蒸汽灭菌原理

2023年12月30日发(作者：)

From PHARMACEUTICAL ENGINEERING, NOVEMBER/DECEMBER 2013, VOL 33, NO 6

©Copyright ISPE 2013

Steam Sterilization Principles

蒸汽灭菌原理

by Marcel Dion and Wayne Parker

This article presents how a good understanding of basic steam sterilization principles can help with avoiding most

common mistakes made when using steam autoclaves.

本文论述了良好地理解基本的蒸汽灭菌原理将有助于避免蒸汽灭菌柜使用中出现的大多数常见错误。

Steam sterilization has been used for more than a

containing liquids may require processes using

century to sterilize items that can withstand moisture

steam-air mixtures or super-heated water-air mixtures.

and high temperature. Steam is water in the vapor state;

These processes, as well as in-situ sterilization of tanks,

therefore, it is non-toxic, generally readily available,

filters, etc., are not addressed in this article.

and relatively easy to control. A good understanding of

蒸汽灭菌用于耐热耐湿物品的灭菌已有一个多basic steam sterilization principles and cycles is

世纪。蒸汽是水的气态形式；因此，它无毒、易制necessary to avoid mistakes that can lead to non-sterile

取且相对易控制。良好地理解基本的蒸汽灭菌原理load items, poor performance of the equipment,

和周期能够避免一些错误，这些错误可能导致装载personnel injury, lower productivity, higher operation

物品非无菌、设备性能低、人员伤害、产量低、运and maintenance costs, and damage to load items.

行和维护成本高，以及装载物品的破坏。蒸汽灭菌Steam sterilizers are used for numerous applications in

广泛应用于制药和医疗器械行业。本文的重点是饱the pharmaceutical and medical device industries. The

和蒸汽的应用，如实验室培养基灭菌、净化和一般focus of this article is saturated steam applications,

器具灭菌。注射剂产品或带液体的器械的最终灭菌such as laboratory media sterilization, decontamination,

可能需要使用蒸汽-空气混合气，或过热水-空气混合and general component sterilization. Terminal

气。本文不涉及这些过程以及储罐、过滤器等的原sterilization of parenteral liquid products or devices

位灭菌。

Steam Sterilization Principles 蒸汽灭菌原理

Six factors are particularly critical to assure successful

steam sterilization:

六个确保蒸汽灭菌成功的关键因素是：

1. Time 时间

2. Temperature 温度

3. Moisture 湿度

4. Direct steam contact 直接蒸汽接触

5. Air removal 排气

6. Drying 干燥

simply because all the organisms do not die at the

same time. A minimum amount of time at sterilization

temperature is required to kill all the organisms.

Geobacillus stearothermophilus (Bst) spores are

generally used to test steam sterilizer cycles because

they are extremely resistant to moist heat sterilization.

They are also non-pathogenic and commercially

readily available. The number of survivors is usually

plotted on a logarithmic scale. A straight line survivor

curve such as the one shown in Figure 1 is typical.

暴露（灭菌）时间之所以关键，是因为不是所有的微生物都同时死去。在灭菌温度下杀死所有微1. Time 时间

The exposure (sterilization) time is a critical factor

生物至少需要一定的时间。嗜热脂肪芽孢杆菌（Bst）孢子常用于测试蒸汽灭菌周期，因为其极度耐受温热灭菌，同时也不致病且容易购买。存活数量通常画成对数坐标，常见的是如图1所示的直线型存活曲线。

，假设露时间，以达到10-4的无菌保证水平（SAL）。这意味着一个Bst起始微生物数量为1百万（106）。在孢子在此过程中存活的概率为万分之一（10-4）121°C (250°F)下每多2min暴露时间，SAL值会下降10倍。不同应用需要不同的SAL值。周期开发时应注意确保设定了正确的SAL值。所灭菌产品的实际生物负载逻辑上会比Bst杀灭得快。这种所造成的“过度杀灭”可以用于耐用物品的灭菌，而且也应尽可能地采用。2

Figure 1. Typical survivor curve.

图1. 典型的存活曲线

The D-value (time to reduce the microbial population

by 90%) for Bst should be 1.5 to 3.0 minutes at

121.1°C (250°F) .1 For the purpose of this discussion, a

D121 value of 2.0 minutes and a sterilization

temperature of 121°C (250°F) is used. A typical

sterilization cycle will include an exposure phase of at

least 20 minutes at 121°C (250°F) for a Sterility

Assurance Level (SAL) of 10-4, assuming a starting

population of one million (106) organisms. This means

there is a one in ten thousand (10-4) chance of a single

viable Bst spore surviving the process. For each

additional two minutes of exposure at 121°C (250°F),

the SAL is decreased by a factor of ten. The required

SAL varies with application. Care should be taken to

assure the correct SAL is targeted prior to cycle

development. The actual bioburden of the products

being sterilized will logically be killed faster than Bst.

The resultant “overkill” is an accepted method for

sterilization of durable items and should be used when

possible.2

121.1°C (250°F) 下Bst的D值（微生物数量减少90%所需的时间）应为1.5～3.0min。1本次讨论中采用D121值2.0min，灭菌温度121°C (250°F)。典型的灭菌周期包括至少20min 121°C (250°F)下的暴2. Temperature 温度

The second critical factor in steam

sterilization is the temperature of the

saturated steam controlled in the

chamber of the sterilizer. Figure 2

clearly demonstrates how increasing the

temperature dramatically reduces the

time needed to achieve sterilization. Figure 2 illustrates

approximately how much time is required to achieve

equivalent microbial lethality (SAL 10° with a starting

population of 106, D121- value 2.0 minutes) at

different moist heat exposure temperatures.3 The

temperature of saturated steam is directly related to the

pressure at which it is controlled. The pressure-

temperature relationship values are shown in saturated

steam tables.4 A typical cycle at 121°C (250°F) will

require 15 to 17 lbs of gauge pressure (103 to 117 kPa)

in the chamber of the sterilizer. The gauge pressure

required will be higher than the pressure shown in the

saturated steam table due to air mixed with the steam

and elevation above sea level. The maximum pressure

in an autoclave is limited by the specifications (ASME

pressure rating) of the pressure vessel (chamber and

jacket).

