Gaseous sterilization uses ethylene oxide and formaldehyde gases to sterilize heat-sensitive medical equipment and supplies. These gases are effective at killing bacteria, viruses, and bacterial spores. Ethylene oxide is more commonly used internationally and can penetrate packaging materials. Both gases work by alkylating proteins and nucleic acids in microorganisms. Sterilization protocols involve exposing items to the gases at specific concentrations and temperatures for extended periods, then removing toxic residues before use. The mechanisms, equipment, and processes used for ethylene oxide and formaldehyde sterilization are described.

1. Gaseous sterilization
The chemically reactive gases ethylene oxide (CH2)2O and formaldehyde (methanal,
H.CHO) possess broad-spectrum bactericidal activity, and used to sterilize re-usable surgical
instruments, certain medical, many heat-sensitive items such as disposable plastics, petri-dishes
and syringes, heart-lung machine components, sutures, and catheters diagnostic and electrical
equipment, and the surface sterilization of powders. Ethylene oxide is most commonly employed
as gaseous sterilant on an international basis than formaldehyde.
Ethylene oxide (EtO) is both microbicidal and sporicidal and kills by combining with cell
proteins. The reason behind its widespread use is its ability to penetrate packing materials, even
plastic wraps.
In the commercial production of disposable medical devices ethylene oxide treatment can
also be considered as an alternative to radiation sterilization. These techniques are generally
reserved for temperature-sensitive items because they do not offer the same degree of sterility
assurance as heat methods.
Mechanism of sterilization
The mechanism of biocidal action of the two gases is proposed to be through alkylation
of sulphydryl, amino, hydroxyl and carboxyl groups on proteins and imino groups of nucleic
acids. The lethality of these gases increases in a non-uniform manner with increase in
concentration, exposure temperature and humidity. For this reason, sterilization protocols have
generally been established by an empirical approach using a standard product load containing
suitable biological indicator test strips. Concentration ranges (weight of gas/unit chamber
volume) employed are generally in the order of 800-1200 mg/L for ethylene oxide and 15-100
mg/L for formaldehyde, with operating temperatures in the region of 45-63⁰C and 70-75⁰C,
respectively. The sterilization processes are lengthy even at the higher concentrations and
temperatures and therefore unsuitable for the re-sterilization of rapid-turnover articles. Further
delays occur because of the need to remove toxic residues of the gases before release of the items
for use. An extended quarantine period may also be required owing to recovery of survivors in
sterility tests is more prolonged with gaseous sterilization methods than with other processes.
In addition, both gases are potentially mutagenic and carcinogenic. For example ethylene
oxide reacts with chlorine to produce ethylene chlorohydrins which are responsible for acute

2. toxicity including irritation of the skin, conjunctiva and nasal mucosa. Therefore, to protect
personnel safe working protocols are required and strict control of their atmospheric
concentrations is necessary. Formaldehyde has one advantage over EtO that it can normally be
detected by smell at concentrations lower than those permitted in the atmosphere, whereas this is
not true for ethylene oxide.
Ethylene oxide
Ethylene oxide gas is highly explosive in mixtures of >3.6% v/v in air. Therefore it is
usually supplied for sterilization purposes as a 10% mixture with CO2, or as an 8.6% mixture
with HFC 124 (2-chloro-1,1,1,2 tetrafluoroethane) to reduce these explosion hazards.
Alternatively, pure ethylene oxide gas can be used below atmospheric pressure in sterilizer
chambers from which all air has been removed. The efficacy of ethylene oxide treatment
depends upon achieving a suitable concentration in each article. As stated before, ethylene oxide
has good penetrating powers and can diffuses readily into many packaging materials including
rubber, plastics, fabrics and paper. After initial exposure the level of ethylene oxide in a sterilizer
starts to reduce due to its absorption during the process and the treated objects must undergo a
desorption stage to remove toxic residues. Complete desorption may take many days because for
natural desorption to occur articles are shelved in open, controlled environment, e.g. for
materials like PVC. Special forced aeration cabinets where heated, flowing air assists gas
removal can reduce desorption times to between 2 and 24 hours.
In dried state organisms are more resistant to ethylene oxide treatment owing to their
inclusion in crystalline or dried organic deposits. Thus, for an efficient ethylene oxide
sterilization cycle, the minimum level of moisture is of prime importance in the immediate
product environment. To meet these requirements, a sterilizer humidity of 30-70% and
frequently a preconditioning of the load at relative humidities of >50% is required.
Sterilizer design and operation
A leak-proof and explosion-proof steel chamber comprises of 100-300 liters capacity,
surrounded by a hot-water jacket to provide a uniform chamber temperature makes up an
ethylene oxide sterilizer. Successful operation of the sterilizer requires evacuated chamber with
controlled humidity and preconditioning of the load by passage of sub atmospheric pressure
steam. Following preconditioning of the load a further evacuation period is given and then
admission of preheated vaporized ethylene oxide from external pressurized canisters or single-

3. charge cartridges. To minimize variations in conditions and to ensure uniform diffusion of
ethylene oxide throughout the sterilizer chamber, forced gas circulation is often employed. A
essential prerequisite for sterilization to be achieved within individual articles in the load is that
all packaging materials must be air-, steam- and gas-permeable.
During the sterilization process the pressure drops due to absorption of ethylene oxide by
the load and is compensated by the introduction of excess gas at the beginning or by the addition
of more gas as pressure drops. The same may also be true for moisture absorption, which is
compensated for by supplementary addition of water to maintain appropriate relative humidity.
After treatment, the gases are evacuated either directly to the outside atmosphere or through a
special exhaust system. Filtered, sterile air is then admitted either for a repeat of the vacuum/air
cycle or for air purging until the chamber is opened. In this way, safe removal of the ethylene
oxide is achieved, reducing the toxic hazard to the operator. The operation of an ethylene oxide
sterilizer should be strictly monitored and controlled automatically.
Formaldehyde
Formaldehyde gas for use in sterilization is produced by heating formalin (37% w/v
formaldehyde) to a temperature of 70-75⁰C with steam, leading to the process known as low
temperature steam formaldehyde (LTSF). Formaldehyde has a similar toxicity to ethylene oxide
and although absorption to materials appears to be lower, similar desorption routines are
recommended. Formaldehyde has low penetrating power and can be employed to principally
paper and cotton fabric this limits use of formaldehyde in gaseous sterilization.
Sterilizer design and operation
An LTSF sterilizer is designed to operate with sub atmospheric pressure steam. Steam is
admitted to the evacuated chamber to allow heating of the load and to assist in air removal. The
sterilization period starts with the release of formaldehyde by vaporization from formalin and
continues through either a simple holding stage or through a series of pulsed evacuations and
steam and formaldehyde admission cycles. The chamber temperature is maintained by a
thermostatically controlled water-jacket. At the end of the treatment period formaldehyde vapour
is expelled by steam flushing and the load is dried by alternating stages of evacuation and
admission of sterile, filtered air.