Microorganisms are subject to destruction in a variety of ways. Aside from the classical methods of steam, dry heat, and radiation, destructive sterilization may also occur by immersion in a chemical solution. This is termed liquid-phase sterilization (1). A number of chemical agents, such as aldehydes, acids, bases, and strong oxidants in solution, under the appropriate conditions, are capable of destroying bacteria and fungi, including both vegetative cells and spores in a quantitative fashion (2,3). Objects to be sterilized are immersed in the solution of the chemical agent, after which the agent must be removed in a manner that preserves the sterilized object from recontamination. Removal of the chemical sterilant from the exposed surfaces that have been sterilized must be accomplished in a manner that maintains the sterility of the item postprocessing. Recontamination falls outside the scope of usual consideration for sterilization processes. However, in liquid chemical sterilization it is customary to include the agent’s removal (whether this is accomplished by physical or chemical means) in the overall process, together with any needed additional steps to avoid recontamination.

A substantial number of liquids in aqueous solution are capable of sterilizing articles during immersion. Examples include the following:

As is the case for gas sterilization, the effectiveness of chemical sterilants varies with concentration and temperature. Other factors that affect antimicrobial activity include pH, extent of mixing (if present), and presence of cellular or other debris. Because of the limited number of variables, process control for sterilization by liquids is relatively simple.

Because there are no widely accepted biological indicators for sterilization by liquids, the use of a common mesophilic sporeformer such as Bacillus atrophaeus or B. subtilis is commonplace because these are the likely worst-case bioburden isolates.

The agents used for sterilization by liquids vary with respect to sterilant stability, effective pH range, concentration, temperature, contact time required, and potential interaction with the materials. When selecting the most appropriate sterilant, manufacturers must consider its effect on the materials, package components, and equipment, as is the case with all other sterilizing processes. The variety of agents, process diversity, and potential applications preclude a material-by-material review of these agents in this chapter. Manufacturers should note that these agents are highly toxic, and appropriate safety measures should be practiced at all times during cycle development, validation, and routine operation.

Experimental evidence has shown that first-order kinetics is appropriate for microbial destruction, which makes validation a simple exercise. The validation of sterilization by liquids can be accomplished using either the half-cycle approach or the bracketing method.

In this method (see Figure 2), analysts evaluate conditions of concentration and temperature that bracket the defined process condition to support both over- and under-treatment of the materials and bioburden, respectively. Users can establish the death rate for the microbial population for each of the conditions that bracket the routine process.

Regardless of the approach used, in order to complete the cycle development and validation manufacturers must identify a rapid neutralization method that inactivates the chemical agent to allow microbial quantification after fractional kill exposure. The exposure periods may need to be brief because many of these agents have rapid kill rates.