BIOLOGICAL STERILITY INDICATOR AND METHOD FOR MAKING AND USING SAME

June 15, 1971 R. R. ERNST 3,585,112

BIOLOGICAL STERILITY INDICATOR AND METHOD FOR

MAKING AND USING SAME

Filed July 14, 1967

United States Patent O ce

3,585,112

Patented June 15, 1971

1

3,585,112

BIOLOGICAL STERILITY INDICATOR AND

METHOD FOR MAKING AND USING SAME

Robert R. Ernst, Rochester, N.Y., assignor to

Sybron Corporation

Application Oct. 23, 1965, Ser. No. 503,987, now Patent No. 3,346,464, which is a continuation-in-part of application Ser. No. 272,035, Apr. 10, 1963. Divided and this application July 14, 1967, Ser. No. 653,414 The portion of the term of the patent subsequent to Oct. 10,1984, has been disclaimed

Int. Cl. GOln 55/00 U.S. Cl. 195—103.5 6 Claims

ABSTRACT OF THE DISCLOSURE A biological sterility indicator including a sealed semipermeable envelope 10 being permeable to water and impermeable to bacteria in either liquids or gases, a selected quantity of suitable unincubated test organism 12, and growth media 18 and dye indicator 20, all preferably sealed within the envelope. The indicator is rendered suitable for testing multiple types of sterilizing media by utilizing multiple types of test organisms, each of which is sufficiently resistant to at least one of such sterilizing media thereby to yield an accurate test of all such sterilizing media.

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This invention relates to a biological sterility indicator and method for making and using same. This application is a divisional application of my copending application 503,987 filed Oct. 23, 1965 now U.S. Pat. 3,346,464 which was a continuation-in-part application of my prior copending application Ser. No. 272,035 filed Apr. 10, 1963 and now abandoned.

In attempting to solve the problem of testing sterility, many inadequate solutions have come to the forefront. Sterility indicators can be basically classified as physical, chemical and biological. While all commercially available types of indicators are constantly being improved, the only acceptable and true indication of sterilization is the biological type. The biological indicator is usually designed to provide a safety factor for the sterilization process. A test organism is selected which is many times more resistant to the sterilization process employed than most organisms which would be present by natural contamination such as pathogens. Thus, by adjusting the contamination level of the test organism, a safety factor is provided which will assure sterility of the product to be sterilized if prescribed sterilization parameters are followed. To further guarantee sterility, indicators are placed in strategically difficult-to-sterilize locations within the selected sterilizer.

After exposure to the sterilization process, biological indicators in the past had to be aseptically transferred by trained personnel to aseptic testing areas, whereby under fastidious, aseptic techniques, the trained personnel subjected the microorganisms to suitable sterile growth media for a suitable incubation period at a proper temperature to determine the effective kill of the sterilization. Visual observation of any growth of the test organisms in the growth media causing turbidity has generally been the indication of lack of effective sterilization.

A chemical acid-base (pH) or oxidation-reductionpotential (O-R) dye indicator has oftentimes been added to the growth media. A change in dye color or bleaching produced by chemical changes in the growth medium by the metabolizing test organisms provides a better and quicker visual check on unsterile results than mere turbidity.

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Known biological sterility testing procedures require trained personnel for competent interpretation of the results and to determine whether or not any contamination may have been introduced in the transfer process. 5 Because of the difficulty involved in acquiring and training personnel skilled both in the mechanical and biological arts to check the effectiveness of sterilization procedures, manufacturers of sterilization equipment have attempted to produce sterilizers and related equipment 10 which would require little or no professional skill by including sufficient safety devices in the sterilizers themselves to prevent mishaps which otherwise result in unsterile goods. Usually excessive sterilization periods are prescribed to insure complete sterilization. 15 Physical indicators have oftentimes been built into the sterilizers, such as thermometers, pressure gauges, and the like; however, such indicators do not indicate completely the physical parameters necessary to achieve sterilization. Superheat or radiant heat may provide a false in"° dication of the proper conditions for steam sterilization with lag thermometers. Superheat and residual air would also provide a false indication on a pressure responsive gauge.

Commercial chemical indicators are chemicals which indicate sterility by color changes, or change from solid to liquid state; however, these also are subject to inaccuracies. Only the living organism can sense the true relationships of physical chemical parameters necessary to effect go sterilization. Neither the physical nor chemical indicators can indicate over the entire range of parameters interrelating time, temperature and concentrations of moisture, chemicals, or radiation dose. Therefore, it is recognized in the art of sterilizing that biological tests are the most 35 accurate sterility tests.

