CA2244125A1 - Indicator systems and material compression and insertion devices for preparing same - Google Patents

Abstract

This invention relates to novel apparatus and methods for inserting and positioning a compressible material into a container and for using the container for detecting a specific environmental parameter or combination of parameters and for determining the effectiveness of a sterilization procedure. Precise positioning of a plug of compressible material in a container provides flexibility for production of indicator systems that vary in their response to sterilizing conditions. These indicators reflect the efficacy of sterilizers based on different modes of sterilization and reproduceability for accurate monitoring of each mode. The invention also relates to test indicators containing controlled volumes of compressed, gas-permeable materials and to methods for using test indicators for determining the efficacy of different types of sterilization processes. The test indicator consists of a plurality of interactive enzymes in a container with at least one opening. The opening is filled with a compressed cylindrical foam insert and the test indicator is placed into the sterilization chamber. The foam insert regulates the amount of sterilant such as steam, gas, chemicals or plasma entering the test indicator. Upon proper sterilization, the sterilant destroys the interactive enzymes and no color product is formed. Inactivation of the enzyme system parallels the inactivation of bacterial spores subjected to the sterilization process. Results are available in from a few seconds to a few hours. The test indicator can also be placed into a container with material such that the design simulates an environmental parameter test of the sterilization process.

Description

CA 0224412~ 1998-07-22 W O 97/26924 PCT~US97/00554 INDICATOR SYSTEMS AND MATERIAL COMPRESSION
AND INSERTION DEVICE~ FOl~ PR~P~NG SAME
Back~round of the Invention Field of the Invention This invention relates to indicator systems comprising a container cont~ining an environmental sensing system and compressible material separating the sensing system from its surroundings. Indicator systems can be used for the determination of sterili7~tion in a variety of sterilization processes or in d~le,~ g the efficacy of an environmental test such as a test for air removal from a sterilization chamber The invention fur~er relates to novel methods for inserting and positioning a compressible material into a container and for using the container for detecting a specific environmental parameter or combination of parameters, or for determining the effectiveness of a sterilization procedure. The invention also relates to novel apparatus for use in these methods.
Description of the Background Iri health care, as well as many other industries, it is nearly always necessary to monitor the effectiveness of processes used to sterilize equipment such as medical devices, instruments and other disposable or nondisposable articles, and often waste. In these settings, sterilization is generally defined as the process of completely destroying all viable microorgs~nismc including structures such as viruses, spores, yeasts and fungus. Standard practice in hospitals is to include a sterility indicator in a batch of articles to be sterilized. The use of sterility indicators allows a direct and sensitive approach to assay the lethality of the sterilization process.
~ A standard type of biological sterility indicator includes a known quantity of test microbial spores. This indicator is placed into the W O 97126924 PCTrUS97/00554 st~rili7~hon charnber and exposed to the sterilization process along with the objects to be sterilized. The test microorg~ni.~mc, for example Bacillus s~earothermophilus or B. sllbtilis spores, are incubated for a specified period of time under conditions which favor proliferation and examined for possible 5 growth, such as turbidity in the growth medium or the presence or absence of certain metabolic produets of any surviving microor~;~ni.cm~. Positive growth, indieating the presenee of a viable spore, indicates that the sterilization process was insuffieient to destroy all of the microor~ni.cm.s.
While the apparatus for cont~ining the spores has varied eontinuously, the 10 general sterility deteetion proeess has not. Many sueh indicators are disclosed in U.S. Patent numbers 3,239,429; 3,440,144: 4,596,773;
4,717,661; 4,732,850 and 5,167,923.
The largest use of sterility indieators oeeurs in researeh and the health care industry. Typieally, sueh facilities have limited resources and 15 must reuse their rnaterials and instruments within 24 to 48 hours after sterilization and often immediately. Conventional sterility indieators normally require that the microorg~ni~m~ be cultured for at least two and often up to seven days to assure adequate detection of any surviving mieroorg~ni.cmc. During this time, items which go through the sterilization 20 process. should not be used until the results of the spore viabiIity test have been determined. Consequently, a holding period for sterility verification is often required. This holding period is both impractieal and ine~fieient and, thus, the major drawback of all conventional sterility indieators.
The use of an enzyme and its subsequent activity as an 25 indicator in detecting sterility has been described in U.S. Patent number 5,073,488. This technology has been greatly advanced with U.S. Patent number 5,486,459 whieh describes the use of a plurality of interactive CA 0224412~ 1998-07-22 W O 97/26924 PCT~US97/00554 enzymes. This technique involves subJecting a set of interactive enzymes to a sterilization cycle. Following completion of the cycle, the set is incubated with a substrate which is acted upon by the enzymes and transformed into a detectable product. Er~rme-modi~led product can be detected, for 5 example, colorimetrically or fluorometrically. This method has been proven to be accurate and detection speeds are greatly accelerated as compared to spore systems. In fact, definitive results using interactive enzyme technology can be determined in less than a few minutes.
Sterility indicators are often placed in special packaging or lû wraps to sim~ te the condition of wrapped goods being processed in a sterilizer. If the articles to be sterilized are in special wrappirlgs or pack~ing, the sterilant needs to effectively pass through the wrappings to destroy microor~ni.sm.c on the article. To test the effectiveness of the sterilant passing through additional materials, sterility indicators are placed 15 in challenge packs. These packs impede the st.~ nt as would the wrappings and thereby represent the conditions of wrapped goods in a sterilizer.
~ here are international standards such as the International Or~ni7~tion for Standardization (ISO~ and the European Standards (EN) that deal with sterilization testing including steam sterilization. International 20 standards dealing with biological indicators and testing procedures are foundin the ISO 11138 series and EN 86û series. Tntern~tional standards for the air removal tests for pre-vacuum steam sterilizers comprises a chemical indicator in a test pack are found in the ISO 11 140 series and EN 867 series.
These packs incorporate the Bowie-Dick test and have similar performance 25 standards as seen in AAMI (American Association of Medical Instrumentation), but use different testing procedures.

CA 0224412~ 1998-07-22 W O 97/26924 PCT~US97/00~54 AAMI has proposed guidelines for challenge packs cont~ining indicators that are assembled by hospital workers to simulate the conditions of wrapped goods in a steam or ethylene oxide sterilizer. Materials re~uired for an ~AMI challenge pack for a steam sterilizer include sixteen freshIy laundered huck towels, autoclave tape and sterility indicators. In one method, each towel is folded length-wise into thirds and then folded width-wise in half. Towels are placed one on top of another with the folds opposite each other. Sterility indicators are placed between the eighth and ninth towels and the pack is secured with autoclave tape. The AAMI steam challenge pack is placed into a steam autoclave for the a~pl~liate arnount of time. Upon completion of a cycle, the indicators are processed to deterrnine if the sterilization process was sufficient to inactivate the indicators buried in the pack.
In the case of ethylene oxide sterilization, AAMI recomrnends placing a sterility indicator into a plastic syringe so that the plunger is not touching the indicator. In this case, the needle end of the syringe is open.
Two such syringes are placed in the center of a stack of folded towels and the stack is wrapped in a single towel. For routine monitoring, the syringe and indicator can be wrapped in a single towel and placed into a peel pouch.
Tests are also perforrned that evaluate the effectiveness of air removal in a prevacuum steam sterilizer. Prevacuum steam sterilizers are used to ...i~i...i,.~ the amount of air present in the sterilization chamber, thus enhancing the penetration of steam into porous loads. A prevacuum sterilizer air removal test is also l~nown as the Bowie-Dick test or a 25 prevacuum sterilizer residual air test.
AAMI guidelines for the Bowie-Dick test pack state that the standard pack is made using folded cotton surgicaI towels. Several towels CA 0224412~ 1998-07-22 W O 97/26924 PCT~US97/00554 are folded to create a stack 10 to 11 inches high with a rectangular border of 9 by 12 inches. A Bowie-Dick test sheet, which comprises a pattern of chemical indicator ink or indicator type on a porous sheet, is placed in the center of the pack. The pack is wrapped in a single cotton wrap and 5 processed in a steam prevacuum sterilizer. The acceptance criterion is that the test sheet or tape (l~rkenc .Iniru~ ly after processing. In other words, thechemical indicator ink changes color upon exposure to steam and if the entire sheet shows a uniform color change, there was no residual air to impede the steam.
AAMI guidelines state that other devices may be used in place of the AAMI challenge packs and Bowie-Dick tests if they provide equivalent results to the AAMI packs. Enclosure of sterilization indicators in various fibrous m~teri~l.c, analogous to textiles such as the towels used in the AAMI challenge packs7 has been proposed in U.S. Patent numbers 15 5,200,147; 5,252,484 and 5,223,401. Packages in which a sterilization indicator is surrounded by porous material to replace some of the towels are described in U.S. Patent number 4,692,307.
Compressible material, such as ~oam, has a wide variety of uses when placed inside a container. For example, compressible material in 20 a container could be used for absorbing shock vibrations or sound, as a barrier for solids, liquid or gases, for separating components, for absorbing liquids and/or for application of liquids such as ink, paint or antiseptics. A
major drawback of using compressed material in a container is the expense of compressing the material into the container. The conventional process for 25 inserting compressed material into a container was for the material to be compressed by hand and forced m~ml~lly into the container. This method is~slow. often L~ oducible and, consequently, expensive.

U.S. Patent number 3,811,242 relates to an apparatus for compressing blocks of compressible material, particularly polyurethane, to a small percentage of their original volume by compressing in the direction of the longest axis of the block, ~llcces.~ively, and in perpendicular directions 5 untiI the block is of the desired si7e. Re~ ing bands are appIied to the block to prevent rebound or expansion in all directions of compression.
U.S. Patent number 5,400,067 relates to an apparatus for inserting a rectangular foam insert into the rectangular ink chamber of an ink jet print head. The apparatus involves two flat pistons opposing two fixed 10 plates which fo~n a right angle. Pressure is exerted by each of the two pistons successively ~g~in~t two adjacent sides of the foam rectangle to compress the foam to a cross-sectional area smaller than the area inside the Ln,k c~.ber. ~e il~ e~r.bel is p~si~ioned over a recf:~n~l~r waiied tube formed by extensions of the pistons and the opposing plates, and then a ram lS moving orthogonally to the two pistons pushes the foam into the ink chamber. This device requires successive compression in perpendicular directi~ns by three moveable components.
U.K. Patent mlmber 2,084,954 reIates to a method for packaging a cylindrical sponge into a tllbe. The sponge is placed on a 20 support plate between two jaws having concave arcuate section. According to this method, one jaw is fixed and a second jaw moves across the plate.
Sponge is first compressed by a platen parallel to the support plate which descends toward the support plate until the separation between the platen and the support plate is equal to the desired diameter of the compressed 25 sponge. Sponge is further compressed by movement of the moveabIe jaw across the plate lmtil the two jaws abut, forming a cylindrical cavity cont~ining the compressed sponge. The tube is axially aligned with the CA 0224412~ 1998-07-22 W O 97/26g24 PCTnJS97/005S4 cavity and the sponge is pushed from the cavity into the tube by a plunger.
This device requires successive compression in perpendicular directions by ~ numerous moveable components that must be kept in perfect alignment.
U.S. Patent number 4,602,472 relates to a device for p~rL ~ging 5 rolls of fiber insulation by compressing them in a compression chamber which employs double stage compression. A pivoting stage compresses the roll in a first direction, analogous to the platen of U.K. Patent number 2,084,954. A ram having a concave semicylindrical surface compresses the roll in a second direction to form a cylinder of the desired size.
10 Subsequently, a discharge ram pushes the roll along the axis of the cylinder into a tubular member suitably sized to receive the roll. A paper or plastic sleeve having a closed end is placed over the tubular member~ and the roll pushed through the tubular member into the sleeve, thereby pulling the sleeve off the tubular member and enclosing the roll in the sleeve material.
U.S. Patent number 5,208,954 relates to a device for inserting, into cavities in masonry building blocks, preformed insulating foam inserts wherein the foam inserts are slightly larger than the cavities. In the device, the foam is positioned over a channel which in turn is aligned with the cavity in the block, the channel having a throat with curved sidewalls. A tamping 20 head pushes the foam insert through the channel where the foam is compressed by the throat to have a cross-section small enough to fit into the cavity.
U.S. Patent number 3,450,036 relates to a device for packing loose granular material such as potting soiI around a plant and depositing the 25 packing material and plant into container, such as a pot or bag. The device includes at least two arcuate sections that fit together to form a cylinder holding the plant and soil. A ram pushes the plant and soil together out of CA 0224412~ 1998-07-22 W O ~7126924 PCTnUS97/00554 the cylinder and into the container. As potting soil is not compressible to any significant degree, the device is a forming apparatus, but not a compressing apparatus.
None of these apparatus and methods are satisfactory for 5 reproducibly inserting and positioning a compressible material such as foam or sponge into a vial.

