EP0706579A1

EP0706579A1 - Sterilisation indicator device

Info

Publication number: EP0706579A1
Application number: EP94920991A
Authority: EP
Inventors: Mongi Mahwachi
Original assignee: Individual
Current assignee: Individual
Priority date: 1993-07-01
Filing date: 1994-06-29
Publication date: 1996-04-17
Also published as: WO1995001451A1; FR2708287B1; FR2708287A1; JPH08511946A; CA2166197A1

Abstract

A device for estimating the effect of a fluid on a microorganism strain or an enzyme, comprising a compartment (1) for contacting the fluid with said strain or enzyme and a compartment (2) for highlighting said strain or enzyme after the contacting step by means of a highlighting medium (15). The device is characterised in that it consists of a movable assembly having at least two configurations (8, 11). The first configuration includes a channel for circulating the fluid within the assembly and contacting it with the strain or enzyme attached to a support (9), while the second configuration includes a member for contacting the highlighting medium with the support to which the strain or enzyme is attached, and optionally a means for isolating the highligting medium containing the support from the atmosphere, as well as a means for viewing any changes in the highlighting medium (18).

Description

STERILIZATION INDICATOR DEVICE

The present invention relates to a device for evaluating the action of a fluid on a microorganism strain or an enzyme, more particularly a sterilization indicator device. Sterilization and disinfection are widely used processes in a wide variety of fields.

Sterilization is in particular implemented in care establishments such as hospitals, clinics, so as to have medical instruments, glassware, linen, generally any material, completely free of microorganism in order to stop the spread of infectious diseases.

However, sterilization is also used in the pharmaceutical industry for medical and surgical equipment such as sutures or generally a large number of single-use objects and also for medicines for which a total absence of microorganism is necessary.

Sterilization is also implemented more and more often in the agro-food industries in order to control the biocontamination of the environment, this is particularly the case in the dairy industries.

It is also, in these same areas, in common use to use disinfection, but it is in the case of care establishments for example, the treatment of surfaces, linens, this to reduce the proportion of germs pathogens to limit the incidence of infections. In the pharmaceutical and food industry, disinfection is also practiced in order to comply with good manufacturing practices; this process also tends to spread in other areas such as for example local authorities, dental offices, hairdressers, etc. The objects subjected to sterilization and disinfection can be of an excessively varied nature, it can be as well glass, as metals or synthetic materials, similarly the geometric configurations can be very varied, flat, hollow shape, smooth or not.

Currently, to judge the effectiveness of sterilization, for example for moist heat, we use external means, it is mainly recording of temperature and pressure as well as sterilization times. Depending on the chart, the time / temperature pair can be determined to obtain sterilization under given conditions. However, this means may prove to be insufficient, which is why in most cases there is also an internal device allowing the demonstration of sterilization.

In its simplest form, it is a device that includes microorganisms in a vegetative or resistance form, in particular in a spore form, which will be checked at the end of sterilization that effectively there is no longer any of viable microorganisms or spores, or at least that the level of viable microorganisms or spores remaining is below a certain threshold. An enzyme can also be used to reveal the effectiveness of sterilization. Depending on the type of sterilization applied, microorganisms known for their resistance in sporulated form will be used, for example for sterilization by moist heat, Bacillus stearothermophilus will be used. for sterilization by dry heat or gases we will use Bacillus subtilis and for sterilization by ionizing radiation we will use Bacillus pumilus.

As regards the disinfection processes, only this second method can be used, in fact there are no charts making it possible to determine disinfecting conditions.

The present invention relates more particularly to indicator devices comprising microorganisms in vegetative or spore form or of enzymes, intended for testing, in general, the action of a fluid on said microorganism or the enzyme, in general in order to test sterilization or disinfection of an enclosure.

The sterilization indicator devices generally comprise, on the one hand, a support on which the spores of the resistant strain are placed which can be brought into contact with the sterilization fluid and, on the other hand, a compartment in which is located a medium allowing the culture of the sporulated microorganism and a developing agent which changes color in the presence of the microorganism in culture. The control process therefore consists in introducing the device into the sterilization enclosure, the spores are then brought into contact with the sterilization fluid, then, at the end of sterilization, the culture medium is introduced, by breaking a bulb. and the developing agent in contact with the spores, and after a determined time a color is visualized in the medium if among the spores some of them were still viable.

