其他生物体更具抗性。If growth of mycobacteria is observed consideration should be given to having the cultures transferred to a specialist laboratory for identification of the mycobacterial strains isolated.
如果观察到分枝杆菌的生长，应考虑将培养物转移到专业实验室，以鉴定分离出的分枝杆菌菌株。
E.4 Test for legionellae E.4军团菌检测Test post-disinfection rinse water for legionelle in accordance with ISO 11731-2.
根据 ISO 11731-2 测试消毒后冲洗水中的军团菌。2)Middlebrook is a trade name. This information is given for the convenience of users of this part of ISO 15583 and does not constitute an endorsement by ISO of the product named. Equivalent products may be used if they can be shown to lead to the same results.
2） Middlebrook 是一个商品名称。此信息是为了方便 ISO 15583 这一部分的用户而提供的，并不构成 ISO 对指定产品的认可。如果可以证明等效产品可以导致相同的结果，则可以使用它们。
本帖最后由 水痘 于 2023-11-17 11:04 编辑
谢谢jerkran老师，谢谢！ jerkran 发表于 2023-11-16 17:19
英文：Annex E(normative)Tests for microbial contamination of post-disinfection rinse water
E.1 Water ...
学习了，谢谢分享 ISO 15883-4:2018(E)
Annex D
(normative)
Methods for microbiological evaluation of disinfection of liquid transport system
D.1 Principle
The following two methods are intended to simulate various incidents that might arise during normal use of the WD, and that could give rise to contamination of the WD (see 4.9.2 and 6.12.5).
Method 1 (specified in D.3.6.1) tests the self-disinfection cycle after a simulated malfunction of the internal water treatment equipment that, although repaired quickly (24 h later), has caused contamination of the WD by the microorganisms present in the supply water.
Method 2 (specified in D.3.6.2) also simulates the case of WD contamination by microorganisms present in the supply water following a malfunction of the internal water treatment equipment. However, in this case, the self-disinfection cycle is only applied one week after a water equipment malfunction, and that during this week the WD has continued to be used (one endoscope washing/disinfection cycle perday). This allows evaluation of the efficiency of the self-disinfection cycle of a potentially contaminated WD after one week of use. Moreover, monitoring the internal level of contamination of the WD during the interval of time between the water treatment equipment failure and the execution of the self- disinfection cycle will allow evaluation of whether the WD's design is effective in limiting the growth of microorganisms and potential development of biofilm in the pipes of the WD.
国际标准化组织ISO15883-4:2018
附件D
(规范性)
液体输送系统消毒的微生物评价方法
D.1原则
以下两种方法旨在模拟WD正常使用过程中可能出现的各种事故，以及可能引起WD污染的事故(见4.9.2
和6.12.5)。
方法1(在D.3.6.1中规定)测试内部水处理设备在模拟故障后的自消毒循环，虽然快速修复(
24小时后)，但已导致WD被供应水中存在的微生物污染。
方法2(在D.3.6.2中规定)也模拟了内部水处理设备故障后，供应水中存在的微生物造成WD
污染的情况。然而，在这种情况下，自消毒循环仅在水设备故障后一周应用，在这一周内继续使用WD(每天一个内镜清洗/消毒循环)。这允许一个潜在的污染WD使用一周后的自我消毒周期的效率的评价。此外，在食水处理设备发生故障至自行消毒程序执行期间，监察水务署内部的污染程度，可评估水务署的设计是否能有效地限制微生物的生长，以及限制水务署喉管内可能形成的生物膜。
D.2 Material
D.2.1Microorganisms
Pseudomonas aeruginosa CIP4) A22 or ATCC5) 25619 (or equivalent) as microorganism.
Bacterial suspension, with 1 x 109 CFU/ml to 1 x 1010 CFU/ml in sterile distilled water.
D.2.2 Culture medium
Soybean casein digest(SCD) agar or Tryptone soya agar (TSA) (see EN 12353 and EN 13727), as maintenance and counting medium.
D.2.3Washer-disinfector
The following cycles shall be available:
一 endoscopeWD cycle;
- self-disinfection cycle;
4)CIP refers to Collection of Institut Pasteur. The CIP numbers are the collection numbers of strains supplied by this reference culture collection. This information is given for the convenience of users of this document and does not constitute an endorsement by ISO of the product named.
