Method of sterilizing an object with atomic nitrogen from a nitrogen plasma

A method of sterilizing an object with atomic nitrogen from a nitrogen plasma comprises the steps of positioning the object in a sterilization chamber, and conditioning the object present in the chamber. The step of conditioning includes a first stage of injecting atomic nitrogen into the chamber, during which a first concentration of atomic nitrogen in the chamber is imposed, a suction stage performed after the first injection stage, during which the chamber is evacuated, and a second stage of injecting atomic nitrogen into the chamber that is performed after the suction stage, during which a second concentration of atomic nitrogen is imposed in the chamber. The method further comprises a sterilization step of sterilizing the object, performed after the conditioning, and includes injecting atomic nitrogen into the chamber, during which step a concentration of atomic nitrogen in the chamber is imposed that is greater than the first and second concentrations.

FIELD OF THE INVENTION

The present invention relates to a method of sterilizing an object by injecting atomic nitrogen from a nitrogen plasma.

BACKGROUND OF THE INVENTION

It is known to sterilize objects by means of an autoclave in which the object that is to be sterilized is raised to a determined high temperature, of about 120° C., with this lasting for determined periods of time and with cycles that are set out by legislation.

Applying a high temperature can raise difficulties and can lead to certain objects being damaged, e.g. when those objects include portions made of polymer material.

Methods that enable sterilization to be performed at lower temperatures have consequently been developed in order to reduce the damage to objects while they are being treated.

In this context, methods of sterilization have been developed by treating the object with a stream of atomic nitrogen from a nitrogen plasma.

Nevertheless, it remains desirable to improve the effectiveness of sterilization by known methods, in particular by reducing the treatment time.

In addition, certain known methods may present a phenomenon of saturation insofar as there may always remain some quantity of microorganisms that are not destroyed by the sterilization treatment, even if the treatment is prolonged. It would be desirable to have a method with improved sterilization effectiveness, without such a saturation phenomenon.

OBJECT AND SUMMARY OF THE INVENTION

In a first aspect, the invention provides a method of sterilizing an object with atomic nitrogen from a nitrogen plasma, the method comprising at least:

positioning the object in a sterilization chamber;

conditioning the object present in the chamber, the conditioning comprising at least:

a first stage of injecting atomic nitrogen into the chamber, during which a first concentration of atomic nitrogen in the chamber is imposed;

a suction stage performed after the first injection stage, during which the chamber is evacuated; and

a second stage of injecting atomic nitrogen into the chamber that is performed after the suction stage, during which a second concentration of atomic nitrogen is imposed in the chamber; and

a sterilization step of sterilizing the object, performed after the conditioning, comprising injecting atomic nitrogen into the chamber, during which step a concentration of atomic nitrogen in the chamber is imposed that is greater than the first and second concentrations.

The term “atomic nitrogen” should be understood as nitrogen obtained after dissociating of dinitrogen N2(i.e. the element N).

The atomic nitrogen concentrations imposed in the chamber during conditioning and during the sterilization step may be measured using a spectrophotometer. By way of example, it is possible to use the method described in the publication by Bockel et al.: “Optical diagnostics of active species in N2microwaves flowing post-discharge” (S. Bockel, A. M. Diamy, and A. Ricard: Surface and coatings technology, 74-75 (1995), 474-478) in order to measure such concentrations of atomic nitrogen. Performing conditioning as described above serves to improve the effectiveness of the sterilization step performed subsequently, while also using a temperature during the method that is limited, lower than 60° C. The inventors consider that the fact of performing the above conditioning in which the sterilizing species (atomic nitrogen) is imposed at a concentration that is lower than the concentration used during the subsequent sterilization step serves to weaken the microorganisms, thereby making the subsequently performed sterilization step more effective.

In an implementation, the conditioning comprises:

a first suction step performed after the first stage of injecting atomic nitrogen, during which the chamber is evacuated;

a stage of injecting molecular nitrogen into the chamber that is performed after the first suction stage; and

a second suction stage performed after the stage of injecting molecular nitrogen, during which the chamber is evacuated, the second stage of injecting atomic nitrogen into the chamber being performed after the second suction stage.

The term “molecular nitrogen” should be understood as nitrogen in the dinitrogen state (i.e. the molecule N2).

Performing such an intermediate stage of injecting molecular nitrogen between the first and second injection stages serves to further improve the effectiveness of the sterilization step.

In particular, the duration of the stage of injecting molecular nitrogen may be shorter than at least one of the durations of the first and second stages of injecting atomic nitrogen. In particular, the duration of the stage of injecting molecular nitrogen may be shorter than each of the durations of the first and second stages of injecting atomic nitrogen.

In an implementation, the conditioning further comprises:

a third suction stage performed after the second stage of injecting atomic nitrogen, during which the chamber is evacuated;

a second stage of injecting molecular nitrogen into the chamber, performed after the third suction stage;

a fourth suction stage performed after the second stage of injecting molecular nitrogen, during which the chamber is evacuated; and

a third stage of injecting atomic nitrogen into the chamber, performed after the fourth suction stage and during which a third atomic nitrogen concentration is imposed in the chamber;

the atomic nitrogen concentration in the chamber that is imposed during the sterilization step being greater than each of the first, second, and third concentrations.

The fact of performing such a third stage of injecting atomic nitrogen serves to still further improve the effectiveness of the sterilization step.

In an implementation, the conditioning further comprises, after its last stage of injecting atomic nitrogen, a transition stage comprising at least one additional suction stage, during which the chamber is evacuated.

The fact of performing such a transition stage serves to still further improve the effectiveness of the sterilization step.

In particular, the transition stage may comprise two additional suction stages separated by a stage of injecting molecular nitrogen into the chamber.

In an implementation, the pressure reached in the chamber during the injection of atomic nitrogen in the sterilization step is higher than the pressure reached in the chamber during each of the first, second, and optional third stages of injecting atomic nitrogen.

The increase in the concentration of atomic nitrogen during the sterilization step may be obtained by increasing the flow rate with which atomic nitrogen is injected into the chamber and/or by reducing the suction of the content from the chamber. This increase in flow rate or reduction in suction leads to an increase in the pressure in the chamber. Nevertheless, as mentioned below, it is also possible to increase the concentration of atomic nitrogen during the method in other ways.

In particular, the pressure reached in the chamber during the injection of atomic nitrogen in the sterilization step may be greater than or equal to 10 millibars (mbar).

In particular, the pressure reached in the chamber during each of the first, second, and third stages of injecting atomic nitrogen may lie in the range 6 mbar to 10 mbar.

In an implementation, the object is a medical instrument.

DETAILED DESCRIPTION OF EMBODIMENTS

FIG. 1is a diagram of a sterilization device1configured to sterilize an object O by treatment with a post-discharge stream of a nitrogen plasma. Such a post-discharge stream comprises a mixture of neutral species, namely atomic nitrogen N and dinitrogen N2.

The device1comprises a duct5having a first segment28putting a compressor3into communication with a plasma generator20. The first segment28is provided with a nitrogen filter element10situated between the compressor3and the plasma generator20.

A stream7of compressed air coming from the compressor3flows through the first segment28to the filter element10. The filter element10is constituted by an element that is itself known and that is configured to separate dinitrogen from oxygen in the stream7of compressed air. After passing through the filter element10, a stream of dinitrogen16flows through the first segment28to the plasma generator20. The oxygen14that has been separated from the nitrogen is discharged via an exhaust duct12.

The first segment28enables the stream16of dinitrogen to be admitted into the plasma generator20. The volume content of dinitrogen in the dinitrogen stream16admitted into the plasma generator20may be greater than or equal to 95%, or indeed greater than or equal to 99%. The dinitrogen stream16admitted into the plasma generator20may include residual oxygen at a volume content that is less than or equal to 1%. In a variant, the dinitrogen stream16admitted into the plasma generator20may have no oxygen. In known manner, the plasma generator20serves to generate a nitrogen plasma from the nitrogen stream16. The plasma generator20comprises an evacuated enclosure24subjected to the action of an electromagnetic field generator that is constituted in this example by a microwave generator22. The electromagnetic field generated in the enclosure24is of sufficiently high intensity to cause the nitrogen to ionize.

The duct has a second segment30that puts the plasma generator20into communication with a sterilization chamber40in which the object O for sterilizing is positioned. The post-discharge stream32from the nitrogen plasma flows to the sterilization chamber40via the second segment30.

The sterilization chamber40defines a treatment zone41including at least one support42on which the object O is positioned during the sterilization treatment. The figure shows a treatment zone41having a single support42and a single object O, however it would naturally not go beyond the ambit of the invention for the treatment zone to have a plurality of supports, each carrying one or more objects. The sterilization chamber40is provided with a door45to enable the object O to be inserted into the treatment zone41, and to enable it to be removed after sterilization.

The object O may be a medical instrument such as an endoscope, a chisel, or a scalpel. The invention is also advantageous for sterilizing objects other than medical instruments, such as electronic cards.

The second segment30presents a proximal end30asituated beside the plasma generator20and in communication therewith. The second segment30also presents a distal end30bdefining an injection orifice34for injecting the post-discharge stream32into the sterilization chamber40. The plasma generated by the plasma generator20penetrates into the second segment30via the proximal end30a.While the plasma that has been formed is flowing through the second segment30, ionic and metastable species are destroyed by colliding with one another or with the walls of the duct5. As a result, an electrically neutral post-discharge stream comprising both atomic nitrogen N and dinitrogen N2is injected into the chamber40via the injection orifice34. The post-discharge stream32flows through the second segment30and is injected into the sterilization chamber40through the injection orifice34. The treatment zone41is in communication with a vacuum pump48. This pump draws the post-discharge stream32into the treatment zone41via a second duct46and discharges the gas to the outside.

