Apparatus and method for dertermining the effectiveness of sterilization.

0001The present invention relates generally to a method and apparatus for determining the effectiveness of sterilization procedures, and more particularly, to an improved method and apparatus for determining the presence or absence of live microorganisms following their passage through a sterilization cycle.

0002Numerous medical items used in hospitals, such as sutures, catheters, gloves, bandages, and the like must be sterilized prior to use, either by the manufacturer or the hospital. Sterilizing media such as steam, dry heat, sterilizing gases and irradiation have been employed for such purpose. Problems have arisen, however, in determining the completeness or efficacy of the sterilization procedure, and the need has long existed for means whereby the effectiveness of a sterilization cycle may be determined quickly and reliably.

0003Heretofore, one method of testing the effectiveness of sterilization has involved the use of a small vial which contains a spore strip (i.e., generally a piece of absorbent paper having a predetermined form of microbial spores thereon) and a sealed ampule of a sterile nutrient media. The vial typically has means, such as a semipermeable membrane or passages in the closure, that permits steam or gas to enter the vial during the sterilization cycle. The vial is placed in the sterilization chamber along with the items to be sterilized so that the spores within the vial are exposed to the sterilization along with, and in the same manner, as the items to be sterilized. Upon completion of the sterilization cycle, the vial is removed, and the inner ampule broken to release the media to mix with any spores that may have survived the sterilization. The vial is then incubated for a period of one to several days, depending on the strain of spores used. If viable spores remain, their germination and growth, utilizing the nutrient of the media, will alter the pH of the solution, causing a visible change in color of the solution or an increase in turbidity, thereby indicating the lack of complete sterilization. The foregoing sterilization detection procedure, however, suffers from various drawbacks, including difficulties in breaking of the media containing ampule without contaminating the media, the relatively long period of time required before results can be determined, the ambiguity in the results that sometimes are obtained, and the inability to make reliable quantitative determinations of the degree of sterilization.

0004While systems have been available for more quickly determining biological activity, such systems, heretofore, have not been viewed as being adaptable for use in sterilization testing procedures. For example, in effecting bacterial determinations in samples of blood, spinal fluid, or the like, it is known to inoculate a test sample containing or believed to contain bacteria or microorganisms into a sealed vial containing a radioisotope labeled nutrient media. The sealed vial is then incubated so that any microorganisms present in the sample will metabolize, oxidize, or otherwise process the nutrient media and evolve or respirate a gaseous carbon dioxide which includes the radioisotope. After a relatively short incubation period, on the order of four to eight hours, the vial can be removed from the incubating chamber and utilized in a known radioactive gas detection apparatus, which quantitatively measures the evolved gas and the rate of evolution to provide an indication of the presence of and the degree of activity of micro- organisms in the test sample. Because such radio- activity media containing vials are typically maintained in a gas-tight sealed condition throughout the procedure, they have not been utilized in sterilization indicator procedures where the sterilizing atmosphere must enter the vial.

0005The invention consists in a test container for use in :determining the effectiveness of sterilization comprising: a vial; and characterised by means for supporting within said vial a test strip having a - predetermined form of micro-organism thereon, and an aqueous growth medium, such that said test strip is normally maintained out of contact with said medium, a closure member mountable in a first position on said vial which permits entry of a sterilizing atmosphere into said vial and into contact with the micro- organisms on said test strip during a sterilization cycle, and means for permitting said test strip to be brought into contact with said aqueous medium in said vial at a selected time after the sterilizing cycle; said closure member being movable to a second position on said vial which seals the interior of said vial so that gases generated by viable micro-organisms in contact with said medium during incubation thereof are contained in said vial to permit their analysis.

0006According to a further aspect, the invention consists in a method of testing the effectiveness of sterilization comprising the steps of: placing with items to be sterilized a vial containing a test strip with a predetermined form of micro-organisms thereon and an aqueous growth medium such that when brought in contact with viable micro-organisms on said test strip and incubated for a predetermined period will result in the evolution of detectable gas, maintaining the test strip in said vial out of contact with the said medium therein while exposing the test strip to the sterilizing atmosphere during a sterilization cycle along with the items to be sterilized, contacting the test strip with the medium in said vial following the sterilization cycle, sealing the medium and test strip in the vial, incubating the vial and its sealed contents for a predetermined period to cause any viable micro-organisms remaining following the sterilization cycle to evolve a gas, and analyzing the gas evolved in said sealed vial to determine the effectiveness of the sterilization cycle.