5)ATCC refers to American Type Culture Collection,10801 University Boulevard,
D.2材料
D.2.1微生物
铜绿假单胞菌CIP4)A22或ATCC5)25619(或同等物)作为微生物。
细菌悬液，在无菌蒸馏水中加入1X109C FU/ml至1X1010C FU/ml。
D.2.2培养基
大豆酪蛋白消化物(SCD)琼脂或胰蛋白胨大豆琼脂(TSA)(见EN12353和EN13727)，作为维持和计数培养基。
D.2.3洗涤消毒器
应提供下列循环:
-内窥镜WD循环;
-自消毒循环;
4) CIP指巴斯德研究所收藏。CIP编号是由该参考培养物集合提供的菌株的集合编号。提供这些信息是为了方便本文件的用户，并不构成LSO对指定产品的背书。
5) ATCC是指美国典型培养物收集中心，地址:美国弗吉尼亚州马纳萨斯大学大道10801号，
一 sampling cycle;
一 contamination cycle.
D.2.3.1Sampling cycle
The sampling cycle shall correspond to a routine endoscope cleaning and disinfection cycle interrupted during the stage before disinfection, and for which the detergent shall be replaced by sterile distilled water. Once the cycle has been interrupted, a sample from the bottom of the tank containing water having circulated in the WD pipe work shall be taken.
NOTE This sampling programme only includes the cleaning and rinsing stage and circulates water throughout the WD's pipe work, without there being any addition of disinfectant or detergent product.
If the cycle cannot be interrupted immediately prior to the disinfection stage then a complete cycle substituting sterile purified (e.g. reverse osmosis) water for all process chemical solutions shall be used.
D.2.3.2Contamination cycle
This special programme corresponds to a routine cleaning and disinfection cycle for which:
一the disinfectant solution heating system (if fitted) is deactivated;
一 the detergent and disinfectant are replaced by sterile distilled water.
During this contamination cycle, the WD is connected to the external tank containing the contamination solution (see Figure D.1), so that during each phase of the contamination cycle, theWD is only fed with the contamination solution contained in the external tank.
D.2.4 Connection of the WD to the external tank The connection of the WD to the external tank shall be as shown in Figure D.1.
采样周期;
污染循环。
D.2.3.1采样周期
取样周期应与消毒前阶段中断的常规内窥镜清洁和消毒周期相一致，清洁剂应更换为无菌蒸馏水。一旦循环被中断，应从水箱底部取样，其中含有在WD管道工程中循环过的水。
注:此取样程序只包括清洁和冲洗阶段，并在整个WD管道工程中循环水，没有添加任何消毒剂或洗涤剂产品。
如果循环不能在消毒阶段之前立即中断，则应使用一个完整的循环，用无菌纯化水(如反渗透)代替所有工艺化学溶液。
D.2.3.2污染循环
这一特殊程序对应于常规清洁和消毒周期，其中:
消毒液加热系统(如已安装)被停用;
-清洁剂和消毒剂被无菌蒸馏水取代。
在这个污染循环中，WD连接到装有污染溶液的外部槽(见图D.1)，因此在污染循环的每个阶段，WD只被供给外部槽中的污染溶液。
D.2.4WD与外部储罐的连接
WD与外部储罐的连接应如图D.1所示。
1
2
Key
1 WD
2water supply
3drainage
4external tank
NOTE
As a function of manufacturer's recommendations, external peripherals can be inserted between the water supply network and the WD (water softener, etc.).
Figure D.1-Connection of the WD to the external tank- Test configuration
1
2
键
1 WD
2供水
3排水
4外部油箱
注意
根据制造商的建议，可以在供水管网和WD(软水器等)之间插入外部外围设备。
图D.1-WD与外部储罐的连接-试验配置
D.3 Procedure
D.3.1 External tank disinfection
Before each test, subject the external tank in which the contamination solution is prepared to a thermal disinfection cycle with an Ao of not less than 600.
D.3.2Verification of absence of microbicidal residue in the external tank after
Disinfection During the last rinsing stage of the external tank, collect 9 ml of the water circulating in the external tank and associated pipework.
Incorporate 1 ml of a bacterial suspension of Pseudomonas aeruginosa at 103 CFU/ml in the previously sampled 9 ml of water.
After mixing thoroughly and 10 min of contact time, determine the number of viable bacteria present in the reaction mixture, CN, by serial dilution and counting on an SCD or TSA plate. After applying Formula (D.1), the rinsing is only considered to be valid if
10x0.8
(D.1)
Where CNis the number of viable bacteria present in the reaction mixture;
Cc is the exact concentration of bacteria in the bacterial suspension (control).
