ASTERILIZATION

Sterilization can be defined as any process that effectively kills or eliminates transmissible
agents (such as fungi, bacteria and viruses) from a surface, equipment, foods, medications, or
biological culture medium. In practice sterility is achieved by exposure of the object to be
sterilized to chemical or physical agent for a specified time. The success of the process depends
upon the choice of the method adopted for sterilization.

Sterilization methods

Heat Sterilization
Heat sterilization is the most widely used and reliable method of sterilization, involving destruction
of enzymes and other essential cell constituents. The process is more effective in hydrated state
where under conditions of high humidity, hydrolysis and denaturation occur, thus lower heat input is
required. Under dry state, oxidative changes take place, and higher heat input is required. This
method of sterilization can be applied only to the thermostable products, but it can be used for
moisture-sensitive materials for which dry heat (160° -180° C) sterilization, and for moisture-
resistant materials for which moist heat (121° -134°C) sterilization is used.

1. Dry Heat Sterilization: Examples of Dry heat sterilization are:

1. Incineration
2. Red heat
3. Flaming
4. Hot air oven
It employs higher temperatures in the range of 160° -180° C and requires exposures time up to 2
hours (heat at 180º for half hour, 170º for 1 hr., or 160º C for 2 hrs.) depending upon the
temperature employed. The benefit of dry heat includes good penetrability and non-corrosive
nature which makes it applicable for sterilizing glassware and metal surgical instruments. It is
also used for sterilizing non-aqueous thermostable liquids and thermostable powders. Dry heat
destroys bacterial end toxins (or pyrogens) which are difficult to eliminate by other means and
this property makes it applicable for sterilizing glass bottles which are to be filled aseptically .

Hot-air oven
Dry heat sterilization is usually carried out in a hot air oven, which consists of the following:
i) An insulated chamber surrounded by an outer case containing electric heaters.
ii) A fan
iii) Shelves Thermocouples
v) Temperature sensor
vi) Door locking controls.

1
Operation
The Hot Air Oven works on the basis of Hot Air inside the chamber which is created due to the
forced air circulation. It is a universal fact that in chambers, the hot air first rises up, and once it
reaches the top of the instrument, it came back to the bottom of the chamber. The circulating fans
and fan motor that are equipped with the instrument helps to create even temperature inside the
chamber which helps to achieve an optimum level of heat inside the Hot Air Oven. After heating
the specimen in the oven at least for two hours, the test specimens are kept inside the chamber
until the temperature comes down and then the sterilized material is removed from the oven.

The instrument is widely used to sterilize glassware in pharmaceutical industries such as Petri
dishes, pipettes, bottles, test tubes, flasks, pestle, etc.

Fig.1: Hot air oven

2. Moist Heat Sterilization: Moist heat may be used in three forms to achieve microbial
inactivation,
1. Dry saturated steam – Autoclaving
2. Boiling water/ steam at atmospheric pressure
3. Hot water below boiling point
Moist heat sterilization involves the use of steam in the range of 121°-134°C. Steam under
pressure is used to generate high temperature needed for sterilization. Saturated steam acts as an
effective sterilizing agent. Steam for sterilization can be either wet saturated steam (containing
entrained water droplets) or dry saturated steam (no entrained water droplets).
Autoclaves use pressurized steam to destroy microorganisms, and are the most dependable
systems available for the decontamination of laboratory waste and the sterilization of laboratory
glassware, media, and reagents. For efficient heat transfer, steam must flush the air out of the

2
autoclave chamber. Autoclaves should be tested periodically with biological indicators like
cultures of Bacillus stearothermophilus to ensure proper function. This method of sterilization
works well for many metal and glass items but is not acceptable for rubber, plastics, and
equipment that would be damaged by high temperatures (Figure 2).

Fig. 2: An Autoclave

Autoclaves, or steam sterilizers essentially consist of following:

i) A cylindrical or rectangular chamber, with capacities ranging from 400 to 800 liters.
ii) Water heating system for steam generating system
iii) Steam outlet and inlet valves
iv) Single or double doors with locking mechanism.
v) Thermometer or temperature gauge
vi) Pressure gauges

Operation
For porous loads (dressings) sterilizers are generally operated at a minimum temperature of
134°C, and for bottled fluid, sterilizers employing a minimum temperature of 121 °C are used.
Ensure that there should be sufficient water in the autoclave to produce the steam. The stages of
operation of autoclaves include air removal, steam admission and sterilization cycle (includes
heating up, holding/exposure, and cooling stages).

Sterilization by Filtration
Microbes can also be removed from liquids by means of filters with very small pores which trap
bacteria. This method is used for sterilizing serum, antibiotic solutions, carbohydrate solutions
and media that are heat labile.

3
 There are two main types of filters currently in use, the asbestos filter and the membrane filter.
The asbestos of Seitz filter has some disadvantages in laboratory work, but due to its cost
advantage, is still used extensively in some industries. The membrane filter has however boomed
in its use in laboratories over recent years.

Fig.3: asbestos filter

The membrane filter has however boomed in its use in laboratories over recent years.
Membrane filters are thin, porous sheet structures composed of cellulose esters or similar
polymeric materials. Pore sizes range from 14µ to 0.25µ and are very uniform (variation of
0.45µ filter being ±0.02µ) and occupy around 80% of the total filter volume. Thus, flow rates are
usually far greater than those of other filters having the same size retention capabilities. They are
not attacked by water, dilute acids and alkalis, aliphatic or aromatic hydro-carbons or non-polar
liquids. If a compatibility problem does exist, solvent resistant filters are available. As they are
integral structures containing no ionic or particulate material, particulate contamination or
alteration of the pH cannot occur.

