Biological indicator recheck test: The ultimate examiner of sterilization effect
一、核心定义 Core Definition
生物指示剂是一种含有特定活微生物的制品，这些微生物通常具有已知的、高度稳定的对特定灭菌过程的抵抗力。它被专门设计用于验证灭菌设备（如高压蒸汽灭菌器、环氧乙烷灭菌柜等）、特定物品的灭菌及用于隔离系统/无菌洁净室除菌效果的评估，来验证灭菌程序是否真正有效。
Biological indicators are products containing specific live microorganisms, which usually have known and highly stable resistance to specific sterilization processes. It is specifically designed to verify the sterilization of sterilization equipment (such as high-pressure steam sterilizers, ethylene oxide sterilization cabinets, etc.), the sterilization of specific items, and to evaluate the sterilization effect of isolation systems/sterile clean rooms, in order to verify whether the sterilization process is truly effective.
1、直接衡量生物杀灭效果： 物理和化学参数只能证明设备“提供了”灭菌条件，但无法证明这些条件“达到了”预期的杀灭微生物的效果。设备可能存在冷点、蒸汽饱和度不够等物理传感器无法完全反映的问题。生物指示剂提供的是生物学证据。
1. Direct measurement of biocidal effect: Physical and chemical parameters can only prove that the equipment "provides" sterilization conditions, but cannot prove that these conditions "achieve" the expected effect of killing microorganisms. The equipment may have problems such as cold spots and insufficient steam saturation that physical sensors cannot fully reflect. Biological indicators provide biological evidence.
2、挑战最极端情况： 生物指示剂内的微生物是精心挑选的、对特定灭菌方式具有最强抵抗力的细菌芽孢。它的死亡，代表了所有抵抗力比它弱的自然污染物也必然被杀死。
2. Challenge the most extreme situation: The microorganisms in the biological indicator are carefully selected bacterial spores with the strongest resistance to specific sterilization methods. Its death means that all natural pollutants with weaker resistance than it will inevitably be killed.
3、法规与标准的强制要求： 在制药、医疗器械等领域，各国药典（如中国药典、USP）和GMP规范都明确规定，必须定期使用生物指示剂对灭菌工艺进行验证，进行微生物纯度和形态的鉴定及测定微生物数量。
3. Mandatory requirements of regulations and standards: In the fields of pharmaceuticals, medical devices, etc., the pharmacopoeias of various countries (such as the Chinese Pharmacopoeia and USP) and GMP norms clearly stipulate that biological indicators must be used regularly to verify sterilization processes, identify the purity and morphology of microorganisms, and determine the quantity of microorganisms.
二、生物指示剂的构成要素 Constituent Elements of Biological Indicators
一个标准的生物指示剂通常包含：
A standard biological indicator usually includes:
1、载体： 承载微生物的物体，如滤纸片、不锈钢片、玻璃片或安瓿的内表面。如下图：
1. Carrier: An object that carries microorganisms, such as filter paper sheets, stainless steel sheets, glass sheets or the inner surface of ampoules. As shown in the following figure
2、活的微生物：这是核心。通常是细菌的芽孢，因为芽孢是生命世界中抵抗力最强的形态，对热、化学药品和辐射都具有极强的耐受性。
2. Live microorganisms: This is the core. It is usually the spores of bacteria, as spores are the most resistant form in the living world and have extremely strong tolerance to heat, chemicals and radiation.
3、常用菌种 Common strains:
嗜热脂肪地芽孢杆菌：用于湿热灭菌（如高压蒸汽灭菌）的验证。它的芽孢对高温饱和蒸汽的抵抗力最强。
Bacillus stearothermophilus:Validation for moist heat sterilization (such as high-pressure steam sterilization). Its spores have the strongest resistance to high-temperature saturated steam.
枯草芽孢杆菌： 用于环氧乙烷灭菌和干热灭菌的验证。
Bacillus subtilis: Validation for ethylene oxide sterilization and dry heat sterilization.
4、已知数量：每份生物指示剂所含的活芽孢数量是已知且稳定的，通常在10⁵ 到 10⁶ CFU（菌落形成单位）之间。这为定量评估灭菌效果提供了基础。
4. Known quantity: The number of viable spores contained in each serving of biological indicator is known and stable, typically ranging from 10⁵ to 10⁶ CFU (colony-forming units). This provides a basis for quantitatively evaluating the sterilization effect.
5、基本包装： 保护载体和微生物，并允许灭菌因子（如蒸汽、气体）穿透。例如，玻璃安瓿或专用的纸塑袋。
5. Basic packaging: Protect the carrier and microorganisms, and allow sterilization factors (such as steam and gas) to penetrate. For example, glass ampoules or dedicated paper-plastic bags.
三、生物指示剂的保持条件Retention Conditions of Biological Indicators
液体生物指示剂：温度必须于 2°C - 8°C 条件下冷藏，严禁冷冻或剧烈震荡，避光保存。使用前操作：从冰箱取出后，应在使用前让其静置恢复至室温。使用前请轻柔地摇匀或涡旋，以确保孢子均匀悬浮。
Liquid biological indicator: It must be refrigerated at a temperature of 2°C to 8° C. Freezing or violent shaking is strictly prohibited. Store it away from light. Pre-use operation: After taking it out of the refrigerator, it should be left to stand and return to room temperature before use. Shake gently or swirl before use to ensure the spores are evenly suspended.
纸片生物指示剂：温度在2°C - 8°C 冷藏。某些产品经特殊稳定化处理后可在阴凉干燥的室温（如15-25°C）下保存，但首选且最可靠的仍是冷藏。严格防潮，必须在干燥器中密封保存。避光。使用前操作：从冰箱取出后，应密封状态下恢复至室温后再打开包装，以防止冷凝水浸湿纸片。
Paper biological indicator: Refrigerate at 2°C - 8°C. Some products can be stored at room temperature (such as 15-25°C) in a cool and dry place after special stabilization treatment, but refrigeration is still the preferred and most reliable method. Strictly prevent moisture and must be stored in a sealed container in a desiccator. Avoid light. Pre-use operation: After taking it out of the refrigerator, it should be restored to room temperature in a sealed state before opening the package to prevent condensation water from soaking the paper.
不锈钢片载体生物指示剂：温度在2°C - 8°C 冷藏。严格防潮，必须在干燥器中密封保存。避光。使用前操作：从冰箱取出后，应在密封状态下恢复至室温后再打开包装，防止冷凝水凝结在冰冷的钢片表面。
Stainless steel plate carrier biological indicator: Refrigerated at 2°C - 8°C. Strictly prevent moisture and must be stored in a sealed container in a desiccator. Avoid light. Pre-use operation: After taking it out of the refrigerator, it should be restored to room temperature in a sealed state before opening the package to prevent condensation water from forming on the cold surface of the steel sheet.
注意：具体操作务必遵循您所使用的生物指示剂产品说明书以及所在机构的质量管理规程。
Note: Specific operations must strictly follow the product manual of the biological indicator you are using and the quality management regulations of your institution.
四、检测过程 Inspection Process
1.液体生物指示剂 Liquid biological indicator
取4只生物指示剂，打开后分别吸取1ml至灭菌玻璃试管中，再加入9ml稀释液，先在水浴锅热击后，立即冰浴2分钟，取出后涡旋1分钟，稀释至所需级别后加入到培养皿后涂布计数。热击和培养条件见下表。热击与培养条件如下表：
Take 4 biological indicators, open them and draw 1ml of each into sterilized glass test tubes. Then add 9ml of the diluted solution. First, heat them in a water bath, then immediately place them in an ice bath for 2 minutes. After taking them out, vortex for 1 minute. Dilute to the required level and add them to petri dishes, then spread and count. The heat shock and culture conditions are shown in the table below. The heat shock and culture conditions are as follows:
|
名称 |
热击条件 |
培养条件 |
|
嗜热脂肪芽孢杆菌 |
95-100℃, 15 min |
55-60℃, 48h |
|
枯草芽孢杆菌 |
65-70℃, 15 min |
30-35℃, 48h |
2.纸片载体生物指示剂 Paper carrier biological indicators
取4片载体分别置4根灭菌玻璃试管中，加入10ml稀释液，再加入适量经过灭菌的玻璃珠，涡旋适宜时间至纸片完全变成糊状，先在水浴锅热击后，立即冰浴2分钟，取出后涡旋1分钟，稀释至所需级别后加入到培养皿上涂布计数。
Take 4 carriers and place them respectively in 4 sterilized glass test tubes. Add 10ml of dilution solution, then add an appropriate amount of sterilized glass beads. Vortex for an appropriate time until the paper pieces are completely paste-like. First, heat them in a water bath, then immediately place them in an ice bath for 2 minutes. Take them out and vortex for 1 minute. Dilute to the required grade and then add them to petri dishes for coating and counting.
3.金属片载体生物指示剂Metal strip carrier biological indicator
取4片载体分别置4根灭菌玻璃试管中，先加5ml稀释液，再加入适量经过灭菌的玻璃珠，先超声7分钟，移动位置后继续超声8分钟，后再加入5ml稀释液，此即为10倍稀释度。再涡旋1分钟后，先在水浴锅中热击15min后立即冰浴2分钟，取出后涡旋1分钟，稀释到所需级别后加入到培养皿上后涂布计数。
Take 4 carriers and place them respectively in 4 sterilized glass test tubes. First, add 5ml of dilution solution, then add an appropriate amount of sterilized glass beads. Ultrasonicate for 7 minutes, move the position and continue ultrasonicate for 8 minutes, then add 5ml of dilution solution. This is a 10-fold dilution. After vortexing for 1 minute, first heat it in a water bath for 15 minutes, then immediately place it in an ice bath for 2 minutes. Take it out and vortex for 1 minute. Dilute it to the required grade and add it to a petri dish, then spread and count.
五、注意事项 Precautions
1、样品前处理：样品需置于冰浴中维持温度在0–3℃；用玻璃试管盛装样品；配合直径约4–6 mm的玻璃珠，在涡旋仪上以3000 r/min进行涡旋处理；超声条件建议设置为功率约500 W，频率约60 Hz。
1. Sample pretreatment: The samples should be placed in an ice bath to maintain a temperature of 0-3℃. Use a glass test tube to hold the sample; In combination with glass beads of approximately 4 to 6 mm in diameter, vortex treatment is carried out at 3000 r/min on a vortexometer. It is recommended to set the ultrasonic conditions at a power of approximately 500 W and a frequency of about 60 Hz.
2、样品支架选择：建议采用不锈钢支架放置样品，以减少对超声能量的吸收与屏蔽。同时应确保样品底部不与超声清洗槽底部直接接触，以免干扰换能器产生超声波。
2. Sample holder selection: It is recommended to use a stainless steel holder to place the sample to reduce the absorption and shielding of ultrasonic energy. At the same time, it should be ensured that the bottom of the sample does not come into direct contact with the bottom of the ultrasonic cleaning tank to avoid interfering with the generation of ultrasonic waves by the transducer.
3、样品摆放方式：样品应均匀、等距分布，避免因驻波形成、样品遮挡以及超声波吸收与衰减等因素导致清洗槽内声场分布不均。样品数量过多或摆放杂乱均会影响超声处理效果。
3. Sample placement method: Samples should be evenly and equidistant distributed to avoid uneven sound field distribution in the cleaning tank due to factors such as standing wave formation, sample obstruction, and ultrasonic absorption and attenuation. An excessive number of samples or their disorderly placement will both affect the ultrasonic treatment effect.
4、液面高度控制：处理过程中应确保液面完全浸没样品，以保证洗脱效果的一致性与充分性。
4. Liquid level height control: During the processing, it is necessary to ensure that the liquid level fully submerges the sample to guarantee the consistency and sufficiency of the elution effect.
5、超声仪预处理：在放入样品前，应先开启超声仪空载运行5分钟进行排气，以消除气泡对超声能量传递的干扰。
5. Pretreatment of ultrasonic instrument:
Before placing the sample, the ultrasonic instrument should be turned on and run unloaded for 5 minutes to exhaust air, so as to eliminate the interference of air bubbles on the transmission of ultrasonic energy.
6、温度控制：超声波在35–60℃范围内空化效应较佳，建议在此温度区间内进行操作以提升处理效率。
6. Temperature control: The cavitation effect of ultrasonic waves is best within the range of 35 to 60℃. It is recommended to operate within this temperature range to enhance processing efficiency.
总结：生物指示剂是一个简单而强大的工具，它将复杂的物理化学参数验证，最终归结为一个明确的生物学问题。它是无菌保证体系的最后一道、也是最关键的一道防线，广泛应用于医院、制药、医疗器械、食品工业和科研等领域，是确保产品安全和公众健康不可或缺的利器。
Summary: Biological indicators are simple yet powerful tools that ultimately boil down complex physicochemical parameter verifications to a clear biological problem. It is the last and most crucial line of defense in the aseptic guarantee system, widely used in hospitals, pharmaceuticals, medical devices, food industry and scientific research, etc. It is an indispensable tool for ensuring product safety and public health.