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Joy T. Kunjappu, PhD, DSc
Disinfectionand sterilization have been favorite recurring themes in infection control. Thisrepetition stems from the importance of the significant "life and death"scenario it embraces. Consequently, these topics play a pivotal role in the management ofsurgical and allied areas.
Ever since Pasteur promulgated the microbiological implications of infection caused bymicroorganisms, many materials and procedures involving germicides, antiseptics, andbacteriostats have been in vogue to control or arrest their growth. However, theseprocedures only served the purpose of eliminating or terminating the microorganisms in alimited sense and were far away from the desirable objective of their total destruction.Thus, the term "sterilization" became popular, and it encompasses "...anyprocess by means of which all forms of microbial life (bacteria, spores, fungi, andviruses), contained in liquids, on instruments and utensils, or within various substances,are completely destroyed."1 In modern times, the inclusion of otherinfections, such as those caused by prions, falls under this definition.2-3
"Sterilization" is a buzzword in Central Service and Infection Controldepartments in any healthcare facility as well as large-scale industrial plants. Varioussterilization processes are in practice today that make use of sterilants such as steam;ethylene oxide; *-ray and electron beam radiations; ultra-violet (UV) light, chemicalslike formaldehyde, glutaraldehyde, and peracetic acid; and low-temperature hydrogenperoxide plasma. Sometimes, filtration through special filters physically removes allmicrobial life.4 These methods have one goal in common: the total removal orkilling of microorganisms present in the material to be sterilized.
Steam sterilization has acquired a special status as a time-tested sterilizationmethod. In spite of its inadequacy in sterilizing thermally labile materials, steamsterilization is still regarded as an ideal choice, primarily due to the non-toxic natureof steam, its low cost, and the highly efficient sterilizing power it embodies. Othersterilization methods described above satisfy the need for overcoming the limitations ofhigh temperature and high pressure steam.
Monitoring thesuccessful sterilization of materials and equipment used in surgical and relatedprocedures is of prime concern since even last traces of microorganisms left alive caninflict infection to a patient. An ideal monitoring protocol will be the one that makescertain that all the microorganisms present in the system are killed (primary monitoring).Such a utopian condition obviously cannot be attained due to practical reasons. Hence,secondary monitoring techniques have been developed where arguments lying in the realm ofinductive and deductive logistics are used to justify the efficacy of the process.
Monitoring steam and other sterilization methods basically are achieved by two generalways: biological monitoring and chemical monitoring. In biological monitoring, killingexternally added microorganisms (resistant bacterial spore preparations) during asterilization cycle is monitored; while in chemical monitoring, the physico-chemicalreactions during a sterilization cycle are monitored. Biological monitoring was consideredto be more realistic than chemical monitoring because, in the former, the efficacy ofsterilization was followed directly by overseeing the system for any traces of addedmicroorganisms left after sterilization.
In biological monitoring of steam sterilization, bacterial spores, deemed to be mostresistant to moist heat, are incorporated into the detection system called indicators.More specifically, a spore-bearing organism like Bacillus stearothermophilus (NCAStrain 1518), which is known to be extremely resistant to moist heat, is used for thispurpose. Total destruction of such a strain is known to occur under steam sterilizationconditions in ~9 minutes at 250Âº F, and in ~1 minute at 272Âº F at 100,000 population ofdried spores. If the system is free from these spores subsequent to a sterilization cycle,it may be inferred that the microorganisms contaminating the sterilized materials will betotally destroyed well before this period of time. And, indeed, a safety margin isinducted into the upper limits of these sterilization cycles to ensure the effectiveelimination of microbial life with a high probability (1/1,000,000).
Biological monitoring, though convincing and reassuring of the absence of livemicroorganisms, is looked at pessimistically due to the inordinate time frames involved ina typical monitoring experiment. To circumvent such a serious limitation, faster andconvenient methods were sought, and the chemical detection systems were the answer.
In a typical chemical monitoring method for steam sterilization, physico-chemicaltransformation of a chemical system on exposure to moist heat is made useful. An idealchemical indicator will indicate the temporal sequences of exposure to steam or othersterilants by distinct color change/s. Time-temperature effects trigger these welldiscernible color changes at a stipulated time period.
Before proceeding further to evaluate the potential of similar chemical monitoringdevices to replace the biological monitors, let's examine the birth and evolution ofbiological indicators in the context of steam sterilization.5 The firstgeneration of biological indicators was the Spore Strip. Strips, are made typically offilter paper, impregnated with known population of bacterial spores and inserted in asterilizable glassine envelope. After sterilization, the spore-strip is sent to amicrobiology laboratory for culture test to detect any surviving microorganism. Such atest usually requires about seven days of incubation and is not generally performed at thesterilization center. The long wait in getting the test results delays the release ofsterilized packs for their purported function. Thus, "fast with the test" wasthe motivating force that led to the invention of biological indicators.