蒸汽灭菌的第二个关键因素是灭菌柜腔室内饱和蒸汽的温度。图2说明增加温度可以显著地减少达到灭菌效果所需的时间。图2表明了在不同湿热暴露温度下达到等效细菌致死率（SAL10°，起始数量106，D121-值2.0min）大概需要的时间。3饱和蒸汽的温度与控制的压力直接相关。压力-温度关系见4 121°C (250°F)下典型的周期需要灭菌饱和蒸汽表。腔室达到15-17lbs表压（103-107kPa）。实际所需的表压会比饱和蒸汽表中的值略高，因为蒸汽中混有

pressure at the desired temperature. Superheat is also

created when saturated steam passes over a surface at a

higher temperature. The sterilizer jacket temperature

should always be set slightly below the

chamber sterilization temperature to avoid

superheating of the steam as it enters the

chamber.

蒸汽湿度对其蛋白灭活或变性能力影响很大，因此需要采用饱和蒸汽。饱和蒸汽在同一压力下与加热的水达到平衡，也就是说它含有最大量的蒸汽且不含冷凝水。蒸汽灭菌推荐采用饱和蒸汽。不是所有的蒸汽都可以用于灭菌柜，推荐采用Figure 2. Sterilization time versus temperature.

专用的洁净蒸汽，应避免使用过热蒸汽、含液态水图2. 灭菌时间与温度。

的蒸汽，以及含过量锅炉添加剂或污染物（如铁锈）。

过热蒸汽是指饱和温度以上的蒸汽。过热发生在蒸3. Moisture 湿度

汽分配系统中，当管路压力经减压阀（PRV）后降Moisture in the steam has a major impact on its ability

to denature, or coagulate proteins; hence the

低的时候。压降越大，造成的过热越严重。过热蒸汽不包含确保灭菌所需的湿气。过热蒸汽的过剩能importance of using saturated steam. Saturated steam

is at equilibrium with heated water at the same

量是暂时的，最终会被灭菌腔室内的物品所消耗，pressure, which means it contains the maximum

但在按EN285要求验证灭菌柜空载温度稳定性时，会造成困难。用于蒸汽灭菌柜的理想洁净蒸汽系统amount of moisture without liquid condensate present.

的输出压力应控制在30-35psig（207-241kPa）。Saturated steam is recommended for steam sterilization.

EN285指出，提供的蒸汽压力应不大于操作温度下Not all steam is acceptable for use in a sterilizer. A

腔室压力的2倍。过热也发生在饱和蒸汽经过高温dedicated clean steam supply is recommended.

表面时。灭菌柜夹套温度应始终稍低于腔室灭菌温Superheated steam, steam containing excessive liquid

度，以避免蒸汽进入腔室时过热。

water, and steam containing excessive boiler additives

or contaminates (such as rust) should be avoided.

4. Direct Steam Contact 直接蒸汽接触

Superheated steam is defined as steam that is above its

saturation temperature. Superheat occurs in steam

Direct steam contact with the surface of the object to

be sterilized is required for the steam to transfer its

distribution systems when the line pressure is dropped

stored energy to the object. Without direct steam

across a Pressure Reducing Valve (PRV). The larger

contact to all surfaces, the item will not be sterilized.

the pressure drop, the more superheat is created.

The amount of energy stored in steam is much higher

Superheated steam does not contain the required

than dry air or water at the same temperature. From the

moisture necessary to assure sterilization. The excess

energy in superheated steam is transient and is

saturated steam table mentioned above, one can see

eventually dissipated by the items in the sterilizer

that it takes 419 kJ/kg (180 Btu/lb) to heat water from

0°C to 100°C (32°F to 212°F). This is the enthalpy of

chamber, but can cause difficulty when validating the

sterilizer to the empty chamber temperature

water (hl). It takes an additional 2,257 kJ/kg (970

Btu/lb) to create steam at atmospheric pressure (100°C

stabilization requirements of the European Standard

EN285.5 The ideal clean steam system for steam

or 212°F). This additional energy stored in the steam is

the enthalpy of vaporization (he), and is the key to

sterilizers is regulated at 30 to 35 psig (207 to 241 kPa)

at the source. EN285 indicates the steam supply

steam sterilization. In order for the steam to transfer its

stored energy, it must condense on the surface of the

pressure should not be more than twice the chamber

空气，且地理位置比海平面高。蒸汽灭菌柜的最大压力由压力容器（腔室和夹套）的规格（ASME压力等级）限制。

object being sterilized.

蒸汽要将潜热传给与被灭菌物品，需要与其表面发生直接蒸汽接触。没有与所有表面发生直接蒸汽接触的话，物品就不能被灭菌。蒸汽所携带的能量比同一温度下的干空气或水高出很多。从上述饱和蒸汽表可以看出，将水从0°C加热到100°C需要419 kJ/kg (180 Btu/lb)。这就是水的焓值(hl)。在大气压力下(100°C or 212°F)，还需要2,257 kJ/kg (970

Btu/lb)的能量才能生成蒸汽。这些储存于蒸汽中的额外能量，就是气化焓(he)，也是蒸汽灭菌的关键。蒸汽为了传递其存储的能量，必须在灭菌物品的表面冷凝。

5. Air Removal 排气

Air is the biggest deterrent to steam sterilization. Air

must be removed from the chamber and the load before

direct steam contact and sterilization can occur. This is

accomplished in a steam sterilizer by a series of

vacuum pulses prior to sterilization (pre-conditioning

phase). A small amount of air will always be present in

the autoclave chamber, but must be minimized.

Insufficient air removal, sterilizer chamber vacuum

leaks and poor steam quality (excess non-condensable

gases) are the most common causes of sterilization

failures.

