Which of the following is a method of moist heat sterilization?

Of all the methods available for sterilization, moist heat in the form of saturated steam under pressure is the most widely used and the most dependable method. Moist heat has better penetrating power than dry heat and, at a given temperature, produces a faster reduction in the number of living organisms. Steam sterilization is nontoxic, inexpensive, rapidly microbicidal, and sporicidal. It rapidly heats and penetrates fabrics.

Table of Contents Show

Principle of Moist Heat sterilization
Biological Indicators
Advantages of Steam Sterilization Method
Disadvantages of Steam Sterilization Method

Sterilization is defined as killing or removal of all microorganisms including bacterial spores.

Moist heat sterilization using autoclave is commonly used for the sterilization of biohazardous trash, heat, and moisture resistant materials such as aqueous preparation (culture media). This method is also used for the sterilization of surgical dressings and medical devices.

The most common type of steam sterilizer in the microbiology laboratory is the gravity displacement type. Another type of autoclave is vacuum/gravity assisted.

Gravity Displacement type Autoclave

Principle of Moist Heat sterilization

Moist heat destroys microorganisms by the irreversible denaturation of enzymes and structural proteins. The temperature at which denaturation occurs varies inversely with the amount of water present. Sterilization in saturated steam thus requires precise control of time, temperature, and pressure.

Pressure serves as a means to obtain the high temperatures necessary to quickly kill microorganisms. Specific temperatures must be obtained to ensure microbicidal activity. Minimum sterilization time should be measured from the moment when all the materials to be sterilized have reached the required temperature throughout.

The recommendation for sterilization in an autoclave is 15 minutes at 121°C (200 kPa). The temperature should be used to control and monitor the process; the pressure is mainly used to obtain the required steam temperature.

Alternative conditions, with different combinations of time and temperature, are given below.

1 1 atm = 325 Pa

Temperature (°C)	Approximate corresponding pressure (kPa)	Minimum sterilization time (min)
126-129	250 (~2.5 atm)	10
134-138	300 (~3.0 atm)	5

In certain cases (e.g. thermolabile substances), sterilization may be carried out at temperatures below 121 °C, provided that the chosen combination of time and temperature has been validated.

Monitoring of steam sterilization process

Like other sterilization systems, the steam cycle is monitored by mechanical, chemical, and biological indicators. Steam sterilizers usually are monitored using a printout (or graphically) by measuring temperature, the time at the temperature, and pressure.

Chemical indicators are affixed to the outside and incorporated into the pack to monitor the temperature or time and temperature. Autoclave indicator tapes are commercially available and a change in color of the tape suggests proper sterilization.

Temperature-monitoring probes should be inserted into representative containers, with additional probes placed in the load at the potentially coolest and least accessible parts of the loaded chamber. The conditions should be within ±2 °C and ±10 kPa (±0.1 atm) of the required values. Each cycle should be recorded on a time-temperature chart or by other suitable means.

Biological Indicators

The effectiveness of steam sterilization is monitored with a biological indicator using an envelope containing spores of Geobacillus stearothermophilus (formerly Bacillus stearothermophilus; e.g. ATCC 7953 or CIP 52.81) for which the D-value (i.e. 90% reduction of the microbial population) is 1.5-2.5 minutes at 121 °C, using about 106 spores per indicator (this is based on a worst-case scenario that an item may contain a population of 106 spores having same resistance as that of Bacillus stearothermophilus). After sterilization is over the strip is removed and inoculated into tryptone soy broth and incubated at 56°C for 5 days. No growth of Geobacillus stearothermophilus indicates proper sterilization.

Table: list of commonly used biological indicators (BIs)

Spores of Bacteria	D Value
Geobacillus stearothermophilus (most common)	1.5-2.5
Bacillus coagulans	0.3
Clostridium sporogenes	0.8-1.4
Bacillus atropheus	0.5

Positive spore test results are a relatively rare event and can be attributed to operator error, inadequate steam delivery, or equipment malfunction.

Advantages of Steam Sterilization Method

Nontoxic to patient, staff, environment
Cycle easy to control and monitor
Rapidly microbicidal
Least affected by organic/inorganic soils among sterilization processes listed
Rapid cycle time
Penetrates medical packing, device lumens

Disadvantages of Steam Sterilization Method

Deleterious for heat-sensitive instruments
Microsurgical instruments damaged by repeated exposure
May leave instruments wet, causing them to rust
Potential for burns

References and further readings

CDC:Guideline for Disinfection and Sterilization in Healthcare Facilities

Applying heat to bacterial media and utensils in research and the medical field as well as to sterilize food is one of the most common methods for control of bacterial growth. To achieve sterilization, different techniques and tools are used.