Because of the recognized superiority of biological tests, manufacturers of sterility indicators have attempted to simplify the biological tests to eliminate the tedious aseptic transfer and treatment during the testing period subse40 quent to sterilization. As for example, one type of biological indicator for steam contains spores of a thermophilic bacterial species in a liquid culture medium within a sealed glass vial. This organism will not grow at ambient temperatures. Incubation of this vial at the proper tem45 perature will initiate growth. With this test, no allowance is made for the quality of the steam used. A high degree of superheat or residual air might render the sterilization process ineffective; however, since this indicator is dependent only on time of exposure to an external heat 50 source at a specific external temperature without regard to saturation of the steam, it is quite possible that a false indication may result. Also, this indicator would not be responsive to chemical sterilization. Although this type of indicator obviates aseptic techniques and media prepara55 tions, it is limited to penetrative heat or radiation and to thermophilic organisms.

Other biological indicators are commercially available, some of which comprise bacterial spores impregnated in filter or blotter paper strips. These require aseptic handling 60 and transfer and the preparation and utilization of sterile growth media. The possibilities of contamination in transfer from one sterile environment to another is not avoided. One commercial preparation incorporates spores and dried media with dye directly on blotter paper strips. It is 65 necessary to provide sterile distilled water in sterile glass tubes to which the strips must be aseptically transferred. Another difficulty with this preparation is that it cannot be used for gaseous sterilization since microorganisms are sometimes encapsulated in crystal structures during the 70 preparation of the indicators, thereby forming a protective barrier against the penetration of necessary moisture and/or gaseous killing agents.

3,585,112

Thus, prior to my invention, there is no satisfactory simple sterility indicator or method for making and using the same.

It is one object of my invention ot provide a more satisfactory biological sterility indicator and method for 5 making and using same.

It is a further object of my invention to provide a simplified biological sterility indicator and method for making and using same.

It is a further object of my invention to provide im- 10 proved biological sterility indicator and method for making and using the same, being adapted for use with all of the common sterilizing media, i.e., steam, dry heat, radiation, ethylene oxide, propylene oxide, methyl bromide and other gaseous agents. 15

It is another significant object of my invention to provide an improved biological sterility indicator and method for making and using the same which does not require aseptic transfer or treatment following sterilization and during the testing period. 20

Other objects and advantages of this invention will be particularly set forth in the claims and will be apparent from the following description, when taken in connection with the accompanying drawings, in which:

FIG. 1 is a planar view of one embodiment of my inven- 25 tion;

FIG. 2 is a sectional view taken along the line 2—2 looking in the direction indicated by the arrows;

FIG. 3 is a planar view of a second embodiment of my invention; and 30

FIGS. 4-7 are planar views of various sequential stages of production of a second and improved embodiment of my invention.

As viewed in FIG. 1, my invention is characterized by a sealed semi-permeable envelope, bag, or container 10 35 being constructed of a dialyzing film permeable to selected sterilizing media, permeable to water and impermeable to bacteria present in either liquids or gases and preferably of plastic composition, such as polyamide-6 sold under the trademark "Capran-77C" by Allied Chemical Company.

Within the semi-permeable envelope 10, is sealed a selected quantity of a suitable unincubated test organism and carrier 12, the test organism 12 being selected because of its responsiveness to the selected sterilizing media. This semi-permeable^envelope 10 might be formed for example, 45 by heat-sealing folded plastic webbing or tubing to form an envelope. As used herein, the term "webbing" is broadly intended to mean a sheet or strip of film, membrane or other non-woven material as well as woven material, but not limited to woven material. For purposes of illustra- 50 tion, I have shown plastic tubing heat-sealed at both ends at 13 of the semi-permeable envelope 10, thereby sealing the selected test organism 12 therein.

Preferably, the envelope or bag 10 is a translucent semipermeable material. As used herein, the word "trans'lu- 55 cent" is intended to be defined as including "transparent." Moreover, translucency is not essential to the concept of my invention. A dye indicator (pH or O-R) could be used which would change color, bleach out, or induce color or stain an opaque or translucent bag or envelope 00 10 responsive to the presence of metabolizing bacteria, i.e., the metabolites formed by the living or viable bacteria. A preferable dye indicator is phenol-red because of its pH range and other properties such as its dynamic color change from red to yellow. The phenol-red stains <ia this particular type of bag in the acid range, thereby providing a more positive visual test.