0007The invention provides quick and reliable quantitative testing of the effectiveness of any suitable sterilizing process.

0008In order that the invention may be better understood, several preferred embodiments of the invention will now be described, by way of example only, with reference to the accompanying schematic drawings, wherein: <ul id="ul0001" list-style="none"><li>Figure 1 is a side elevational view of a sterilizing test container embodying the present invention and for use with the method of the present invention;</li><li>Figure 2 is an exploded perspective of the test container shown in Figure 1;</li><li>Figure 3 is an enlarged vertical section of the container shown in Figure 1, taken in the plane of line 3-3, showing the closure member thereof in a first locked position which permits entry of a sterilizing atmosphere into the interior of the container;</li><li>Figure 4 is a vertical section, similar to Figure 3, but showing the closure thereof in a second locked position sealing the interior of the container in gas-tight relation to the outside atmosphere;</li><li>Figures 5 and 6 are horizontal sections taken in the planes of lines 5-5 and 6-6, respectively in Figure 3;</li><li>Figure 7 is a horizontal section taken in the plane of line 7-7 in Figure 4;</li><li>Figure 8 is a perspective of a radio-active gas detecting apparatus in operative engagement with a container such as shown in Figure 1;</li><li>Figure 9 is a representation of the detecting apparatus shown in Figure 8;</li><li>Figure 10 is a vertical section of an alternative embodiment of test container according to the present invention;</li><li>Figure 11 is a horizontal section taken in the plane of line 11-11 in Figure 10;</li><li>Figure 12 is a verticval section of another alternative embodiment of a test container according to the present invention;</li><li>Figure 13 is a vertical section of still another alternative embodiment of test container, showing the closure member thereof in a vented position;</li><li>Figure 14 is a vertical section of the test container shown in Figure 13, but showing the closure member thereof in a vial sealing condition; and</li><li>Figure 15 is a vertical section of still a further alternative embodiment of test container.</li></ul>

0009While the invention is susceptible of various modifications and alternative constructions, certain preferred embodiments have been shown in the drawings and will be described below in detail. It should be understood, however, that there i no intention to limit the invention to the specific forms described but, on the contrary, the intention is to cover all modifications, alternative constructions and equivalents falling within the scope of the invention.

0010Referring more particularly to FIGS. 1-7 of the drawings, there is shown an illustrative test container 10 adapted for use in testing the effectiveness of sterilization procedures in accordance with the present invention. The container 10 includes an outer vial 11 that is closed at the bottom end, a sealed media containing ampule 12 disposed in upstanding relation within the vial 11, an annular disc-shaped test strip 14 positioned on the upper end of the ampule 12, and a closure member 15 positionable onto the upper open end of the vial 11. The illustrated vial 11 has a generally cylindrical configuration and preferably is made of a transparent, relatively rigid plastic material.

0011The ampule 12 may be of a conventional configuration, in this instance having a generally cylindrical shaped central portion with a spherical bottom end 12a and a narrowed upwardly extended top end 12b. The ampule 12 preferably is made of a frangible glass material and contains in hermetically sealed condition a predetermined volume of an aqueous nutrient media 16. For supporting the ampule 12 concentrically within the vial 11, the vial is formed with a plurality of ribs 18 extending upwardly and outwardly from a central location in the bottom of the vial. A pair of inwardly recessed, channel- shaped ribs 19 are formed in opposed sides of a lower portion of the vial for supporting the sides of the ampule 12 in an upright position.

0012The test strip 14 may be a strip of absorbent material of a known type bearing a predetermined form of microorganisms for the sterilization procedure to be tested, preferably in the form of bacterial spores. As is known in the art, the spore bearing microorganism Bacillus stearotheromophilus may be utilized when detecting the effectiveness of steam sterilization. Bacillus substilis commonly is employed in testing the effectiveness of ethylene oxide and dry heat sterilization cycles, and Bacillus pumilus is used when verifying irradiation sterilization cycles. It will be understood that combinations of such microorganisms may be utilized on a single spore strip. While reference may be made herein to spores as the test microorganisms, it will be understood that microorganisms other than spore formers may be used in conjunction with the test strips.