About a quarter century back, a new version of biological indicators came intoexistence. They were referred to as "self-contained indicators" because thegrowth medium was incorporated in an isolated vial within the outer plastic tube thatcontained the spore strip. A pH indicator was used for detection of bacterial growth. Thissystem responded to bacterial growth in one to two days. Also, it gave the flexibility ofincubating the steam processed biological indicator strip within the department aside fromthe short duration needed for the test compared to the classical spore strip. But, eventhese reduced time scales were considered to be too long to provide practical feasibility.
Another formof biological indicator was introduced in the first half of this decade to reduce theresponse time using the denaturing of certain enzymes present in the bacterial spore. Sucha procedure improved the time profile for detection and yielded the results in one tothree hours depending on the sterilization cycle. A fluorogenic biochemical reactionserved as the detection system, and a dedicated fluorescence instrument was needed forthis test. A direct correlation between spore killing and enzyme denaturation processformed the focal point of this method. Moreover, one product was provided with a regularself-contained spore strip also in the same system for further confirmation of bacteriallife.
A recent addition to this evolutionary saga of biological indicators is an enzyme-basedproduct with a response time of about 20 to 30 seconds. The indicator system contained apure enzyme that underwent denaturing during the sterilization cycle. A reagent added tothe sterilized indicator tube developed an end color in the event of sterilizationfailure. This cannot be viewed as a biological indicator since the enzyme itself may beregarded as a chemical, notwithstanding the fact that its origin may be biological. Somepeople consider this type of indicator as a distinct class referred to as "enzymeindicators." But here, they will be included under chemical indicators, though itlacks the important characteristic of a chemical indicator, viz. an instant readout.
Chemical indicators make use of either a physical or chemical transformation of achemical under the sterilization conditions leading to color changes. They may be"process indicators," the type used in autoclave tape or printed onsterilization bags and pouches, which indicate whether an item is processed or not. Or,they may be the "tell-tale" type as in a Bowie-Dick pack, which monitors theprogress of the sterilization process manifested in the form of color changes, functioningas a diagnostic tool. Some chemical indicators are tailor made to detect a particularsterilization cycle. The chemical indicators are available under different names,6such as integrators, emulators, multi-parameter indicators, etc. based on the theirfunction-performance relationship. Some of the terms have their origin in theclassification systems of various enforcement agencies.
Recently, there is a renewed interest in exploring the wide possibilities of chemicalmonitoring. It is practically feasible to arrive at an appropriate chemical system thatwill respond to all the critical parameters of a specific sterilization cycle for any modeof sterilization. This is because the immense potential of multimillion chemical reactionscan be adapted, in principle, to suit the requirement of any kind of sterilant.Furthermore, the abundant variations of color shades that can be generated from manychemical reactions make it possible to attain any desired color to mark the end point of asterilization cycle. Usually, a chemically active system consisting of a thermochromicmaterial is incorporated in a special ink formulation that is printed on a suitablesubstrate to form a chemical indicator. Thermochromism refers to the phenomenon of colorchange by the agency of heat.7
Thus, there is a renaissance in the application of chemical indicators in sterilizationtechnology. A chemical indicator, suitably calibrated, respecting all the criticalparameters of a sterilization cycle can function as an effective substitute of biologicalmonitoring. A case in point is the chemical indicators belonging to the class ofintegrators. These integrating indicator devices for steam respond to the time andtemperature parameters integrating their effects on the performance of the indicator. Thisis achieved by regulating the trigger of the color changing chemistry or the physicalprocess that indicates the end point.
In spite of the inconveniences associated with the use of biological indicators, theyhave high status in sterilization technology since recommendations of many professionalbodies make its periodic or frequent use mandatory. For example, the Association ofperiOperative Registered Nurses (AORN)8 recommends biological monitoring on adaily basis; whereas, the Association for the Advancement of Medical Instrumentation(AAMI)9 stipulates weekly monitoring. However, both the associations recommendbiological monitoring with every load that contains implantable materials.
Some time back, the concept of parametric release was put forward as a means ofsterilization monitoring.10 This is based on the principle that the parameterssuch as temperature, pressure, and time related to a (steam) sterilization cycle, ifmonitored under controlled conditions for each load, can by itself provide a qualityassurance parameter of successful sterilization and may substitute other modes ofmonitoring. Though this concept has obtained respectability in a few European countries,it has not been acceptable to a vast majority. Steam sterilization monitoring is acontinually evolving topic waiting to absorb novel concepts and technology.
Joy T. Kunjappu, PhD, DSc, is a consultant for Chemicals and Consulting (New York,NY). He may be reached at (212) 948-4828 and JKUNJAPPU@aol.com.The author would like to thank Dr. Jane Schuman for inoculating useful ideas.
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