Summary of the Invention The present invention overcomes the problems and 10 disadvantages associated with current strategies and designs and provides novel methods and apparatus for inserting and positioning a compressible material into a container and novel methods and test indicators for det~rrnining the effectiveness of a sterilization procedure or measuring a pararneter of the sterilization process.
One émbodiment of the invention is directed to apparatus for consistently positioning a compressible material in a container. Precise positioning of a plug of compressible material in a container has been discovered to provide the flexibility necessary for production of indicator systems that vary in their response to sterilizing conditions to reflect the 20 efficacy of sterilizers based on different modes of sterilization and the reproduceability necessary for accurate monitoring of each mode.
Another embodirnent of the invention is directed to methods for reproducibly compressing a gas-permeable, compressible material and positionally inserting the compressed material into a container. The 25 apparatus is particularly useful for m~nllf~cture of adjustable indicator systems for the determination of the effectiveness of sterilization processes ~sing steam, gas, chemical and plasma sterilizers. These systems can be CA 0224412~ 1998-07-22 W O 97/26924 PCT~US97/005~4 used for many types of tests in hospitals, laboratories and clinics, as well as in research institutions, in food and environment~l technology and in technologies that utilize sterilization in m~nllf~cturing, production or waste disposal.
Another embodiment of the invention is directed to test indicators co~ ,g a biological material separated from the sterilizer environment by a control~ed volume of compressed, gas-permeable material.
The material controls access of the sterilizing medium to the biological material. Volume, size, shape or density of compressed. gas-permeable material in the indicator is ~le~ ed by the particular sterilization process.
Another embodiment of the invention is directed to a test indicator for del~ g the effectiveness of a sterilization procedure. Test indicators comprise an outer container having liquid impermeable and substantially gas non-absorptive walls, at least one opening leading into a chamber which contains one or more components of an interactive enzyme system, and a liquid impermeable or liquid semi-permeable gas-transmissive barrier between the components and the opening. These components may be fixed to a solid support or free-floating in a non-aqueous or partially-aqueous solution. After ster~ization, the user simply mixes the components in the container with the rem~ining components of the enzyme system. If any enzyme activity is present, the enzymes plus any necessary coenzymes, cofactors and catalysts will interact with the substrate fom~ing detectable product which can be assayed to delrl ,.~ e the effectiveness of the sterilization procedure.
Another embodiment of the invention is directed to methods - for de~ g the efficacy of di~e~ L types of sterilization processes. The test indicator consists of a plurality of interactive enzymes in a container CA 0224412~ 1998-07-22 W O 97126924 PCT~US97/005~4 with at least one opening. The opening is filled with a compressed cylindrical foam insert. The test indicator is placed into the sterilization chamber. The foarn insert regulates the arnount of sterilant such as steam, gas, chemicals or plasma entering the test indicator to achieve a response S that can be equivalent to indicators collt~inin~ bacterial spores. After the st~riT;7~ion cycle is complete, the foam insert is removed and the rem~in;n~
components of the enzyme system are combined. If the proper sterilization conditions were not met, the interactive enzyme system remains active, and a color product forms upon the addition of the rem~inin~ components of the 10 enzyme system. If the proper sterilization conditions were met, the sterilant has destroyed components of the interactive enzyme system and no color product is formed. Inactivation of the enzyme system parallels the inactivation of bacterial spores sub~ected to the sterilization process. Resultsare available in from a few seconds to a few hours.
Another embodiment of the invention is directed to methods for adjusting the sensitivity of sterility indicators of the invention to one ormore predel~ ed environm~nt~l parameters. Test in~ tors, substantially identical to the sterility indicators, are exposed to a sterilization procedure and the effectiveness of that procedure determined. The position andlor composition of the gas-tr~ncm;cc;ve plug of another test indicator is adjusted and the another test indicator is exposed to the sterilization process. From the effectiveness of each test indicator for reacting to the environmental parameter, the sensitivity of the sterility indicator can be accurately and quanLiL~Li~ely adjusted.
Another embodiment of the invention is directed to methods for creating a challenging environment for the penetration of sterilant. Using an enzyme, spore or chemical indicator in a container with a controlled CA 0224412~ 1998-07-22 volume of gas-permeable material for creating a reproducible resistance for the steri~ant penetration. a test pack for evaluating sterilant penetration or air removal is created.
Other embodiments and advantages of the invention are set 5 for~, in part, in the description which follows and, in part, will be obvious from this description or may l~e learned from the practice of the invention.

Description of the Drawin~s Figure I Diagram of the construction of a container for a rapid sterility I O i~idicator.
Figure 2 A preferred embodiment of the rapid sterility indicator unit.
Figure 3 Diagram of the ~lefe~l~d operation of a multiple-component container of a rapid sterility indicator.
Figure 4 Diagram of a challenge pack.
1~ Figure 5 A two container design challenge pack.
Figure 6 Rapid sterility indicator test unit challenge pack design.
Figure 7 A vertical front view of a machine incorporating a slide with a loop of flexible material to compress the foam and a plunger for insertion into the container.~0 Figure 8 (A) A vertical front view of a machine with a horizontal crusher slide to compress the material and a vertical plunger and staging nozzle used for insertion, and (B) a side view of the ~ng~ r forming die to compress the material prior to insertion.~5 Figure 9 A front view of an a~p~Lus with a rotating container held by a flexible tu~e used for insertion of compressible materials.

W O 97/26924 PCTrUS97/00554 Description of the Invention As embodied and broadly described herein, the present invention is directed to methods and apparatus for inserting and positioning a compressible material into a container, to test indicators cont~inin~
controlled volumes of compressed, gas-permeable material for det~rrnining the effectiveness of a sterilization procedure and to methods for using the test indicators.
Conventional sterility indicators typically comprise viable spores which are exposed. along with the objects to be sterilized, to a sterili7ing condition. After exposure, the indicator is removed and the spores cultured under defined conditions. Culturing takes days to a week for any sort of definitive results. Indicators often require post-sterilization incubation at higher than ambient temperatures to provide detectable results.
It had been discovered that in~1ic~tors comprising enzymes and preferably interactive enzyme systems are a suitable substitute for spores.
Inactivation of an enzyme system by a stenli7~tion process mimics the death of viable spores. It has also been discovered that an insert design comprised of a compressible material such as foam provides accurate as well as reproducible results with these steriIity indicators.
Indicators comprising multiple interacting enzymes and adJustable foam inserts overcome the problems and disadvantages present with current strategies and ~1esign.~ for evaluating sterilization processes andcan provide nearly in~t~nt~neous and reproducible results. The indicator system is simple to use and requires minim~l training. Reliable results are achieved without specialized instructions or equipment. Surprisingly, indicators are also useful for monitoring many types of sterilization p~ocesses. The amount and/or length of the insert is simply adjusted during CA 0224412~ 1998-07-22 W O 97/26924 PCTrUS97/00~54 manufacture according to the requirements of the process. In all cases, the results that can be achieved are rapid as well as reliable and reproducible.
One embodiment of the invention is directed to a test ndicator device (the rapid sterility indicator or RSI) for the rapid determination of theefficacy of different types of sterilization processes (e.g., steam heat, dry heat, chemical sterilant, plasma). Indicators comprise a compressible material placed as a plug into an opening or sleeve of a container wherein the container or sleeve has a smaller cross-sectional area than the cross-section of the article when not compressed. The container is made of non-adsorptive m~t~-ri~1 such that the only pathway for the sterilant is through thefoam insert. That foam insert regulates the amount of sterilant (e.g, steam, gas, chemicals or plasma) entering the test indicator and the amount of foam utilized may be regulated according to the steri1i7:ing process. Indicator reagents are placed in the container with at least one opening and the opening is filled with the compressed cylindrical insert.
The container may be a vial in which a foam stopper is to be inserted or a cylindrical tube in which the plug is partially permeable to liquid. This foam stopper forms a filter for fluids passing through the tube.
The compressible m~t~ri~1 is a gas-permeable, open-celled natural or ar~ficial (plastic) foam or sponge which may be comprised of, for example, polyu~ e, polyester, polyether, cellulose, melamine or a combination of these materials. Foam density, pore size, cell structure (the percent of opened cells), size, shape, amount of foarn, stiffness and tensile strength can be chosen to fit the particular situation.
For a container with a cross-sectional area of from about 0.03 to about 0.2 square inches, the compressed material has a non-compressed c~oss-sectional area of from about 0.2 to about 3.5 square inches. The inside CA 0224412~ 1998-07-22 W O 97/2692~ PCTrUS97/005~4 portion of the plug is from about 0.4 to about 2.0 inches in length, preferably about 1.2 to 1.5 inches, with an overhang portion. Preferably, the overhang portion is less than about 0.5 inches in length. These areas and lengths can be adjusted accordingly for larger and smalIer sized containers. The inside S leng~h and/or the length of the overhang portion can be adiusted very easily during m~mlf~cture. Optimal lengths can be determined empirically by one of ordinaly skill in the art according to the parameters of a particular sterilization process. Shorter lengths tend to be most useful for chemical process whereas longer plug and overhang lengths are typical for steam 10 steriIization. Adjustments can also be made to the distance of the plug from the sensing system and the density, the degree of compactness and the composition of the plug. All of these factors affect the sensitivity of the indicator to the sterilization process.
As indicators are easily adjusted, another advantage is that 15 in~1ic~t~ rs can be modified to meet all major as well as minor alterations of a s~erilization process. It is not necessaly to switch to another type of sterilization indicator upon chz~ngin~ sterilization processes or sterilants.
The insert can be varied to optimize the sterility indicator and thereby meet multiple situations and different sterilants as well as different sterilization 20 protocols. It is also not necessary to change the type of sterility indicatorupon ch~n~ing the sterilization process. As an adJustment can be as simple as ch~n~np; the length of the plug, it is a very straight forward matter to irnplement a change during manufacture with little to no added expense.
Sterility indicators fu~er contain spores, enzymes, an enzyme 25 system or combinations thereof, as sensing reagents that provide an indication of sterility. These reagents may be a liquid or solid. Liquids are preferably in a non-aqueous or partially aqueous medium. Solids may be CA 0224412~ 1998-07-22 W O 97t26924 PCT~US97/00554 membranes such as disks and are preferably powders or tablets that contain granularized reagents. Such reagents can be made into a ~ranulation by fluid-bed granulation. Fluid-bed granulation takes different components and coimmobilizes these components into clusters. Clusters comprise dirrt;~ t components dried onto a seed particle. The granulation process begins by suspending a seed material in air and spraying a liquid material onto the seed. Other components are added either to the liquid solution or to the fluidized particles. Particles adhere to the liquid and form clusters of different components and, finally, moisture is removed from the clusters.
The granulation process can be used to m~nllf~cture enzymes coimmobilized in a granulation or pressed into a tablet with little moisture as enzymes are typically most stable when packaged without water.
Granulation begins with a dry powder, referred to as the seed, which functions as a solid support. Seed material can be an inert substance or one of the components of the granulation and is placed into the process chamber. Controlled airflow into the chamber creates an air suspension of the particles and, thus, particles are suspended or fluidized. Once the particles are suspended in the air, a liquid solution is sprayed onto the solid particles.
The humidity, temperature and air velocity are controlled in the chamber. Humidity is kept very low and the temperature is increased to approximately 35~C. The liquid, a~ter being sprayed onto the seeds, evaporates and a granulation is ~ormed. Seeds are coated with the dirrt;
ingredients fo~ning the clusters and water is removed.
There are several ways two enzymes can be formed into a granulation product. For example, each enzyme can begin as a liquid solution. Using an inert solid seed material such as cellulose, one enzyme CA 0224412~ 1998-07-22 W O 97/269~4 PCTAUS97/0~554 is sprayed onto the tluidized cellulose seeds. A second granulation is made of the second enzyme and the two granulations are blended together.
Alternatively, the two enzymes could be mixed together as one liquid solution and sprayed onto the seed material. Altern~hvely, one or both S en~ymes could begin as a solid material. The solid material would be used as the seed material and a liquid binder solution is sprayed onto the seeds.
Liquid solution is needed to create granulation and the solid, dry components adhere to the liquid solution. While the material is being fluidized, the high temperature and lo-Y humidity remove water from the granulation product 10 and the enzymes are coimmobilized onto the seed material.
Granulations can also be pressed into tablets. For example, several granulations can be blended together using mechanical blenders and pressed into a single tablet. ~hen working with several granulations, each can be tested for activity and then the final composition of the tablets activity I 5 can be adjusted by altering the amounts of each granulation component. The final enzyme tablet will contain very little water, typically less than about 5% and preferably less than about 3%.
Indicator reagents suitable for some applications comprise a single enzyme, such as that described in U.S. Pat. No. 5,~73,488, aIong with 20 the substrates, re~gt?nts, catalysts, co-factors, etc., necessary to produce a detectable product. Indicator reagents may also comprise multiple components of an interactive enzyme system. The enzyme system preferably comprises a known mix of enzymes, coenzymes, catalysts, cofactors, substrates, other reaction reagents or combinations such as those 25 provided in U.S. Patent number 5,486,459. Enzyme systems comprise a plurality of enzvmes that rapidly catalyze a series of coupled reactions which to~gether produce a detectable product.