0013For securing the closure member 15 on the end of the vial 11, the closure member 15 is formed with internal threads 20 which are adapted for threadable engagement with external threads 21 formed on the upper portion of the vial 11. To facilitate turning of the closure member 15 relative to the vial 11, and thus advancement of the closure member onto the vial, the closure member in this instance is formed with a knurled outer peripheral gripping surface 22.

0014In one embodiment of the invention, the closure member and vial threads define a gas venting passageway, and the closure member is positionable between a first predetermined locked position that permits a sterilizing atmosphere to pass through the passageway into the interior of the vial and a second predetermined locked position which simultaneously seals the interior of the container and effects the breakage of the ampule to"release the media. To this end, in the illustrated embodiment the threads 21 of the vial 11 are formed with a plurality of longitudinally aligned recesses or vents 24 at circumferentially spaced locations about the outer periphery of the vial. Since the threads 20 of the closure member 15 engage the vial threads 21 in outwardly spaced relation to the outer perimeter of the vial, and since the vents 24 in the vial threads are recessed flush with the outer perimeter of the wall, it can be seen that with the closure member in threaded engagement on the end of the vial, as shown in FIG. 3, the vents define a plurality of longitudinal passageways which permit sterilizing atmosphere from outside the container to pass upwardly from under the closure member, through the vent passageways, and into the interior of the vial.

0015In response to further threaded engagement of the closure member onto the vial, means are provided for simultaneously fracturing the ampule contained therein and sealing the interior of the vial. For this purpose, a ram 30 is disposed in the vial concentrically about the ampule 12. The ram 30 in this instance comprises an annular collar portion 30a disposed concentrically about the upper portion of the ampule 12 and a pair of pincher arms 30b extending in depending fashion from collar 30a on opposed sides of the ampule, the pincher arms 30b being located in an annular space defined between the ampule and the peripheral wall of the vial, as shown in FIG. 3. The ram 30 preferably is made of a plastic material which permits some degree of flexibility of the pincher arms 30b, as will become apparent. For supporting the ram 30 in a raised, normally stored condition, the upper ends of the recessecl-ribs 19 are formed with upwardly and outwardly tapered ramps 31 which extend from a location in close relation with the ampule 12 to the peripheral wall of the vial 11. The ramps 31 are disposed on opposed sides of the vial, with lower most ends of the ram pincher arms 30b supported thereon. For retaining the pincher arms 30b on the ramps 31 in proper circumferentially spaced relation to the ampule 12, the peripheral wall of the vial is formed with pairs of ribs 32 within which the pincher arms 30b are retained for relative vertical movement (FIGS. 5 and 6). To facilitate movement of the pincher arms 30b along the ramps, as will become apparent, the pincher arms have lower most rounded ends 30c.

0016For moving the ram 30 from the raised, stored position, shown in FIG. 3, to an ampule breaking ^t position, shown in FIG. 4, the closure member 15 is formed with a depending, concentrically-disposed, annular bearing member 35 which upon advancement of the closure member 15 into further threaded engagement with the vial 11 by relative rotation thereof, the bearing member 35 is brought into engagement with an upper end of the ram and forces it in a downward direction. Such downward movement of the ram 30 causes the pincher arms 30b to be cammed downwardly and inwardly by the ramps 31 to a position which ultimately fractures the ampule 12, thereby releasing the media 16 and allowing the test strip 14 to fall into contact with the media.

0017In order to seal the interior of the vial 11 upon breakage of the ampule 12, the closure member 15 may be threaded tightly onto the vial until the upper end of the vial engages an annular sealing rib 36 formed on the underside of the closure member. The sealing rib 36 preferably is formed with the same flexibility and is directed inwardly at an angle to the longitudinal axis of the closure member such that when the upper end of the vial engages the side of the sealing rib 36 (as shown in FIG. 4), a gas-tight seal is effected about the entire upper end of the vial.