This type of apparatus is described for example in French patent 2,045,855. Most of the devices of the prior art have a certain number of drawbacks: first of all, the support for the spores is mostly paper, gold the materials to be sterilized in practice are almost never made of paper but plastic, metal, rubber, glass, etc. ; - In general, the supports have a flat shape which is unrelated to the geometrical conformations of most of the materials to be sterilized which are generally of extremely varied shape; finally, the support easily absorbs sterilizing agents in certain cases, which leads to easier destruction of the spores when in reality the microorganisms contaminating the material to be sterilized are carried on non-absorbent surfaces.

Thus, some of the devices depending on the way they are implemented can lead to falsely reassuring results.

In addition, most of the biological indicator devices currently proposed involve the breaking of a glass bulb, which is generally not desirable, in particular because the small pieces of glass can create microcracks in the outer container, there is therefore a risk of contamination.

Finally, in certain cases it may be advantageous to be able to control the sterilization during the operation without having to intervene directly in the sterilization enclosure, this is generally not possible with the devices currently existing.

This is why the present invention relates to a device intended for evaluating the action of a fluid on a microorganism strain or on an enzyme, of the type comprising: a compartment (1) allowing the contact of the fluid and said strain or the enzyme and a compartment (2) allowing the detection of said strain or the enzyme after contact with the fluid, using a development medium (15), characterized in that it consists of a mobile assembly which may have at least two configurations (8, 11) having, in a first configuration, a passage allowing the circulation of the fluid to the interior of the assembly and its contacting with the strain or the enzyme fixed on a support (9) and in a second configuration, a means allowing the development medium to be brought into contact with the support on which is fixed the strain or the enzyme and, optionally, means making it possible to isolate the development medium containing the support from the external atmosphere, and means allowing the observation of possible modifications of the development medium (18).

Preferably, this device is a biological indicator of sterilization and / or disinfection.

Under these conditions, the fluid in question is water vapor, a gas or a liquid, but also very generally dry heat or ionizing radiation, even if it is not strictly speaking a fluid.

As regards the revealing medium, it is most of the time a medium allowing the culture of the strain from the spores or from the vegetative form comprising a developer demonstrating the presence of viable microorganisms.

Finally, the microorganisms will most of the time be in the form of spores.

Preferably, the device according to the present invention will also include means allowing the observation of possible modifications of the developer liquid.

In general, the device according to the invention will consist of two compartments movable relative to one another, one of the compartments comprising the support (9) and at least one orifice (14) allowing an external fluid to be brought into the interior in contact with the support, and the other compartment comprising the revelation medium, said compartment being arranged so that the displacement of one relative to the other brings the support of the strain (9) in contact with the development medium and possibly in that another displacement leads to the isolation of the development medium containing the support from the external medium.

The displacement of the two mobile compartments can be done both by translation and by rotation about an axis.

In general, the support of the strain is a mobile element which can have any configuration intended in particular to allow the best representation of the surfaces which will have to be sterilized with decontaminated outside.

It may in particular be a flat support or else a support comprising recesses or corrugations, but in most cases it will rather be a device of tubular shape, preferably having the shape of a small tube and this support will be movable between a first position in contact with the fluid and a second position in contact with the development medium.

In the preferred embodiment, the device consists of two mobile compartments which rotate relative to each other about an axis and which have two facing surfaces perpendicular to said axis, the upper compartment comprises a tubular conduit (6) substantially parallel to the axis of rotation opening out of the compartment through two orifices, a support is placed in said tubular conduit and the support rests on the surface located opposite the compartment below the surface which opens a tubular conduit opposite the previous one and the other end of which opens to the outside, a channel is thus created which crosses the entire device parallel to the axis and a part of which is formed by the support of the strain, the lower compartment further comprises, on its surface facing an orifice, the dimension of the orifice being such that, by rotation of the two compartments, the tubular conduit re from the upper compartment is brought into coincidence with the orifice and the support falls through this orifice into the revelation medium located in the lower compartment, as well as by bringing the assembly back to its position initially, the medium containing the support of the external medium is isolated, the lower compartment comprising, in addition, one or two transparent windows or being completely transparent, making it possible to see the color of the development medium. In addition, the lower compartment may include, in addition to the culture medium, in the lower part of the fluid conduit, a physicochemical indicator (making it possible to see the effect of sterilization in depth or at a distance on a physic- chemical).

