Physical agents act by imparting energy in the form of heat or radiation or by removing organisms through filtration.
HEAT
Heat energy can be applied in the form of moist heat (either boiling or autoclaving), or dry heat, or by pasteurization. In general, heat kills by denaturing proteins, but membrane dam age may also be involved. Moist heat sterilizes at a lower temperature than dry heat because water aids in the disruption of noncovalent bonds (e.g., hydrogen bonds), which hold protein chains together in their secondary and tertiary structures.
Moist heat sterilization (autoclaving) is the most frequently used method of sterilization. Because bacterial spores are resistant to boiling (100°C at sea level), they require a higher temperature to inactivate them. This cannot be achieved unless the pressure is increased. An autoclave chamber, in which steam, at a pressure of 15 lb/in2, reaches a temperature of 121°C and is held at that temperature for 15 to 20 minutes is used to kill organisms. To test the effectiveness of the autoclaving process, spore-forming organisms, such as members of the genus Clostridium, are used. Sterilization by dry heat requires temperatures in the range of 180°C for 2 hours. It is used primarily for glassware.
Pasteurization, used primarily for milk, consists of heating the milk to 62°C for 30 minutes followed by rapid cooling. (“Flash” pasteurization at 72°C for 15 seconds.) This kills vegetative cells of milk-borne pathogens (e.g., Mycobacterium bovis, Salmonella, Streptococcus, Listeria, and Brucella), but does not sterilize the milk.
RADIATION
The two types of radiation used to kill microorganisms are ultraviolet (UV) light and X-rays. The greatest antimicrobial activity of UV light occurs at 250–260 nm, which is the wavelength region of maximum absorption by the purine and pyrimidine bases of DNA. UV irradiation induces the formation of thymine dimers. As a result, DNA replication is inhibited and the organism cannot grow. Cells have repair mechanisms that involve either cleavage of dimers in the presence of visible light (photoreactivation) or excision of damaged bases, which is not dependent on visible light (dark repair). UV radiation can damage the cornea and skin, and its use in medicine is limited. However, it is used in hospitals to kill airborne organisms, especially in operating rooms when they are not in use. Bacterial spores are quite resistant and require a dose up to 10 times greater than do the vegetative bacteria.
X-rays have higher energy and penetrating power than UV radiation and kill mainly by the production of free radicals (e.g., production of hydroxyl radicals by the hydrolysis of water). These highly reactive radicals can break covalent bonds in DNA, thereby killing the organism. X-rays kill vegetative cells readily, but spores are remarkably resistant. X-rays are used in medicine for sterilization of heat-sensitive items, such as sutures and surgical gloves, and plastic items, such as syringes.
FILTRATION
Filtration is the preferred method of sterilizing certain solutions (e.g., those with heat-sensitive components). In the past, solutions for intravenous use were autoclaved, but heat-resistant endotoxin in the cell walls of the dead gram-negative bacteria caused fever in recipients of the solutions. Therefore, solutions are now filtered to make them pyrogen-free prior to autoclaving.
The most commonly used filter is composed of nitrocellulose and has a pore size of 0.22 micrometer. This size will retain all bacteria and spores.