Dry or otherwise inactive or unincubated growth media 18 such as trypticase-phytone is preferably sealed in the ,-0 envelope 10 with the selected dry test organisms and carrier 12 and a dry or otherwise inactive or unincubated acid base (pH) or oxidation-reduction (O-R) potential dye indicator 20 may also be incorporated within the sealed envelope as shown in FIGS. 1 and 2. Leucine assay 75

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(Difco broth) has also proved a successful growth media.

It will be understood that my invention contemplates the arrangement whereby the growth media 18 is on the outside of the envelope 10 and will penetrate the dialyzable envelope to initiate growth of any living bacteria inside the envelope 10 when the envelope 10 is submerged in a solution including the growth media 18. However, experiments have proved that when the growth media is outside of the semi-permeable envelope initially, it is difficult to control growth due to external contamination which confuses the results and this is particularly true if a dye indicator is incorporated outside of the semi-permeable envelope with the growth media.

As stated in the preamble, one of the disadvantages of the commercial preparation including spores and dry media on blotter paper strips is the ineffectiveness to test gaseous sterilization. I have found that impregnating liquid media on bibulous paper in high concentration proved very successful since this could be sterilized and dried simultaneously by ethylene oxide presterilization prior to incorporation in the heat-sealed pouch. Thus, this media-impregnated paper ISA (FIG. 3) could be used in lieu of the dry nutrients or growth media 18, thereby providing excellent growth.

It will be understood that indicators other than pH and O-R indicators can be used. For example, a fat soluble Lipase indicator such as victoria blue and night blue. Lipase-releasing microorganisms such as some Bacillus species are capable of metabolizing lipids resulting in the release of the dye into the water solution turning a vivid blue.

Another phenomenon which could be utilized as a visual indicator is the ability of some bacteria to liquify gelatin. A mixture of gelatin and carbon-black can be denatured with formaldehyde to form a stable complex until gelatinase by bacterial metabolism breaks down the gelatin, releasing the carbon-black into the solution giving a very vivid indication. Bacillus subtilis var. globigii produce enzymes which convert tryosine to melanine (brown-black) pigments.

Also certain organisms are capable of breaking down glucosides. A dye complex such as indican (a colorless glucoside) can be split at the glucose-dye bond by the metabolism of the living bacteria releasing the indigo blue dye to the media.

Therefore, it is obvious that the dye indicator need only be some form of indicator which is responsive to the presence of metabolizing bacteria.

In carrying out the process, the envelope 10 and contents are preferably placed in the most remote area of the sterilizing chamber and subjected to the sterilizing conditions for the selected sterilizing period. After completion of the sterilizing cycle, the operator may use his hands or unsterile instruments to remove the envelope 10 from the sterilizer and submerge the envelope and contents into ordinary tap water free of chlorine and preferably distilled water. Since bacteria present in either water or air will not permeate the semi-permeable envelope, there is no problem of aseptic transfer. In view of the fact that distilled water is usually slightly acidic, preferably a pH dye indicator solution is mixed with a suitable quantity of NaOH to render the distilled water solution slightly akaline, preferably in a pH range of 9.5 to 11.00 in order to produce the desired initial color from a pH indicator such as phenol-red and assure stability of color during the incubation period, thereby to assure accuracy of the desired visual color indication at the completion of incubation.

An impermeable outer envelope 14 (FIG. 3) of clear plastic can be provided for this purpose which would serve to keep the envelope 10 submerged and obviates the use of glassware for field use. Outer envelope 14 is heatsealed at the bottom at 15 and open at the top 16 through which water is introduced and provided with a grommet 17 for hanging. The top of the outer bag 14 preferably

3,585,112

is closed at the top after the water is introduced, as for example by mastic tape, or it may be heat-sealed if desired, thereby to prevent evaporation during incubation of the test organism. The envelope 10 being semi-permeable permits the water to readily permeate the envelope 10 5 and dissolve the growth media and activate any reaction which will occur if the otherwise inactive or unincubated test organisms were not completely killed during the sterilization period.

If the sterilization was ineffective to kill the selected 10 test organisms, then the dye indicator changes color responsive to the growth of the living bacteria of the test organisms in the presence of the growth media. In the absence of a dye indicator, the turbidity change in the growth media may be observed through a translucent en- 15 velope. During the incubation period, the water and growth media are maintained at a proper temperature for optimal growth of the test organism.