D.3.3 Preparation of the contamination solution
Fill the external tank with 30 1 of tap water and 30ml of a Pseudomonas aeruginosa suspension containing 109 CFU/ml. After thorough mixing, take a sample in order to determine, by serial dilution and counting on a SCD agar plate or TSA plate, the exact concentration of microorganisms in the contamination solution.
D.3程序
D.3.1外罐消毒
在每次测试之前，应对制备污染溶液的外部罐进行Ao不小于600的热消毒循环。
D.3.2外罐消毒后无杀菌剂残留的验证
在外缸的最后冲洗阶段，收集9毫升在外缸和相关管道中循环的水。
在先前取样的9毫升水中加入1毫升绿脓杆菌菌悬液，浓度为103C FU/ml。
在充分混合和10分钟接触时间后，通过连续稀释和在SCD或TSA板上计数来确定反应混合物中存在的活细菌的数量Cn。在应用公式(D.1)后，只有在以下情况下才认为冲洗有效
10xC≥0,8
CC
(D.1)
在哪里
Cy是反应混合物中存在的活细菌的数量;
cc是细菌悬浮液(对照)中细菌的确切浓度。
D.3.3污染溶液的配制
用301自来水和30 ml含109 CFU/ml的铜绿假单胞菌悬浮液填充外部水箱。充分混合后，取样，通过连续稀释和在SCD琼脂平板或TSA平板上计数，确定污染溶液中微生物的确切浓度。
D.3.4 Contamination of the WD via the water supply network
After having prepared the contamination solution and deactivated the WD water treatment unit, connect the WD subjected to the tests to the external tank (FigureD.1). Then start the WD contamination cycle in order to ensure circulation of the contamination solution in all the internal piping of the WD.
D.3.5 Determination of the WD contamination level
During the different tests, determine the contamination level of the WD by running a sampling cycle and then establishing the concentration of microorganisms in the water having circulated in all the piping of the WD during this cycle. For this, during the sampling cycle collect 2 1 of the water in the chamber of the WD. Filter 10ml, 100 ml and 1 000 ml of the 2 1 of water through 0,2 μm membranes.
Then rinse the membranes with 3 x 50 ml of sterile distilled water, place on counting medium and incubate at 37 °C for 24 h.
After incubation, count and identify the number of colony forming units, and express the results as a number of colony forming units per litre.
D.3.4WD通过供水网络受到污染
配制污染溶液并停用WD水处理装置后，将接受测试的WD连接到外部水箱(图D.1)。然后启动WD污染循环，以确保WD所有内部管道中的污染溶液循环。
D.3.5 WD污染水平的确定
在不同的测试中，通过运行一个采样周期来确定WD的污染水平，然后确定在此周期中WD所有管道中循环的水中微生物的浓度。为此，在取样周期内收集WD室中的水的2-1。过滤器10毫升，100毫升和1000毫升的水通过0.2微米的膜。然后用3X50ml无菌蒸馏水冲洗膜，放置在计数培养基上，在37°C下孵育24小时。
培养后，计数和鉴定菌落形成单位数，并将结果表示为每升菌落形成单位数。
D.3.6 Establishment of the efficacy of the disinfection of the liquid transport system
D.3.6.1Method1
Proceed as follows:
1)install the WD;
2)run a self-disinfection cycle;
3)run a sampling cycle;
4)determine the WD contamination level;
5)deactivate the water treatment system (i.e. remove filter, deactivate heating system);
6)disinfect the external tank;
7)prepare the contamination solution;
8) contaminate the WD via the water supply network;
9) leave the WD at room temperature (not less than 20 °C) to incubate for 24 h;
10) connect the WD normally;
11)re-activate the water treatment system;
12) run a self-disinfection cycle;
13) run a sampling cycle;
14) determine the contamination level of the WD in accordance with D.3.5;
15) if the analysis of the results shows a total viable count of 10 CFU/100ml or more or presence of
Pseudomonas aeruginosa in the sample taken during step 14), repeat steps 12), 13) and 14).
NOTE
It is not necessary to determine the contamination level before the disinfection cycle (see step 12)
since the extent of contamination that can occur is specific to the design of the WD liquid transport system.