0018To facilitate reliable selective locating of the closure member 15 in a vented condition (FIG. 3) or in a sealing condition (FIG. 4), means are provided for positively establishing such positions in a manner that permits the user of the container to see, feel, and hear when the closure member is properly located. To this end, the closure member 15 is formed with a pair of depending locking legs 39 on opposed sides thereof and the vial 11 is formed with pairs of outwardly extending lugs 41, 42 on opposed sides, the lugs 41, 42 defining recesses for positively receiving and holding the respective closure member locking legs in the first predetermined or venting condition, as shown in FIGS. 3 and 6. To facilitate positioning of the closure member locking legs in such first locked position as an incident to rotation of the said closure member onto the end of the vial, the locking legs preferably have limited flexibility and the first lug 41 of each pair is formed with a chamfered side 41a that permits the respective locking leg to be cammed up over the lug 41 and then snapped into the recess. The closure member locking legs 39 are thereby positively retained in the recesses, maintaining the closure member 15 in predetermined desired orientation on the vial for enabling reliable passage of a sterilizing atmosphere through the vented passageway in the vial threads and into the interior of the container. To further facilitate entry of the sterilizing atmosphere into the container, the annular bearing member 35 of the closure member 15 is formed with circumferentially spaced openings 35a which are aligned with the vents 24 in the vial threads when the closure member is locked in such vented position (FIG. 5).

0019To locate the closure member 15 in the sealed condition (FIG. 4), the closure member may be rotated such that the legs are forced over the second lugs 42 of each pair so as to free the closure member for further rotation and threaded engagement onto the vial. As the closure member 15 approaches the sealed condition, shown in FIG. 4, the closure member locking legs 39 will engage second position lugs 44, in this case located on diametrically opposed sides of the vial 11 in spaced axial relation below the lugs 41 of the first sqt. To facilitate movement of the locking legs 39 into this locked position, the lugs 44 again are formed with a forward chamfered surface 44a over which the legs 39 are cammed and then snapped into locked position behind the lugs 44 which positively retain the closure member in the sealed condition. It will be appreciated that the locking legs 39 and lugs 41, 42, 44 enable the user of the container 10 to easily establish the proper venting position of the enclosure member 15, as well as the sealed position, by visually observing the positioning of the locking legs, as well as by hearing and feeling when the closure member is properly located.

0020From the foregoing, it can be seen that with the closure member 15 locked in a first or venting position (FIG. 3), the test container 10 may be placed in a sterilization chamber along with items to be sterilized so that the microorganisms on the test strip 14 within the vial 11, are exposed to the sterilization cycle along with, and in the same manner, as the items being sterilized. Upon completion of the sterilization cycle, the test container 10 can be removed and the inner ampule 12 easily broken by simply turning the closure member 15 into further threaded engagement with the vial 11, which will automatically force the ram 30 downwardly into the ampule, breaking the ampule to release the media 16 and allowing the test strip 14 to fall into and mix with the media (FIG. 4). As an incident to such further threaded engagement of the closure member 15 on the vial 11, the upper end of the vial will be moved into engagement with the sealing ribs 36 of the closure member, sealing the contents of the test container 10 from the outside environment with the closure member legs being forced over the lugs 44 and snapped into locked position, positively retaining the closure member in such sealed condition.

0021In keeping with the invention, the aqueous media 16 contained in the ampule 12 is a sterile solution of a bacterial growth nutrient which contains a component or companents having a radioactive isotope such that when brought in contact with viable microorganisms on the test strip 14 and incubated for a determined period will result in the evolution of detectable radioactive carbon dioxide gas. Preferably the radioactive component is a sugar, such as glucose, in which all of the carbons are the 14C radioactive isotope. It will be understood by one skilled in the art that other radioactive labeled bacterial growth nutrient medias could be used. Because the closure member 15 is brought into sealing relation with the vial 11 as an incident to fracturing of the media ampule 12, gases generated within the test container during subsequent incubation of the contents of the container are retained therein. Typically, incubation periods from two to several hours are sufficient to cause the evolution of detectable radioactive gases from viable microorganisms.

0022Following incubation of the sealed test container, the container is utilized in a radioactive gas analysis apparatus 50 that is adapted to remove from the container 10 gases generated during incubation, analyze the gases, and make a quantitative determination as to the degree of biological activity that survived the sterilization. To facilitate removal of the gas from the test container 10 by the analysis apparatus 50, the closure member 15 has an upper end formed from a rubber septum 51, in this case being mounted at the top of the closure member 15 and retained in position by an annular closure ring 52. The septum 51 is made of a suitable rubber material which effectively maintains the gas-tight integrity of the closure member 15, while permitting insertion therethrough of appropriate hypodermic needles to enable the withdrawal of accumulated gases from the interior of the container without entry of the outside environment. To maintain the septum 51 in a clean condition prior to use, a removable end cap 54 is fixed on the top of the closure member 52, as shown in FIG. 3, and can be removed from the container, as shown in FIG. 4.