CA 0224412~ 1998-07-22 Another embodiment of the invention is directed to a method for determining the effectiveness of a sterilization process. The basic process comprises subjecting at least one and pre~erably multiple components of an enzyme system to a sterilization procedure. The enzyme 5 system comprises a known mix of enzymes, coenzymes, catalysts, cofactors, substrates, other reaction reagents or combinations thereof, which is housed in a test indicator. The components have an interdependent activity which correlates with the viability of the microorg~ni.~m.~ used in state-of-the-art biological indicators.
According to this method, a test indicator is placed into the sterilization chamber and subjected to a sterilization process. After the sterilization cycle is complete, the foam insert may be removed and the r~m~ining components of the enzyrne system added to form a mixture. The ll~Lulc~iS incubated, if necessary, for a period of time sufficient to allow for15 product formation from the interaction of the enzymes with the substrate.
Incubation times range from a few seconds to a minute and are preferably less than about 15 minl~tes, more preferably less than about 10 minutes and even more preferably less than about 3 mimltes. If desirable, incubation can be elimin~te-l and the product detected almost immediately or in less than 20 about 20 seconds. A detectable product will form if all of the components of the enzyme system, including the plurality of enzymes, are present and active. A posi*ve result is observed when each exposed component survives denaturation and is able to fimction interactively to produce a detectable enzyme-modified product. The enzyme-modified product as an indicator of 25 residllal ac*vity is visually detectable within 1 to 60 mimltes and preferably within seconds. Any change detected, which is preferably a color change, is an in(lic~*on to an observer that the sterilization cycle had not inactivated certain components and, thus, was insufficient to assure sterilization of other articles exposed to the sterilization procedure. Conversely, an absence of a color change indicates that the sterilization procedure had inactivated at leastone of the components thereby preventing the interactive reaction from taking place and thus, an equivalent of rapidly and directly detecting the survivability of bacterial spores in a similar conventional test.
~ack of detectable enzyme-modi~led product within the established period of time indicates a sterilization cycle which has been lethal to the function of the interactive enzyme system as well as lethal to a viable 1 o6 population of Bacillus stearothermophilus spores. Generally, these values are expressed as D-values, which is the time taken at a given temperature to reduce the viable population of test microor~ni~nlc to ten percent of its original value. Inactivation of the enzyme system paralIels the inactivation of b~cteti:~l spores subjected to the sterilization process, exceptthat the result may be available in minutes or seconds as compared to at least overnight incubation required for detection of bacterial growth from spores.
Product can be detected using a variety of procedures. For example, substrate can be labeled and the resulting radioactivity or enzyrnatic, electrical or fluorometric activity of the product detected using conventional devices such as, for example, those utilized for det~ ~ing the effectiveness of sterilization procedures. Preferably, product is detected visually as visual detection is simple and inexpensive recluiring little training and no specialized inst~-ment~tion outside of what would be found in a typical working environment.
The relationship between the components is very relevant to a d~te~ tion of sterility because it is not simply a chemical or enzyme reaction, but an enzyme interaction reflective of the p-e~,un-~tive CA 0224412~ 1998-07-22 W O 97/26924 PCT~US97/00554 physiological state of microor~ni.cm.~ within the chamber. The ability of the methods of the invention to rapidly determine the efficacy of a sterilization - cycle is based upon the discovery that the survival of functional capability of an enzyme system is necessaIy for the production of an enzyme-modified 5 product. The rapidity of formation of the enzyme-modified product from the interacting enzymes is due, at least in part, to coimmobilization wherein the close proximity of two or more components of the enzyme system on a common solid support such that diffusion controlled exchange with buL~
solution is limited. This process is further supplemented by component 10 channeling or, the bringing together of two or more components of sequential reactions at a surface or microenvironment to further limit diffusion-controlled exchange with buL~ solution. Component channeling with regard to enzymes is described in I. Gibbons et al. (Meth. Enzymol.
136:93-103, 1987~.
The ability of the components of an enzyme system to survive conditions which only partially kill test microorg~ni.~m~ is dependent, at least in part, upon the use of a semi-permeable barrier between the sterilant and the enzymes, and that the interactive enzyme system will remain active following a sterilization cycle which is insuf~lcient to kill the test 20 microorg~ni~m~. ~t is not necessaTy that the barrier be impermeable to microor~ni~m~ such as bacteria, only that it be fluid permeable to permit exposure of the indicator components to the sterilizing ellv~ mllent. Such as through open cells of a compressible material or around the sides of a closed cell compressible material. This provides a direct correlation of 25 spore viability with the interactive activity of the enzymes of the system which, following an inadequate sterilization cycle, is sufficient to convert a s~bstrate system for those enzymes to a visually detectable concentration of W O 97/26924 PCTrUS97/00554 product within a relatively short time, preferably I to 60 minutes. The basis for the correlation between the activity of the enzymes and other components to the germin~hon and growth of rnicroorg~ni~m~ is due to the commonaIity of both in their reliance upon systems of biologically derived interacting S enzymes and coenzymes to function. The sterility indicator demonstrates that there is a direct correlation between the conditions to kill a microorganism and the conditions to inactivate a component of a network of interacting enzymes. In fact, the interactive system can be considered to mimic a bacterial spore in that there is a semi-permeable membrane, the 10 spore wall, that encases a collection of interactive enzymes. In the case of an amplification interactive enzyme system, if any one of the key enzymes, coenzymes, cofactors, substrates, cataIysts, or other reagent components of the system are totally inactivated when an indicator solution is added, no color change will occur, thus, mimicking conventional spore systems, but IS able to provide results at much faster speeds.
Using the test indicators of the invention, steriIity verification is de~elll.i.,ed from completion of the test results which, surprisingly, can bevery rapidly achieved because the reliability of conventional biological ;n~ ~rs is combined with the speed of techni~ues closer to that utilized by 20 enzymatic and chemical inf~iC~tr~rs Further, and unlike spores, resistance is correlated with activity, and in enzyme systems conf~;ning enzymes, coenzymes, catalysts, substrates or other reagents of an interactive system, stability can be very precisely q~ ed individually as well as in multiple enzyme systems. Therefore, using interactive enzyme systems not on~y is 25 speed increased, but a level of standardization can be achieved which is far superior to that obtained with conventional biological or other enzymatic tec~niques.