空气是蒸汽灭菌的最大障碍。灭菌腔窒和装载物必须在直接蒸汽接触前排除空气，才能进行灭菌。在蒸汽灭菌柜中，这是通过灭菌前的一系列真空脉动操作完成的（预调节阶段）。少量空气会始终存在于蒸汽灭菌腔内，但必须尽量减少。排气不充分、灭菌腔真空泄漏及蒸汽质量差（不凝气过量）是最常见的灭菌失败因素。

6. Drying 干燥

Wrapped items must be dry before they can be

aseptically removed from the sterilizer. Condensation

is the natural result of steam contact with the cooler

surfaces of the load during the heating and exposure

phases. The presence of condensation (wet packs or

pouches) can cause re-contamination of the load when

removed from the sterilizer. A steam sterilizer dries the

load after sterilization by drawing a deep vacuum in

Steam Sterilization Basic Cycles

the chamber (post- conditioning phase). A vacuum

level of 1.0 to 2.0 psia (6.9 to 13.8 kPa) is

recommended for efficient drying. At 1.0 psia (6.9 kPa)

chamber pressure, water boils at 38.7°C (101.7°F).

Therefore, the condensate will boil and be removed as

steam through the sterilizer’s vacuum system. The

energy required to boil the condensate comes from the

load itself. As the temperature of the load cools due to

evaporation of the condensate, evaporation (drying)

decreases. When the load temperature cools to the

boiling point of water at the drying vacuum level,

drying is negligible. Adding further drying time past

this point will not provide any further drying. Optimal

load drying times depend primarily on load density and

packaging. Due to their low density, plastic and rubber

items may require additional drying, as they cool

rapidly (pulsed air or heated pulsed air drying

post-conditioning processes). The amount of residual

moisture in a package can be determined by weighing

the package before and after the sterilization process.

Typically, verification of the absence of visible water

droplets on or in the package is sufficient.

包裹物品必须经过干燥后才能从灭菌柜内无菌地取出。冷凝是加热和暴露阶段中蒸汽接触物品冷表面的自然结果。从灭菌柜取出物品时，冷凝水的存在（湿包装或呼吸袋）可能引起二次污染。蒸汽灭菌通过在腔室内形成深度真空来进行灭菌后的干燥（后调节阶段）。有效干燥推荐的真空水平为1.0 -

2.0 psia (6.9 - 13.8 kPa)。腔室在在1.0 psia (6.9 kPa)下，水的沸点为38.7°C (101.7°F)。因此，冷凝水将气化，并以蒸汽形式从真空系统排出。使冷凝水气化的能量来源于物品本身。随着物品在冷凝水气化过程中降温，气化（干燥）变缓。当物品温度降到干燥真空水平对应的水沸点时，干燥速率就很慢，可忽略了。在此之后延长干燥时间意义不大。最优装载干燥时间主要取决于装载密度和包装。由于其低密度，塑料和橡胶物品可能需要额外的干燥，因为它们冷却迅速（脉动空气或加热脉动空气干燥，后调节过程）。通过对包装在灭菌前后的称重，可以计算出包装的剩于湿分量。通常来说，肉眼检查包装上或内部不存在可见水滴就足够了。

蒸汽灭菌基本周期

Steam sterilization cycles typically consist of three

phases:

蒸汽灭菌周期通常包含三个阶段：

1. Pre-Conditioning: during this phase, air is

removed from the chamber and the load is

humidified by means of alternating vacuum and

pressure pulses.

预调节：此阶段时，空气从腔室排出，通过真空和压力脉动交替使装载加湿。

2. Exposure: during this phase, the chamber

temperature is raised to and held at the

programmed sterilizing temperature for the

programmed exposure time (both are user

selectable). The exposure also may be controlled

by accumulated F0 for liquids if a load probe and

appropriate sterilizer controls are used. Refer to

point #7 in common mistakes section below for

more information on F0.

暴露：此阶段时，腔室温度升高至程序设定的灭菌温度并保持设定的暴露时间（二者均可由用户选择）。如果采用了装载探头和适当的灭菌控制，液体的暴露也可以通过累积F0值来控制。更多F0值的信息参照以下常见错误部分的第#7点。

3. Post-Conditioning: during this phase, dry goods

loads are cooled and dried or a liquids load is

cooled. The chamber pressure is brought to

atmospheric.

后调节：此阶段时，干燥物品装载被冷却、干燥，或者液体装载被冷却。腔室压力回到常压。

Over the years, various cycles have

been developed for different

applications. It is critical that the

proper cycles be used.

过去几年间，针对不同的应用开发出了不同的周期。采用适当的周期也很关键。

• A basic gravity cycle (cycle without pre-vacuum) can

be used for items such as unwrapped metal

components, glassware, or non-porous items that do

not entrap air.

对于那些不滞留空气的物品，如未包裹的金属器件、玻璃器皿或非多孔物，可以采用基本重力循环（无预真空循环）。

• Liquids require modified gravity cycles to prevent

liquid loss from boiling over. Liquids in open or vented

containers or in bottles with loose caps can be

processed in a “basic” liquid cycle (with slow exhaust).

The cooling (exhaust) phase of this cycle allows for the

chamber to slowly return to atmospheric pressure to

prevent boil-over as seen in Figure 3. Nominal liquid

loss due to evaporation during the slow exhaust phase

is typically 10 to 15%. The time required for the slow

exhaust phase can vary considerably depending on the

volume of liquid per container and per load. Larger

volumes require slower exhaust rates. Use of a load

probe and F0 exposure control is recommended.

Vented containers only are to be used with this

process.

液体物品需要采用改进的重力循环来防止沸腾溢出造成液体损失。开口或通气容器中的液体，或松盖的瓶装液体可以采用”基本”液体循环（缓慢排气）。这个循环的冷却（排气）阶段使腔室缓慢地回到常压，以防止图3所示的沸腾溢出。缓慢排气阶段的气化造成的名义液体损失通常在10-15%。根据各容器液体体积和装载不同，缓慢排气阶段的时间大不相同。更大体积需要更慢的排气速率。推荐采用装

载探头和F0暴露控制。通气容器仅用于这种过程。Figure 3. Typical liquid cycle chamber pressure at

121°C (250°F).