D.3.6液体输送系统消毒效能的建立
D.3.6.1方法一
进行如下操作:
1)安装WD;
2)2)运行自消毒循环;
3)3)运行一个采样周期;
4)确定WD污染水平;
5)关闭水处理系统(即拆去过滤器、关掉暖气系统);
6)外罐消毒;
7)配制污染溶液;
8)通过供水网络污染WD;
9)将WD置于室温(不低于20℃)下孵育24h;
10) 连接WD正常;
11)重新启动水处理系统;
12)2)运行自消毒循环;
13)3)运行一个采样周期;
14)根据D.3.5确定WD的污染水平;
15)如果结果分析显示在步骤14)期间采集的样品中总活菌计数为10CFU/100ml或更高或绿脓杆菌的存在，则重复步
骤12)、13)和14)。
注:在消毒循环之前没有必要确定污染水平(见步骤12)，因为可能发生的污染程度取决于WD液体输送系统的设计。
D.3.6.2Method 2
Proceed as follows:
1)install the WD;
2)run a self-disinfection cycle;
3)run a sampling cycle;
4)determine the WD contamination level;
5) deactivate the water treatment system (i.e. remove filter, deactivate heating system);
6)disinfect the external tank;
7)prepare the contamination solution;
8)contaminate the WD via the water supply network;
9)leave the WD at room temperature (not less than 20 °C) to incubate for 48 h;
10) connect the WD normally;
D.3.6.2第二种方法
进行如下操作:
1)安装WD;
2) 2)运行自消毒循环;
3)3)运行一个采样周期;
4)确定WD污染水平;
5)关闭水处理系统(即拆去过滤器、关掉暖气系统);
6)外罐消毒;
7)配制污染溶液;
8)通过供水网络污染WD;
9)将WD置于室温(不低于20℃)下孵育48小时;
10)连接WD正常;
11) re-activate the water treatment system;
12) run a standard endoscope cleaning and disinfection cycle;
13) run a sampling cycle;
14) determine the contamination level of the WD in accordance with D.3.5;
15) leave the WD at room temperature (not less than20°C) to incubate for 24 h;
16) run a standard endoscope cleaning and disinfection cycle;
17) run a sampling cycle;
18) determine the contamination level of the WD in accordance with D.3.5;
19) leave the WD at room temperature (not less than 20°C) to incubate for 24 h;
20) run a standard endoscope cleaning and disinfection cycle;
21) run a sampling cycle;
22) determine the contamination level of the WD in accordance with D.3.5;
23) leave the WD at room temperature (not less than20 °℃) to incubate for 24 h;
24) run a standard endoscope cleaning and disinfection cycle;
25) run a sampling cycle;
26) determine the contamination level of the WD in accordance with D.3.5;
27) leave the WD at room temperature (not less than 20 °C) to incubate for 24 h;
28) run a standard endoscope cleaning and disinfection cycle;
29) run a sampling cycle;
30) determine the contamination level of WD in accordance with D.3.5;
31) leave the WD at room temperature (not less than 20 °C) to incubate for 48 h;
11)重新启动水处理系统;
12)12)运行标准内窥镜清洗消毒循环;
13)3)运行一个采样周期;
14)根据D.3.5确定WD的污染水平;
15)将WD置于室温(不低于20°℃)下孵育24小时;
16) 16)运行标准内窥镜清洗消毒循环;
17)7)运行一个采样周期;
18)根据D.3.5确定WD的污染水平;
19)将WD置于室温(不低于20°℃)下孵育24小时;
20) 20)运行标准内窥镜清洗消毒循环;
21)21)运行采样周期;
22)根据D.3.5确定WD的污染水平;
23)将WD置于室温(不低于20°℃)下孵育24小时;
24)24)运行标准内窥镜清洗消毒循环;
25) 25)运行采样周期;
26)根据D.3.5确定WD的污染水平;
27)将WD置于室温(不低于20°℃)下孵育24小时;
28) 28)运行标准内窥镜清洗消毒循环;
29)29)运行一个采样周期;
30)根据D.3.5确定WD的污染水平;
31)将WD置于室温(不低于20℃)下孵育48小时;
32) run a self-disinfection cycle;
33) run a sampling cycle;
34) determine the contamination level of the WD in accordance with D.3.5;
35) if the analysis of the results shows a total viable count of 10 CFU/100ml or more or presence of
Pseudomonas aeruginosa in the sample taken during step 33), repeat steps 32), 33) and 34).
D.4 Results/acceptance criteria
For both methods (Method 1and Method 2), record the number of self-disinfection cycles needed to
reduce the contamination to less than 10 CFU/100ml with absence of Pseudomonas aeruginosa.
32)
32)运行自消毒循环;
33)33)运行采样周期;
34)根据D.3.5确定WD的污染水平;
35)如果对结果的分析显示在步骤33)期间采集的样品中总活菌计数为10 CFU/100ml或更高或存在绿脓杆菌，则重复步骤32)、33)和34)。
D.4结果/验收标准
对于两种方法(方法1和方法2)，记录在不含铜绿假单胞菌的情况下，将污染降低到10 CU/100 ml以下所需的自消毒周期数。
EUROPEAN STANDARD
EN ISO 15883-1:2009+A1
NORME EUROPÉENNE
EUROPÄISCHE NORM
July 2014
ICS 11.080.10
English Version
Washer-disinfectors - Part 1: General requirements, terms and
definitions and tests(ISO 15883-1:2006)
Laveurs désinfecteurs - Partie 1: Exigences générales,
Reinigungs-Desinfektionsgerate - Teil 1: Allgemeine
termes et définitions et essais (ISO 15883-1:2006)
Anforderungen, Begriffe und Prüfverfahren (ISO 15883-
1:2006)
This European Standard was approved by CEN on 16 May 2009.
CEN members are bound to comply with theCEN/CENELEC Internal Regulations which stipulate the conditions for giving this European
Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such national
standards may be obtained on application to theCENManagement Centre or to any CEN member.
This European Standard exists in three official versions (English, French, German). A version in any other language made by translation
under the responsibility of a CEN member into its own language and notified to theCEN Management Centre has the same status as the
official versions.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland,
France, Germany, Greece, Hungary, Iceland, lreland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal,
Romania, Slovakia,Slovenia, Spain, Sweden, Switzerland and United Kingdom.
欧洲标准规范
欧盟ENISO 15883-1:2009年版
2014年7月
国际商品分类
中文版
洗涤消毒器第1部分:一般要求、术语和定义及试验
洗涤器消毒。第1部分:一般要求术语、定义和试验(ISO
Reinigungs-Desinfektionsgeräte第1部分:一般
15883-1:2006)
要求、术语和测试方法(ISO 15883-1:2006)
本欧洲标准于2009年5月16日获得CEN的批准。
CEN成员必须遵守CEN/CENELEC的内部规定，该规定规定了给予本欧洲标准国家标准地位而不作任何修改的条件。可以
向CEN管理中心或任何CEN成员申请获得有关这些国家标准的最新清单和参考书目。
本欧洲标准有三个正式版本(英语、法语、德语)。由CEN成员负责翻译成自己的语言并通知CEN管理中心的任何其他
语言的版本与正式版本具有同等地位。
CEN成员是奥地利的国家标准机构，比利时、保加利亚、塞浦路斯、捷克共和国、丹麦、爱沙尼亚、芬兰、法国、德国、希腊、匈牙利、冰岛、爱尔兰、
意大利、拉脱维亚、立陶宛、卢森堡、马耳他、荷兰、挪威、波兰、葡萄牙、罗马尼亚、斯洛伐克、斯洛文尼亚、西班牙、瑞典、瑞士和英国。
6.4 Tests on water quality and water volume
6.4.1 General
When specified in subsequent parts of ISO 15883, or required by the WD manufacturer (see 5.23.3), the tests
described in 6.4.2 to 6.4.4 shall be applied.
These tests can also be used for initial assessment of the available water supply.
6.4.2 Quality of final rinse water
6.4.2.1 Sampling
The sample shall be taken from the supply line as close as practicable to the WD. When the rinse water is
stored in a tank within the WD, heated in a calorifier in the WD or otherwise treated within the WD, samples
shall also be taken from the discharge point into the chamber.
6.4水质和水量测试
6.4.1一般情况
当ISO 15883的后续部分有规定时，或WD制造商有要求时(见5.23.3)，应进行6.4.2至6.4.4中所述的试验。
这些测试也可用于对可用水供应的初步评估。
6.4.2最终冲洗水的水质
6.4.2.1
取样
样品应尽可能地从靠近WD的供应管线处取样。当淋洗水储存在WD内的水箱中，在WD内的加热器中加热或在WD内进行其他处
理时，也应将样品从排放点取到室内。
ISO 15883-1:2006+A1:2014(E)
6.4.2.2Tests for chemical purity
Tests for chemical purity shall include tests for those determinands known to influence the efficacy of the
process.
NOTE
This can include, but is not limited to, tests to determine the value of the following:
一conductivity;
- pH;
一 oxidizable substances [determined by the European Pharmacopoeia (EP) method or as redox potential determined
by the United States Pharmacopoeia (USP) method];
一total hardness (salts of Ca2+, Mg2+, Sr2+ expressed as mmol CaCO3);
一total dissolved solids(TDS) determined as evaporative residue;
- inorganic phosphate and inorganic silicate , determined as the molybdate reactive species;
一chloride .
6.4.2.3
Tests for bacterial endotoxins
If a requirement for the level of bacterial endotoxins in the final rinse water is given in other parts of ISO 15883,
determine the level by the limulus amoebocyte lysate(LAL) test with a sensitivity of 0,25 EU/ml, or better,
using the method given in the European Pharmacopeia (EP) or United States Pharmacopeia (USP).
6.4.2.4 Tests for microbial quality
Make a total viable count by membrane filtration of not less than 100 ml final rinse water sample. Place the
filter on R,A-medium in accordance with Annex D, or other suitable low nutrient medium and incubate at
28°℃ to 32 °C for a minimum of 5 days to determine the aerobic mesophillic viable count.
国际标准化组织ISO 15883-1:2006
6.4.2.2化学纯度的试验
化学纯度的测试应包括那些已知会影响工艺效率的确定物的测试。
注意
这可以包括但不限于，测试以确定以下各项的值:
电导率;
一pH;
可氧化物质【按欧洲药典(EP)方法测定或按美国药典(USP)方法测定的氧化还原电位];
总硬度(以mmolCaCO表示的Ca2+、Mg2+、Sr2+的盐);
总溶解固体(电话和数据系统)测定为蒸发残留物;
无机磷酸盐和无机硅酸盐【，被确定为钼酸盐反应物种;
氯化物
6.4.2.3
细菌内毒素检查
如果ISO 15883的其他部分对最终冲洗水中的细菌内毒素水平有要求，使用欧洲药典(EP)或美国药典(USP)中给出的方法，通过灵敏度为0.25 EU/ml或更高的鲎变形细胞溶解物(LAL)试验确定水平。
6.4.2.4微生物质量的测试
用薄膜过滤法对不少于100ml的最终冲洗水样进行总活菌计数。将过滤器置于符合附录D的R、A培养基或其他合适的低营养培养基上，在28℃至32C下孵育至少5天，以确定需氧中温活菌计数。
6.4.3 Quality of water used during testing
Prior to carrying out operational qualification and performance qualification testing,determine the quality of
water used at each stage of the operating cycle other than the final rinse (see also 6.4.2). Tests for chemical
purity shall include tests for those determinants known to influence the efficacy of the process.
NOTE
This can include, but is not limited to, tests to determine the value of the following:
- conductivity;
一pH;
oxidizable substances [determined by the European Pharmacopoeia(EP) method or as redox potential determined
by the United States Pharmacopoeia(USP)method];
一total hardness (salts of Ca2+, Mg2+, Sr2+expressed as mmol CaCO3);
total dissolved solids(TDS) determined as evaporative residue.
6.4.3试验过程中使用的水的质量
在进行操作确认和性能确认测试之前，确定操作循环中除最后冲洗外的每个阶段所用水的质
量(也见6.4.2)。化学纯度的测试应包括那些已知影响工艺效能的决定因素的测试。
注意
这可以包括但不限于，测试以确定以下各项的值:
电导率;
一pH;
-可氧化物质【按欧洲药典(EP)方法测定或按美国药典(USP)方法测定的氧化还原电位];
总硬度(以mmol CaCO表示的Ca2+、Mg2+、Sr2+的盐);
总溶解固体(电话和数据系统)测定为蒸发残留物。
6.4.4Volume of water used per stage
Measure the volume of water used at each stage of the operating cycle using suitable volumetric measuring
vessels. The accuracy of the vessels shall be equal to, or better than, 1 % of the volume to be measured, as
specified by the manufacturer.
Alternatively the volume may be measured by interposing a total volume flow meter(s) in the pipe(s) supplying
the WD and determining the volume used from readings taken immediately before and after each stage of the
operating cycle.
The meter should be in a known state of calibration, suitable for the operating pressure range of the WD and
designed for connection within a supply pipe of the diameter used on the WD. The meter shall be located on a
straight section of pipe with no less than 20 pipe internal diameters from the nearest bend or obstruction on
either side of the meter.
Volume/time flow meters should not be used since the calculation of the total volume from measurements of
time and varying flow are unlikely to be sufficiently accurate.
