FR2708287A1 - Indicating device for sterilization. - Google Patents

Abstract

The present invention relates to a device intended to evaluate the action of a fluid on a strain of microorganism or on an enzyme, of the type comprising: - a compartment (1) allowing the fluid to be brought into contact with said strain or the enzyme. and - a compartment (2) allowing the detection of said strain or the enzyme after coming into contact with the fluid, using a development medium (15), characterized in that it consists of 'a mobile assembly which 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, a means allowing the contacting of the development medium with the support on which the strain or the enzyme is fixed and, optionally, means allowing the medium to be isolated revelation containing the support of the external atmosphere e, and means allowing the observation of possible modifications of the revelation medium (18).

Description

The present invention relates to a device for evaluating the action

  of a fluid on a strain of microorganism or an enzyme, more

  particularly a sterilization indicator device.

  Sterilization and disinfection are processes that are widely used in a wide variety of fields. The sterilization is implemented in health care institutions such as hospitals, clinics, so as to have medical instruments, glassware, linen, generally any material, totally free of microorganism to stop the disease.

  spread of infectious diseases.

  But, sterilization is also used in the pharmaceutical industry for medical-surgical equipment such as sutures or generally a large number of disposable objects and also for drugs for which a total absence of microorganism

is necessary.

  Sterilization is also being used more and more often in the agri-food industries in order to control the biocontamination of the environment, particularly in the dairy industries. It is also, in these same areas, common use to use disinfection, but it is in the case of care institutions for example, the treatment of surfaces, towels, this to reduce the proportion of germs pathogens to limit the incidence of infections. In the pharmaceutical industry and the food industry, disinfection is also practiced to respect good manufacturing practices; this process tends to spread in other areas such as local authorities, dental offices, hairdressers, etc. Objects subjected to sterilization and disinfection may be of an excessively varied nature, it may be as well glass, metal or plastic, the same configurations

  geometries can be very varied, flat, hollow, smooth or not.

  Currently, to judge the effectiveness of sterilization, for example for moist heat, using external means, it is essentially the recording of temperature and pressure and sterilization times. As a function of the abacus, it is possible to determine the time / temperature couple that makes it possible to obtain a

  sterilization under given conditions.

  However, this means may be insufficient, which is why in most cases there is also an internal device

  allowing the demonstration of the sterilization.

  In its simplest form, it is a device that contains microorganisms in a vegetative or resistance form, especially in a spore form, which will be controlled at the end of the sterilization that actually no longer remains viable microorganisms or spores, or at least the remaining level of viable microorganisms or spores is below a certain threshold. We can also use

  an enzyme to reveal the effectiveness of sterilization.

  According to the type of sterilization applied, microorganisms known for their resistance in spore form will be used, for example for sterilization by moist heat, Bacillus stearothermophilus will be used, for sterilization by dry heat or gases Bacillus subtilis will be used and for sterilization by radiation

  ionizing agents Bacillus pumilus will be used.

  With regard to disinfection processes, only this second method is usable, in fact there are no abacuses allowing

  to determine disinfecting conditions.

  The present invention more particularly relates to indicator devices comprising microorganisms in vegetative or sporulated form or enzymes, intended to test, in a general manner, the action of a fluid on said microorganism or the enzyme, in general so as to

  to test the sterilization or disinfection of an enclosure.

  The sterilization indicator devices generally comprise, on the one hand, a support on which are placed the spores of the resistant strain which may be brought into contact with the sterilizing fluid and, on the other hand, a compartment in which there is a medium allowing the culture of the spore-forming microorganism and a revealing agent which changes color in the presence of the microorganism in question.

culture.

  The control method therefore consists of introducing the device into the sterilization chamber, the spores are then brought into contact with the sterilization fluid, and then, at the end of the sterilization, the culture medium is introduced by breaking an ampoule. and the revealing 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 the patent

French 2,045,855.