图3. 121°C (250°F)下典型的液体循环腔室压力

Liquids are at or near boiling temperature at the end of

a slow exhaust cycle and must be allowed to cool

before the load can be safely removed from the

sterilizer. Liquids in sealed containers require an air

overpressure cooling cycle to prevent explosion of the

container(s) during the cooling phase or unloading

process as seen in Figure 3. Clean, dry compressed air

(process air) is admitted to the sterilizer chamber at the

end of the exposure phase and controlled at a pressure

higher than the pressure of saturated steam at the

temperature of the load probe. As the air flows over the

load, the load is cooled and the chamber pressure starts

to drop due to condensation of steam in the chamber.

The supplied compressed air flow rate must be

sufficient to maintain overpressure during the entire

cooling phase. This “Air Cooling” process is highly

recommended for sterilization of liquids in sealed OR

vented containers because it eliminates evaporation

and boil-over during the cooling phase. In addition,

liquids can be cooled to a temperature safe for

handling (60°C to 80°C (140°F to 176°F)) during the

process by flowing water through the sterilizer jacket

during the cooling phase. The load can be safely

removed immediately upon cycle completion. The

American Society of Mechanical Engineers (ASME)

pressure rating of the sterilizer limits the

amount of overpressure than can be

utilized. Fill volume has a significant effect

on the internal pressure of the sealed

container. The lower the fill volume, the

lower the internal pressure will be due to

compression of the air in the head space of

the container. The approximate internal

pressure of a sealed container can be

calculated using Robert Beck’s equation.6

液体在缓慢排气循环结束时温度接近或在沸点，因此在从灭菌柜中安全地取出物品前，必须可以进行冷却。密封于容器的液体需要加压冷却循环，以防止容器在冷却阶段或卸载过程中爆裂，如图3。

洁净、干燥压缩空气（工艺用气）在暴露终点通入灭菌腔室，并控制在比装载探头温度下饱和蒸汽压更高的压力。随着空气流经装载物品，物品被冷却，并且由于蒸汽在腔内冷凝，腔室压力开始下降。压缩空气的供气流速必须足以维持整个冷却阶段的过压。密封或通气容器中液体灭菌强烈推荐采用这一“空气冷却”过程，因为它可以避免冷却阶段的气化和沸腾溢出。另外，在冷却阶段，通过从灭菌夹套中流经冷却水可以将液体冷却至可安全操作的温度(60°C to 80°C (140°F to 176°F))。循环结束时，装载物品可以立即安全地取出。美国机械工程师协会（ASME）的灭菌柜压力等级给出了可以采用的过压限制。密封容器的灌装体积对其内部压力有重大影响。灌装体积越小，由于空气挤压产生的容器顶空内部压力也越小。密封容器的大致内部压力可以由Robert Beck方程计算。6

• Since air is generally a deterrent to sterilization, a

“Prevacuum” cycle (alternating vacuum and pressure

preconditioning pulses) is recommended for all loads

other than liquids (Figure 4).

由于空气会阻碍灭菌，除了液体的灭菌，所有其它物品的灭菌都推荐采用“预真空”循环（真空和压力预调节脉动交替）（图4）。

Figure 4. Typical prevacuum cycle chamber pressure at

121°C (250°F).

图4. 121°C (250°F)下典型预真空循环的腔室压力

Measuring Performance 性能测定

Several methods can be used to verify the efficacy of Biological Indicators (BIs) and Chemical Indicators

the sterilization process. Typical methods use (CIs) that are placed in worst case positions in the load

and/or in test packs.

灭菌过程的效果可以用几种方法来确定。典型的方法是将生物指示剂（BIs）和化学指示剂（CIs）置于装载和/或测试包装中的最差位置。

• Biological indicators provide the best test for

sterilization and are used to establish the efficacy of

the cycle. In this category, we can find:

生物指示剂是灭菌测试最好的方式，用于确立循环的效果。在这个类别中，我们可以找到：

- Inoculated spore test strips. The strips must be

aseptically transferred to an incubated growth media

soon after the sterilization process is complete.

- 接种孢子试纸。试纸必须在灭菌过程结束后以无菌操作迅速转移至培养基并培养。

- Self-Contained Biological Indicators (SCBI) (Figure

5). Because they are self-contained, SCBI’s reduce

chances for false positives due to poor aseptic transfer

technique. They are typically used to monitor the

effectiveness of steam sterilizing process.

-自含式生物指示剂（SCBI）（图5）。因为它们是自含式的，SCBI降低了由于无菌转移技术不当造成的假阳性机率。它们通常用于监测蒸汽灭菌过程的有效性。

- Glass ampoules are also used when the indicators

must be placed in a liquid product to be sterilized

(culture media as an example).

- 当指示剂必须置于液体产品中进行灭菌时（例如培养基），也可以采用玻璃安瓿瓶。

Figure 5. Self-Contained Biological Indicators (SCBI).

图5. 自含式生物指示剂（SCBI）

• Chemical indicators provide immediate proof of

steam penetration (not necessarily of sterilization). In

this category, we can find:

化学指示剂提供了蒸汽穿透的即时指示（不一定是灭菌）。在这个类别中，我们可以找到：

- Autoclave tapes that show the process has occurred

with no correlation to time/temperature.

湿热灭菌试纸，表明灭菌过程已经进行了，但不说明时间/温度的情况。

- Chemical integrators that are correlated to time and

temperature. These particular indicators can help

reduce cycle development time by providing

immediate indication of sterilization efficacy.

与时间和温度关联的化学指示剂。这种专用指示剂可以提供灭菌效果的即时指示，从而有助于减少循环开发时间。

- Steam penetration studies: temperature sensors can be

placed in hard to reach locations to provide indication

of steam penetration.

蒸汽穿透研究：温度探头可以置于难以到达的位置，以指示蒸汽穿透情况。

Prevacuum sterilizers should be tested routinely for air

leaks and air removal capability. Automatic chamber

leak tests (vacuum hold tests) are typically provided in

the software of modern prevacuum sterilizers, and

should be run daily after a warm-up cycle. The

sterilizer chamber is evacuated to the limit of the

vacuum system (<1.0 psia or 6.9 kPa) and the chamber

and associated piping are isolated (valves closed) for a

hold period. The difference between the absolute

pressure at the beginning and end of the hold period is

the total leak rate. The leak rate should be < 1.0 mm

(0.039 inches) Hg/minute(2). Hold time varies per

procedures, from 10 to 30 minutes. It should be noted

that a pressure rise during the hold phase is not always

indicative of a chamber vacuum leak. Wet steam can

cause condensate to be introduced into the chamber

during the test preconditioning pressure pulses. Any

condensate in the chamber will evaporate at the test

vacuum level, causing a rise in chamber pressure. One

practical way to determine the source of the pressure

rise is to observe the leak rate during the vacuum hold

phase with an absolute pressure gauge connected to the

sterilizer chamber. An air leak rate will be fairly

constant over the vacuum hold period. A pressure rise

from evaporation of condensate will result in a high

rate at first, and then will diminish as the condensate is

evaporated.

预真空灭菌柜应进行日常的空气泄漏和排气能力检测。现代化预真空灭菌柜通常在软件中提供了自动腔室泄漏检测（真空保持测试）程序，并且应该在每天热机循环后进行。灭菌腔室排空至真空系统极限（<1.0 psia或6.9 kPa），然后腔室和相连管路被隔离（阀门关闭）并保持一段时间。保持时间开始和结束时的绝对压差就是总的泄漏速率。泄漏速率应 <1.0 mm (0.039 inches) Hg/min(2)。不同程序的保持时间不同，一般从10-30 min。需要注意的是，保持期间压力上升并不一定预示着腔室泄漏。在测试预调节压力脉动时，湿蒸汽可能带入冷凝水至腔室内。任何腔室内的冷凝水在真空测试压力下都会气化，造成腔室压力上升。一种确定压力上升原因的方法是，用一支与灭菌腔室相连的绝对压力表观察真空保持阶段的泄漏速率。空气泄漏速率在真空保持期间是恒定的。冷凝水气化造成的压力上升起初速率较快，然后随着冷凝水的气化，速率会逐渐降低。

In addition to the vacuum hold test, a challenge test

such as the Bowie-Dick test should be run periodically

as seen in Figure 6. The challenge test is different from

a vacuum hold test in that it challenges the sterilizer to

remove the air from within a dense package and

displace the air with steam. It is fairly uncommon for a

sterilizer to pass a vacuum hold test and fail a

challenge test, but it has been observed. Insufficient air

removal during the prevacuum phases and/or poor

steam quality (excess entrained non-condensable gases,

superheated steam or wet steam) can cause this

anomaly. Challenge tests are temperature specific, and

tests designed for 132°C (270°F) will not function

properly in a 121°C (250°F) test cycle.

除真空保持测试外，也应定期进行挑战测试，如Bowie-Dick测试，如图6所示。挑战测试不同于真空保持测试，它挑战的是灭菌柜从密集包装中排除空气并用蒸汽置换这些空气的能力。灭菌柜通过真空保持测试但通不过挑战测试的情况并不多见，但是有这种情况。预真空阶段排气不足和/或蒸汽质量差（不凝气体过量，过热蒸汽或过湿蒸汽）可能造成这种异常。挑战测试是有特定温度的，设计于132°C (270°F)的测试在121°C (250°F)的测试循环中不会奏效。

Figure 6. Bowie-Dick test pack.

图6. Bowie-Dick测试包

The Ten Most Common Mistakes in Steam Sterilization

蒸汽灭菌中最常见的十种错误

Most mistakes regarding the programming and

operation of typical steam sterilizers are related to the

basic principles of steam sterilization.

常见蒸汽灭菌柜的大多数程序和操作错误都与蒸汽灭菌的基本原理有关。

1. Containers with closed valves, empty glass bottles

with tightened screw caps or secured aluminum

foil are placed in the sterilizer.

灭菌柜中放入关闭阀门的容器、旋紧瓶盖或紧扣铝盖的空玻璃瓶。

As a result, steam cannot directly contact the inside

surfaces and sterilization does not occur. This problem

can be resolved by assuring that all items in the

sterilizer have a way for the steam to get in and the air

to get out. If there is uncertainty about whether an

item’s configuration, set-up, packaging, or orientation

will allow adequate steam penetration, a thermocouple,

chemical and/or biological indicator can be placed

inside the item to be certain.

因此，蒸汽不能直接接触到内表面，不能进行灭菌。这一问题可通过确保灭菌柜中的物品有蒸汽进入和空气排出的通道来解决。如果不确定物品的配置、设置、包装或方位是否允许充分的蒸汽穿透，那么可以通过在物品内部放置热电偶、化学和/或生

物指示剂来确定。

2. Pouched and/or heavily wrapped items are tightly

packed in the chamber.

袋装和/或严密包裹的物品紧实地装载在腔室内。

As a result, air may remain trapped in the items after

the preconditioning phase and prevent sterilization.

Items should not be overwrapped, and sufficient space

should be maintained between load items. The

preconditioning vacuum and pressure pulses must be

set correctly to attain complete air removal from the

load. Typically, four (or more) preconditioning vacuum

pulses should be programmed to reach at least 28 in

(711 mm) Hg vacuum ((1.0 psia or 6.9 kPa (absolute))

to assure sufficient air removal for worst case loads.

Some very dense loads may require a short (2 to 5 min)

hold phase at peak preconditioning vacuum to allow

time for trapped air to be removed. Preconditioning

pressure pulses should be programmed for 3 to 5 psig

((21 to 34.5 kPa (gauge)). Higher pressures set for

prevacuum pressure pulses can result in an excessive

amount of superheat and difficulties with temperature

stabilization during the first few minutes of the

exposure phase.

因此，预调节阶段后空气可能仍然滞留在物品中，阻碍了灭菌。物品不应过度包裹，且应在物品间留有足够的空间。预调节真空和压力脉动必须正确地设定，以完全排除装载内的空气。通常，应该程序设定达到28 in (711 mm)汞柱((1.0 psia or 6.9

kPa (绝对压力))真空且四次(及以上)预真空脉动，以确保最差装载情况下空气排除充分。有些非常密集的装载在预调节真空峰值时可能需要一段较短（2-5

min）的保持时间，从而排除滞留的空气。预调节真空脉动应设定为3~5 psig ((21~34.5 kPa (表压))。预调节压力脉动可能造成暴露阶段前几分钟时的过热和温度稳定困难。

3. Heavier items are placed on top shelves.

较重的物品置于货架顶层。

Water droplets and/or stains are observed on the

outside of wrappers of items placed on the mid to

lower shelves after the sterilization cycle is complete.

Because the items are not dry, they cannot be

aseptically removed from the sterilizer. Condensation

is the natural result of steam contact with the cooler

surfaces of the load. The condensate will fall from

shelf to shelf. The denser the load item, the more

condensate is created. Therefore, place heavier items

on the bottom shelf. In addition, consider placing a

cotton sheet or lint free towels on each sterilizer

loading cart shelf prior to loading to allow the

condensate to be absorbed. This also aids in drying. As

the condensate wicks into the sheet or lint free towels,

the condensate surface area is greatly increased and

evaporates much more rapidly during the drying phase

than the same amount of condensate in a droplet or a

puddle.

灭菌循环结束后，置于中间和低层的物品包装外表面可以看到水滴和/或污迹。由于物品不是干燥的，不能从灭菌柜中无菌地取出。冷凝是蒸汽接触物品冷表面时的自然结果。冷凝水会从货架上滴下。物品的放置越密，产生的冷凝水越多。因此，将更重的物品放置于低层。另外，在装载前考虑在各灭菌装载货架上放置纯棉布或无绒毛巾来吸收冷凝水。这也有助于干燥。由于冷凝水浸入棉布或无绒毛巾，冷凝水表面积大大增加，与同量冷凝水滴相比干燥阶段的气化速率也大大提高。

4. Load is too dense or items are positioned

incorrectly in the load.

装载过于密集或物品在装载中摆放位置不对。

As a result, wet or damp items are observed at the end

of the cycle. Wrapped items positioned so that

condensate is allowed to collect will not be dried.

Items should be positioned so that the condensate is

allowed to flow downward. Items (wrappers, pouches,

filters, or other porous biological barriers) that remain

wet at the end of cycle cannot prevent contamination

of the load when removed from the sterilizer. As the

load cools outside the sterilizer, the water in the

wrapper will be drawn into the wrapped item. Any

contamination that is present in the environment can be

drawn through the sterile barrier along with the water.

There are numerous other possible causes for wet loads.

The most common are:

因此，循环结束时发现物品变得潮湿。放于会收集冷凝水的位置的包裹物品不会被干燥。物品的放置应允许冷凝水向下流动。物品（包裹、呼吸袋、过滤器或其它多孔生物滤膜）由于在循环结束时仍是湿的，在从灭菌柜取出时不能避免装载的污染。由于在灭菌柜外冷却，包裹上的水会浸入到被包裹

的物品中。任何环境中的污染源都可能随水一起穿过无菌屏障。还有许多其它原因可能会造成装载潮湿，最常见的是：

a. Insufficient drying vacuum level or time

programmed

程序设计干燥真空水平或时间不够

b. Rubber or plastic items in pouches (i.e., rubber

stoppers, plastic tubing) may require additional

drying (a pulsed air or heated pulsed-air drying

process is recommended for these items)

袋装的橡胶或塑料制品（如橡胶塞、塑料管）可能需要额外的干燥（对于这些物品，推荐采用脉动空气或脉动热空气干燥工艺）

c. Wet steam

湿蒸汽

While there is no single solution to eliminating wet

loads, it’s likely that experimenting with drying time,

repositioning items, reducing load density, modifying

cycle settings, and investigating steam quality will

resolve the problem.

由于没有哪个方案一定能有效消除装载潮湿，很可能通过试验干燥时间、重新放置物品、降低装载密度、改进循环设置和调查蒸汽质量可以解决这个问题。

5. Pouches are placed flat on the sterilizer shelves or

stacked on top of one another.

呼吸袋平放于灭菌柜货架上或叠放在一起。

As a result, pouches may have water droplets inside

and cannot be aseptically removed from the sterilizer.

Typical cause is when the condensate naturally created

when steam penetrates the pouch and contacts the

surface of the item within is not removed during the

post-conditioning drying phase. Pouches should be

spaced properly and placed in rack that holds the

pouch on its edge (Figure 7) to prevent pooling of the

condensate inside the pouch. Pouches should not be

placed flat on the sterilizer shelf. Pouches should not

be overloaded. Remember that more mass means more

condensate.

因此，呼吸袋可能会有水滴在内部，不能从灭菌柜中无菌地取出。通常原因是蒸汽穿透呼吸袋和接触物品表面时会自然形成冷凝水，这些冷凝水在后调节干燥阶段没能除去。呼吸袋应留有足够的空隙，并放置于搁架上（图7），以防止呼吸袋内部冷凝水聚集。呼吸袋不应平放于灭菌架上。呼吸袋不应叠放。记住，物品越多，冷凝水越多。

Figure 7. Proper position for pouches.

图7. 呼吸袋正确的放置方式

Sufficient drying vacuum level and time should be

programmed to allow for complete evaporation of the

condensate. Wet steam should be corrected. Double

pouching may require additional prevacuum pulses

with dwell time at maximum vacuum and increased

drying time. Doubled pouches should never be

assembled so that the items inside cannot be seen.

Pouch flaps should not be folded over.

程序应设定足够的干燥真空水平和时间，使冷凝水完全气化。湿蒸汽应调节为饱和蒸汽。双层呼吸袋可能需要额外的预真空脉动，在最大真空时保持一定时间并增加干燥时间。双层呼吸袋绝不应以看不到内部物品的方式组装。袋口不应折叠起来。

6. Liquids in vented containers are placed in a deep

pan to catch boil-over (slow exhaust cycle).