I have discovered that plastic films comprising poly amide resin commonly sold under the trademarks "Cap- 20 ran-77C," "Portex" and "nylon-6" are very desirable materials out of which to manufacture the envelope 10. These materials have the desirable characteristics of being: semi-permeable plastic, readily permeable to the common sterilizing agents such as steam, dry heat, radiation 25 and the gaseous sterilizing agents such as ethylene oxide, propylene oxide, methyl bromide; readily permeable to water; but impermeable to all bacteria and most microorganisms hereinafter in the claims cumulatively referred to as "bacteria"; whether present in a gas or liquid. This 30 film is heat and adhesive-sealable, and is readily sterilizable by steam without unfavorable effects on the film.

Preferably, the growth media in the dry form is included in the sealed envelope 10 as illustrated at 18 in FIGS. 1 and 2 or dried on blotter paper as shown at ISA in 35 FIG. 3; in FIG. 3 the dye is dried on the blotter paper 20A and contained in the outer impermeable envelope 14 with the selected test organism 12 and growth media ISA inside of the semi-permeable envelope 10. Depending on the composition of the envelope 10, ions and/or small 40 molecules of metabolites of the living bacteria would diffuse out of the envelope 10 into a water-dye solution externally of the envelope 10 causing a color change in the water-dye solution or staining the envelope 10 externally, thereby indicating lack of sterility. Since some dyes are 45 bacteriocidal or bacteriostatic, it is preferable to have reaction of the dye taking place externally of the inner envelope 10 and separated from the metabolizing bacteria. Another variation of this invention is to provide the dye indicator in an envelope or compartment adjacent the envelope 10 rather than in an outer envelope which completely envelopes the inner envelope 10; however, it is important that the common wall between the organism and media compartment and the dye indicator compartment be semi-permeable so that water and ions and/or 55 small molecules of metabolites of living bacteria will diffuse through the common wall to the dye indicator thereby to give the desired visual indication of sterility or lack thereof.

I have successfully used many dyes and combination 60 of dyes and mordants for fixing the dyes on the envelopes.

Thus, I have provided a new and improved biological sterility indicator and a vastly simplified method of making an accurate biological test of sterility by the use of my indicator.

These sterility indicators can be adapted to most sterilizing techniques now being used on a large scale, including steam, dry heat, boiling water or other liquids, radiation, gaseous agents including but not limited to ethylene oxide, propylene oxide, methyl bromide, and the like. Neither expensive equipment nor sterile transfer areas are necessary with the use of my invention.

By the way of illustration, the following are examples of successful sterility tests utilizing my invention:

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EXAMPLE 1

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Conditions: The test spores were Bacillus stearothermophilus. Incubation temperature was 54° C. All spore strips were incubated in sterile trypticase soy broth (BBL) . The invention included Castle spore strips and separate bibulous paper strips impregnated with tryptone-phytone. These paper strips were heat-sealed in the nylon bag (10 FIG. 2). To the outer bag (14) of FIG. 1, water was added to the fill mark and several drops of Phenol-red indicator solution were added. Color change was noted in the external solution in 24 hours.

Results:

Average No. spores/unit — 200,000 Growth observed — 24 hours Control 0-min. — -1/1

No. Unsterile 8 „ , ._.„ „ , „ .

-j= — TM — — j -- „ Exposure to 250° F. steam for 10 mm.

No. Unsterile 0 _, r,rTM-r-. , *

Exposure to 250° F. steam for lo mm.

TM — -— r

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As a further improvement of my prior invention, I have discovered certain advantages of encapsulating the selected dye indicator and/or the selected growth media and/or the selected test organisms in a readily water soluble film-forming substance permeable to selected sterilizing media, preferably a cellulose substratum such as methyl cellulose or hydroxyethyl cellulose. I have also found polyvinyl pyrollidone to be a suitable film-forming substance for this purpose. All of these film-forming substances desirably produce a flexible, thin, encapsulating material. The dye indicators, media and/or test organism is mixed with the readily water soluble, permeable, filmforming substance along with other ingredients hereinafter described.

(I) Dye indicator

The following is the formulation for a 100 milliliter (ml.) volume of stock solution of one embodiment of my improved dye indicator mixture:

For 100 ml. volume of stock solution of formulation— used 1 drop (0.1 ml. volume) dried for each 2-5 ml. H2O in "outer bag solution"

7-8 g. hydroxyethyl cellulose

5 g. sodium citrate (buffer)

2 g. glycerine (humectant)

1.0 ml. NaOH—50% concentration

0.05 g. Phenol-red (phenolsulfonthalein)

100.0 ml. distilled H2O

While in the liquid state, the dye indicator mixture of film-forming substance is preferably sterilized, thereby to preclude mold forming on the dried film which might otherwise occur.

The film-forming substance mixed with whichever of the selected dye, media or test organism incorporated therein may be dry processed in one of the following two ways: