Steam sterilization: Review of autoclaves, validation, instrument packing, and sterility failures

Abstract

Surgical care for the disease is required in a significant number of people worldwide. In India, approximately 4% of the population needs surgical care. In ophthalmology, lens-based surgeries account for a significant contribution to the list. In this massive volume of surgeries, a proper sterilization process is vital for the successful outcome of these surgeries. Healthcare professionals must have an in-depth understanding of the sterilization process, which helps reduce complications and prevent terminal events.

The backbone of any operating theater is the effectiveness of the Disinfection and Sterilization protocol that the hospital follows. Selecting the appropriate autoclave for the load and choosing the correct autoclave cycle parameters is vital for a complete microbiological kill. The activities of the Central Sterile Supply Department (CSSD) must be continuously monitored to ensure a steady supply of sterile goods required for surgeries. The physical, chemical, and biological parameters are monitored in the autoclaving process based on recommendations from the International Standards Organization, the Association for the Advancement of Medical Instrumentation, and European Norms. Although plasma sterilization is emerging, steam remains the dominant form of sterilization in hospital settings worldwide.[1] This review article outlines the various types of steam autoclaves used in practice and discusses validation methods to address common causes of sterilization failures.

Overview of Steam Sterilization

Most hospitals worldwide still prefer steam sterilizers for sterilization because steam has been well documented to destroy all forms of microorganisms, including spores effectively. Steam also has a significant penetrating ability into fabrics and surgical instruments with a lumen.[2] In a steam sterilizer, the physical parameters of time, temperature, and pressure determine the sterilization quality. The atmospheric pressure at sea level is 1.01 bar or 14.7 pounds per square inch (psi). As water is heated in a closed vessel, the vapor pressure gradually increases. When vapor pressure equals atmospheric pressure, water begins to boil, generating saturated steam at a higher temperature. Steam produced at 1 bar (14.7 psi) and 100 degrees Celsius is less effective compared to steam generated under an increased pressure of 15 psi and 121 degrees Celsius as the higher temperature of the steam is necessary to kill microorganisms.[3] There are two main variables, namely, temperature and pressure, in steam sterilization, listed in Table 1. The dryness fraction is defined as the amount of water present in the steam. An ideal dryness fraction of 97% is optimal for effective microbial kill, meaning only 3% water is available in this steam.[4,5]

Table 1.

Parameters for steam sterilization

Steam Quality		Dry Saturated Steam (Dryness fraction >97%)		Dry Saturated Steam (Dryness fraction >97%)
Temperature		121 degrees Celsius		134 degrees Celsius
Pressure		15 psi		21 psi
Time		21 min		5 min

Steam Sterilization – Mechanism of Action

When saturated steam condenses on the surface of instruments, it generates latent heat that kills microorganisms by destroying their cell walls, enzymes, and other structural proteins.[6]

Steam Sterilizers

Depending upon the hospital’s needs and volume of the load to be sterilized, autoclaves are available in various volumes and shapes. They are available in horizontal double-door or single-door models. The double-door models are helpful as they open directly into the sterile zone of the operating theater, facilitating the unidirectional flow of instruments. The sterilizers can also be tailored to meet the needs and space available in the CSSD area, as well as the type of packaging materials used. Using rigid metal containers requires more space than using individual packing units.

Generally, steam sterilizers are classified based on the mechanism used to create a vacuum inside the autoclaves. The three types are Class B, N, and S autoclaves. Creating a vacuum in the autoclave chamber is crucial for the sterilization process as residual air prevents saturated steam from condensing on the surface of the instruments, thereby rendering the sterilization ineffective. In Class N autoclaves, there is no active mechanism to create a vacuum; the steam generated is allowed to enter the chamber. As steam enters the chamber, it gradually pushes the air out of the autoclave through a venting system of valves placed at the bottom of the chamber (as air is heavier than steam). The main disadvantage of a Class N type sterilizer is that the steam cannot effectively evacuate the air trapped inside the lumens of surgical instruments or air trapped in the folds of densely packed linen. Hence, this type is used only for laboratory glassware and solid “naked” instruments. Class N modes are typically vertical units as the gravitational effect also facilitates air removal. In the Class S autoclave, vacuum is created by using a vacuum pump to evacuate air and steam from the inner chamber of the autoclave. Here, the vacuum pump is used twice: once before the sterilization phase of the autoclave cycle and once during the drying phase, to remove air and steam, respectively, from the chambers, thereby preventing the formation of wet packs. Class B autoclaves utilize a vacuum pump to generate multiple steam-pressure pulses during the presterilization, sterilization, and drying phases of the autoclave cycle. This process ensures the complete removal of air and steam from the autoclave, allowing steam to penetrate the porous/hollow instrument load[4,5] [Table 2].

Table 2.

Classification of steam sterilizers[4,5]

		Class N Autoclaves		Class S Autoclaves		Class B Autoclaves
Air removal		Steam pushes the air to the bottom of the autoclave, where it exits through the outlet. (Gravity Displacement)		The vacuum pump sucks the air/steam out of the inner chamber of the autoclave. (Vacuum-assisted)		Multiple Steam-Pressure Pulses remove air/steam from the inner chamber in the sterilization process. (Vacuum-assisted)
Instruments		Solid instruments only		Solid/Porous/Hollow instruments		Solid/Porous/Hollow instruments
Wrapping		Unwrapped only		Wrapped or unwrapped		Wrapped or unwrapped
Settings		121 degrees C, 15 psi for 21 min		121 degrees Celsius, 15 psi for 21 min (or) 134 degrees Celsius, 21 psi for 5 min

Ophthalmic surgical instruments have lumens; hence, a Class B or S-type autoclave is the preferred choice due to its ability to effectively create a vacuum and ensure steam penetration, thereby providing reliable sterilization. The multiple Steam Flush-Pressure pulses above atmospheric pressure created by the Class B Autoclave Vacuum pump ensure rapid, adequate, and complete removal of trapped air from the hollow instruments and linen in the load. The positive pressure created in the chamber ensures that no air enters the chamber in the sterilization phase. The typical sterilization parameters are 121°C at 15 psi for 21 minutes or 132°C at 21 psi for 5 minutes.[6,7]

It is good practice to use Class B autoclaves for ophthalmic surgeries as these procedures involve several hollow instruments such as phacoemulsification tubing and I/A handpieces. Although Class S autoclaves are capable of air removal, there is considerable variability in their manufacturing, and their validation processes are not yet standardized.

Autoclave Cycle

Every clinician/paramedical staff member must be aware of the autoclave cycle, which will help them troubleshoot during autoclave failure and maintain the system. The three stages of a steam sterilization cycle are as follows:

Purge phase: The saturated steam generated by the boiler gradually enters the chamber, thus increasing the pressure and temperature inside the chamber, and forcing the air out. In a Class B or S autoclave, the steam flush-pressure pulses are activated here to remove the air.

Sterilization phase: In a Class N and S autoclave, once the pressure reaches 15 psi, the outlet valve is closed, allowing the temperature to rise to 121 degrees Celsius and be maintained for 21 minutes. This can be done either manually or through the automated program built into the autoclave. In a Class B autoclave, the sterilization phase consists of multiple steam flush-pressure pulses, which enable the steam to penetrate all areas of the surgical load.

Exhaust (Dry) phase: Once the desired temperature, time, and pressure are reached, the chamber outlet is opened, allowing the steam to exit the chamber. In a Class N autoclave, the pressure inside the chamber displaces the air. In contrast, in Class B and S types, the active vacuum pump sucks the residual steam out of the chamber to ensure that no steam condenses on the surface or inside the pack, thereby preventing wet packs.

Validation, Monitoring, and Verification of Steam Sterilization

Validation and verification refer to a process that shows, with evidence, that a particular device can achieve its intended purpose and perform to the standards specified by the manufacturer. For example, the sterilizer can create a complete vacuum, or the required temperature and pressure can be attained.[8,9] Sterilization is a process whereby just looking at a pack will not indicate whether the pack is sterile or not; hence, a system of verification is needed to ensure the process has been completed, such as a chemical indicator fixed on the outside of the pack.[10]

The Principles for Validation of Sterilization are[8,9,10]

1. Installation Qualification: Performed by the manufacturer during the installation of the autoclave to check the electrical supply for the unit and inspect the overall installation of the autoclave, including physical inspection of the door hinges, valves, and gaskets. The manufacturer issues an installation certificate after completion of the checks.

2. Operational Qualification: This ensures that the critical measurement devices in the autoclave are working well. The manufacturer performs this check after the autoclave is installed to verify and to confirm the accuracy of the vacuum, pressure, and temperature gauges. The manufacturer then issues a Calibration certificate.

3. Performance Qualification: This demonstrates if the desired sterilization is achieved in the entire load by monitoring the physical and microbiological parameters. The physical parameters consist of monitoring the mechanical parameters and chemical indicators. During every autoclave cycle, mechanical parameters, including time, temperature, and pressure, must be recorded using either an analog or digital system, and this information must be documented in the logbook. The various performance indicators for sterilization are listed in Table 3, with the biological indicator serving as the ultimate verification of the sterility of the load.

Table 3.

Performance Indicators for Steam Sterilization

Indicator Type		Instructions
Class 1 indicator		Applied to the exterior of every sterilization load, it serves only to distinguish between processed and unprocessed packs. It does not confirm the adequacy or success of sterilization.
Class 2 indicator: (process challenge device - bowie-dick test)		Ensures adequate air removal from the Autoclave inner chamber
Class 5 chemical integrator		Integrator of three parameters, namely Time, Temperature and Pressure.
Class 6 indicator :(process challenge device-helix test)		Ensures adequate Stream penetration into hollow instruments.
Biological indicator		The most definite indicator, ensuring the complete success of sterilization

Bowie–Dick Indicator

The Bowie–Dick (BD) test is classified as a Class 2 indicator, ensuring that a proper vacuum has been achieved in an autoclave before it is used for the day. The BD test is applicable only for autoclaves with a dynamic air removal process, such as Class B and S types, and is not applicable in Class N autoclaves. The BD test strip detects whether the vacuum pump of the sterilizer is working efficiently to remove air from the autoclave, enabling steam to condense evenly on the load. The test strip is placed in the center of a predetermined quantity of folded, clean, and preconditioned cotton surgical towels, which are positioned in the most challenging part of the autoclave for steam exposure, typically near the doors or at the bottom racks, and run at 134°C for 5 minutes.[4,11] The test should be done every day before processing a load in the autoclave. More miniature disposable test packs are now available to replace the traditional method of placing paper into a stack of linen to test the efficiency of the vacuum pump in a Class B or S autoclave.[12] These devices are developed to simulate a load of surgical instruments that is to be sterilized.[11,13,14] A positive BD test means that the test sheet inside the pack changes color evenly, indicating that there has been complete removal of air from the autoclave. The occurrence of an uneven color change on a BD strip or black spots appearing in the test paper indicates incomplete removal of air, and this residual air has prevented the steam from condensing on the test strip. In the event of a failed BD test, the autoclave should not be used, and the cause of the negative test result must be investigated and corrected before it is put into use.[4,11,15] The various causes of BD test failure[16] are outlined in Table 4.

Table 4.

Bowie–Dick test troubleshooting[16]

Cause for Failure		Description		Solution
Air Leak		An air leak is present; the steam cannot penetrate the load		Run a vacuum leak test
Unwanted Condensation		Condensation in the steam jacket of the autoclave can lead to cold spots.		Check the steam valves in the autoclave.
Faulty pack		The pack is faulty		Check the expiry date and storage conditions
No warm-up cycle		A warm-up cycle enables the sterilizer to reach the temperature		Run a 5-minute sterilization cycle before the BD test
Incorrect procedure		BD pack works only in an empty autoclave chamber		Ensure an empty chamber and proper temperature

Class 5 Chemical Integrator

Steam sterilization is an integral process that requires the correct parameters of steam pressure, time, and temperature[17] to achieve a complete kill of microorganisms. The Class 5 Chemical Integrator is designed to react with all three variables of pressure, temperature, and time to indicate whether the load has been thoroughly sterilized. The chemical in the strip gradually changes color and enters the pass zone as all the variables in the autoclave cycle have reached the essential limits of sterilization standards.

The three variables work in a synchronous manner to achieve complete sterilization, and even a slight change in any one of the parameters would result in a sterilization rate less than 100%.[18] For example, a decrease in temperature would mean an increase in exposure time, and vice versa. If the temperature is entered incorrectly by personnel without a corresponding increase in time, the sterilization process would be faulty. Understanding the link between the triad of pressure, temperature, and time is crucial and should be shared by all personnel in the CSSD. The cause of a faulty class 5 chemical integrator is listed in Table 5. It is good practice to ensure that every pack that goes into a sterilizer has a class 5 integrator. For example, if you have five surgical packs in a rigid metal container, there should be one integrator in each pack placed in the rigid container.

Table 5.

Class 5 Chemical integrator troubleshooting[18]

Cause for Failure		Description		Solution
Improper packing		Practices such as placing a folded peel pouch within another peel pouch, inserting peel pouches into instrument trays or container systems, preparing textile packs that are excessively dense for the selected cycle parameters, using oversized packs for small instruments, and using artificial linen fabrics—can adversely affect effective sterilization.		Packing should be performed using recommended materials, with appropriately sized packs matched to the instruments, and exclusively using 100% cotton linen.
Improper Loading		Overloading or stacking packs, improper placement of peel pouches with plastic sides facing in different directions, and positioning packages too close to one another can impede effective air removal and steam penetration during sterilization.		Instruments to be stacked vertically (especially hollow instruments) with the plastic side facing the paper side and adequate space between trays
Equipment Malfunctioning: Sterilization failure may result from incomplete air removal, inadequate cycle temperature, insufficient exposure time at the required temperature, and poor steam quality or quantity.		Trapped air acts as a barrier to steam penetration, the use of hard water in autoclaves compromises steam quality, and inadequate exposure time or temperature results in ineffective sterilization.		Routine monitoring should include checking the vacuum pump and pressure gauges and ensuring selection of the appropriate sterilization cycle.

Packing of Instruments to be Sterilized

Packaging materials can be either wrapped or a container system. The packing material should allow steam to pass freely during the sterilization cycle. After sterilization, it should prevent microorganisms from entering the pack to maintain sterility throughout storage time. It should also be compatible with other aspects of the sterilization process, such as its ability to withstand high temperatures and drying. Surgical instruments can be packed in either perforated polycarbonate or stainless-steel boxes, or wrapped using woven or unwoven medical-grade sheets or plastic-paper peel pouches. Packaging materials should be designed to accommodate the type of sterilization process being used and be suitable for the items being sterilized. Characteristics of an ideal packing material and the corresponding validation test are listed in Tables 6 and 7, respectively.[19] Packing materials can be either reusable or disposable materials as listed in Table 8. Reusable woven linen must be inspected under light to look for punctures/tears/free threads, and any that are found must be discarded. The instruments packed in linen must be kept at room temperature (20–24 degrees Celsius) for 2 hours with a relative humidity of 35–70% before they are packed in the autoclave to avoid overheating of the fabric during the sterilization process. Due to the high regulatory requirements, it is best to avoid using linen and opt for alternative materials for packing instruments. Rigid container systems should have an inner basket/tray (SS/Polycarbonate) to hold the instruments (unpacked) and an outer rigid container that acts as a barrier against microorganisms, allowing sterilant to pass inside through valves or filters. A common mistake made when using a rigid container system is keeping the instruments packed in a linen or paper-plastic pouch within the rigid container, which adds an additional barrier to the steam. This results in inadequate steam penetration and removal, leading to condensation and wet packs. Although the rigid container system provides safety for the instruments, it has some disadvantages, as listed in Table 9.

Table 6.

Steam sterilization packing material[19]

Packaging material requirement		Material to be used
Sterilization packaging should prevent microbial penetration and maintain pack sterility, resist liquid penetration (water, alcohol, blood, oil, and body fluids), allow effective steam penetration, and be flexible yet puncture resistant.		Commonly used sterilization packaging materials include paper (73–153 GSM), plastic (PVC or polycarbonate), cloth (100% cotton), paper–plastic peel pouches, wrapped perforated cassettes, and SMS (spunbond–meltblown–spunbond polypropylene).

Table 7.

Characteristics of the packing material[19]

Property of the material to be tested		Validation Test
Linting		Resistance to linting when opened
Sterilant Penetration		Routine BI Monitoring
Strength		Grab Tensile - ASTM Test Method D 5034-90; Federal Test Method Standard No. 191A, Method 5100. (Manufacturer test certificate)
Microbial Barrier Effectiveness		Time-related, event-related, and shelf-life-related sterility maintenance studies were conducted by independent laboratories and are reviewed in the literature.
Has the ability of the microorganism to penetrate the wrap been tested using standardized procedures?		Dry Spore Talc Challenge Test (Manufacturer test certificate)
Does the wrap provide an adequate barrier to penetration by liquids?		Hydrostatic Head Test - Federal Test Standard 191A, Method 5514. (Manufacturer test certificate)
Drapeability		Drape Stiffness ASTM D 5732-95. (Manufacturer test certificate)
Disposal		As per the Local BMW disposal norms

Table 8.

Classification of packing materials

Reusable		Disposable
Woven Linen (100% cotton) or Polycarbonate/Stainless steel grade 304 containers		Plastic-Paper peel pouches or Non-woven paper (75GSM)

Table 9.

Rigid container system

Advantages		Disadvantages
Protect instruments from damage, eliminate the need for packing materials, provide an excellent barrier when equipped with a reliable latching system, and are easy to use.		Bulky design, the need for additional drying time to remove residual moisture, increased storage space requirements, and greater labor for cleaning and maintenance. Damage to valves or latching mechanisms may lead to air or steam leaks and subsequent contamination, and validation can be challenging due to variations in container materials, shapes, sizes, and latching or valve systems.

SMS is a three-layered nonwoven fabric manufactured through a process of blending a melt-blown fiber layer sandwiched between spun-bond layers, creating a versatile material. This nonwoven fabric features high tensile strength, durability, resistance to wear and tear, water resistance, and breathability, making it suitable for wrapping instruments for sterilization. It is also cost-effective and can be manufactured in different thicknesses, providing a suitable resistance to the penetration of microorganisms into the material. A surgical load that needs to be kept sterile for a longer duration should be double wrapped with either a plastic paper pouch or an SMS fabric. There are multiple double wrapping techniques available, like the envelope or square methods, which are equally efficient. Double wrapping can be done with a single sheet of fabric or using two separate sheets.[15] Double-layer packing is also adopted when the surgical load has to be transported to another sterile environment that is far from the CSSD area.

The salient features to be followed when using disposable packing material are listed in Table 10.

Table 10.

Salient points on using Disposable Packing materials

SMS material provides good safety It can be self-sealed or heat-sealed, avoiding gaps that can trap air or harbor microorganisms. Must be tamper-proof to prevent re-sealing Sharps and pointed instruments are to be double-packed. While using SMS to wrap the instruments, use Class 1 indicator tape to completely wrap the pack. When using plastic paper pouches, ensure the contents are easily visible, and the pack is of adequate size to contain the instrument.

Plastic-paper seal pouches are also used, with either single- or double-layer packing, depending on their storage time. Single-layered packing instruments should be used preferably early, and instruments that need to be stored for a longer duration should be double-packed. One side of these pouches is made of a transparent plastic material, manufactured using polythene (PE), polypropylene (PP), and polyethylene terephthalate (PET). The paper is made of medical-grade paper with a 75 GSM thickness, allowing adequate steam penetration and resisting dampness while preventing the penetration of microorganisms.[19] Process indicators for steam sterilization are applied to the paper surface, enabling the differentiation between sterilized and unsterilized packages. Stringent quality checks, such as verifying uniform seal width, peel strength, and external and internal pouch dimensions, should be carried out on all pouches to ensure they are appropriately sealed to maintain sterility.

All packs must be labeled before sterilization, which helps identify the instruments, sterilizers, and the cycle used. The instructions on the packing label are listed in Table 11. A sterile pack is considered sterile until it is compromised or suspected to be compromised by breakage of the seal, damage to the pack, wetness, soiling, or reaching its expiry date. Certain events, such as improper storage conditions, can render the sterile pack unsterile even before its expiry date has been reached. The proper storage conditions of a sterilized pack are listed in Table 12.

Table 11.

Package Labeling

Package labeling should include a description of the contents, date of sterilization and expiry, sterilizer identification, sterilization cycle used, requesting department, storage conditions, and the initials of personnel responsible for packing and running the sterilization cycle.

Table 12.

Storage conditions for the sterilized load

Sterile items should be stored at room temperature (18–24°C) with the relative humidity maintained between 35% and 75%, away from damp areas or walls. The storage room air should be dust-free, conforming to ISO Class 5 cleanroom standards, with positive-pressure ventilation, restricted traffic, and location within the clean or sterile zone of the operating theater complex.

Helix Test (Class 6)

The Helix test is a process challenge device test that determines the ability of the sterilizer to remove the trapped air on the inside of hollow instruments and tubing’s and the ability of the autoclave to penetrate steam through the lumens of the instruments.

This is an essential test in ophthalmic surgeries because the instruments have long lumens that can trap air, causing inadequate steam penetration and resulting in sterilization failure. Hence, the Helix test is used to check the ability of steam to penetrate hollow lumens and monitor the temperature and steam quality in the sterilizer. Since dynamic vacuum generation is available in Class B and S-type sterilizers, the helix can be used only in these types.

The BD test and the Helix test should preferably be done in an empty autoclave before the sterilizer is used. When the load to be sterilized contains solid instruments or linen, a BD test is sufficient; however, if the surgical load includes hollow instruments, it is mandatory to perform both the BD and Helix tests.

Procedure

A Helix test tube is made of either silicon or medical-grade 304 Stainless steel and is 150 cm long with a diameter of 0.002 m. The other side of the tube is closed by a hermetically sealed capsule that contains a strip printed with an indicator ink, known as the helix strip. The helix tube is sealed in a similar packing as the hollow instrument that is to be sterilized. The Helix test is carried out after the autoclave has passed the BD test. The Helix test tube should be placed in an empty autoclave, preferably in the most challenging part of the autoclave for steam penetration, which is near the entry or exit or in the lowest rack of the autoclave. The autoclave is run at a temperature of 121 degrees Celsius for 21 min or 134 degrees Celsius for 5 min to simulate the sterilization performance of an autoclave. The helix test operates on the principle that the sterilizer’s vacuum pump has been able to remove all the air inside the chamber. It thus allows steam to enter the interior of a deliberately narrow coiled tube and trigger a color change in the indicator ink printed on the strip kept inside. The Helix test is considered positive when the test strip is placed inside the case and changes color wholly and uniformly. If there is a patchy color change or no color change, the test is considered a failure, indicating that the sterilizer has not been able to obliterate all air. This residual air in the tube prevents the strip from coming into contact with the steam, causing a negative test.

The reasons for a failed Helix Class 6 PCD test are outlined in Fig. 1, which enables CSSD personnel to take appropriate action based on the specific reasons for the failed test.

Figure 1.

Helix strip troubleshooting

Biological Indicator

Despite other indicators showing that the physical parameters are being met in the sterilization process by the autoclave, the Biological Indicator (BI) is the only test that proves the sterilized load is sterile. This is because BI demonstrates the ability of steam sterilization to destroy microorganisms during the process completely. Biological indicators must be used in every sterilizer and in every cycle for routine monitoring of the autoclave cycle. The organism used in BI is the endospores of Geobacillus stearothermophilus as they are highly resistant to killing, due to their D-value being the highest. A fixed number of these endospores is placed inside a small glass tube that is enclosed in a silicon tube containing the growth medium and dye. The D-value of the endospores is listed in Table 13. The major drawback of the BI is that it requires a maximum of 56 hours of incubation to read the results; however, the self-contained biological indicator overcomes this. The self-contained biological indicator utilizes an automated reader to determine microbiological growth by detecting color changes based on enzymatic activation in a shorter period than the traditional BI.[20] The results of the Class 5 Integrator most often match those of the BI, meaning that 99% of the time, a BI will show no growth when the sterilizer has passed a Class 5 Integrator test. This gives the surgeon confidence to proceed with the surgery without waiting for the results of the BI.

Table 13.

Bacterial Endospores and their resistance[21]

Organism 106 spores per indicator (This is based on a scenario that an item to be sterilized may contain a population of 106 spores having the same resistance as that of Bacillus stearothermophilus)		D 121 Value (Time needed@ 121*C to kill bacterial load by 90%=1 log)
Geobacillus stearothermophilus		1-2.8 minutes
Bacillus coagulans		0.3 minutes
Clostridium sporogens		0.8-1.4 minutes
Bacillus atropheus		0.5 minutes

How to use

Take two vials of the biological indicator from the same manufacturer with the same batch number. The name of the sterilizer and the surgical load in which it is kept should be indicated on both BI. One BI should be packed using the same packing material as that used for the surgical load to be sterilized and placed alongside the instruments. Care should be taken to ensure that BI does not come in contact with other chemical indicators or instruments to prevent damage to the device. The other BI should be labeled as “Control” and kept for later incubation and comparison with the sterilized BI that is placed in the sterilizer.

After the completion of the drying phase of the autoclave cycle, the BI processed in the autoclave is removed, allowing for adequate cooling time. The change in color of the Class 1 chemical indicator on the label of the BI indicates that it has been processed in the autoclave, which differentiates it from the BI that is kept outside, unprocessed as a “control.” The glass ampoule, which contains the endospores of the bacillus, is crushed using a mechanical crusher without damaging the outer silicon tube. This allows the endospores to mix with the growth medium and the dye in the surrounding silicon tube, giving it a purple appearance in both BI tubes. This mixing is performed for both the processed and control biological indicators, and they are then placed in a BI incubator at 58°C for 48 hours. Alternatively, one can use the Rapid Readout Biological Indicators, which can detect the color change in 1 hour before the visible color change occurs after 48 hours of incubation. This color change is facilitated by the enzyme indicator included in the BI, which becomes activated once the bacillus spore regenerates.

The BI, which acts as a control, should be stored under proper physical parameters, including temperature, humidity, and proximity to chemicals, to ensure that the viability of the spores has not been altered.

Once inside the incubator, the BI vials should be checked at convenient time intervals such as 6, 12, and 18 hours for early signs of a color change to yellow, indicating bacterial growth. If the autoclave had attained the desired temperature, pressure, and time, all the endospores would have been destroyed, and the BI media would have remained purple, indicating that no growth had occurred. This final negative reading, where the color remains purple, is made after 48 hours as sometimes one can detect growth between 48 and 56 hours. The “Control” vial, which also has been incubated, will exhibit a yellow color change in the media as early as 12–24 hours as the bacillus starts to proliferate once the right environment has been achieved in the incubator. If growth is detected in the sterilized BI vial, indicated by a color change from purple to yellow, the sterilization process is considered a failure. Most often, if a BI test has failed, the class 5 integrator test should also have failed the sterilization process.

Immediate Use Steam Sterilization (IUSS)

IUSS[21] and flash sterilization are often used interchangeably in the field of sterilization. This method should be used only in specific, exceptional situations and should not be employed as a routine method of sterilizing the surgical load. In ophthalmic surgeries, it is used to process a critical surgical item for immediate use when other forms of sterilization are unavailable, such as in situations where an accidental contamination of a nonreplaceable instrument requires immediate replacement in the sterile field or emergency surgical situations. Instruments that undergo IUSS must be cleaned and decontaminated in the same way as the routine sterilization process. At the same time, care should be taken to ensure that the guidelines from the manufacturer are strictly followed, or the instruments may be damaged. IUSS should be performed only in dynamic air removal sterilizers (Class S or Class B) with physical parameters set at 20 psi and 131 degrees Celsius for 5 minutes. The instruments should be placed in the sterilizer unpacked on an open, perforated tray made of either medical-grade 304 Stainless steel or Polycarbonate, with all the necessary validating chemical and microbiological indicators. After the sterilization process, the indicators are checked, and the instruments should be used immediately, allowing only adequate time for them to cool down. As the instruments are placed unpacked, the sterilizers must be placed in the sterile zone of the operating theater complex with direct access to the main operating room. IUSS should not be used as a routine method of sterilizing surgical items or as a substitute for an instrument shortage. It should be used only in emergencies when alternative sources of sterile instruments are unavailable. IUSS should not be used for sterilizing implants. The instruments processed by IUSS cannot be stored for later use as they do not have packaging. IUSS should not be used for phacoemulsification handpieces as the rapid heating and cooling would damage the crystals. IUSS should not be used for any heat-labile instruments, including tubing, endo laser probes, light pipes, and vitrectomy cutters.

Frequency of validation of sterilizer and sterilization loads

The frequency of validating the sterilization process depends on the load being processed, as listed in Table 14. Performance qualification monitoring of the sterilizer must be conducted during routine monitoring[21] (as listed in Table 15), during installation of the sterilizer, during relocation, after routine maintenance, and after a malfunction or failure[22] (as listed in Table 16).

Table 14.

Validation for sterilization loads

Linen Loads		Surgical Instruments/Implants
Physical parameter monitoring of every sterilization cycle. Temperature/Time/Pressure		Physical parameter monitoring of every sterilization cycle. Temperature/Time/Pressure
External and Internal Class 1 Chemical Indicator		External and Internal Class 1 Chemical Indicator
Monitoring of any one of the following BI or with BI + Class 5 Integrator or with Class 5 Integrator or with a Class 6 PCD		Monitoring Every Load with BI + Class 5 Integrator or with Bowie–Dick when the load has porous instruments

Table 15.

Routine sterilizer efficacy monitoring

Sterilizers >2 cubic feet and Tabletop sterilizers		IUSS cycles and Dynamic air removal Sterilizers
Fully loaded chamber		Empty chamber
Physical parameter monitoring of every sterilization cycle. Temperature/Time/Pressure		Physical parameter monitoring of every sterilization cycle. Temperature/Time/Pressure
External and Internal Class 1 Chemical Indicator		External and Internal Class 1 Chemical Indicator
Weekly/Preferably whenever the sterilizer is used, BI + Class 5 Integrator		Weekly/Preferably whenever the sterilizer is used, BI + Class 5 Integrator Bowie-Dick when the load has porous instruments

Table 16.

Performance qualification monitoring. (During Installation/Relocation/Malfunction/Failure of the sterilizer)

Sterilizers >2 cubic feet and Tabletop sterilizers		IUSS cycles and Dynamic air removal Sterilizers
Empty chamber		Fully loaded chamber
3 consecutive cycles of physical parameter monitoring. Temperature/Time/Pressure		3 successive cycles of physical parameter monitoring. Temperature/Time/Pressure
3 consecutive cycles of BI + Class 5 Integrator and Bowie–Dick Test		External and Internal Class 1 Chemical Indicator
		3 consecutive cycles of BI + Class 5 Integrator

Wet Packaging After Sterilization: Causes and Management

Wet packs[23] are defined as those that exhibit the presence of moisture, on either the inside or outside of the pack, in the form of droplets or a visibly damp appearance after the drying phase of the autoclave cycle. It is normal to have a few droplets of water on the surface of the pack that usually dry in 5–10 minutes. Still, if they are present for more than this allowed time, they are considered unsterile, and the sterilization process was not successful. When the linen or pack is wet, it will not function as an effective barrier to prevent microorganisms from entering the pack. Wet packs can be due to external moisture, moisture on the surface of the pack, or due to internal moisture, moisture inside the packing.

Poor quality packaging material

Reasons for moisture in the packs can be attributed to the use of poor-quality packaging materials. It is good practice to avoid using reusable linen to wrap instruments for sterilization as it can trap moisture and cause wet packs, making the load unsterile. As linen retains moisture more than SMS material, some amount of moisture is expected to be present on the outside of the pack at the end of the drying phase of the autoclave cycle. This moisture will dry up within 10–20 minutes. When we use linen to pack surgical instruments, the moisture is retained for a longer duration by the cotton fibers, resulting in unsterile wet packs.[23]

Rigid Container System

Autoclave bins made of stainless-steel grade 304 or polycarbonate material are still used to pack instruments for sterilization. The malfunction of the valves in the bins can lead to the steam getting trapped inside the container, causing it to condense on the instrument surface. Even with a good vacuum pump, it would be challenging to evacuate the steam inside bins, resulting in wet-packs.[23] It is still common practice in many institutions to wrap instruments and then place them inside rigid metal bins, which makes it difficult for steam to penetrate and hinders complete steam evacuation, resulting in wet packs. Due to these disadvantages, it is a good practice to avoid rigid bins and linen when packing surgical loads as better alternatives are readily available.

Faulty packing technique

Any material that can retain moisture, such as linen, gauze, eye pads, and cotton wicks, must be packed separately and should not be combined with other surgical instruments in the same pack. It is also advisable to avoid placing these items inside a closed container system due to their inherent ability to retain moisture. Linen must be placed crosswise, preferably in the upper racks, to prevent condensation and wet packs.[24] When a large volume of metal instruments is overloaded into a surgical tray, the excess mass can cause the instruments to overheat. During the cooling phase, this can lead to condensation. Sometimes, moisture may get trapped inside the lumen of the instrument before packing due to incomplete drying. Instruments with hinges should not be left open inside the pouches as this can trap moisture during the drying phase. A pack should not contain items made of similar materials, and combining items of different materials in the same pack would result in moisture being trapped inside, leading to internal humidity. Combining linen with metallic surgical instruments in a rigid container system typically results in internal wet packs.

The surgical pack should adhere to defined standards to ensure uniform sterilization efficacy. Linen packs should weigh less than 5 kg each, and the dimensions of any pack loaded into any sterilizer should not exceed 300 mm × 300 mm × 450 mm. Surgical instrument packs should weigh less than 11 kg per pack, and individual surgical trays should not exceed dimensions of 300 mm × 300 mm × 600 mm. The total number of trays per sterilization cycle is determined by the chamber volume of the autoclave.

Loading technique

Insufficient space between the packs can trap moisture, leading to wet packs. Surgical loads should be arranged such that a minimum clearance of 3 cm is maintained between adjacent items. Paper–plastic pouches are preferably placed in a vertical orientation within the autoclave to facilitate optimal steam penetration. For rigid container systems, instruments should remain unwrapped inside the container, the total container weight should not exceed 3 kg, and sufficient spacing should be maintained around the container to allow effective steam access; these containers should be positioned near the rack edges rather than deeper within the load. In the case of mixed loads, linen materials should be placed on the upper shelf, while metal instruments should be positioned on the lower shelf to minimize condensation.[24]

Sterilizer Malfunction

A sterilizer malfunction frequently results in both internal and external wet packs. To minimize risk, the sterilizer should be located in an area with appropriate ventilation, a stable ambient temperature between 24°C and 40°C, and a relative humidity of 40–60%. Common causes of sterilizer malfunction include inadequate drying time in linen packs, excessive load weight or overpacking, and autoclave cycle errors, such as preset parameter changes secondary to a power interruption. Additional factors include faulty valves leading to water reflux into the inner chamber, compromised autoclave gaskets or jacket integrity, and poor maintenance practices. Steam quality also plays a critical role: Steam containing more than 3% liquid water by mass leads to excessive condensation and wet packs. Water with low total dissolved solids (<100 ppm)—produced by reverse osmosis or distillation—is ideal for generating dry, saturated steam. Any process deviation that disrupts dry saturated steam generation at approximately 97% quality can result in moisture retention within packs. Excessive steam demand—caused by an undersized boiler relative to chamber volume or by overloading—may lower system pressure and lead to water-logged steam. Variation in the temperature of incoming steam across multiple pipeline connections from a central boiler may further promote condensation.[25] Finally, a malfunctioning vacuum pump can trap air and noncondensable gases (NCGs), impair steam penetration, and contribute to wet packs.

Prevention of wet packages

Prevention of wet packages relies on careful control of loading practices, autoclave function, and environmental conditions. Regular preventive maintenance should be undertaken to inspect electrical sockets for thermal damage; ensure proper functioning of door hinges, gaskets, and valves; and verify that physical parameter gauges are checked and calibrated at least annually. Avoiding autoclave overloading helps reduce steam entrapment between packs, while ensuring sufficient drying time and proper vacuum pump performance prevents residual condensation. Instrument tip protectors that retain moisture should be avoided, and surgical packs must remain within the recommended size and weight limits. The use of high-quality SMS wrapping materials is preferred over linen, and instrument hinges must be kept in an unlocked and open position—such as packing scissors with blades open—to ensure uniform steam penetration. Wrapping material dimensions should be appropriate for the contents, and metallic bins, valves, hinges, and gaskets should be routinely inspected for malfunction.[25] Environmental parameters in the storage and CSSD areas must be monitored and maintained at room temperature with 40–60% humidity; if needed, humidity control may be achieved using dehumidifiers or an HVAC system. Instrument trays should be oriented correctly within the sterilizer, especially packs containing hollow instruments (e.g. phacoemulsification or I/A handpieces), which should be placed vertically and spaced adequately to allow optimal air removal and steam distribution. Wet packs are considered unsterile as they may allow microbial survival and proliferation. The additional time, labor, and cost associated with reprocessing such items impose unnecessary operational burdens; therefore, every effort must be made by CSSD personnel to prevent their occurrence.

Traceability

Tracing the source of error is essential when a patient develops an infection. Following the validation, monitoring, and verification protocols, the CSSD department would be better equipped to identify the root cause of the error during the sterilization process. To find the source of the error, the autoclave indicators, including the BI test results, should be affixed in the register adjacent to the corresponding autoclave cycle. Details such as the number and name of the surgical instruments in each pack must be listed clearly for every autoclave cycle in the register.

Storage of Sterilized Goods

The duration of storage of sterilized goods has always been a question of debate. Hospitals employ various protocols for storing steam-sterilized goods; however, there is no clear consensus or scientific evidence to confirm the validity of these protocols. A literature review reveals that multiple studies have different periods of validity for sterilized packs, depending on the type of packing material used. The conclusion from these studies is that, if the surgical packages are intact without any damage before they are opened and the storage conditions, including physical parameters of temperature, humidity, and pressure, are ideal, the sterilized packages can be stored for up to 96 weeks.[26] An anecdote in sterilization is that in this study, linen-wrapped sterilized packages were also sterile for 96 weeks, compared to double-layered paper-plastic pouches. It is generally believed that linen retains moisture and should not be used to store instruments for a prolonged period. However, more extensive trials are needed with the latest generation of packing materials to determine the validity of steam-sterilized goods.

Summary

Class B autoclaves should be used for the sterilization of ophthalmic instruments as they ensure complete air removal and effective sterilization of hollow devices. The manufacturer’s recommended guidelines for sterilization parameters, loading practices, and preventive maintenance must be strictly followed. A “one-tray–one-case” protocol is advisable, with double-wrapping using SMS or paper–plastic pouches, while instruments sterilized in metallic bins should be left unwrapped and used immediately. Immediate-Use Steam Sterilization (IUSS) must be reserved exclusively for emergencies and should not form part of routine practice, with instruments processed by IUSS remaining unwrapped and used immediately. Every tray should incorporate chemical indicators, including an externally applied Class 1 indicator and an internally applied Class 5 integrator. The Bowie–Dick test should be performed in an empty chamber—preferably before each load—to confirm adequate air evacuation. Steam penetration for hollow instruments should be verified using a Process Challenge Device (PCD) or Helix test, which is particularly important in ophthalmic settings. Biological indicators serve as the definitive proof of sterilization, and although Class 5 integrators correlate well with BI performance, BI testing remains preferable for cycle verification. Wet packs should be regarded as unsterile and must not be used. The maintenance of sterility is dependent on the characteristics of the storage environment, particularly temperature, humidity, handling frequency, and wrapper integrity.

Conclusion

The success of a hospital is primarily determined by the success of its operating theater and sterilization process. It is of paramount importance that the hospital administration dedicates time, finances, and resources to training their staff on the importance of sterilization and their validation, monitoring, and verification protocols. This review article would be helpful to all healthcare professionals involved in CSSD activities.

Conflicts of interest:

There are no conflicts of interest.

Funding Statement

Nil.