6.4.4每级用水量
使用合适的容积测量容器测量操作循环每个阶段的用水量。容器的精度应等于或优于制造商规
定的被测体积的1%。
可替换地，可以通过在供应WD的管道中插入总体积流量计并从紧接在操作循环的每个阶段之前和之后获取的读数确定所使用的体积来测量体积。
流量计应处于已知校准状态，适合WD的工作压力范围，并设计用于连接WD所用直径的供水管。仪表应该安装在直管段上，从仪表两侧最近的弯头或障碍物开始，其内径不小于20倍。
不应使用体积/时间流量计，因为通过测量时间和变化的流量来计算总体积不太可能足够准确。
ISO 15883-1:2006+A1:2014(E)
Annex D
(normative)
Microbiological recovery medium for estimation of bacterial
contamination of water
D.1 Constituents
The microbiological recovery medium for the estimation of bacterial contamination of water(R2A medium)
shall consist of:
一Yeast extract
0,50 g
一Proteose peptone
0,50 g
一Casein hydrolysate0,50 g
一 Glucose
0,50 g
-Starch
0,50 g
-Na-pyruvate
0,30 g
- K₂HPO4
0,30 g
- MgSO4
0,024 g
- Agar
15,00g
-Purified water
1000,00 ml
国际标准化组织ISO15883-1:2006
附件D
(规范性)
水质细菌污染评价用微生物回收培养基
D.1成分
用于估计水的细菌污染的微生物恢复培养基(R2A培养基)应包括:
酵母提取物
0.50克
蛋白质肽
0.50克
酪蛋白水解物0.50g
葡萄糖
0.50克
-淀粉
0.50克
-丙酮酸盐
0,30克
-磷酸二氢钾
0,30克
-硫酸镁
0.024克
-琼脂
15,00克
纯净水
100万毫升
D.2 Preparation
Adjust the pH so that after sterilization it is pH 7,2 with crystalline K2HPO4 or KH2PO4before adding agar.
Add agar, heat medium to boiling to dissolve agar, and autoclave for 15 min at 121℃.
D.2准备
在加入琼脂之前，用结晶K，HPO4或KH，PO4灭菌后调节pH值为7,2。加入琼脂，加热介质至沸腾使琼脂溶解，并在121℃下高压灭菌15分钟。
ISO15883-4:2018 附件D(规范性)<液体输送系统消毒的微生物评价方法>,通过运行一个采样周期来测试确定WD的污染水平,过滤器10毫升，100毫升和1000毫升的水通过0.2微米的膜。然后用3X50ml无菌蒸馏水冲洗膜，放置在SCD或TSA板计数培养基上，在37°C下孵育24小时。培养后，计数和鉴定菌落形成单位数，并将结果表示为每升菌落形成单位数。 国际标准化组织ISO15883-4:2018 附件E（规范性）<消毒后冲洗水微生物污染检测>, E.2好氧嗜温菌试验,根据ISO 15883-1：2006第6.4.2.4节和附录C，对消毒后冲洗水进行嗜温好氧菌检测。6.4.2.4微生物质量的测试.<欧盟ENISO 15883-1:2009年版>说，用薄膜过滤法对不少于100ml的最终冲洗水样进行总活菌计数。将过滤器置于符合附录D的R2A培养基培养基或其他合适的低营养培养基上，在28℃至32C下孵育至少5天，以确定需氧中温活菌计数。 个人理解与困惑：1、欧盟ENISO 15883-1:2009年版，引用是否准确。2、从粗糙的翻译看，内镜终末漂洗水微生物检测应按欧盟ENISO 15883-1:2009年版标准，用薄膜过滤法对不少于100ml的最终冲洗水样，R2A培养基或其他合适的低营养培养基上，在28℃至32C下孵育至少5天进行总活菌计数。3、《药典》中的纯化水微生物检测是否适用？ 《中华人民共和国药典》（简称《中国药典》)2010年第二部P411页
