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# 定义
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含有活性微生物并对特定灭菌工艺具有明确耐热性的测试系统,简写BI
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# 用途
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能够定量测定和评价灭菌效果的生物检测器
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适用于灭菌程序的设计、开发、验证、确认,灭菌程序的日常监测
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# 种类
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根据不同灭菌方式的特点,生物指示剂也有不同的形态
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![1671112839029](https://file+192-002e168-002e168-002e108.vscode-resource.vscode-cdn.net/Note/%E5%88%B6%E8%8D%AF/%E5%B7%A5%E8%89%BA%E6%96%B9%E6%B3%95%E7%B1%BB%E9%AA%8C%E8%AF%81/%E7%81%AD%E8%8F%8C%E5%B7%A5%E8%89%BA/%E7%94%9F%E7%89%A9%E6%8C%87%E7%A4%BA%E5%89%82/image/%E7%94%9F%E7%89%A9%E6%8C%87%E7%A4%BA%E5%89%82/1671112839029.png)
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![1671112878739](https://file+192-002e168-002e168-002e108.vscode-resource.vscode-cdn.net/Note/%E5%88%B6%E8%8D%AF/%E5%B7%A5%E8%89%BA%E6%96%B9%E6%B3%95%E7%B1%BB%E9%AA%8C%E8%AF%81/%E7%81%AD%E8%8F%8C%E5%B7%A5%E8%89%BA/%E7%94%9F%E7%89%A9%E6%8C%87%E7%A4%BA%E5%89%82/image/%E7%94%9F%E7%89%A9%E6%8C%87%E7%A4%BA%E5%89%82/1671112878739.png)
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## 湿热灭菌固体生物指示
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![1671112938637](image/生物指示剂/1671112938637.png)
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![1671112952709](image/生物指示剂/1671112952709.png)
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![1671112963896](image/生物指示剂/1671112963896.png)
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# 特性
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具有较高的耐受性、贮存稳定性、批与批之间的一致性、无致病性、易于培养和制备-细菌芽孢
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耐受性要高于灭菌前产品中污染菌的耐受性
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符合地方、国内、国际相关法规要求
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可根据灭菌程序的设计要求而制备
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# BI耐受性的影响因素
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## 生物学种类
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![1671113088853](image/生物指示剂/1671113088853.png)
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## 制备孢子的培养条件
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* 温度
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* 培养基成分
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## 孢子悬浮液的组份
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## 孢子载体材料
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## 灭菌后的培养条件
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* 温度
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* 培养基质量
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# BI耐热性:D值
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## 测定方法:ISO 11138-1
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## 残存概率法
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- 多采用水/油浴+毛细管
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## 阴性分数法
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- SMC法
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- HSK法
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## 生物指示剂耐热性测定仪
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* 方法:ISO 18472
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![1671113254238](image/生物指示剂/1671113254238.png)
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# 生物指示剂的关键质量参数
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## 孢子数量
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- 复核数量结果要在标示结果的50%~300%范围内
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- 计数前需要热处理
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## D值
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- 复核结果要求在标示结果的±20%范围内
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## 最大存活时间(用户不要求测定)
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- 存活时间≥(lgN0-2)×D值
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## 最小灭活时间(用户不要求测定)
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- 灭活时间≤(lgN0+4)×D值
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# 物理灭杀时间Fphy
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* 采用物理数据(热穿透温度和时间)预测一个灭菌程序所赋予装载的灭菌效果
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* 一般采集产品最冷点部位的温度(但不包括干热位置)
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* 仅适用于饱和湿热条件,即不包括无饱和蒸汽的相对干燥位置
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* Fphy = Σ(10^((T-121)÷Z)×Δt)
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* Δt:穿透温度下的过渡时间(通常为1min)
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* Z:生物指示剂的温度系数
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# 标准灭菌时间F0
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F0 = Σ(10^((T-121)÷10)×Δt),Z值取10℃
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* 在121℃灭菌一分钟,可看作标准状态下1分钟(1个单位)
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* F0 = Σ(10^((121-121)÷10)×1)=1.0min
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* 在118℃灭菌一分钟,与121℃灭菌的等效灭菌时间为:
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* F0 = Σ(10^((118-121)÷10)×1)=0.5min
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* 用于计算F0的温度,应取自物品内
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# 生物灭杀时间Fbio
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* 表示一个灭菌程序在一定温度下杀灭某种微生物的实际效果
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* Fbio = D × LR
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* D:微生物的耐热参数
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* LR:在灭菌过程中微生物数量的对数下降值,即LR= Lg(N0)-Lg(NF)
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# Fbio和Fphy的一致性
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* 在理想状态下,Fbio=Fphy
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* 加热和冷却曲线为矩形波
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* 每个BI的数量/耐热性和孢子的受热程序均没有差异
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* 每个产品所受到的热效应均相同
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* 实际情况下,Fphy>Fbio
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* 每个产品内的热穿透总有一些差异,会导致Fphy的差异
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* 如果估算出的Fphy与Fbio之间有很大的差异,那么将会导致BI的阳性结果
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* 验证时,可以运行最差灭菌程序条件,如降低温度或缩短保温阶段时间,最低Fphy>Fbio
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* 仅凭物理数据不能完全保证灭菌程序所赋予产品足够的灭菌效果
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* 灭菌效果与具体产品组份相关
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* 有些点可能无法采集热穿透数据,需要BI监控
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* 同样,仅凭生物指示剂数据也不能保证灭菌程序的适当性和合理性
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* 物理数据还可以用来评价灭菌程序对产品质量的稳定性考察
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* 生物数据与物理数据之间要相互支持
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# LVP产品灭菌的工艺开发
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* 问题:如何有效且科学地对以下具有相同包装(如250ml/袋)的大容量注射剂产品,进行灭菌工艺程序的开发与验证
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* 可选取其中一种(或两种)溶液作为主溶液,代表同组产品进行灭菌程序的开发和验证。主溶液的选择标准
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* BI在其中具有最高耐受性-BI验证
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* 加热速度(热穿透)最难(如高粘度)-热穿透研究
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* BI主溶液和热穿透主溶液可以不同
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* 本次讨论主题:BI主溶液
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* BI(B.subtills)在各个产品溶液中的耐热参数
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![1671115161032](image/生物指示剂/1671115161032.png)
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* LVP产品灭菌程序评估
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* 选择0.9%氯化钠注射液为同组产品灭菌工艺验证的主溶液
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* 接种枯草芽孢杆菌孢子至0.9%氯化钠注射液(100ml),形成至少108孢子/袋的含量
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* 将接有BI的容器紧挨在差有温度探头的容器
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* 运行最差灭菌程序条件,随机采集热穿透数据、F0值、Fphy值
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* 对接有BI的所有容器进行无菌检查
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* 0.9%氯化钠注射液在最差灭菌条件下的热穿透数据、Fphy和Fbio
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![1671115175014](image/生物指示剂/1671115175014.png)
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* 计算Fbio值
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* 经检查,20个接有BI的产品均呈阴性,采用阴性分数法计算每个产品内的残存孢子时,需要假设有一个产品呈阳性
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* N0=10^8孢子/袋
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* 计算
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* NF= ln(n÷r) = ln(20÷19) = 0.0513
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* LR = LgN0 - LgNF = Lg(1×10^8)-Lg0.0513 = 9.3
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* Fbio = D ×LR = 0.66 ×9.3 = 6.1min
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* F0 = 8.4min(121℃,Z=10℃) = Σ(10^((T-121)÷10)×Δt)???
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* Fphy = 10.4min(121℃,Z=12.8℃) ???怎么得来的
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* 赋予BI的估计效果
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* LR = Fphy÷D121℃ = 10.4÷0.66 = 15.75
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* 数据对比
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* 如果产品的灭菌前含菌量限度控制在1000 CFU/袋,污染菌的耐热性(D121℃)空载在0.66min之内,那么,F0值为8.4min的湿热灭菌程序在产品灭菌后非无菌品的概率可估算为
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* LgNF = LgN0 - Fphy/D121 = 3.0-15.7 = -12.7
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* PNSU = NF = 10^-12.7 <10^-6
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* 嗜热脂肪芽孢杆菌BI评价-物品特性灭菌程序
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* 产品中含有细菌芽孢的最大可能数
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* 10个孢子/产品,计算时可用100个孢子/产品(最差状况/安全空间)
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* 产品中含有戏剧芽孢的最高耐热性
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* Fbio = D121 × (LgN0 - LgNF)= 0.5 ×[lg100-lg(10^-6)] = 0.5 × [2 - (-6)] = 4min,增加安全系数,取5min作为对灭菌工艺F0值要求
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* 嗜热脂肪芽孢杆菌BI特性
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* 孢子数量,N0=2X10^6孢子/BI
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* 当孢子置于F0为5.0min的灭菌程序后,估算出其残存孢子数量
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* Fbio = D121 × (LgN0 - LgNF)
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* LgNF = lgN0 - F0 ÷ D121 = 6.3 -5 ÷ 2 = 3.8
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* NF= 10^3.8=~6.3×10^3孢子
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* 问题:只要BI孢子残存数量不超过6.3X10^3孢子,灭菌程序符合无菌保证要求?
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* BI状况:N0 = 2×10^6孢子/BI,D121 = 2.0min
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* 计算使BI数量下降至6.3×10^3孢子/BI所需的Fbio值
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* Fbio = D121 × (LgN0 - LgNF)=2.0 × [lg(2×10^6)-Lg(6.3×10^3)] = 5.0min
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* 产品污染菌状况:N0 = 100,D121 = 0.5min
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* 计算产品经F0为5.0min程序后,残存的孢子数量
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* LgNF = lgN0 - F0 ÷ D121 =2 - 5.0 ÷ 0.3 = -14.7
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* NF = PNSU = 10^-14.7<10^-6(无菌保证标准)
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# 嗜热脂肪芽孢杆菌BI用于过度灭菌工艺效力评价
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## 过度杀灭嗜热灭菌工艺
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- 用于带孔/固体物品的灭菌
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- 灭菌效力要求
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- F0和Fbio≥12min
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- 温度探头必须摆放在蒸汽最难穿透的部位
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- 嗜热脂肪芽孢杆菌BI的要求
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- 孢子含量≥1X106孢子/BI
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- 耐热性D121℃≥1.5min
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## 初始评价
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- 物理灭杀值(20支探头),F0值16~20分钟
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- 20个BI在灭菌、培养后,全部呈阴性结果
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- BI孢子起始数量2.3×10^6/BI
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- D121℃=1.8min
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- 计算Fbio值
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- Fbio=D121℃×(LgN0-LgNF)=1.8×{Lg(2.3×10^6)-Lg[ln(20/19)]}=13.8min
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- 最小F0值和Fbio值均大于12min
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## 再评价
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- 物理灭杀值(20支探头),F0值16~20分钟
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- 20个BI在灭菌、培养后,18个阴性,2个阳性
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- BI孢子起始数量2.3×10^6/BI
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- D121℃=1.8min
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- 计算Fbio值
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- Fbio=D121℃×(LgN0-LgNF)=1.8×{Lg(2.3×10^6)-Lg[ln(20/18)]}=13.2min
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- 最小F0值和Fbio值均大于12min
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- 如果要求或希望BI被全部灭杀,则
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- 延长暴热时间
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- 检查BI的z值是否小于10℃?
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- 如果允许有阳性结果
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- 在每个探头至少两个BI
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- 对有阳性结果出现的位点,根据阴性分数法计算出孢子的残存量
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# Z值对计算F0值的影响
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- Z值是D值变化一个对数单位时,灭菌温度变化的度数,随微生物种类受热环境而变化
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- 用于换算杀灭率,从而估算等效灭菌值(等效灭菌时间,Fphy值)
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- 采用热穿透计算Fphy值
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- Fphy = Σ(10^((T-121)÷Z)×Δt)
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- 标准等效灭菌时间F0以121℃参照温度,Z值取10℃
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- 用F0值来估测灭菌程序对BI的杀灭效果
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- 当Z值不等于10℃时,Fphy值会出现什么样的变化?
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# 总结
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- 开发、评价和确认一个灭菌工艺程序对灭菌物品的灭菌效果时,需要同时测定Fphy值和Fbio值,并且Fphy值≥Fbio值
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- Fphy值由物理和数学方法计算出来
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- 当Z值取10℃,得F0值
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- 当Z值取测试用BI的实际值,得到Fphy
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- Fbio值由BI测试而计算出来
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- 对组份、特性和包装相似的产品,可采用统筹法(主溶液)对全组产品的灭菌程序效果进行评价和确认
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