0023For removing and analyzing gases in the sealed container, the radioactive gas detecting apparatus 50, shown in FIGS. 8 and 9, may be of a known type, such as Bactec models 301 or 460 sold by Johnson Laboratories, Inc. and described in U.S. Patent No. 3,935,073. Such apparatus includes an ion chamber 55, and electron meter and meter display 56, a sequence controller 58, a C0₂ absorber 59, a vacuum pump 60, and control valves 61 and 62. A test container 10 may be brought into position such that sterile hypodermic needles 64 and 65 can be caused to penetrate the rubber septum 51 of the test container closure member 15 so that gas is drawn from the container through needle 64 and valve 66 and into ion chamber 55 for measurement of radioactivity. The amount of activity, in this instance, is displayed on the display meter 56, as well as on a printout 68. It will be understood that by appropriate electronics, in the event the measurement exceeds a predetermined level, a light 69 may be actuated. The amount of measured radioactivity, thereby, will reflect the existence of viable microorganism remaining in the test container following sterilization, as well as quantitatively indicate the extent such sterilization was effective.

0024After the gaseous atmosphere in the container has been sampled and analyzed, the valve 66 can be actuated to connect the ion chamber 55 with filtered air 70 to allow the ion chamber 55 to be flushed with clean air so that the apparatus is in condition to analyze the next sample. In practice, it has been found that determinations as to the effectiveness of the sterilization cycle can be made by the apparatus 55 in a matter of minutes following completion of incubation, which typically takes only between about two to several hours.

0025Referring now to FIGS. 10 and 11, there is shown an alternative design of the test container embodying the present invention, wherein items similar to those described above have been given similar reference numerals with the distinguishing suffix "w" added. The test container lOw includes a closure member 15w, test strip 14w, and a radioisotope media containing ampule 12w, all identical to those described above. The vial llw in'this instance has a uniform, cylindrical configuration, again having a closed bottom with ampule supporting ribs 18w and an externally threaded upper end. Hence, with the closure member 15w in a first locked position, as shown in FIG. 10, the test strip 14w within the test container may be exposed to a sterilization cycle.

0026To effect breakage of the ampule 12w upon threaded advancement of the closure member 15w onto the vial llw following a sterilization cycle, the lower half of the ampule 12w in this case is supported within a generally U-shaped pincher 75 having upstanding pincher arms 76 disposed on diametrically opposed sides of the ampule. The pincher arms 76 are formed with ampule engaging beads 78 at their upper terminal ends. For moving the pincher arms 76 into ampule breaking engagement, a ram 30w is concentrically disposed about the upper half of the ampule 12w and is formed with depending legs 30(b)w having wedge-shaped terminal ends 80. The ram legs 30(b)w in this case are located in closely adjacent relation to the peripheral wall of the vial llw. For maintaining the pincher arms 76 and ram legs 30(b)w in proper relation on opposite sides of the ampule, the inside wall of the vial llw is formed with pairs of inwardly extending ribs 81 which define recesses within which the pincher arms and ram legs are disposed. Upon threaded advancement of the closure member 15w onto the vial llw as described previously, a bearing member 35w of the closure member 15w will engage the ram 30w and force it in a downward direction such that the wedge-shaped terminal ends 80 are forced between the pincher arms 76 and vial wall, thereby forcing the beaded terminal ends 78 of the pincher arms 76 inwardly against the ampule 12w. The inward forces are concentrated at a central, relatively vulnerable area of the ampule, so as to cause reliable fracturing of the ampule. As an incident to fracturing of the ampule 12w, the closure member 15w is moved into sealing relationship with the upper end of the vial llw as previously described, enabling the test container lOw to then be processed through incubation and radioactive gas detection process steps to quantitatively determine the effectiveness of the cycle sterilization in which the test container was used.