  Most devices of the prior art have a number of disadvantages: - first of all the support of spores is mostly paper, gold materials to be sterilized in practice are almost never paper but plastic , metal, rubber, glass, etc .; - In general, the supports have a flat shape which is unrelated to the geometric conformations of most materials to be sterilized which are generally of extremely varied shape; - Finally, the support absorbs, in some cases, easily sterilizing agents, resulting in easier destruction of spores when in fact the microorganisms contaminating the material to

  sterilize are worn on non-absorbent surfaces.

  So, some of the devices depending on how they are put

  may lead to falsely secure results.

  In addition, most biological indicator devices currently proposed include the breaking of a glass bulb, which is not generally desirable, especially because the small pieces of glass can create microcracks of the outer container, so there is

a risk of contamination.

  Finally, in some cases it may be interesting to be able to control the sterilization during operation without having to intervene directly in the sterilization chamber, this is generally not

  possible with currently existing devices.

  This is why the present invention relates to a device for evaluating the action of a fluid on a strain of microorganism or on an enzyme, of the type comprising: a compartment (1) allowing the fluid to come into contact with and said strain or the enzyme and - a compartment (2) allowing the detection of said strain or of the enzyme after contacting the fluid, with the aid of a revealing medium (15), characterized in that it consists of a movable assembly that 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 bringing it into contact with the strain or the enzyme fixed on a support (9) and - in a second configuration, a means allowing the contacting of the revelation medium with the support on which the strain or enzyme is attached and, optionally, means to isolate the revealing medium containing the support of the external atmosphere, and means for observing the modifications

  possible from the medium of revelation (18).

  Preferably, this device is an indicator

  biological 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

of a fluid.

  With regard to the revelation medium, it is usually a medium allowing the culture of the strain from spores or vegetative form comprising a developer

  evidence of viable microorganisms.

  Finally, microorganisms will mostly be under

spore form.

  Preferably, the device according to the present invention will also comprise means making it possible to observe the

  possible modifications of the revealing liquid.

  In general, the device according to the invention will consist of two compartments movable relative to each other, one of the compartments comprising the support (9) and at least one orifice (14) for bringing an external fluid inside into contact with the support, and the other compartment comprising the revealing medium, said compartment being arranged so that the displacement of one with respect to the other brings the support of the strain (9 ) in contact with the revealing medium and possibly in that another displacement leads to

  isolate the revealing medium containing the support from the external medium.

  The displacement of the two mobile compartments can be done

  both by translation and by rotation around an axis.

  In general, the support of the stump is a movable element which can have any configuration intended in particular to make it possible to better represent the surfaces which will have to be sterilized at

decontaminated outside.

  It may be in particular a flat support or a support having recesses or corrugations, but in most cases it will rather be a device of tubular shape, preferably having the form 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

medium of revelation.

  In the preferred embodiment, the device consists of two compartments rotatable relative to each other about an axis and having two surfaces facing perpendicular to said axis, the upper compartment comprises a tubular conduit (6) substantially parallel to the axis of rotation opening out of the compartment by two orifices, a support is placed in said tubular duct and the support rests on the surface located opposite the lower compartment to the surface which opens a tubular conduit vis-à-vis the previous and the other end opens outwardly, thereby creating a channel which passes through the entire device parallel to the axis and a part is constituted by the support of the stump, 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 the upper compartment is brought into coincidence of the orifice and that the support falls through this orifice into the revealing medium in the lower compartment, and by bringing the assembly back to its initial position, the medium containing the support of the external environment, the lower compartment having, in addition, one or two transparent windows or being completely transparent, allowing to see the

color of the revelation medium.

  In addition, the lower compartment may comprise in addition to the culture medium, in the lower part of the fluid conduit, a physicochemical indicator (allowing to see the effect of a sterilization in

  depth or distance on a physicochemical indicator).