In general, the support of the microorganism will consist of a surface which may be identical to the surfaces to be sterilized and / or to be decontaminated, in practice, in particular it may be metallic surfaces, paper surfaces if necessary, plastic surfaces, but preferably the surfaces will be glass, in fact as will be seen in the examples which follow, the use of a glass support makes it possible to have results which are the most reliable.

The microorganisms are preferably present in their most resistant form, that is to say in the form of spores which are fixed on the support of the microorganism by techniques which are known. It is also possible to use non-resistant strains, for example a real flora or even an enzyme having the same resistance as the reference spores.

As indicated above, in its preferred form the support consists of a glass tube inside which spores of microorganisms have been deposited according to methods which will be described in the examples and the fluid will be caused to circulate through this tube.

The development medium is preferably constituted by a culture medium allowing the growth of the spores when these are still viable, said culture medium comprising an indicator, most of the time colored, whose coloration changes or appears during growth. microorganisms, for example by modifying the pH with products of the phenol red type or products which react to redox such as for example methylene blue, it can also be products which are colored by revealing an enzyme specific for the strain in question, it will in most cases be a colored substrate of the specific enzyme. In the preferred embodiment of the invention, the support, after being brought into contact with the fluid to be tested, will be caused to fall into the development medium and therefore incubate in its presence, however it is necessary to be able to unhook the spores fixed on the support, this can be done mechanically, in particular by adding a stirring element in the medium and / or in the tube, in particular a small metal ball which will allow by stirring to scrape the spores, it is it is also possible to carry out a passage through ultrasound, but, as will appear in the examples, the most rational solution is to introduce a surfactant, such as Tween 80 for example, into the revelation medium, which will make it possible to easily detach and completely microorganisms as spores, but has no influence on cell viability.

Preferably the result can be read through one or two transparent plastic windows or a completely transparent lower compartment, or any other transparent material, in which case it is advantageous to use glass as a support, which allows 'have a greater visible thickness, while a metal support will be at least from this point of view less effective.

One of the advantages of the device of the invention is that, given the fact that the fluid to be tested can circulate in a pipe in which it is brought into contact with the support carrying the spores or the microorganisms in vegetative form or an enzyme , we can provide at the ends of this pipe two nozzles on which we can come fix tubes of suitable material, non-porous, to be able to test the sterilization in depth or sterilization at a distance, that is to say that the you can test the state of sterilization in a place where the indicator is not located directly by immersing the two tubes in the place to be tested and placing the device outside, it is also possible of test the sterilization of an enclosure by keeping the device according to the invention outside provided that two tubes have been adjusted in which the atmosphere circulates which must be sterilized.

Among the various devices making it possible to test the sterility of an atmosphere, the device according to the present invention is the only one which makes it possible, in a simple manner, to test sterility in depth by means of the tubing plunging deep into the product or the atmosphere to be tested and connected to the device by the nozzle (s), said device not itself being placed in depth. The present invention therefore also relates to a device as described above to which are fixed by the nozzles of the pipes making it possible in particular to test sterility in depth, for example in a large container of elongated type or in a place which is difficult to access, by placing the 'one end of the pipe at the place to be tested. It is also possible to also provide for aspirating the atmosphere at one of the ends by means of a pump connected to the other nozzle.

The following examples are more particularly intended to demonstrate other characteristics and advantages of the present invention. The examples are implemented using a device of the type shown in the appended figures in which:

- Figure 1 shows a section along a plane passing through the axis of rotation of a device according to the invention in the fluid passage position; - Figure 2 shows the same device but in another position where the microorganism support is placed in the middle;

- Figure 3 shows the device after returning to the initial position

FIG. 4 is a view of the device in FIGS. 1 to 3.

Figure 1 shows a device according to the present invention in a section through a plane passing through the axis of rotation of the two compartments relative to each other. This device consists of two mobile compartments (1 and 2) connected by a connecting piece (5), each of the compartments consists of identical or different casings (3 and 4) in which is placed an insertion element which, for the upper compartment, comprises a tubular conduit (6) substantially parallel to the axis of rotation, opening to the outside through an orifice (7) and through an orifice (8) opposite the lower compartment. The support (9) which is substantially cylindrical and applied in the tubular conduit (6) is coated on its inner part with spores (10). The support (9) rests on the surface located opposite the lower compartment (12) at the surface of which opens a tubular conduit opposite the previous one (11) and whose end (13) leads to the outside. The tubular conduit (11) may contain a physico-chemical indicator. In this configuration, a channel has thus been formed which crosses the entire device parallel to the axis and part of which is formed by the support of the strain.

The lower compartment also has an orifice (14), the dimension of which is such that, after 180 ° rotation of the two compartments (1 and 2) relative to each other to arrive at the configuration shown in the Figure 4, the tubular conduit (6) is brought into coincidence with said orifice and the support (9) falls into the revelation medium (15) located in a reservoir (20) of the lower compartment.

After a second 180 ° rotation, the two compartments are returned to the initial position shown in FIG. 3, but the support (9) is then placed in the development medium (15) but isolated from the outside and can then be restless.

It should be noted that in order to avoid external contamination, the through orifices (7 and 13) each have a filter (21 and 22).

In addition, the tubular channel (11) is narrower at its upper part to allow the blocking of the support (9).

Finally, the ends of the channel (7 and 13) are provided with nozzles such as 16 and 17 in order to allow the adaptation of tubes if necessary in order to test deep sterilizations or in order to test atmospheres in which one does not wish to introduce the device.

FIG. 4 represents a view of the device according to the present invention in which there are two compartments (1 and 2) connected by the connecting piece (5), one of the tubular conduits emerging with its nozzle (16) as well as the transparent reading window (18) which makes it possible to see the variations in color of the development medium (15). EXAMPLE 1

The tests were only carried out with the spores of Bacillus stearothermophilus CIP 5281 = ATCC 7953 in the case of sterilization with water vapor.

1) Preparation of the spore suspension, concentration of the suspension and microbial count

1.1 Material - Broth for freezing the strain (1)

. Beef extract 3 g

. Tryptic casein peptone 5 g

. Glycerol 150 g. Distilled water QSP 1,000 1 - First culture broth (2)

. Yeast extract 2.5 g

. Tryptone 5.0 g

. Glucose 1.0 g

. Distilled water QSP 1000 1 - Sporuation agar divided into vials of Roux (3)

. Meat extract 10.0 g

. Yeast extract 2.0 g

. MnSO ₄ , H ₂ O 0.04 g

. Agar 23.0 g. QSP distilled water 1000 ml

1.2 Preparation of the spore suspension

Inoculation of the broth (2) with approximately 106 spores originating from a freezing broth at - 20 ° C (1) in which the strain of Bacillus stearothermophilus CIP 5281 is stored - Incubation at 56 ° C for 24 hours. Transfer of approximately 3 ml of this culture to a vial of

Ginger containing sporulation agar. Subject the Roux flask to various inclinations so that the inoculum comes into contact with the entire surface of the agar; reject excess inoculum - Incubate the vial at 56 ° C for 20 days.

Observation of the state of the culture under a microscope: observe the spores with diluted fuschine or under a phase contrast microscope. The vegetative cells must be lysed and the spores are colored in very pale pink. If sporulation has not started, repeat the test.

If sporulation has started, continue incubation until the vegetative cells are lysed.

Recover the solid culture with sterile distilled water, scraping the surface of the agar if necessary. 1.3 Concentration of the suspension

Distribute the recovery liquid (colored yellow-brown) in centrifuge tubes. Carry out three successive washes of 15 minutes each by centrifugation at 2,700 rpm: at each wash, discard the supernatant and take up the pellet in 5 or 10 ml of sterile distilled water. Transfer the last suspension (which should no longer be colored yellow-brown) in a Pyrex bottle with a screw cap. Heat 30 minutes at 100 ° C to eliminate any remaining vegetative forms. Store at + 4 ° C.

Shake the suspension well before each sampling, as the spores settle at the bottom of the bottle.

1.4 Enumeration of the spore suspension

Enumeration of spores after suitable dilution in sterile distilled water, inclusion in Mueller Hinton Agar agar (Diagnostic Pasteur, ref. 64884), 35 g / 1, prepared immediately; incubation at 56 ° C. The spore suspension should titrate at least 10 ⁷ spores / ml.

Check for the absence of bulky spores or organic debris by staining with diluted fuschine. 2) fixing of the spores on the support

We tested different supports: flat, glass, stainless steel, rubber and fabric, cylindrical, glass, stainless steel and rubber. 5 The deposits were 100 μl of the spore suspension on the surface of the flat supports and in the lumen of the cylindrical supports and the spores are dried at 37 ° C. 3) Recovery of spores fixed on the supports

After depositing 100 μl of a suspension of spores calibrated 10 on the various supports and drying as indicated above, the support is placed in a defined volume of sterile distilled water. The implementation of different methods shows that the use of 0.5% Tween 80 with stirring is sufficient to be able to take off most of the spores while the other methods prove to be either difficult to implement or insufficient in their results.

The implementation of the device as described above with different types of support allows the determination of the decimal reduction time called DT at a given temperature. This decimal reduction is the time necessary to inactivate 90% of the microorganisms 20 present before sterilization, that is to say also the time necessary to reduce the value of the initial microbial population by 10 times. In the context of sterilization, the Di ₂ i ° c of the paper, glass, stainless steel, rubber and fabric supports of flat geometry is determined by exposure of the supports in the device according to the present invention at different times. sterilization.

We find the following results: Di ₂ i ° c paper = 1.9 min

Di ₂ i ° c glass = 2.3 min

Di ₂ rc stainless steel = 2.4 min 30 Di ₂ i ° c fabric = 2.3 min

Hard rubber = 2.4 min

When instead of the flat supports we take supports of cylindrical geometry, glass, stainless steel and rubber, we find the following results: 35 Di ₂ i ° c glass = 2.5 min

Di ₂ i »c stainless steel = 2.4 min Di ₂ i ° c rubber = 2.3 min

In conclusion, we can retain in the new biological indicator supports whose nature and configuration are representative of the objects actually subjected to sterilization, hollow supports in glass, steel or rubber, but it seems appropriate to preferably use cylindrical supports glass which give the most important decimal reduction times and therefore the greatest safety in terms of sterilization. 4) Sensitivity study

The technique used in the device according to the present invention is an "all or nothing" technique. To test the sensitivity of the device, a culture medium consisting of a trypto-casein-soy broth modified by the addition of 0.5% Tween 80 is used. Phenol red is added as a pH indicator. This pH indicator by acidification of the medium changes from red to yellow when the culture is positive.

Tests are carried out with a very reduced number of spores which have led to the following results: for 30 tests with 2 + 1 spores, approximately 80% of positive cultures in 24 h of incubation, for 30 tests with 5 + 1 spores, 100 % of positive cultures in

24 h incubation. The sensitivity threshold is therefore 5 ± 1 spores.

Thus this technique makes it possible to measure a reduction in base 10 logarithms of the number of spores after sterilization, which could be verified by tests in real situations.

Cylindrical glass support carrying 5,106 spores: - exposure time 3 min: 100% of positive responses exposure time 6 min: 100% of positive responses exposure time 9 min: 100% of positive responses exposure time 12 min: 90% of positive responses exposure time 15 min: 0% of positive responses The device according to the present invention makes it possible to obtain much more satisfactory results than the biological indicators of the prior art, in particular those described for example in Technique Hospitalière n ° 493, October 1986, p. 52-56.

Claims

CLAIMS 1) Device intended to evaluate the action of a fluid on a microorganism or enzyme strain, of the type comprising: a compartment (1) allowing the contact of the fluid and said strain or the enzyme and a compartment (2) allowing the detection of said strain or the enzyme after contact with the fluid, using a development medium (15), characterized in that it consists of a mobile assembly can have at least two configurations (8, 11) having, in a first configuration, a passage allowing the circulation of the fluid inside the assembly and its contacting with the strain or the enzyme fixed on a support ( 9) and in a second configuration, means allowing the development medium to be brought into contact with the support on which the strain or the enzyme is fixed and, optionally, means making it possible to isolate the development medium containing the support the outdoor atmosphere ieure, and means allowing the observation of possible modifications of the revelation medium (18). 2) Device according to claim 1, characterized in that it consists of two compartments movable relative to each other, one of the compartments comprising the support (9) and at least one orifice (14 ) allowing an external fluid to be brought into the interior in contact with the support, and the other compartment comprising the revelation medium, said compartment being arranged so that the displacement of one relative to the other brings the support of the strain (9) in contact with the development medium and possibly in that another displacement leads to isolating the development medium containing the support from the external medium.

3) Device according to one of claims 1 and 2, characterized in that the two compartments are movable in translation or in rotation.

4) Device according to one of claims 1 to 3, characterized in that the support of the strain is movable between a first position where it is in contact with the fluid and a second position in which it is in contact with the liquid revelation. 5) Device according to one of claims 1 to 4, characterized in that the first and the second compartments are capable of rotating or being moved in translation relative to each other to bring, after the support has been brought into contact with the external fluid in a first position by rotation or translation, said support facing an opening formed in the second compartment containing the development medium so as to be able to fall into said development medium.

6) Device according to one of claims 4 and 5, characterized in that, in the first configuration, the first compartment has two orifices bringing the interior of the compartment into contact with the exterior, in that in the compartment is located the support so as to be in contact with a fluid coming from the outside through the orifices (7, 8) and in the second configuration the support is brought opposite an orifice formed in the second compartment (14) which contains the revelation medium 15) and falls into said revelation medium, and in that when the assembly is returned to the first configuration, the revelation medium containing the support is isolated from the external atmosphere.

7) Device according to one of claims 1 to 6, characterized in that the microorganism support (9) is of substantially tubular shape and that each end of said tube is in contact with the orifices of the first compartment in order to ensure the passage of the external fluid inside said tube.

8) Device according to claim 7, characterized in that it consists of two compartments movable in rotation relative to each other about an axis and having two opposite surfaces perpendicular to said axis, the compartment upper comprises a tubular conduit (6) substantially parallel to the axis of rotation opening out of the compartment through two orifices, a support is placed in said tubular conduit and the support rests on the surface located opposite the lower compartment on the surface of which opens a tubular conduit opposite the previous one and the other end of which opens to the outside, a channel is thus created which crosses the entire device parallel to the axis and one of which part is formed by the support of the stump, the lower compartment further comprises, on its surface facing a orifice, the dimension of the orifice being such that, by rotation of the two compartments, the tubular conduit of the upper compartment is brought into coincidence with the orifice and the support falls through this orifice into the revelation medium located in the lower compartment , as well as returning the assembly to its initial position, the medium containing the support of the external medium is isolated, the lower compartment further comprising a transparent window allowing the color of the development medium to be seen.

9) Device according to claim 8, characterized in that the revelation medium is contained in a tubular element substantially parallel to the axis.

10) Device according to one of claims 8 and 9, characterized in that the openings opening to the outside comprise a filter (21, 22).

11) Device according to one of claims 8 to 10, characterized in that the external orifices include nozzles (16, 17).

12) Device according to one of claims 8 to 11, characterized in that the two compartments consist of a housing having the same structure.

13) Device according to one of claims 8 to 12, characterized in that there is in the lower part of the fluid conduit a physicochemical indicator.

14) Device according to one of claims 1 1 to 13, characterized in that pipes are arranged on the nozzles to allow testing the sterility in depth.