A 0.22µ membrane filter should normally be used in sterile filtration operations as all reported
bacteria are larger than 0.22µ and will therefore be physically retained under all conditions by
the filter.

Gas sterilization

Sterilization by means of a bactericidal gas, frequently used for items that are heat and moisture
sensitive Ethylene oxide is the gas most often used; it is highly explosive and flammable in the
presence of air, but these hazards are reduced by diluting it with carbon dioxide or fluorinated
hydrocarbons. Gas sterilization is a chemical process resulting from reaction of chemical groups
in the bacterial cell with the gas.

4
Factors influencing gas sterilization include time of exposure, gas concentration, penetration of
the gas, and temperature and humidity in the sterilizing chamber. Automatically controlled
ethylene oxide sterilizers are usually heated to a temperature of 54°C (130°F). A humidity level
of 35 to 70 percent is recommended.

Chemical sterilization
Is typically used for devices that would be sensitive to the high heat used in steam sterilization,
and for devices that may be damaged by radiation (rubbers and plastics can become more brittle
after irradiation.)

Often chemical sterilizers function by using low temperature, highly reactive gases that come
into direct contact with the test article (often through a semi-porous membrane or package.)
Liquids - for example, bleach - are also used for sterilization.

Considerations in Chemical Sterilization

A primary concern in using chemical sterilization is ensuring that the item to be sterilized is
compatible with the sterilant. Some sterilant can be chemically damaging to certain materials;
you may wish to consult with your materials manufacturer for more information.

Other concerns regarding chemical sterilization include the potential harm to humans exposed to
the sterilization chemicals or residuals from the sterilization process. The sterilization process
must be monitored to ensure the safety of workers performing the sterilization.

CHEMICAL METHODS OF DISINFECTION:

Disinfectants are those chemicals that destroy pathogenic bacteria from inanimate surfaces.
Some chemical has very narrow spectrum of activity and some have very wide. Those
chemicals that can sterilize are called chemi-sterilants.
Those chemicals that can be safely applied over skin and mucous membranes are called
antiseptics.
An ideal antiseptic or disinfectant should have following properties:
􀂃 should have wide spectrum of activity
􀂃 should be able to destroy microbes within practical period of time
􀂃 should be active in any pH
􀂃 should be stable
􀂃 should have long shelf life
􀂃 should be non-toxic, non-allergenic, non-irritative or non-corrosive
􀂃 should not leave non-volatile residue or stain
􀂃 Efficacy should not be lost on reasonable dilution
􀂃 should not be expensive and must be available easily

5
ALCOHOLS:
Mode of action: Alcohols dehydrate cells, disrupt membranes and cause coagulation of protein.
Examples: Ethyl alcohol, isopropyl alcohol and methyl alcohol
Application: A 70% aqueous solution is more effective at killing microbes than absolute
alcohols. 70% ethyl alcohol (spirit) is used as antiseptic on skin. Isopropyl alcohol is preferred
to ethanol. It can also be used to disinfect surfaces. It is used to disinfect clinical thermometers.
Methyl alcohol kills fungal spores, hence is useful in disinfecting inoculation hoods.
Disadvantages: Skin irritant, volatile (evaporates rapidly), flammable

PHENOL:
Mode of action: Act by disruption of membranes, precipitation of proteins and inactivation of
enzymes.
Examples: 5% phenol, 1-5% Cresol, 5% Lysol, hexachlorophene, chlorhexidine,
chloroxylenol (Dettol)
Applications: Phenols are coal-tar derivatives. They act as disinfectants at high concentration
and as antiseptics at low concentrations. They are bactericidal, fungicidal, but `are inactive
against spores and most viruses. The corrosive phenolics are used for disinfection of ward
floors, in discarding jars in laboratories and disinfection of bedpans.
Chlorhexidine can be used in an isopropanol solution for skin disinfection, or as an aqueous
solution for wound irrigation. It is often used as an antiseptic hand wash. 20%
Chlorhexidinegluconate solution is used for pre-operative hand and skin preparation and for
general skin disinfection. Chlorhexidinegluconate is also mixed with quaternary ammonium
compounds such as cetrimide to get stronger and broader antimicrobial effects (eg. Savlon).
Chloroxylenols are less irritant and can be used for topical purposes and are more effective
against gram positive bacteria than gram negative bacteria. Hexachlorophene is chlorinated
diphenyl and is much less irritant. It has marked effect over gram positive bacteria but poor
effect over gram negative bacteria, mycobacteria, fungi and viruses. Triclosan is organic
phenyl ether with good activity against gram positive bacteria and effective to some extent
against many gram-negative bacteria including Pseudomonas. It also has fair activity on fungi
and viruses.

Quality Assurance
Quality assurance is an ongoing process, and everyone using any sterilization method should
follow the recommended practices as defined by the Association for the Advancement of
Medical Instrumentation (AAMI) and the policies and procedures of their facility.
AAMI and the policies and procedures of their facility. Though the AAMI Standards and
Recommendations are not law, they are the recognized industry standards for sterilization and
would be relevant in any legal proceeding. It is very important that all users of these methods of

6
sterilization know how to read and interpret the information presented by the quality assurance
monitors in order to ensure compliance with AAMI recommendations and with each healthcare
facility's own policies and procedures.

The efficacy of every sterilization method must be monitored. The quality assurance of each
process includes physical, chemical, and biological monitors.