CA 0224412~ 1998-07-22 Another embodiment of the invention is directed to methods for the m~m-f~cture of adjustable indicator systems for the determination of the effectiveness of sterili7~tion processes using steam, gas, radiation, chemical and plasma sterilizers, which are used in many hospitals, 5 laboratories, and clinics, as well as in research institutions, in food and environrnental laboratories, and in all technologies which utilize sterilizationin m~nllf~cturing, production or waste disposal.
Sensitivity of sterility indicators can be adjusted quickly and easily for the manufacture of sterility indicators reactive to one or more 10 predeterrnined parameters. For exarnple, a test indicator substantially identical to the sterility indicator is exposed to a sterilization procedure andthe effectiveness of that test indicator for reacting to the predetermined environmental parameter de~ led. The position and/or composition of the, for example, gas-tr~n.cmi.~sive plug of another test indicator is adjusted 15 and another test indicator is exposed to the sterilization process. From the results determined for each test indicator, the sensitivity of the sterility indicator can be adjusted to Op~lLi~ detection to the specific environmental condition or conditions.
Adjl-~tment~ can be as simple as repositioning the gas-20 tr~n.~mi.csive plug such as, for example, by extending or retracting anoverhang portion of the plug, or by altering the composition of the plug by, for example, increasing or decreasing plug density, pore size or composition.
The overhang portion could be extended to increase sensitivity of the indicator to a com~ination of increased temperature, humidity and pressure.
25 A plug comprised of a compressible material can be adjusted by increasing the density of the compressible material to decrease sensitivity of the W O 97/26924 PCTnUS97/0055 indicator to a combination of increased sterilant, chemicals, temperature, humidity and pressure.
A sterility indicator includes a biologically relevant material, such as bacterial spores or preferably a source of multiple interacting 5 enzymes, in a container having a liquid impermeable and substantially gas non-adsorptive wall and at least one opening filled with a gas-transmissive barrier, said opening leading into a chamber which contains one or more components of the interactive enzyme system~ with the gas-tr~n~m;~cive barrier between the components and the opening. Interacting components 10 are preferably loca~ized within close proximity to one another such as withinthe matrix of a cellulose filter disk or granulation product, and/or within a defined medium and are thus, coimmobilized. One or more enzymes, substrates, coenzymes or catalysts may be included on the solid matrix.
Within the container is an effective amount of a gas-~n~mi.~sive material to l S form the barrier which is semi-permeable, but not freely or wholly permeable to the tr~n.cmi.csion of liquids and gases, and an effective means for m~int~inin~ a finite distance between the semi-permeable opening and the enzyrnes. The barrier may be liquid permeable or impermeable, but is preferably a sponge which reduces the likelihood of slippage that may 20 sometimes occur with plungers and stoppers. Also p lerel~ble is a barrier which is a plug that is constructed of a polymer such as a synthetic, a plastic,a rubber, Gore-Tex (a gas tr~n.cmi~sive and liquid impermeable polymer) or a combination thereof. A Gore-Tex barrier would be liquid impermeable whereas an open cell foarn barrier, such as a sponge, would be liquid semi-25 permeable.
A rapid multiple enzyme sterility indicator of the invention is ill~strated in Figure 1. The indicator comprises cylindrical tube 10 having CA 0224412~ 1998-07-22 W O 97/26924 PCTrUS97/00554 liquid impermeable walls with single opening 11 at one end. Cylindrical tube 10 contains solid support disk 12 upon which multiple interacting enzymes are coimmobilized. Cylindrical tube 10 also contains non-aqueous medium 13 covering solid support disk 12. Single opening 11 is covered 5 with cap 14 having a plurality of holes 15 allowing unimpeded access of sterilant through single opening 11. The apparatus of Figure I is assembled by placing solid support disk 12, upon which multiple interacting enzymes are coimmobilized, into the bottom of cylindrical tube 10. Non-aqueolls medium 13 is added to cover solid support disk 12. A cylinder of heat 10 resi.ct~n~ foam material 17 is compressed into cylindrical tube 10 providing a structural framework for the containment of non-aqueous medium 13.
Foam material 17 also serves to m~int~in a fixed distance between the multiple interacting enzymes coimmobilized upon solid support disk 12 and single opening 11. Cap 14 is placed on top of cylindrical tube 10 covering 15 single opening 11.
A preferred indicator unit of the invention is the rapid-multienzyme sterility indicator shown in Figure 2. This multiple-enzyme sterility indicator comprises a test unit and indicator solution. The test unit is comprised of cylinder tu~e 22 having li~uid impermeable walls with an 20 opening at one end. Cylindrical tube 22 contains granulized tablet 21 comprising the coimmobilized interacting enzymes. The opening of the tube is filled with compressed foam insert 20. The foam material regulates the amount of sterilant reaching the tablet cont:linin~; the interacting enzymes.
The dispenser of the indicator solution is shown in Figure 3.
25 A boffle cont~ins indicator solution 31 which prodalces a visual color changewhen added to active multiple interacting enzymes coimmobilized on solid support disk 32. The bottle contains eyedropper 33 with premeasured W O 97/26924 PCTnUS97/On554 volurne line 34. Filling eyedropper 33 to premeasured volume line 34 with indicator solution 31 assures that the correct volume or number of drops of solution, is dispensed into the tube.
A method for conducting the sterility test is also illustrated in 5 Figure 3. The sterility indicator is placed into the sterilizer along with other materials which are to be sterilized. The sterility indicator is exposed to the sterilant during the course of a sterilization cycle. After the completion of the sterilization cycle, the sterility indicator is removed from the sterilizer and allowed to cool to room temperature. Cap 35 and foam material 36 are 10 removed and can be safely discarded. Indicator solution 31 is drawn into eyedropper 33 using the premeasured volume line 34 to assure that the correct volume of in~ tor solution is used and dispensed into the tube. The resl-lting ~ is incubated, if necessary, at room temperature for seconds to minl~te~, preferably for less than about 10 mimltPs and more preferably for 15 less than about 3 minutes. The solid support disk is visually inspected at the end of the incubation period. An absence of red coloration on the solid support disk (e.g, white) indicates negative result 37 and signifies a successful sterilization cycle. The presence of red coloration on the solid support disk indicates positive result 38 and signifies an unsuccessful 20 st~rili7~tion cycle.
The sterilization procedure useful in the practice of the invention may be, fior example, a steam-pressure procedure or autoclaving (121 ~C or higher, such as 132 ~C or 134~C), a chemical procedure lltili7:in~
ethylene oxide or another ap~3l0~liately lethal chemical or dry heat of 25 temperatures between about 50~C to about 200~C, or a plasma-phase sterilization procedure. These procedures are practiced in the health care industry, but also in industries having to do with environmental technology, , CA 0224412~ 1998-07-22 W O 97/26924 PCTrU$97/00554 food m~nl1f~cturing, waste disposal and in those technologies where sterility is required.
Another embodiment of the invention is directed to an indicator for determining the effectiveness of a sterilant to pass through a S tortuous path such as a challenge pack. Challenge pack testing, can make use of the same design and the same adjustable features. An AAMI steam ch~l1enge pack consists of a biological indicator such as bacterial spores on an inert carrier, wrapped in 16 surgical towels. The towels create a tortuous path for the steam to reach the indicator. This .~im~ tes the wrapped goods l O processed in a steam sterilizer in a hospital setting.
Challenge packs are used to test the effectiveness of the sterilant to pass through the pack and reach the indicator. This simulates wrapped goods processed in the sterilizer. The foam insert design can be used for challenge pack testing. A sterility indicator, either the enzyme-l S based indicator described previously or a conventional spore-based indicatormay be used in this type of challenge pack. An example of a challenge pack is shown in Figure 4.
Sterility indicator 40 is placed into container 41 which has at least one opening fil1ed with a prec~t~. " ~ ed amount of foam 42. Container 20 41 has substantially gas non-adsorptive walls so the sterilant has to enter through foam 42 to reach sterility indicator 40 and thereby regulates the amount of sterilant entering the container. Sterility indicator 40 contains either spores or enzymes 43. Foam 42 regulates the amount of stearn or sterilant entering the container. After the challenge pack has been exposed 25 to the sterili~ation process, the indicator is removed from the challenge pack and processed. If the indicator is positive, proper sterilization conditions were not achieved within the pack. A negative result means proper W O 97/26924 PCTAUS97/005~4 conditions were met. The rapid sterility indicator described above or a standard biological indicator can be used in conjunction with the challenge pack. The challenge pack is simple to use and provides reproducible results.
The desired amount of challenge can be easily reproduced to mimic the S challenge described by standards such as AAML ~so or EN for a steam or ethylene oxide challenge pack.
Another embodiment of the invention is directed to the foam insert design for the air removal test. The air removal test consists of a container with a Bowie-Dick test sheet or a chemical indicator on a carrier.
10 The transparent container consists of at least one opening which is filled with foam. After the test cycle is complete, the air removal test is removed from the stt~n~i7~ti~-n chamber. The user observes the uniformity of the coIor ch-ange of the chemical indicator. Since the material of the container is transparent, the user would sirnply observe the uniformity of the chemical 15 indicator ink. Thus, there is no need to unwrap the device.
The air removal test is also based on the similar design. By placing a chemical indicator into a transparent container with a foam insert, the prevacuum air removal test equivalent is made. The air removal test is placed into a prevacuurn steam st~rili7:er. After the cycle is complete, the 20 user can simply view the uniforrnity of the color change of the chemical indicator in the transparent container or simply open the container and remove the chemical indicator.
I~e foam insert design overcomes many disadvantages of the current designs for testing the effectiveness of sterilization processes. The 25 foarn insert design can be used as a component of a rapid sterility indicator composed of interactive enzyme systems which can provide nearly in~t~nt~neous results. A sterility indicator with the foam insert design also CA 0224412~ 1998-07-22 W O 97/26924 PCT~US97/00554 offers the advantage of being adjustable to suit various types of sterilization process. Presence of foam also allows the sterility indicator to effectively control the amount of sterilant entering the device in a standardized manner.
The enzyme content of the rapid sterility indicator and the foam 5 specifications can be easily controUed to provide reproducible results during manufacturing. Conventional biological indicators that are based on the inherent resistance of bacterial spores can not be as easily controlled.
The foam insert design also overcomes disadvantages of chaUenge pack and air removal test designs. Assembly of AAMI test packs 10 is very time consuming. AAMI packs are not standardized in the sense that di~elences in how individuals make the packs and different types of towels can result in packs with differing characteristics. The advantages of the foam insert design is that it can be used for a sterility indicator for many types of sterilizers as well as challenge packs and air removal tests, it is 15 simple to use and it is standardized and reproducible. The fact that the samedesign can be used for multiple tests (e.g, sterility, challenge packs, air removal) offers simplicity to the users. The challenge pack and air removal test designs also allow the user to quickly and easily retrieve the indicator.
There is no need to unwrap many towels to retrieve the indicator. The 20 transparent container also permits the user to confirm that an indicator is present in the pack.
Another embodiment of the invention is directed to a method for dete~ g the effectiveness of a sterilant to pass through a tortuous path compri.~es a chaUenge pack that employs the foam and container design 25 that allows the users to easily open and close the challenge pack for repeated use. The chaUenge pack consists of two containers, two foam inserts and a sterility indicator, as shown in Figure 5. Each container has at least two CA 0224412~ 1998-07-22 openings, one opening is the same size as the diameter of the container, the second opening is much smaller. One container 50 has a slightly larger diameter than the other container 51. Each container has a small hole 52 opposite the larger opening and a piece of foam 53 placed near the small opening. A sterility indicator 54 is placed into the smaller diameter container and the second container with a slightly larger diameter is placed over the first container's large opening. The containers fit together tightly due to their similar diameters. Instead of relying on the similar diarneters to hold the two tubes together, a screw closure, a snap locking device or a twist 10 locking device could be used. The containers have su~st~nti~lly gas non-adsorptive walls so that the sterilant has to enter through smalI holes 52 and pass through the foam inserts S3 to reach sterility indicator 54.
After the challenge pack has been exposed to the sterilization process, the two cont~iners are dislodged from one another and the indicator 15 is retrieved and processed as usual. This test pack can be used again by placing an unexposed indicator into one of the containers and replacing the second container over the first container as shown in ~igure S. The sterility indicator can be the enzyme-based indicator or a conventional spore-based indicator.
Another embodiment of a reusable challenge pack design consists of a single container with one opening and a cap. A sterility indicator is placed into the container. The cap is made from a porous plastic material which allows steam to pass through. The capping device can be easily opened and closed by the user to retrieve the exposed indicator and add an unexposed indicator for the next test. The materials are a non-absorptive plastic which can withstand multiple exposures to sterilization.

CA 0224412~ 1998-07-22 W O 97/26924 PCT~US97/00554 Another embodiment of a reusable challenge pack designs comprises a single container with a closure device, foam and a test indicator as shown in Figure 6. The bottom portion of cylindrical container 60 has two holes 61 in the sides of the container. Tubular foam insert 62 fits tigh~tly5 into the container. Foam insert 62 has a hole in the center which conforms to the shape of sterility indicator 63 which fits tightly into foarn insert 62.
Screw cap 64 is placed over the large opening of the container. When screw cap 64 is secured onto the container Co~ g sterility indicator 63, sterilant would pass through the small openings in the sides of the container and 10 through foam insert 62, before reaching sterility indicator 63. This is a tortuous path for the sterilant. This design would perform equivalent to other challenge packs. The materials are a non-absorptive plastic which can with~t~n~1 multiple exposures to sterilization. The sterility indicator can be the enzyme-based indicator or a conventional spore-based indicator. By 15 using a larger container and corresponding larger foam insert and replacing a chemical indicator (test sheet covered with unexposed chemical indicator ink) for the sterility indicator, this design could be used for air removal tests in pre-vacuum sterilizers. After processing in a pre-vacuum sterilizer, the uniformity of the chemical indicator color change would be used to 20 dt;~ e if any air was present in the chamber. If air was present, the color change of the chemical indicator would not be uniform.
Another embodiment of the invention is directed to an a~al~Lus for inserhng compressible materials. Accuracy of the sterilization - indicators depends on consistent placement of the foarn plug in the25 con~iner. Prior to the present invention, foam was placed in the container by hand, but unacceptably high incidence of illlpl~o~er placement results frorn this method. This invention provides apparatus for controlled W O 97/26924 PCTrUS97/00554 positioning of foam in a container such as that present in the rapid sterility indicator. Various forms of the apparatus of this invention are shown in the drawings and described herein.
Figure 7 is a vertical front view of an insertion apparatus that 5 compresses the resilient compressible material such as foam with a flexible strip of m~tPri~l 70 held in a loop configuration. The strip of material for theloop can be made from any flexible material such as Teflon, nylon, mylar or a thin sheet of steel preferably about .010 inches thick for a tight wrap.
Initially, the two ends of the flexible material are nearly touching, forming 10 a large loop. One end of the loop material is held at a fixed point the otherend is attached to holl~o~ slide 72. The large loop is the initial receptacle for the non-compressed material. The slide is pushed all the way to the left creating the loop with the largest diameter.
Non-compressed foam material 73 is placed into the large loop 15 70. Vial 74 is placed into holder 75. Slide 72 is pulled to the right by hand, cams. air cylinders or electric linear motion. As the slide is moving to the right. the loop becomes smaIler and smaller in diameter, compressing the foam material into approximately l/2 inch to 1/4 inch or less of the original non-compressed diameter size. The foam is compressed in an efficient and 20 reproducible manner. Compressed foam is plunged into the vial by ver~ical plunger 76 actuated by hand, cams, air and/or hydraulic cylinders or electric linear motion actuator. Plunger 76 backward extrudes the compressed foam material 73 into vial 74. The drawing shows holder 75 that indexes 90 degrees. In this manner, foam material 73 is efficiently deposited into vial 25 74. The apparatus can be adjusted to deposit the foam all the way into the vial, to a desired depth, or to leave a desired amount of foam protruding past the opening. The depth of the stroke of the vertical plunger regulates the ;

CA 02244l2~ l998-07-22 W O 97/26924 PCTAUS97tO~S54 position of the foam. The path of the vertical plunger is within the flexible loop regardless whether the loop is open or closed.
Figure 8A is a vertical front view of a machine to compress resiIient material and deposit it into a container. A foam cylinder is placed 5 into slide chamber 81 and the vial is placed into vial block 82. ArlgUlar crusher slide 83 strokes to compress resilient material 84. As the ~n~ll~r crusher slide assembly advances, the foam is rolled into a fraction of the original diameter (Figure 8~3). Angular crusher slide 83 provides a single moving part which compresses resilient material 84 and ultimately forms a 10 compression chamber holding the material in com~ssed state. Plunger cylinder 85 stokes downward, pll-n~ng the compressed material into staging nozzle 86 which is a hollow tube. Staging nozzle 86 with the compressed foam is partially inserted into the vial by stripper cylinder 87. Staging nozzle 86 is retracted while plunger cylinder 85 remains stationary causing 15 the foam to be deposited into the vial. Plunger cylinder 85 retracts as a blast of air is forced into crusher slide cylinder 88 clearing any debris.
AdJustments can be made to place the foam at any depth within the vial, including leaving a portion of foam protruding from the vial opening.
The apparatus of Figure 7 and Figure 8 represent ~Ttern~tive 20 devices for carrying out the same method of inserting foam into a vial, whereby a foam insert is compressed into a cylinder in a first step and the compressed foam is positioned irl a vial in a second step. Horizontal slide 72 can be replaced with ~ng~ r crusher slide 83 that is a more positive m~ch~ni~m. The reverse ofthe above is also true as angular crusher slide 83 25 can be replaced by horizontal slide 72.
Figure 9 depicts another method of inserting a compressible resilient material into a vial, by rotating the vial over the stationary piece of foam. One end of foam 91 is placed slightly into vial 92 which is held by flexible tube 93 connected to a rotating device comprised of bearing block and fixture base 94. The other, opposite end of the foam is held and fed into the rotating vial. As vial 92 rotates, foam 91 is inserted into the vial with a helix effect. The device can be rotated with crank 95 by hand, by air cylinders or by electric linear motion actuator. An indexing machine can be added to the above combined fixtures and/or stations. The fixture for rotation can be applied to the index disc which may have an ~nm~l~r slot enabling a rubber wheel to rotate the vial contained on the circumference of 10 the indexing disc.
The following examples illustrate emboflimentc of the invention, but should not be viewed as limi~ing the scope of the invention.

Examples 15 Example I Ranid Sterilitv Indicator for Monitorin~ Steam Sterilization Process.
A rapid sterility indicator for steam sterilization is composed of a test unit and indicator solution. The test unit is composed of a cylindrical glass vial, a tablet co~ g components of a plurality of 20 interacting enzyme systems, a foam insert, and a label. The glass vial is al l ~ux-lnately 1/4 inches in diameter and 1 inch high, open on one end.
The tablet co~ ; two interactive enzymes of the enzyme system is placed into the vial. The tablet is a granulation of glucose dehydrogenase and diaphorase. The preferred en2yme concentration for 25 each enzyrne is 8 to 15 units per 20 mg tablet. The opening of the vial is filled with a compressed foam insert. The foam insert is ~lc:fel~bly a cylinder, with a diameter range of 1/4 to 1 inch, preferably approximately l/2 CA 02244l2~ l998-07-22 W O 97t26924 PCTAUS97/00554 inch and the length range 1/8 to 3 inches preferably approximatelv 1 I/2 inches in length. Foa~n is partially open celled with a density of approximately 6 pounds per cubic foot and the foarn material is polyurethane. The detailed specifications include: polyester foam, open cell, 6 lbs, charcoal color, 5 density is 5.00-6.60 p.c.f., strength 20.0-40.0 p.s.i., elongation 300-500%, tear resistance is 3.0-5.0 p.l.i., compress set 3.0 to 10.0%, load defection, 0.50 to 0.90 p.s.i., fl~mm~bility HF-l, and cell size 50-70 c.d.i. On the outside of the vial was a label cont~in;n~ steam-sensitive indicator ink.
This test unit was placed into the sterilization chamber of a 10 steam sterilizer operating at 121~C along with the items to be sterilized.
After the cycle is complete, the test unit was removed from the sterilization chamber. The color change of the steam-sensitive indicator ink serves to identify the processed units from unprocessed units. The foam insert was removed and five drops of the clear, colorless indicator solution was added 15 to the vial cont~ining the white enzyme tablet. The indicator solution was packaged in an arnber glass bottle with a dropper dispenser. The indicator solution contains p-iodonillolell~oliurn violet within a range of 32 ,uM to 16 mM, preferably 3.2 mM; NAD (,B-nicotinamide adenine dinucleotide) within a range of 1 ~LM to 5.5 rnM, preferably 0.11 mM; glucose within a 20 range of 1% to 90% preferably 10%; ethanol within a range of 1% to 95%
(by volume), preferably 5.5%; citric acid within a range of 0.0032 mm to 3.2 m, preferably 17 mm. The ~le~~ d buffer was 0.05 M Tris~ pH 6.0-8.5.
The predele,l~ led survival cycle in a BIER vessel operating at 121 ~ C was 5 min~1te~, the kill cycle was 15 mimltes for sterility 25 indicators. Survival cycles were cycles with short exposure times in which indicators should test positive, indicating proper sterilization conditions weren~t met. Kill cycles were usually the standard cycles times in which the in~lic~t )r should test negative indicating proper sterilization conditions weremet. A~ter indicator soIution was added to the white tablet, the color of the tablet was visually observed for 1 to 20 seconds. After a S minute autoclave survival cycle at 121~C, which represents inadequate sterilization 5 conditions, a positive result was expected and observed, in which the enzyrnes are active and a red colored product was formed on the surface of the tablet at or before 20 seconds. After a 15 minute autoclave kill cycle at 121 ~C which ~ sellL~ an adequate sterilization cycle, a negative result was expected and observed, in which one or more enzymes were inactivated and 10 no red colored product was formed. These positive and negative results parallel the results of bacterial spores exposed to similar conditions in a steam autoclave.
Table 1 Steam Sterilization Test in a BIER Vessel Survival Time Kill time (5 minlltes) (15 mimltes) Rapid Sterility Tn~1ic~t-~r10/10 0/10 Sportrol spore strips (106 B. stearot*ermophilus) 10/10 0/10 Table 1 shows results from a typical experiment. Results are recorded as the number of positives over the nurnber tested (121 ~C) in a BIER vessel. Rapid steriIity indicators provided both positive results after the survival cycles and negative results after the kill cycles. These results 25 demonstrate the at least equivalence of rapid sterility indicators to conventional bioIogical indicators.

ExampIe 2 Effects of Foam Structure~ Size and Position.
Test units as described in Example 1 were prepared by 30 inserting into ~e vial foam inserts from various suppliers, aIl of which were CA 0224412~ 1998-07-22 W O 97/26924 PCT~US97/00554 6 lbs. per cubic foot foam. In some of the test units, foam was inserted until none pro~uded above the end of the vial, while in others, foam was left to extend outside the vial from 0.125-0.5 inches. Other parameters were as in Example 1, and test units were exposed to 121 ~C for 5 minutes in a BIER
5 vessel. After the cycle, foam was removed, indicator solution was added and color read after 30 seconds.
Table 2 Foam Positioning Effects at 121 ~C
Foam cylinders, approximately: I.5" long, 0.5" diameter, 6 lbs density Positives after 5 min.
' Exposure to 121~CPercentPositives no overhang 43/59 73%
/41' overhang 37/58 64%
l/2" overhang 5/21 24%
Results in Table 2 show the effect of the foam position within the vial. The number of positive results (pink tablet) was recorded over the total number tested. As the amount of foam protruding past the vial increased, the amount of foam compressed within the vial decreased. As the 20 amount of compressed foam decreased, more sterilant is able to pass through the foam and mactivate the enzymes in the tablet. Fewer positives are seen as the overhang increases. These tests were performed using several different types of 6 lbs. foam and the foam was often cut into cylinders by hand. The consistency of the results increased greatly when the foam was 25 from a single supplier and was cut into cyl~nders using reproducible methods such as using a water jet or using dies, though foam position still has an ~ effect on the results as seen in Table 5.
Test units as described in Example 1 were prepared by inserting foam cylinders approximately 1.5 inches long and 0.5 inches in 30 ~ diameter. Two different foam densities were tested, a 6 lbs. per cubic foot W O 97~6924 PCTAUS97/00554 foam and a 2 Ibs. per cubic foot foam, both open celled with a l/4 inch overhang. Test units were exposed to 121 ~C in a BIER vessel for a 5 minute (survival) cycle. The number of positive results (pink tablet) was recorded over the total number tested. Color was recorded after 20 seconds.
Table 3 l~ffects of Foam Density Foam cylinders of a~ 0~c.mately 1.5 inches long, 0.5" diameter Survival after 5 min.
Exposure to I2 1 ~C Percent Positives 6 lbs foam 20/20 100%
2 lbs foam 0/20 0%
The results of Table 3 show that foam density also plays a key role in reg~ ting the amount of sterilant that reaches the tablet. The 6 lbs.
15 foam, used in the prescribed configuration, produced all positives. The same shaped foam of a 2 lbs. density allowed the steam to pass through and inactivate the tablet. Foam density as well as the foam length and overhang amount can be adjusted to provide the desired results for the intended type of sterilization process.
Example 3 Prevacuum Sterilization.
An ~lt~ ive sterilization protocol involves pre-evacuation of the sterilization chamber followed by a shorter period of exposure to steam at a higher temperature (132~C or 134~C). The pred~L~ .ined 25 survival/kill cycle times for a 132~C and 134~C pre-vac sterilizer is 20 seconds/3.0 minutes, respectively. Test units according to Example 1 were processed in the prevacuum mode, and the results are shown in Table 4.

CA 02244l25 l998-07-22 W O 97/26924 PCT~JS97/00554 Table 4 Steam Sterilization Test in a Prevacuum Sterilizer using 132~C and 134~C Steam Survival Time Kill time Rapid Sterility Indicators (132~C) 10/10 0/10 Sportrol spore strips (132~C) ( 1 o6 B. stearothermophilus) 10/10 0/10 Rapid Sterility Indicators (134~C) 10/10 0/10 Sportrol spore strips (134~C) 0 (106 B. stearothermophilus) 10/10 0/10 Results in Table 4 are recorded as the number of positives over the number tested. The indicator reaction was read at 20 seconds. These results show that the rapid sterility indicators met the predetermined 15 sulvival/kill parameters for a 132~C and 134~C prevacuum steam sterilizer, at least equivalent to biological indicators. The Rapid Sterility Indicators, as described in Example 1, can effectively monitor both 121 ~C gravity and 132~C/134~C pre-vac sterilization cycles.
In a subsequent test, foam inserts of the same size were placed 20 into Test Units with 1/8",l/41',~/8", 1/2" and 9/16" overhang and were then subjected to the 132~C pre-vacuum survivalJkill test. Forty test units with each specified overhang (total of 200 test units) were assembled For each overhang test leng~, 2 sets of 10 test units were exposed to a survival cycle, and two sets of 10 were exposed to a kill cycle.

CA 02244l25 l998-07-22 W O 97/26924 PCTrUS97/00554 Table 5 Positioning effect for 132~C
Resu1ts after exposureResults after exposure Foam Positionto a survival cycleto a kill cycle (overhang)(20 seconds) ~3 min11tes~
1/8" I0/I0 0/lO
1/8" 10/10 0/10 I/4" 10/10 0/I0 1/4" 10/10 0/10 3/8" 10/10 û/10 3/8" 10/10 0/10 1/2" 0/10 0/lO
1/2" 0/10 0/10 - 9/16" 3/10 0/10 9/16" 4/10 0/10 Table 5 shows that the foam insert overhang of the rapid sterility indicator can range from l/8" to 3/8" without the results being significantly affected. The speci~lcation for assembly of the rapid sterili~
20 indicator is set at l/4 ~ 1/8" (1/8" to 3/8"). ~fter a survival cycle the enzyme tablet is positive (pink to red) and after a kill cycle the enzyrne tablet is negative (white).
If the foam overhang length is greater than 3/8", the indicator does not provide I00% survival after exposure to the 20 second survival 25 cycle. ~he foam position plays a critical role. The ~oam overhang must be within the def~ed specif;cations for the indicator to func~on properly.

Example 4 Foam Structures to Mimic AAMI Challen~e Packs.
AAMI challenge packs provide a tortuous path for steam 3V penetration. An analogous challenge pack for a steam sterilizer can be created using a container, foam, and a rapid sterility indicator (as desc~bed in Example 1) or a biological indicator. The challenge pack container is a plastic or glass tube, preferably plastic, measuring approxim~te1y 1.125 inch CA 0224412F, 1998-07-22 4 PCT/US97/Oû554 diameter and 5 inches in length with one opening in the container. The container contains a heat sink material such as a metal object. In this case a rapid sterility intli~tor test unit, as described in Example 1, is placed intothe container. The one opening of the container is filled with a foam insert.
S Foam insert is a~loxi~ fely 2 to 4 inches in diameter and 1 to 4 inches long (non-compressed measurements). Foam is partially open celled with a density of approximately I to 6 pounds per cubic foot and foam material is preferably polyurethane.
~h~llenge packs are placed into a steam sterilizer operating at 10 121~C or 132~C and exposed to the predetermined survival and kill time intervals. Survival cycles are cycles with short exposure times in which indicators should test positive, indicating proper st~rili7~tion conditions werenot met. Kill cycles are the standard cycles times in which the indicator should test negative indicating proper sterilization conditions were met. The 15 rapid sterility indicators are processed as described in Example 1, the results are obtained at 20 seconds or less after adding indicator solution.
The AAMI challenge pack is constructed and tested for comparison. The materials required for a AAMI challenge pack for a steam sterilizer are 16 freshly laundered huck towels, autoclave tape, and sterility 20 indicators. Each towel is folded lengthwise into thirds and the folded width- wise in half. Towels are placed one on top of another with the folds opposite each other. Rapid sterility indicators and conventional biological indicators are placed between the eighth and ninth towels. The pack is secured with autoclave tape. The AAMI steam challenge packs are placed 25 into a steam autoclave at 121 ~C for the ap~lu~l iate amount of time. After the cycle, indicators are processed as in Example 1.
.

Table 6 Challenge Pack Analog Exposure time to 121 ~C steam Survival Time for Kill time for Challenge Packs Challenge Packs ~10 minutes) (30 min-ltes) Rapid Sterility Indicators inFoarn Challenge Packs 6/6 0/6 Rapid Sterility Indicators in AAMI Challenge Packs 6/6 0/6 Sportrol Spore Strips ( I o6 B. stearothermophilus) inAAMI Challenge Packs 6~6 0/6 The results in Table 6 are recorded as the number of positives over the number tested. As shown, when exposed to the survival cycle time, all indicators tested positive. When exposed to the kill cycle time, all indicators tested negative. This table also shows the equivalence of the conventional biological indicators in AAMI challenge packs to the rapid sterility indicators in AAMI challenge packs. These results demonstrate the equivalence of the rapid sterility indicator in the foam challenge pack to the AAMI stearn challenge pack.

Example S Rapid Sterilitv Indicator in a Plasma-Phase Hvdro~;en Peroxide Sterilizer.
The rapid sterility indicator as described in Example 1 was used with one morlific~tion, the ~3le~ d foam length was changed firom 1.5 inches to 0.375 inches. Rapid sterility indicators were exposed to survival and kill cycles in the plasma-phase sterili7:~r. Survival cycles are cycles withshort sterilant diffilsion times in which indicators should test positive, indicating proper sterilization conditions were not met. Kill cycles are the .

W O 97t26924 PCTrUS97/00554 standard cycles times in which the indicator should test negative indicating proper sterilization conditions were met.
The survival time for the plasma-phase hydrogen peroxide sterilizer was d~e~ led to be a 6 minute diffusion time. Plasma time was 5 held constant at 15 mimltes and vapori7er time was 4 minutes. Kill time was determined to be a 50 minute diffusion time. Rapid sterility indicator test units were placed into the sterilizer and exposed to the survival and kill cycles and processed as in ~xample l. Results were recorded 10 seconds after adding the indicator solution to the test unit.
Table 7 Plasma-phase H2Oz Sterilizer - SurvivallKill Times Foam Len~th Survivall Kill2 No Foam 0/5 0/5 0.375" 5/5 0/5 150.75" 5/5 5/5 1.50" 5/5 5/5 Table 8 Plasma-phase H2O2 Sterilizer - Partial Cycle Times 20Foam Len~;thSurvival3 Ki~4 No Foam 0/5 0/5 0.375" 2/5 0/5 0.75" 5/5 5/5 l.50" 5/5 5/5 I = Diffusion time 6 minutes: Plasma time 15 minutes; Vaporizer time 4 minutes; and Readout time 10 seconds.
2 = Diffusion time 50 minutes; Plasma time 15 minutes; Vaporrzer time 4 minutes; and Readout time 10 seconds.
30 3 = Di~usion time 8 minutes; Plasma time 15 minutes; Vaporizer time 4 minutes; and Readout time 10 seconds.
4 = Diffusion time 10 minutes; Plasma time 15 minutes; Vaporizer time 4 minutes; and Readout time 10 seconds.
In the tests shown in Tables 7 and 8, the rapid sterility indicator showed a graded response to the STERRAD sterilizer. By adjusting the foam length, the desired survival/kill times could be met. This W O 97/26924 PCT~US97tO0554 demonstrates that the rapid sterility indicator can be used to test the effectiveness of the STE~RAD hydrogen peroxide sterilizer. The foam length of 0.375 inches provided the proper survival/kiill results at 10 seconds after addition of the enzyme system reagents. Further testing would further 5 define the optimal foam density, foam length and foam insert overhang for a sterility indicator.
These tests demonstrate that the rapid sterility indicator is effective for molliLol.-lg the plasma-phase hydrogen peroxide sterilizer. All positives were observed after the survival time and all negatives were 10 observed after the kill time.

Example 6 A Two Container Reusable Pack to Mimic AAMI Challen~e Packs.
A reusable challenge pack can also be used to mimic an AAMI
15 Challenge pack. A challenge pack can be created from two containers, two pieces of foarn and a sterility indicator. A reusable challenge pack is made of material which can withstand multiple exposures to sterilization and can be easily opened and closed. The containers are made from plastic or glass, preferably plastic. One cnnt~iner is approximately 7/8" diameter, 3 .5" long.
20 The diameter of one opening is 7/8", the diameter of the second opening at the opposite end of the container is 13/64". The second container is ~lu~ lately I1/8~ diameter, 4" long. The diameter of one opening is 11/a", the diameter of the second opening at the opposite end is 13/64". A
cylindrical piece of open cell, 2 pound polyurethane foam approximately 1 "
25 long and I " diameter is placed into each c(mt~iner, not more than I " from the smaller opening. The rapid sterility indicator test unit as described in E~ample I is placed into one of the containers, on top of the foam. The 7/8"

W O 97/26924 PCT~US97/00554 diameter opening of the one container is placed into the 11/B~I diameter opening of the second container. The containers fit together tightly so that the only pathway for steam to enter the challenge pack is through the small openings, passing through the foam inserts to reach the indicator. The pack may also contain a heat sink material such as a metal object.
Challenge packs were placed into a steam pre-vac sterilizer operating at 132~C and exposed to the predetermined survival and kill cycles. Survival cycles are shorts exposures times in which the indicators should test positive indicating the proper sterilization conditions were not met. A 30 second cycle time in a 132~C pre-vacuum sterilizer is an example of a survival time for indicators in challenge packs. An example of a kill cycle time in a 132~C pre-vacuum sterilizer is 3.5 minutes. After the kill cycle indicators should test negative indicating proper sterilization conditions were met. After the cycles were complete, the containers were dislodged from one another and the indicator retrieved. The rapid sterility indicators were processed as described in Example 1, the results are obtained at 20 seconds or less after adding the indicator solution.
Table 9 Two Container Reusable Challenge Pack Exposure time to 132 ~C steam, pre-vac. sterilizer Survival Time for Kill Time for Challenge Packs Challenge Packs (30 seconds) (3.5 minutes~
Rapid Sterility Indicators 7/7 0/7 in two container reusable challenge pack The results in Table 9 are recorded as the number positive over - the number tested. As shown, when exposed to a survival cycle, all30 indicators tested positive. When exposed to the kill cycle time, all indicators tested negative. This table shows that the challenge pack designs provides W O 97/26924 PCT~US97/005~4 acceptable results. This challenge pack also has the unique feature such that the user can reassemble the pack by placing a new indicator into the container, close the container, and use it for another test. This design could be reused multiple times.

Example 7 A Sin~le Cont~int~r Reusable Pack to Mimic AAMI Challen~e Packs.
A reusable challenge pack can be created from one container with a closure device, one piece of foam and a steriiity indicator. A reusable 10 challenge pack is made of material that can withstand multiple exposures to sterilization and can be easily opened and closed The container is made from plastic or glass, preferably plastic. The container is approximately 1 1/2"diameter, 2l/2" long with a screw cap. The two steam entry holes are approximately 1/4~ in diameter and the holes are located approximately 1/4~
15 below the cap. A tubular piece of open cell, 2 pound polyurethane foam a~o~.lately 1 1/2" long, 11/~" outer diameter (inner diameter 3/8")iS placed into the container. The rapid sterility indicator test unit as described in Fx~mrle 1 is placed into the foam insert. The foam fits tightly into the container and the test unit fits tightly into the foam. The cap of the container20 is screwed into place and creates a tight seal so that the only pathway for steam to enter the challenge pack is through the small openings, passing through ~e foam to reach the indicator. The pack may also contain a heat sink material such as a metal obJect.
These challenge packs were placed into a steam sterilizer 25 o~ g at 132~C (pre-vac sterilizer) and 12I ~C (gravity) and exposed to the predetermined survival and kill cycles. After the kill cycle indicators should test negative indicating proper sterilization conditions were met and -CA 0224412~ 1998-07-22 W O 97/26924 PCT~US97/00554 the indicators should be positive after the survival cycles. After the cycles were complete, the container was opened and the indicator retrieved. The rapid sterility indicators were processed as described in Example 1, the results are obtained at 20 seconds or less after adding the indicator solution.
Table 10 One Container Reusable Challenge P~ck Survival tirne/Kill time Survival tirne/Kill tirne 132~C Stearn Sterilizer 121~C Steam Sterlizer (1 min.! (4 min.) (10 min.) (30 min.
Rapid Sterility 7/7 0/7 3/3 0/7 Indicators in one container reusable challenge pack 132~C Steam Sterilizer 121~C Steam Sterilizer 1 min. 4 min. 10 min. 30 min.
Rapid Sterility 20/2 0/20 20/20 0/20 Indicator in one container reusable challenge pack The results in Table 10 are recorded as the nurnber positive over the number tested. As shown, when exposed to a survival cycle, all indicators tested positive. When exposed to the kill cycle time, all indicators tested negative. This table shows that the challenge pack design provides 25 acceptable results. This ch~llenf~e pack also has the unique feature such that the user can reassemble the pack by placing a new indicator into the container, closing the container, and using it for another test. This design can reused multiple times.

30 Example 8 Automated Compression and Insertion.
- Using the ~ Lus depicted in Figure 8A, a labeled glass vial (a~out 8 mm in diarneter and about 30 mm in length) containing a test tablet (about 5 mrn in diarnetel and about 2 mm in length) is placed into the vial W O97/26924 PCTrUS97/005S4 block. A polyurethane foam cylinder (about 15 rnrn in diameter and about 40 mm in length) is placed into slide chamber 81. Angular crusher slide assembly 83 advances, the foam is compressed to about one fourth the diameter of the non-compressed size. Plunger cylinder 85 stokes downward 5 plunging the compressed foam into staging nozzle 86 which is a hollow tube.
Staging nozzle 86 with the compressed foam is partially inserted into the vial by stripper cylinder 87. Plunger cylinder 85 pushes ~e foam into the vial while staging nozzle 86 is retracted and foam is deposited into the vial.
Plunger cylinder 85 retracts as a blast of air is forced into the crusher slide 10 chamber, clearing any debris. The foarn is protruding past the vial a~ploxi~ tely one quarter inch. The amount of foam inserted into the vial can be adjusted.

Example 9 Comparison of Manual and Mechanical Insertion.
lS The Test Unit of the rapid sterility indicator for steam sterilization consists of a glass vial, enzyme tablet and foam insert.
Historically, the foam insert was placed inside the glass vial manually.
Manual assembly is achieved by twisting and pushing the foam insert inside the vial opening until the foam overhang is within the required specification 20 of 0.125-0.375 inches. To facilitate faster production of the test units, an automated foam stuffer has been m~nl-f~ctured by Custom Machine, Inc.
(Kansas, OH).
The a~u~us of Figure 8 placed a hollow stainless steel tube ins}de the glass vial (already cont~inin~ the enzyme tablet), squeezed the 25 foam vertically and, via forced air, pushed the condensed foam inside the stainless steel tube. The stainless steel tube was removed from its original position leaving the foam. Condensed foam occupied the space allowed W O 97/26924 PCT~S97/W554 inside the glass vial. The automated machine did not use twisting action as the means for foam insertion.
Performance of the test units, when packaged manually, was compared to the performance of the test units packaged via the automated 5 procedure. Testing involved steam exposure in a BIER vessel at 5, 7, 8, 9, 10, 11, 12, 13 and 15 minutes on three dirfelellt lots of test units packaged both m~ml~lly and using the automated procedure. The product packaged automatically also had 5 minute and 15 minute testing in an autoclave. In addition, the foam overhang of the test units packaged automatically were 10 measured to ensure they met the required specification of 0.125-0.375 inches.
Three lots of test mits were manually packaged, 100 per lot.
Each lot consisted of a diLrel-ellt con~lguration of tablet lot and foam lot.
Test units were labeled Lot A, Lot B and Lot C. Two hundred glass vials 15 were labeled as Lot A, Lot B and Lot C. These were the glass vials used for the ~lltf)m~tçrl procedure. Using the automated procedure, 200 test units for each lot (A, B and C) were paclcaged.
The overhang was measured, using calibrated calipers, on 80 ~ut-)m~tic~lly packaged test units from each lot, and the results are shown in 20 Table 1 l . Measured foam overhang of the sampled test units were all within the range of 0.125-0.375 inches.

Table 11 2S Measurement of Foam Overhang in Inches Lot A Lot B Lot C
0.2570.302 0.312 0.248 0.242 0.247 0.289 0.254 0.257 03710.336 0.337 0.254 0.247 0.266 0.299 0.295 0.255 0.288 0.278 0.320 0.249 0.240 0.239 0.278 0.303 0.290 CA 0224412~ 1998-07-22 W O 97/26924 PCT~US97/00554 Table Il Measurement o~ Foam Overhan~ in Inches 0.274 0.3040.312 0.241 0.2430.252 0.298 0.319 0.299 0.307 0.2850.296 0.253 0.2380.244 0.319 0.2830.257 0.326 0.3120.300 0.241 0.2410.240 0.274 0.2500.250 0.302 0.3110.287 0.235 0.2530.23s 0.313 0.3000.266 0.28s 0.2940.284 0.25~ 0.2470.238 0.268 0.2910.258 0.322 0.2580.308 0.254 0.2320.250 0.275 0.2810.312 0.271 0.2880.320 0.257 0.2460.245 0.278 0.3200.261 0.277 0.3130.319 0.251 0.2510.257 0.314 0.2s80.243 0.287 0.3360.372 0.269 0.2470.249 0.288 0.3250.298 0.344 0.3060.306 0.25 1 0.2490.254 0.266 0.2970.279 0.315 0.3230.317 0.2s4 0.2530.243 0.271 0.2690.259 0.346 0.3240.321 0.249 0.2520.245 0.281 0.2610.37I
0.291 0.3080.30s 0.244 0.2420.241 0.275 0.2ss0.274 0.264 0.2910.288 0.2s9 0.2540.254 0.260 0.2680.251 0.292 0.3340.271 0.253 0.2510.245 0.266 0.2610.319 0.313 0.2780.331 0.247 0.2570.246 0.272 0.2820.255 0.319 0.2980.314 0.245 0.2530.259 0.296 0.2610.252 0.29s 0.3400.290 0.257 0.2530.248 0.322 0.2660.247 0.349 0.3130.312 0.247 0.2s80.245 0.284 0.2580.249 0.344 0.3480.340 0.240 0.2680.248 0.272 0.2790.311 0.316 0.3150.326 0.237 0.2530.252 0.321 0.3100.2s2 0.350 0.3150.293 0.243 0.2440.233 0.246 0.2810.268 0.328 0.2990.316 0.251 0.2340.254 0.264 0.2560.287 0.311 0.329NA 0.240 0.2s3NA 0.269 0.277NA
A BIER vessel was set at 121 ~C and 10 test u~its were run in each of the followillg cycles for each of the three lots packaged m~nll~lly:

5, 7, 8, 9, 10, 11, 12, 13 and 15 minlltes with 90 units tested for each lot.
Analysis was performed as described in Example 1. The number of positives over number tested was recorded with a read-out time of 20 seconds.
s _ W O 97126924 ~CT~US97/OV5~4 Table 12 BIER Vessel Tests LotA Lot B ~ot C
S Exposure m~nll~l auto mAtln~l auto m~n~ l auto Time 5 minutes 10/10 10/10 10/10 10/10 10/10 10/10 7 minutes 10/10 10/10 10/10 10/l0 10/10 10/10 8minutes I0/10 10/10 10/10 10/10 10/10 10/10 109 minlltes 5/10 9/10 8/10 10/10 7/10 6/10 10 m;mltes 3/10 5/10 6/10 10/10 5/10 4/10 11 minutes 3/l0 4/10 2/10 10/10 2/10 3/10 12 mimltes 0/10 2/10 0/10 3/10 1/10 2/10 13 mimltes 0/10 0/10 1/10 3/10 0/10 0/10 1515 mim-tes 0/10 0/l0 0/10 0/10 0/10 0/10 Overall, the enzymatic activity of the tablets decreased as the time of exposure increased. Five minute exposure in an autoclave showed 100% positive (30/30) and the 15 minute exposure showed 100% negative 20 (0/30).
Automated assembly of test units does not adversely affect perfolmance of the test unit ~i. e., 100% positive for a 5 minute exposure to steam in a BIER vessel and 100% negative for a 15 minute exposure to steam in a BIER vessel). Performance of the automated test unit does not 25 significantly differ from the performa~ce of the test unit m~nll~ctured m~n~lly, showing that the automated foam stuffer is acceptable for use.
Five test units prepared by m~nll~lly inser~g foam and the same number from automatically packaged lots A, B and C were placed in CA 0224412~ 1998-07-22 W O 97/26924 PCTrUS97/00554 a conventional 121~C gravity autoclave. The autoclave was run for a 5 minute cycle. Analysis was performed according to Example 1. All indicators tested positive.
Five test units from each of the above lots were placed in a 5 conventional 121~C gravity autoclave. The autoclave was run for a 15 minute cycle. Analysis was performed as above. All indicators tested negative.

Example 10 Rapid Sterilitv Indicator with a closed-cell foam in a Plasma-Phase Hvdro~en Peroxide Sterilizer.
The rapid sterility indicator as described in Example 5 was used with some modifications, the preferred foam length was changed from 0.375 inches to 1.0 inches, diameter is approximately 0.5". The plerell~d foam type was changed to a polyethylene closed-cell foam, referred to as 15 Fireflex or Melamine. The density of the foam as approximately 0.7 lbs/cubic foot, elongation = 10%, compression at 50% =6.2%, compression at 75% = 8.5%, compression at 90% = 14.4%, and heat resistant to 302~F.
Rapid sterility indicators were exposed to survival and kill cycles in the plasma-phase hydrogen peroxide sterilizer. Survival cycles a~e cycles with 20 short sterilant diffusion times in which indicators should test positive, indicating proper sterilization conditions were not met. Kill cycles are the standard cycles times in which the indicator should test negative indicating proper sterilization conditions were met.
- The survival time for the plasma-phase hydrogen peroxide 25 sterilizer was ~lPtermined to be a 6.5 minute diffusion time. Plasma time washeld constant at 15 mimltes and vaporizer time was 0.5 minutes. Kill time was determined to be a 35 minute diffusion time. Rapid sterility indicator -W 097/26924 PCTrUS97/00554 test units were placed into the steriIizer and exposed to the survival and kill cycles and processed as in Example 1. Results were recorded 20 seconds after adding the indicator solution to the test unit.
Table 13 S Plasma-phase ~2~2 Sterilizer - Survival/Kill Times Foam Len~th STTrvival' = Kill2 1.0" 10/10 0/10 I = Diffilsion time 6.5 minutes; Plasma time 15 minut~s; Vaporizer time 0.5 minutes; and Readout time 20 seconds.
2= Diffusion tirne 35 minutes; Plasma tirne 15 minutes;
Vaporizer time 0.5 rninutes; and Readout time 20 seconds.
In the tests shown in Table 13, the rapid sterility indicator showed the proper survival/kill performance in a STER~AD steriIizer. By adjusting the foam length or the type of foam, the desired survival/kill times could be met. This demonstrates that the rapid sterility indicator can be used to test the effectiveness of the STERRAD hydrogen peroxide sterilizer. The foam length of 1.0 inches provided the proper survival/kill results at 20 seconds after addition of the er~yme system reagents.
These tests demonstrate that the rapid steriIity indicator is effective for mo~ ~e plasma-phase hydrogen peroxide sterilizer. All positives were observed after the survival time and all negatives were 2~ observed after the kiIl time. The RSI challenge pack can also be modified to work in this sterilizer.

Example 11 A Sin~le Container Reusable Pack Challen~e Packs with Uni~ue Hi~h Resistant Parameters.
The ch~ nge pack described in example 7 can be modified to create an extremely resistant challenge. By increasing the foam density and/or m~kin~ the steam ently holes smaller, the challenge pack will enable CA 0224412~ 1998-07-22 W O 97/26924 PCTrUS97/00554 the indicator inside the pack to survive very long exposures to sterilization.
A reusable challenge pack can be created from one container with a closure device, one piece of foam and a sterility indicator. A reusable challenge pack is made of material that can withstand multiple exposures to 5 sterilization and can be easily opened and closed. The container is made from plastic or glass, preferably plastic. The container is approximate~y I l/2"diameter, 2l/2" long with a screw cap. The two steam entry holes are approximately 1/8" in diameter and the holes are located approximately l/41~
below the cap. A tubular piece of open cell, 4-6 pound polyurethane foarn 10 approxi~nately 1 1/2" long, 11/2" outer diameter (inner diameter 3/8")iS placed into the container. The rapid sterility indicator test unit as described in Example 1 is placed into the foam insert. The foam fits tightly into the container and the test unit fits tightly into the foam. The cap of the containeris screwed into place and creates a tight seal so that the only pathway for 15 steam to enter the challenge pack is through the small openings, passing through the foam to reach the indicator. The pack may also contain a heat sink material such as a metal object.
These challenge packs were placed into a steam sterilizer operating at 134~C (pre-vac sterilizer) and exposed to the predeLelll~led 20 survival and kill cycles. For this high resistant challenge pack the survivalcycle included exposure to 4 pre-vacuum cycles of temperature range of 60-130~C for 8 min~ltes, then 1 minute o~exposure to 134~C. The kill cycle included exposure to the 4 pre-vacuum cycles and a 4 minute exposure to - 134~C. A~ter the kill cycle indicators should test negative indicating proper 25 ct~nli7~tion conditions were met and the indicators should be positive after the survival cycles. After the cycles were complete, ~e container was opened and the indicator retrieved. The rapid sterili~ indicators were _ W O 97/26924 PCTrUS97/00554 processed as described in Example 1, the results are obtained at 20 seconds or less after adding the indicator solution.
Table 14 One Container Reusable High Resistant Challenge Pack 134~C Pre-Vacuum Steam Sterilizer (Total steam exposure time, the first 8 min~ltes temperature is 60-130~C) Survival time Kill time 9 min. 12 min.
Rapid Sterility 10/10 0/10 Indicator in one container reusable challenge pack Tlle results in Table 12 are recorded as the number positive over the number tested. As shown, when exposed to an extremely long survival cycle, includîng high temperatures, all indicators within the high r~cict~nt ch~llen~e pack tested positive. When exposed to the kill cycle time, all indicators tested negative. This table shows that the challenge pack design provides acceptable results. This resistant challenge pack has the unique feature of surviving very long cycles. It is actually testing the over-kill parameters built into the sterilizer. This also allows the user to do a validation in the standardized hospital cycles. The user can expose the indicator to the 3-4 pre-vacuums in a conventional autoclave and observe positive results. Tn the past, positive results of sterility indicators were only seen in BIER vessels or special research and development sterilizers that were able to perfo~n one quick vacuum rather than the 3-4 long vacuums.
This challenge pack also has the unique feature such that the user can reassemble the pack by placing a new indicator into the container, closing the container, and using it for another test.

W 097/26924 PCT~US97/OOS54 Other embo-1iment.~ and uses of the invention will be apparent to those skilled in the art from consideration of the specification and practiceof the invention disclosed herein. All U.S patents and other documents referenced herein, for whatever reason, are specifically incorporated by 5 reference. It is intended that the specification and examples be considered exemplary only, wi~ the true scope and spirit of the invention being indicated by the following claims.

Claims (56)

We Claim:

1. A test indicator for determining the effectiveness of a sterilization procedure comprising:
a container having liquid impermeable walls and at least one opening communicating with an interior chamber, the chamber containing biological material used as an indicator of destruction of living organisms by the sterilization procedure;
a gas-transmissive plug positioned in the at least one opening such that movement of gas between the environment surrounding the test indicator and the interior chamber occurs through the gas-transmissive plug wherein the gas-transmissive plug has an inside portion inside said at least one opening and an overhang portion extending outside of said at least one opening.

2. The test indicator of claim 1 wherein the gas-transmissive plug is compressible.

3. The test indicator of claim 2 wherein the gas-transmissive plug has an inside portion inside said at least one opening and an overhang portion extending outside of said at least one opening.

4. The test indicator of any of claims 1, 2 and 3 wherein the overhang portion is less than or equal to about 0.5 inches.

5. The test indicator of any of claims 1 to 4 wherein the at least one opening has a cross-sectional area of from about 0.03 to about 0.20 square inches.

6. The test indicator of any of claims 1 to 5 wherein the gas-transmissive plug has a non-compressed cross-sectional area of from about 0.2 to about 3.5 square inches, an inside portion of from about 0.4 to about 1.2 inches.

7. The test indicator of any of claims 1 to 6 wherein the sterilization procedure comprises steam heat, chemical sterilant, plasma, dry heat or a combination thereof.

8. The test indicator of any of claims 1 to 7 wherein the biological material comprises microorganisms, bacterial spores, enzymes, at least one component of an interactive enzyme system or a combination thereof.

9. The test indicator of any of claims 1 to 8 wherein the biological material comprises a granulation product.

10. The test indicator of any of claims 1 to 9 wherein the interactive enzyme system comprises a granulation product of glucose dehydrogenase and diaphorase.

11. The test indicator of any of claims 1 to 10 wherein the walls are substantially gas non-absorptive.

12. A sterility indicator for determining the effectiveness of a sterilization procedure comprising:
a container having liquid impermeable walls and at least one opening communicating with an interior chamber, the chamber containing biological material used as an indicator of destruction of living organisms by the sterilization procedure;
a gas-transmissive insert adjustably positioned in the at least one opening such that movement of gas between the environment surrounding the sterility indicator and the interior chamber occurs through the gas-transmissive insert wherein the gas-transmissive insert has an inside portion inside said at least one opening and an overhang portion extending outside of said at least one opening.

13. The sterility indicator of claim 12 wherein the gas transmissive insert is compressible.

14. The sterility indicator of any of claims 12 to 13 wherein the gas transmissive insert is between about 0.75 to 1.5 inches in length and extends from about 0.1 to about 0.5 inches outward from said at least one opening.

15. A method for adjusting sensitivity of the sterility indicator of claims 12 or 12 to a predetermined environmental parameter comprising the steps of:
a) exposing a test indicator substantially identical to the sterility indicator to the sterilization procedure;
b) determining the effectiveness of the test indicator for reacting to the predetermined environmental parameter;
c) adjusting the position or composition of the gas-transmissive insert of another test indicator;
d) exposing said another test indicator to the sterilization process and determining the effectiveness of the another test indicator for reacting to the predetermined environmental parameter and from the effectiveness determined for each test indicator; and e) adjusting the sensitivity of said sterility indicator.

16. The method of claim 15 wherein the sterilization procedure comprises exposing the test indicator to steam at 121°C, 132°C or 134°C.

17. The method of any of claims 15 to 16 wherein the sterilization procedure comprises exposing the test indicator to a sterilant.

18. The method of claim 17 wherein the sterilant is ethylene oxide or plasma-phase hydrogen peroxide.

19. The method of any of claims 12 to 18 wherein the predetermined environmentalparameter is selected from the group consisting of temperature, time, pressure, humidity, concentration of sterilant, penetration of sterilant, air removal or a combination thereof.

20. The method of any of claims 12 to 19 wherein the gas-transmissive insert comprises an overhang portion and the position of said insert is adjusted by extending or retracting an overhang portion that extends outwardly beyond said at least one opening.

21. The method of claim 20 wherein the overhang portion of the gas-transmissive insert is extended to increase sensitivity of the indicator to a combination of increased temperature, humidity and pressure.

22. The method of any of claims 12 to 21 wherein the gas-transmissive insert is composed of a compressible material and the composition of said insert is adjusted by increasing or decreasing the density of said compressible material.

23. The method of claim 22 wherein the density of the compressible material is decreased to increase sensitivity of the indicator to a combination of increased temperature, humidity and pressure or the presence of chemical sterilants.

24. An indicator system for determining the effectiveness of a sterilization parameter comprising:
an outer container having liquid impermeable walls and at least one opening communicating with a chamber containing a test indicator suitable for indicating effective sterilization by the sterilization parameter;
a gas-transmissive insert positioned in the at least one opening such that movement of gas between the environment surrounding outer container and the chamber containing the test indicator occurs through the gas-transmissive insert wherein said gas-transmissive insert is compressible.

25. An indicator system for determining the effectiveness of a sterilization parameter comprising:
an outer container having liquid impermeable walls and at least one opening communicating with a chamber containing a test indicator suitable for indicating effective sterilization by the sterilization parameter;
a gas-transmissive insert positioned in the at least one opening such that movement of gas between the environment surrounding outer container and the chamber containing the test indicator occurs through the gas-transmissive insert wherein said gas-transmissive insert has an inside portion inside said at least one opening and an overhang portion extending outside of said at least one opening.

26. The indicator system of any of claims 24 to 25 wherein the test indicator contains biologically relevant material which is used as an indicator of destruction of living organisms by the sterilization procedure.

27. The indicator system of any of claims 24 to 26 wherein the test indicator contain a thermal-sensitive or chemical-sensitive ink.

28. The indicator system of any of claims 24 to 27 wherein the outer container is transparent, the at least one opening has a substantially circular cross-sectional of from about 1 to about 3 inches in diameter, and the gas-transmissive insert consists of a compressible material having non-compressed diameter from about 2 to about 4 inches and length from about 1 to about 4 inches.

29. The indicator system of any of claims 24 to 28 wherein the gas-transmissive insert is a plug consisting of a partially open-celled foam having a density of from about 1 to about 10 pounds per cubic foot.

30. A method for determining the effectiveness of the sterilization procedure by exposing the indicator system of any of claims 24 to 29 to said sterilization procedure, observing the test indicator within the chamber after exposure and determining the effectiveness of said sterilization procedure.

31. A test pack for detecting a predetermined environmental parameter of a sterilization process comprising:
a container having liquid impermeable walls and at least one opening communicating with an interior chamber, said chamber containing;
a test indicator comprising another container having liquid impermeable walls and at least one opening communicating with an interior chamber;
wherein gas-transmissive inserts are positioned over each of said openings such that said environmental parameter can move from the exterior of said test pack to said interior chamber of said test pack and to said interior chamber of said test indicator.

32. The test pack of claim 31 wherein the environmental parameter is the presence of a vacuum, the concentration of a sterilant, pressure, penetration of sterilant, radiation or an amount of heat.

33. The test pack of any of claims 31 to 32 wherein the gas-transmissive inserts comprise foam that conform to the inside dimension of said containers.

34. The test pack of any of claims 31 to 33 which can be reused upon substitution of said test indicator.

35. The test pack of any of claims 31 to 34 wherein the gas-transmissive insert is a foam cylinder with an outer diameter of about 1 to about 2 inches, an inner diameter of about 1~
inches, and a height of about 1 to about 3 inches.

36. The test pack of any of claims 31 to 35 wherein the container is about 1 to about 4 inches in height and about 1 to about 3 inches in diameter, has a removable cap, and contains one or more holes of about ~ to ~ inches in diameter in said container.

37. A method for inserting compressed material into a container of a smaller area than the non-compressed material, said container having at least one opening, comprising the steps of:
a) placing the non-compressed material into a flexible loop having two ends wherein one end is attached to a slide and the other end being held in a fixed position;
b) axially aligning the container having at least one opening with the flexible loop;
c) moving the slide to cause the diameter of the flexible loop to decrease compressing the material; and d) advancing a plunger to extrude the compressed material from the flexible loop into the container.

38. A method for inserting compressed material into a container of a smaller area than the non-compressed material, said containing having at least one opening, comprising the steps of:
a) placing the compressible material on a crusher slide;
b) advancing the crusher slide to compress the compressible material;

c) axially aligning the container having at least one opening with a longitudinal axis of the compressed material;
d) advancing a plunger to deposit the compressed material into a staging nozzle;e) inserting the staging nozzle into the container; and f) retracting the staging nozzle while the plunger pushes the compressed material into the container.

39. The method of claim 38 wherein the plunger and the slide move in orthogonal planes.

40. The method of any of claims 37 to 39 herein the crusher slide has a tapered opening.

41. A method for inserting compressed material into a container of a smaller area than the non-compressed material, said container having at least one opening, comprising the steps of:
a) placing one end of a compressible material having two opposite ends slightly into a container having at least one opening;
b) securing the other end of the compressible material against rotation; and c) rotating and advancing the container to cause the material to be inserted into the container with a helix effect.

42. The method of any of claims 37 to 41 wherein the at least one opening contains a sterility sensing system.

43. The method of any of claims 37 to 42 wherein the compressible material is foam, sponge, plastic or a combination thereof.

44. The method of any of claims 37 to 43 wherein the compressible material is polyurethane, polyester, polyether, cellulose or a combination thereof.

45. The method of any of claims 37 to 44 further comprising the step of placing the container in a flexible tube.

46. The method of any of claims 37 to 45 further comprising the step of inserting the compressible material to a preselected depth within the container.

47. The method of claim 46 wherein the preselected depth is from about 0.5 to about 1.5 inches.

48 The method of any of claims 46 to 47 wherein the step of inserting the compressible material to a depth within the container leaves one end of the compressible material protruding past the opening of the container.

49. The method of any of claims 37 to 48 wherein at least one end of the compressible material and at least one end of the container is round, square, elongated, ellipse or a multi-sided geometric shape.

50. An apparatus for inserting a compressible material into a cylindrical sleeve of smaller cross-sectional area than the non-compressed material, said apparatus including:a flexible strip having a first end fixed and a second end attached to means for sliding so that the flexible strip forms a cylindrical loop which decreases in size upon movement of the sliding means;
a plunger disposed coaxially with an axis of the cylindrical loop so that the plunger slides through the cylindrical loop; and a holder for positioning a cylindrical sleeve which receives cylindrically compressed material displaced from the cylindrical loop by the plunger.

51. The apparatus of claim 50 wherein the flexible strip comprises material selected from the group consisting of Teflons, mylars, nylons, metals or a combination thereof.

52. An apparatus for inserting a compressible material into a cylindrical sleeve of smaller cross-sectional area than the non-compressed material, said apparatus including: a support plate;
a first forming device fixed to the support plate and having an arcuate section with an axis parallel to the support plate;
a second forming device slidably opposed to said first forming device, said second forming device having an angular shaped opening towards said first forming device so that sliding said second forming device toward said first forming device forms a cavity elongated in a direction perpendicular to the direction of sliding and parallel to the support plate;
a plunger slidably disposed parallel to the support plate so that the plunger slides through the elongated cavity formed by the first and second forming devices; anda holder for positioning a cylindrical sleeve to receive cylindrically compressed material displaced from the elongated cavity by the plunger.

53. The apparatus of claim 52 wherein the plunger precisely positions cylindrically compressed material in the cylindrical sleeve.

54. The apparatus of any of claims 52 to 53 further comprising means for advancing the plunger, said advancing means selected from the group consisting of by hand, cams, air cylinders, hydraulic or electric linear motion actuator.

55. An apparatus for inserting a compressible material into a cylindrical sleeve of a smaller cross-sectional area than the non-compressed material said apparatus including:
means for rotating a cylindrical sleeve; and means for transporting a block of compressible material along the axis of rotation of the cylindrical sleeve so that the compressible material moves progressively into the sleeve.

56. The apparatus of claim 55 wherein the transporting means precisely positions a cylinder of compressible material in the cylindrical sleeve.