0027Referring now to FIG. 12, another alternative embodiment of test container is shown, wherein items similar to those described above have been given similar reference numerals with the distinguishing suffix "x" added. The test container lOx includes a closure member 15x similar to that previously described, a generally cylindrical shaped vial llx, and a radioactive isotope labeled media containing ampule 12x again similar to that previously described but disposed in inverted fashion in the vial with the neck portion 12(b)x thereof oriented downwardly. The test strip 14x in this case is supported on the spherically-shaped upper portion of the ampule 12x and is confined within the bearing member 35b of the closure member. The underside of the ampule 12x is supported at points adjacent the neck 12(b)x by relatively sharp corners 84 of a plurality of rectangular-shaped ribs 18x integrally formed in the bottom of the vial llx. The bearing member 35x of the closure member 15x in this instance is formed with an enlarged lower end having a relatively sharp annular edge 85 that is engageable with the upper end of the ampule 12x.

0028With the closure member 15x in the position illustrated in-FIG. 12, sterilizing atmosphere can enter the vial in the manner previously described during a sterilization cycle. Upon completion of a sterilization cycle, the closure member 15x can be threadedly advanced onto the vial llx causing the bearing member 35x to engage and axially compress the ampule, llx, with high stress concentrations occurring at the points of contact between the opposed ends of the bearing member 35x and the vial ribs 18x. The application of such concentrated forces on the ampule 12x again will cause reliable breakage of the ampule as an incident to moving the closure member 15x into sealing engagement on the end of the vial llx.

0029Referring now to FIGS. 13 and 14, there is shown still another embodiment of test container, wherein items similar to those described above have been given similar reference numerals with the distinguishing suffix "y" added. The test container 10y includes a vial lly having a cylindrical body portion 86 which directly contains a quantity of aqueous radioactive isotope labeled media 16y, a reduced diameter neck portion 88, and an enlarged externally threaded upper end 89 with vented threads for receiving an internally threaded closure member 15y in a manner similar to that previously described. For isolating the media 16y from a test strip 14y and the outside environment during a sterilization cycle, a rubber stopper 90 is disposed in the neck portion 88, and the test strip 14y located on top of the stopper 90.

0030For simultaneously sealing the vial from the outside environment following sterilization and for enabling the test strip 14y to become immersed in the aqueous media 16y, the closure member 15y includes a depending annular-shaped bearing member 35y, similar to that described above but somewhat longer in length, which as an incident to threaded engagement of the closure member 15y from the vented position, shown in FIG. 13, to the vial sealing condition, shown in FIG. 14, is moved into engagement with and forces the rubber stopper 90 through the reduced neck portion 88 of the vial 14y, causing the stopper 90 and test strip 14y both to fall into the media 16y, as shown in FIG. 14. The test container 10y may then be incubated and utilized in radioactive gas detection apparatus as previously described.

0031Referring now to FIG. 15, still another test container is illustrated, wherein items similar to those described above have been given similar reference numerals with the distinguishing suffix "z" added. Test container lOz includes a cylindrical- shaped vial llz, in this instance made of sufficiently flexible material to permit manual manipulation thereof. The vial llz has an upper end formed with vented threads 21z which receive an internally threaded closure member 15z similar to that described above. Contained within the vial is a radioactive media containing ampule 12z having a test strip 14z supported on an upper neck portion thereof. With the closure member 15z in a vented position, as shown in FIG. 15, sterilizing atmosphere is permitted to pass into the interior of the vial llz and into contact with the test strip 14z. Upon completion of the sterilization cycle, the closure member 15z may be threadedly advanced into sealing engagement with the end of the vial llz, and the ampule 12z may be broken by manually manipulating the flexible body portion of the vial llz, thereby causing the test strip to be dropped into contact with the aqueous media 16z. The test container may then be processed through incubation and the gas analysis steps as previous described.

0032From the foregoing, therefore, it can be seen that the method and apparatus of the present invention are adapted for quickly and reliably determining the effectiveness of sterilization procedures, which heretofore has taken between one and several days. Moreover, the method and apparatus are adapted to provide quantitative determinations as to the degree of sterilization. The test container of the present invention, furthermore, is easy and reliable to use during sterilization, incubation, and gas analysis, and may be employed in connection with steam, dry heat, gas, and irradiation sterilization procedures.