通气容器中的液体放置于深托盘内，以收集沸溢的液体（在缓慢排气循环）。

The pan will hold water and it will hold air. The steam

cannot contact the surfaces within the pan because of

the trapped air, and they will not be sterilized. The

solution is to eliminate the pan and adjust the sterilizer

slow exhaust rate to prevent boil-over. A shallow pan,

less than 1” (25 mm) deep, can be used in the event

that a small amount of boil-over cannot be eliminated

by adjusting the slow exhaust rate.

托盘会容纳水也会滞留空气。蒸汽不能在托盘内受滞留空气的阻碍而不能接触到表面，因此达不到灭菌效果。解决方法是去除托盘，调事灭菌柜的缓慢排气速率，防止沸溢。浅托盘，少于1” (25 mm)深，可用于少量沸溢不可避免时的情况。

7. “Overcooked” Media

培养基“加热过久”

Over sterilization of media will caramelize the sugars

and render the media useless. The typical overkill

approach is not recommended for sterilization of media.

The exposure phase should be programmed to achieve

the desired SAL and no longer. Use of a load probe and

F0 exposure control is recommended for sterilization of

media in containers larger than 100 ml (3.4 oz). As

illustrated in Figure 8, F0 is a calculation of the

equivalent exposure at temperatures other than

121.1°C (250°F). As the liquid is heated, the calculated

F0 (from the load probe temperature) is accumulated

until the selected F0 exposure value (minutes) is

achieved, at which point the cycle proceeds to the

exhaust/cooling phase. For example, on the graph, the

kill rate on the same population of organisms is half as

effective at 118°C (245°F) as at 121°C (250°F).

Therefore, at 118°C (245°F), it will require twice the

exposure time to kill the same number organisms.

100 ml (3.4 oz)的容器内的培养基推荐采用装载探头和F0值暴露控制灭菌。如图8所示，F0是121.1°C

(250°F)以外的温度下计算等效暴露。随着液体被加热，计算出的F0（从装载探头温度）逐渐累积，直，此时循环进行至到达到所选的F0暴露值（分钟）排气/冷却阶段。例如，从图上可以看出，在118°C

(245°F)时同样数量微生物的杀死率是在121°C

(250°F)时的一半。因此，在118°C (245°F)时，需要两倍的暴露时间才能杀死同样数量的微生物。

A common formula for calculating the F0

value is:

计算F0值的常见公式是：

F0=∫Ldt 其中

L=100t(T−121.1)2

Figure 8. F0 as a function of temperature.

图8. 温度函数F0

培养基过度灭菌会使糖分焦化，使培养基失效。培养基灭菌不推荐采用典型的过度杀灭方法。程序应设计暴露阶段，以恰好达到所需的SAL。在大于where:

• L is lethal rate of bacterial spores

• t is exposure time, [s]

• T is exposure temperature, [°C]

• z is a constant, [°C]

其中，

• L是细菌孢子致死率

• t是暴露时间，[s]

• T是暴露温度，[°C]

• z是常数，[°C]

The constant z describes the slope of the thermal death

curve. The widely accepted value for z is 10°C (18°F)

in steam sterilization.

常数z描述热力致死曲线的斜率。对于蒸汽灭菌，广泛认同的z值是10°C (18°F)。

8. Using cold water for vacuum

pump that is too hot.

真空泵使用的冷却水过热

As a result, the vacuum pump may

not be able to reach 1.0 psia (6.9

kPa). The heart of the prevacuum

sterilizer is the water-ring vacuum

pump. The efficiency and

maximum vacuum capability of a

water-ring vacuum pump are

adversely affected by higher water

temperatures typically encountered during the summer

months. During operation, the water within the pump

is heated by mechanical friction and heat energy from

the sterilizer chamber. If the temperature of the water

inside the pump reaches 39°C (102°F) during the

preconditioning or post conditioning vacuum peak, the

water inside the pump will boil at ≤ 1.0 psia (6.9 kPa)

and cause cavitation. In this case, the recommended

preconditioning vacuum level of 1.0 psia (6.9 kPa)

cannot be achieved in the sterilizer chamber. A

common “work-around” for this situation is to change

the set point of the prevacuum pulses to a level that can

be achieved. Insufficient air removal can be the result

unless the number of vacuum pulses is increased,

causing longer cycle times and less effective air

removal. Internal pump temperatures higher than 39°C

(102°F) are often observed during the summer months

if the water supplied to the pump is not cooled. Chilled

water is ideal, but typically too expensive to use in a

sterilizer vacuum pump arrangement in which the

water flows from the vacuum pump to drain. The

recommended solution is a recirculation/cooling

system for the vacuum pump water that uses chilled

water in a closed loop heat exchanger. This

configuration is eco-friendly as it saves a significant

amount of water. In addition, the vacuum pump

efficiency is not subject to seasonal water temperature

fluctuations.

因此，真空泵可能达不到1.0 psia (6.9 kPa)。真空灭菌的核心是水环真空泵。在夏季期间经常发生水温过高，这可能会对水环真空泵的效能和极限真空度造成不良影响。在操作期间，泵内的水由于机械摩擦和灭菌腔室的热量被加热。如果预调节或后调节真空期间泵内的水温达到39°C (102°F)，泵内的水在≤ 1.0 psia (6.9 kPa)真空下会沸腾，造成气蚀。这种情况下，灭菌腔室内就达不到推荐的真空水平1.0 psia (6.9 kPa)。这种情况常见的“应对措施”就是改变预真空脉动的真空设定值，降为可达到的水平。这可能造成排气不足，除非增加真空脉动次数，从而造成循环周期延长，排气效率下降。如果真空泵进水未经冷却，夏季时经常发现泵内温度高于39°C

(102°F)。冷冻水是理想选择，但对于灭菌柜真空泵来说成本过高，因为真空泵的出水直接排放。推荐措施是真空泵配循环冷却水系统，采用冷冻水和闭路循环换热器。这种配置可节省大量的水，经济环保。此外，真空泵效率不受水温季节性波动的影响。

9. Load probe is available, but not used.

有装载探头但没有使用。

Most modern sterilizers include (optional) an RTD

load probe and F0 exposure control for use in liquids

sterilization, but many times the probe is not used. If

equipped with a load probe, the exposure can be

controlled by the temperature of the liquid rather than

the temperature in the drain line. Without the load

probe, the temperature of the liquid is not known and

can only be estimated, resulting in inadequate

(non-sterile) or excessive F0 (overcooked). The load

probe should be placed in a container of water

approximating the volume of the largest volume of

liquid being sterilized. Load probe control/F0 must

then be selected in the sterilizer control settings.