  In general, the carrier of the microorganism will be constituted by a surface that may be identical to the surfaces to be sterilized and / or decontaminated, in practice, in particular it may be metal surfaces, paper surfaces if necessary, to plastic surfaces, but preferably the surfaces will be made of glass, as will be seen in the following examples, the use of a

  Glass holder helps to have results that are the most reliable.

  The microorganisms are preferably present in their strongest form, i.e. in the form of spores which are attached to the microorganism support 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 circulated through

this tube.

  The revelation medium is preferably constituted by a culture medium allowing the growth of the spores when they are still viable, said culture medium comprising an indicator, most of the time colored, whose color changes or appears during growth. microorganisms, for example by changing the pH with products of the type of phenol red or products that react to oxidation-reduction such as for example methylene blue, it may also be products that are colored by setting evidence of an enzyme specific to 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 test fluid, will be caused to fall into the revelation medium and thus incubate in its presence, however it is necessary to be able to unhook the spores. fixed on the support, to do this one can proceed 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 an ultrasonic pass but, as will appear in the examples, the most rational solution is to introduce into the revealing medium a surfactant, such as Tween 80 for example, which will make it easy to pick up and completely microorganisms in the form of spores, but has no influence on the

cell viability.

  Preferably the reading of the result can be done through one or two transparent plastic windows or a totally transparent lower compartment, or any other transparent material, in which case it is advantageous to use the glass as a support, which allows have a greater visible thickness, whereas a support

  at least from this point of view is less effective.

  One of the advantages of the device of the invention is that, in view of the fact that the fluid to be tested can circulate in a pipe in which it is brought into contact with the carrier carrying the spores or microorganisms in vegetative form or an enzyme at the ends of this pipe, two nozzles may be provided on which tubes of suitable non-porous material may be fixed in order to be able to test the sterilization in depth or the sterilization at a distance, that is to say that the it is possible to test the sterilization status of a place in which the indicator is not directly by plunging the two tubes to the place to be tested and placing the device outside, it is also possible to test the sterilization of a chamber by keeping the device according to the invention on the outside, provided that two tubings have been fitted in

  which circulates the atmosphere that must be sterilized.

  The following examples are more particularly intended to highlight other features and advantages of the present invention. The examples are implemented using a device of the type shown in the accompanying figures in which: - Figure i shows a section along a plane passing through the axis of rotation of a device according to the invention in a crossing position; fluid; FIG. 2 represents the same device but in another position where the microorganism support is placed in the medium; - Figure 3 shows the device after return to the initial position; - Figure 4 is a side view of the device of Figures 3 there. FIG. 1 represents a device according to the present invention according to a section through a plane passing through the axis of rotation of the

  two compartments relative to each other.

  This device consists of two movable compartments (1 and 2) connected by a connecting piece (5), each of the compartments consists of identical or not (3 and 4) housings in which is placed an insertion element which, for the upper compartment comprises a tubular duct (6) substantially parallel to the axis of rotation, opening outwards through an orifice (7) and an orifice (8) vis-à-vis the lower compartment. The support (9) which is substantially cylindrical and applied in the tubular duct (6) is coated on its inner part with

spores (10).

  The support (9) rests on the surface facing the lower compartment (12) on the surface of which opens a tubular conduit vis-à-vis the previous (11) and the end (13) leads to

  outside. The tubular duct (11) may contain a physical indicator

  In this configuration, a channel has been formed which crosses the entire device parallel to the axis and a part of which is

  constituted by the support of the strain.

  The lower compartment further comprises an orifice (14) whose size is such that after 180 rotation of the two compartments (1 and 2) relative to each other to arrive at the configuration shown in FIG. 4, the tubular duct (6) is brought into coincidence with said orifice and the support (9) falls into the revealing medium (15)

  located in a reservoir (20) of the lower compartment.

  After a second rotation of 180, the two compartments are brought back to the initial position shown in FIG. 3, but the support (9) is then placed in the revealing medium (15) but isolated from

  outside and can then be shaken.

  It should be noted that in order to avoid external contaminations, the opening openings (7 and 13) each comprise a filter

(21 and 22).

  In addition, the tubular channel (11) is narrower at its part

  upper 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 to allow the adaptation of tubings if necessary to test deep sterilizations or to test atmospheres in which we do not want not introduce the device. FIG. 4 represents a cavalier view of the device according to the present invention on which the two compartments 1 and 2 connected by the connecting piece 5 are distinguished, one of the tubular ducts opening out with its nozzle 16 as well as the reading window (18 ) transparent that allows

  to see the color variations of the medium of revelation 15.

EXAMPLE 1

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

water vapour.

  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 10. Trypsic casein peptone 5 g Glycerol 150 g Distilled water QSP 1000 1 - Broth of first culture (2) Extract of yeast 2.5 g 15. Tryptone 5.0 g Glucose 1.0 g Distilled water QSP 1000 1 - Spread of sporulation spread in vials of Roux (3) Extract of meat 10.0 g 20. Yeast extract 2.0 g MnSO4, H2O 0.04 g Agar 23.0 g Distilled water QSP 1 000 ml - 1.2 Preparation of the spore suspension Seeding of the broth (2) with approximately 106 spores from a -20 C (1) freeze broth in which the strain of Bacillus stearothermophilus CIP 5281 - Incubation at 56 C during

24 hours.

  Transfer about 3 ml of this culture into a vial of Roux containing the sporulation agar. Make the vial of Roux undergo various inclinations so that the inoculum comes in contact with

  the entire surface of the agar plate; reject the excess inoculum -

  Incubation of the flask at 56 C for 20 days.

  ii Observation of the state of the culture under the microscope: observe the spores with diluted fuchsin or phase contrast microscope. Vegetative cells should be lysed and spores colored pink

very pale.

  If sporulation has not started, repeat the test. In the case where 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 Spread the recovery liquid (colored in yellow-brown) in centrifuge tubes. Carry out three successive washes of 15 minutes each by centrifugation at 2700 revolutions / minute: with each wash, reject 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 brown-yellow) into a screw-cap Pyrex bottle. Heat

  minutes at 100 C to eliminate any remaining vegetative forms.

Keep at + 4 C.

  Shake the suspension well before each

  spores sediment at the bottom of the bottle.

  1.4 Enumeration of the Spore Suspension Counting the Spores after Proper Dilution in Sterile Distilled Water, Inclusion in Mueller Hinton Agar Agar (Diagnosis

  Pasteur, ref. 64884), 35 g / l, prepared extemporaneously; incubation at 56 C.

  The spore suspension should titrate at least 107 spores / ml.

  Check for absence of spores or organic debris

  bulky by diluted fuchsin staining.

  2) fixation of spores on the support Various supports were tested: - planar configuration, glass, stainless steel, rubber and fabric, - cylindrical configuration, glass, stainless steel and rubber. Deposits were 100 [l of the spore suspension on the surface of flat supports and in the light of cylindrical supports and spores

are dried at 37 C.

  3) Recovery of spores fixed on the supports After the deposition of 100.I1 of a spore suspension calibrated 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 are revealed 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 named DT at a given temperature. This decimal reduction is the time required to inactivate 90% of the microorganisms present before sterilization, that is to say also the time necessary to reduce by 10 times the value of the initial microbial population. In the context of sterilization, Dl2loc is determined from paper, glass, stainless steel, rubber and flat geometry substrates by exposing the supports in the device according to the present invention to

  different sterilization times.

  The following results are found: Dl2loc paper = 1.9 min D12loc glass = 2.3 min D121oc stainless steel = 2.4 min Dl2loc fabric = 2.3 min D121oc rubber = 2.4 min When instead of flat supports takes cylindrical, glass, stainless steel and rubber geometry supports, the following results are obtained: Dl2loc glass = 2.5 min Dl21C stainless steel = 2.4 min Dl2loc rubber = 2.3 min In conclusion, it is possible to to retain in the new biological indicator supports whose nature and configuration are representative of objects actually subjected to sterilization, hollow supports glass, steel or rubber, but it seems appropriate to use preferably cylindrical glass supports that give the decimal reduction time the most important and therefore the greatest security on the

plan of sterilization.

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

the culture is positive.

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

24 hours of incubation.

  The sensitivity threshold is therefore 5 + 1 spores.

  Thus, this technique makes it possible to measure a log 10 change in the number of spores after sterilization, which could be

  verified by real-world tests.

  Cylindrical glass holder carrying 5.106 spores: - exposure time 3 min: 100% positive responses - exposure time 6 min 100% positive responses - exposure time 9 min: 100% positive responses - time of exposure exposure 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 by example in Technique

  Hospitaliere n 493, October 1986, p. 52-56.

Claims (10)

  1) Device for evaluating the action of a fluid on a strain of microorganism or an enzyme, of the type comprising: a compartment (1) allowing the fluid to come into contact with 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 developing medium (15), characterized in that it consists of a set mobile device having at least two configurations (8, 11) having, in a first configuration, a passage allowing the circulation of the fluid inside the assembly and bringing it into contact with the strain or the enzyme fixed on a support (9) and - in a second configuration, means for contacting the revelation medium with the support on which the strain or the enzyme is attached and, optionally, means for isolating the revelation medium containing the support of the outside atmosphere, and means allowing the observation of modifications   possible from the medium of revelation (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). ) for bringing an external fluid inside into contact with the support, and the other compartment comprising the revealing medium, said compartment being arranged so that the displacement of one with respect to the other brings the support of the strain (9) in contact with the revelation medium and possibly in that another displacement leads to   isolate the revealing 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 of revelation.   Device according to one of claims 1 to 4, characterized   the first and second compartments are rotatable or displaceable relative to one another to bring about, after the support has been brought into contact with the external fluid in a first rotational position or translation, said support facing an opening in the second compartment containing the medium of   revelation in order to fall into said revelation 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 inside of the compartment into contact with the outside, in that in the compartment there is the support in such a way as to be in contact with a fluid coming from the outside by 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 revealing medium 15) and falls into said revealing medium, and in that when the set is put back into the first configuration, the   revealing medium containing the support of the external atmosphere.   7) Device according to one of claims 1 to 6, characterized   in that the microorganism support (9) is substantially tubular in shape and that each of the ends of said tube is in contact with the orifices of the first compartment in order to ensure the passage of the fluid   outside 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 surfaces facing perpendicular to said axis, the compartment upper comprises a tubular duct (6) substantially parallel to the axis of rotation opening outwardly of the compartment by two orifices, a support is placed in said tubular duct and the support rests on the surface located vis-à-vis the lower compartment on the surface of which opens a tubular conduit vis-à-vis the previous and the other end opens out, thus creating a channel that passes through the entire device parallel to the axis and a part is constituted 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 compartment of the upper compartment is brought into coincidence of the orifice and the support falls through this orifice in the revealing medium located in the lower compartment, and by bringing the assembly back to its initial position, the medium containing the support of the external environment, the lower compartment further comprising a transparent window for viewing the color of the revealing medium. 9) Apparatus according to claim 8, characterized in that the revealing medium is contained in a tubular element substantially parallel to the axis.   Device according to one of claims 8 and 9, characterized   in that the openings opening outwards comprise a filter (21, 22).   11) Device according to one of claims 8 to 10, characterized   in that the external openings comprise nozzles (16, 17).   12) Device according to one of claims 8 to 11, characterized   in that the two compartments consist of a casing having the same structure.   13) Device according to one of claims 8 to 12, characterized   in that in the lower part of the fluid duct there is physicochemical indicator.