纯 化 水
本品为饮用水经蒸馏法、离子交换法、反渗透法或其他适宜的方法制得的制药用水，不含任何添加剂。微生物限度取本品，采用薄膜过滤法处理后，依法检查(附录XI J)，细菌、霉菌和酵母菌总数每lml不得过100个。
附录XI J 微 生 物 限 度 检 查 法
微生物限度检査法系检查非规定灭菌制剂及其原料、辅料受微生物污染程度的方法。检查项目包括细菌数、霉菌数、酵母菌数及控制菌检查。
计数方法的验证
当建立产品的微生物限度检査法时，应进行细菌、霉菌及酵母菌计数方法的验证，以确认所采用的方法适合于该产品的细菌、霉菌及酵母菌数的测定。若产品的组分或原检验
除另有规定外，本检査法中细菌及控制菌培养温度为30～35°C;霉菌、酵母菌培养温度为23～28。检验结果以lg,lml、10g、10ml或10cm 2 为单位报告，特殊品种可以最小包装单位报告。
2. 薄膜过滤法
采用薄膜过滤法，滤膜孔径应不大于0. 45pm,直径一般为50mm，若采用其他直径的滤膜，冲洗量应进行相应的调整。选择滤膜材质时应保证供试品及其溶剂不影响微生物的充分被截留。滤器及滤膜使用前应采用适宜的方法灭菌。使用时，应保证滤膜在过滤前后的完整性。水溶性供试液过滤前先将少量的冲洗液过滤以润湿滤膜。油类供试品，其滤膜和滤器在使用前应充分干燥。为发挥滤膜的最大过滤效率，应注意保持供试品溶液及冲洗液覆盖整个滤膜表面。供试液经薄膜过滤后，若需要用冲洗液冲洗滤膜，每张滤膜每次冲洗量为100ml。总冲洗量不得超过1000ml ，以避免滤膜上的微生物受损伤。取相当于每张滤膜含lg，lml或10cm 2 供试品的供试液，加至适量的稀释剂中，混匀，过滤。若供试品每lg、lml或10cm 2 所含的菌数较多时，可取适宜稀释级的供试液lml进行试验。用PH7.0无菌氯化钠-蛋白胨缓冲液或其他适宜的冲洗液冲洗滤膜，冲洗方法和冲洗量同"计数方法的验证"。冲洗后取出滤膜，菌面朝上贴于营养琼脂培养基或玫瑰红钠琼脂培养基或酵母浸出粉胨葡萄糖琼脂培养基平板上培养。每种培养基至少制备一张滤膜。
阴 性 对 照 试 验 取试 验 用 的 稀 释 液 lm l， 照 上 述 薄 膜 过滤法操作，作为阴性对照。 阴性对照不得有菌生长。
培养和计数 培养条件和计数方法同平皿法，每片滤膜上的菌落数应不超过100cfu。(平皿法:培养和计数除另有规定外，细菌培养3天，霉菌、酵母菌培养5天，逐日观察菌落生长情况，点计菌落数,必要时，可适当延长培养时间至7天进行菌落计数并报告。菌落蔓延生长成片的平板不宜计数。点计菌落数后，计算各稀释级供试液的平均菌落数，按菌数报告规则报告菌数。若同稀释级两个平板的菌落平均数不小于15，则两个平板的菌落数不能相差1倍或以上。一般营养琼脂培养基用于细菌计数；玫瑰红钠琼脂培养基用于霉菌及酵母菌计数；酵母浸出粉胨葡萄糖琼脂培养基用于酵母菌计数。在特殊情况下，若营养琼脂培养基上长有霉菌和酵母菌、玫瑰红钠琼脂培养基上长有细菌，则应分别点计霉菌和酵母菌、细菌菌落数。然后将营养琼脂培养基上的霉菌和酵母菌数或玫瑰红钠琼脂培养基上的细菌数，与玫瑰红钠琼脂培养基中的霉菌和酵母菌数或营养琼脂培养基中的细菌数进行比较，以菌落数高的培养基中的菌数为计数结果。)
菌数报告规则 以相当于lg，lml或10cm 2 供试品的菌落数报告菌数;若滤膜上无菌落生长，以<1报告菌数（每张滤膜过滤lg、lml或10cm 2 供试品），或<1乘以稀释倍数的值报告菌数。
营养琼脂培养基、营养肉汤培养基、硫乙薛酸盐流体培养基、改良马丁培养基及改良马丁琼脂培养基照无菌检査法（附录XI H〉制备。
附录XI H无 菌 检 查 法
营养肉汤培养基
胨 10. Og 氯化钠5.0g牛肉浸出粉 3. Og 水1000ml
取上述成分混合，微温溶解，调节pH为弱碱性，煮沸，滤清,调节pH值使灭菌后为7. 2±0. 2分装，灭菌。
营养琼脂培养基
按上述营养肉汤培养基的处方及制法，加人加人14.0g琼脂，调节pH值使灭菌后为7. 2±0. 2，分装，灭菌。
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