多数现代灭菌柜配备（可选）了一个RTD装载探头和F0暴露控制，用于液体灭菌的控制，但是许多情况下这一探头都没有被使用。如果配备了装载探头，暴露可以依据液体温度来控制，而不是排放管路内的温度。如果没有装载探头，液体温度是未知的，只能估算，因此造成灭菌不充分（非无菌）或过度（加热过久）。装载探头应置于含水容器内，容器内盛水体积约为待灭菌液体的最大体积。装载探头控制/F0必须在灭菌柜控制设置中选择。

10. Pressure/vacuum rate control is available, but not

used.

卸压/抽真空速率控制可用，但没有使用。

Most modern sterilizers include (optional) rate control

for the vacuum and pressure ramps, but many times the

rate control is not used. When no pressure rate control

is applied steam will enter the chamber at maximum

velocity during the preconditioning pressure pulses,

which creates a superheat problem and EN285

compliance problems as discussed earlier. Slowing the

pressure rate allows time for superheat to dissipate

during the ramp up.

多数现代灭菌柜具备（可选）抽真空和卸压速率的控制功能，但许多情况下该速率控制并没有被使用。如果没有采用卸压速率控制，在预调节卸压阶段，蒸汽将以最大速率进入腔室，从而造成过热的问题和EN285符合性问题。减缓卸压速率使卸压过程中的过热有时间分散。

When no vacuum rate control is applied the chamber

will depressurize at the maximum rate of the vacuum

pump. The typical problem associated with this is burst

pouches. Slowing the vacuum rate allows time for the

pouch internal pressure to equilibrate and prevents

bursting during the preconditioning and post

conditioning vacuum phases.

如果没有采用抽真空速率控制，腔室将以真空

泵的最大速率形成真空。这造成的常见问题是爆袋。在预调节和后调节抽真空阶段，减缓抽真空速率使呼吸袋内部压力有时间平衡，并防止爆袋。

Conclusion结论

Steam sterilization is a process that is dependent on

basic principles that are sometimes unknown or

disregarded by the sterilizer user. A large percentage of

steam sterilizer failures can be solved by logical and

practical application of these basic principles. It should

be noted that proper training for sterilizer users should

include this education. Proper wrapping and loading

techniques are critical for safe and successful

sterilization. As with any critical process equipment,

proper maintenance and calibration is essential.

蒸汽灭菌是一个遵循基本原理的过程，而这些原理常常为灭菌柜使用者所不知或忽略。大部分蒸汽灭菌问题都可以通过合理、实际地应用这些基本原理来解决。需要强调的是，灭菌柜使用者的培训应囊括这些内容。适当的包裹和装载技术对安全成功的灭菌也至关重要。与其它关键工艺设备一样，适当的维护和校准也必不可少。

References 参考资料

1. USP 35 <1035>, Biological Indicators for Sterilization, Table 1.

2. Lewis, R.G., “Practical Guide to Autoclave Validation,” Pharmaceutical Engineering, July/August 2002 for

further discussion of SAL.

3. Principles and Methods of Sterilization in Health Sciences, John J. Perkins, M.S. LL.D., F.R.S.H, Second

Edition, Eighth Printing, 1983, Chapter 6, p. 137.

4. /saturated-steamproperties-d_.

5. The European Standard EN285: Sterilization – Steam Sterilizers - Large sterilizers: 2006 + A2:2009; 8.

Performance Requirements, 8.3.1.3, pp. 15-16.

6. Beck, R.E., “Autoclaving of Solutions in Sealed Containers: Theoretical Temperature – Pressure Relationship,”

Pharmaceutical Manufacturing, June 1985, pp. 18-23.

About the Authors 关于作者

Marcel Dion is Director of Marketing for Washing and Steam Sterilization Systems in the Life

Sciences Division of Steris Corporation. Dion holds a diploma in instrumentation and control

from Levis-Lauzon CEGEP in Quebec, Canada. He was involved in designing and

manufacturing washing systems for the life sciences industry during the first 20 years of his

career. For the past 13 years, he has been responsible for developing and bringing to the market

innovative and efficient cleaning and steam sterilization systems for critical parts/components

that are involved in the drug manufacturing process. Dion has been a member of ISPE, PDA, LAMA and AALAS

organizations for several years. He can be contacted by tel: +1-418-664-7512.

Steris, 490 Armand-Paris, Quebec G1C8A3, Canada.

Wayne Parker is an independent consultant for moist heat sterilization to the pharmaceutical,

biotech, and research industries. Parker graduated from Hillsborough High School in Tampa,

FL in 1965 as a member of the National Honor Society. He attended the University of South

Florida in Tampa from 1965 to 1968. Parker served in the U.S. Air Force from 1968 to 1972 as

a Radar Maintenance NCO and continued his education through the U.S. Air Force Extension University. He was

employed by the American Sterilizer Co. (now Steris Corporation) from 1972 to 2012, when he “retired.” During

his career, Parker held numerous positions, including; field service technician, field service cGMP specialist, area

sales manager and key pharmaceutical accounts manager. He has provided training and consulting services to

numerous companies, and continues as a guest lecturer several times per year at the University of Tennessee,

Memphis, College of Pharmacy post-graduate course in aseptic processing. He has been a member of ISPE, PDA,

and AALAS organizations for many years. He can be contacted by email: autoclaveman2007@.

翻译：袁利，天津大学制药工程2014届硕，ISPE学生会员，email: scuyuanli@