Moist heat causes destruction of micro- organisms by denaturation of macromolecules, primarily proteins. Autoclaving (pressure cooking) is a very common method for moist sterilization. It is effective in killing fungi, bacteria, spores, and viruses but does not necessarily eliminate prions. When sterilizing in this way, samples are placed into a steam chamber. The chamber is closed and heated so that steam forces air out of the vents or exhausts. Pressure is then applied so that the interior temperature reaches 121°C. This temperature is maintained for between 15 and 30 minutes. This elevated temperature and pressure is sufficient to sterilize samples of any commonly encountered microbes or spores. The chamber is then allowed to cool slowly or by passive heat dissipation.

Pressure sterilization is the prevailing method used for medical sterilization of heat-resistant tools. It is also used for sterilization of materials for microbiology and other fields calling for aseptic technique. To facilitate efficient sterilization by steam and pressure, there are several methods of verification and indication used; these include color-changing indicator tapes and biological indicators. For any method of moist heat sterilization, it is common to use biological indicators as a means of validation and confirmation. When using biological indicators, samples containing spores of heat-resistant microbes such as Geobacillus stearothermophilis are sterilized alongside a standard load, and are then incubated in sterile media (often contained within the sample in a glass ampoule to be broken after sterilization). A color change in the media (indicating acid production by bacteria; requires the medium to be formulated for this purpose) or the appearance of turbidity (cloudiness indicating light scattering by bacterial cells) indicates that sterilization was not achieved and the sterilization cycle may need revision or improvement. Other moist methods are boiling samples for certain period of time and Tyndallisation. Boiling is not efficient in eliminating spores. Tyndallisation inactivates spores as well, but is a more lengthy process.

Figure: Autoclave: Large autoclave used for moist sterilization of media and equipment

Dry heat destroys microorganisms by causing coagulation of proteins. The dry heat sterilization process is accomplished by conduction; that is where heat is absorbed by the exterior surface of an item and then passed inward to the next layer. Eventually, the entire item reaches the proper temperature needed to achieve sterilization. The time and temperature for dry heat sterilization is 160°C for 2 hours or 170°C for 1 hour. Instruments should be dry before sterilization since water will interfere with the process. Other heat sterilization methods include flaming and incineration. Flaming is commonly used to sterilize small equipment used to manipulate bacteria aseptically. Leaving transfer loops in the flame of a Bunsen burner or alcohol lamp until it glows red ensures that any infectious agent gets inactivated. This is commonly used for small metal or glass objects, but not for large objects (see Incineration below). However, during the initial heating infectious material may be “sprayed” from the wire surface before it is killed, contaminating nearby surfaces and objects. Therefore, special heaters have been developed that surround the inoculating loop with a heated cage, ensuring that such sprayed material does not further contaminate the area. Another problem is that gas flames may leave residues on the object, e.g. carbon, if the object is not heated enough. A variation on flaming is to dip the object in 70% ethanol (or a higher concentration) and merely touch the object briefly to the Bunsen burner flame, but not hold it in the gas flame. The ethanol will ignite and burn off in a few seconds. 70% ethanol kills many, but not all, bacteria and viruses. It has the advantage that it leaves less residue than a gas flame. This method works well for the glass “hockey stick”-shaped bacteria spreaders. Incineration will also burn any organism to ash. It is used to sanitize medical and other bio hazardous waste before it is discarded with non-hazardous waste.

Different methods are used to achieve sterilization. One of the most common is applying moist heat which includes autoclaving (pressure cooking), boiling, and Tyndallisation.
Dry heat sterilization is accomplished by conduction and is used widely for instruments.
Other heat methods include flaming and incineration. Flaming is commonly used to sterilize small equipment used to manipulate bacteria aseptically.

sterilization: Any process that eliminates or kills all forms of microbial life present on a surface, solution, or solid compound.
Tyndallisation: Tyndallisation is the process of three successive steam treatments to achieve sterilization over the course of three days. This works by killing vegetative cells, allowing germination of surviving spores, and killing the resulting vegetative cells before they have time to form further spores.