The example device1shown inFIG. 1has a single injection orifice34. Naturally, it would not go beyond the ambit of the invention for the post-discharge stream to be injected through a plurality of injection orifices34.

The example device1shown inFIG. 1serves to inject a post-discharge stream from a nitrogen plasma into the sterilization chamber40. Under such circumstances, the atomic nitrogen injected during conditioning and during sterilization forms part of a post-discharge stream from a nitrogen plasma. Nevertheless, it would not go beyond the ambit of the invention for the injected atomic nitrogen to be part of a nitrogen plasma.

Two examples of methods of the invention are described with reference toFIGS. 2 and 3. The two examples shown differ by the nature of the sterilization step that is performed after conditioning. In each of these examples, the inventors have observed that performing the conditioning of the invention makes it possible to obtain improved effectiveness of sterilization (see experimental results ofFIGS. 4 and 5).

Prior to conditioning C, the method may include a preliminary step EP of reducing pressure, during which the pressure inside the chamber40is reduced from atmospheric pressure Pa to a vacuum pressure Pv. This pressure reduction may be performed in non-monotonic manner and may include brief increases of pressure in the chamber40, as shown. The vacuum pressure Pv reached at the end of this preliminary step EP may be less than or equal to 1 mbar.

The conditioning C performed in the examples ofFIGS. 2 and 3comprises initially a first stage IA1of injecting atomic nitrogen into the chamber40, during which a first non-zero concentration of atomic nitrogen is imposed in the chamber40.

In the example of conditioning C that is shown, the pressure in the chamber40increases from the vacuum pressure Pv to a level value P1during the stage IA1. The pressure in the chamber40is then stabilized at this level value P1during the first injection stage IA1. This level value may lie in the range 6 mbar to 10 mbar. By way of example, the duration d11of the first injection stage IA1may be greater than or equal to 5 minutes (min), e.g. it may lie in the range 5 min to 15 min.

Thereafter, the injection of atomic nitrogen into the chamber40is interrupted.

Thereafter, a first suction stage A1is performed during which the chamber40is evacuated. In this example, the pressure in the chamber40decreases progressively from the level value P1that was reached during the stage IA1, down to the vacuum pressure Pv. The vacuum pressure Pv reached at the end of this first suction stage A1may be less than or equal to 1 mbar.

The conditioning C is then continued by injecting dinitrogen into the chamber40(first stage ID1of injecting molecular nitrogen). During the stage ID1, the nitrogen is injected in the form of dinitrogen and no longer in atomic form. During the stages of injecting molecular nitrogen, the dinitrogen stream16is injected directly into the chamber40, with the plasma generator20being switched off during these stages, unlike stages of injecting atomic nitrogen in which the plasma generator20is activated.

In this example, the pressure in the chamber40increases from the vacuum pressure Pv to a level value P1during the stage ID1. The pressure in the chamber40is then stabilized at this level value P1during the first stage ID1of injecting molecular nitrogen. This level value may lie in the range 6 mbar to 10 mbar. In this example, the level value reached during the stages of injecting molecular nitrogen is shown as being identical to the value reached during the stages of injecting atomic nitrogen (value P1), however it would not go beyond the ambit of the invention if that were not so. The duration d21of the first injection stage ID1may for example be less than or equal to 5 min, and for example may lie in the range 1 min to 5 min.

Thereafter, the injection of molecular nitrogen into the chamber40is interrupted.

A second suction stage A2is then performed during which the chamber40is evacuated. In this example, the pressure in the chamber40decreases progressively from the level value P1reached during the stage ID1to the vacuum pressure Pv. The vacuum pressure Pv reached at the end of this second suction stage A2may be less than or equal to 1 mbar.

The conditioning C is then continued by performing a second stage IA2of injecting atomic nitrogen into the chamber40during which a second non-zero concentration of atomic nitrogen is imposed in the chamber40. The second concentration may be identical to or different from the first concentration.

In the conditioning example C shown, the pressure in the chamber40initially increases from the pressure Pv to a level value P1during the stage IA2. Thereafter, the pressure in the chamber40is stabilized at this level value P1, which by way of example may lie in the range 6 mbar to 10 mbar, during the second injection stage IA2. In this example, the level value reached for the pressure are shown to be identical for each of the stages of injecting atomic nitrogen, however it would not go beyond the ambit of the invention if that were not so. The duration d12of the second injection stage IA2may for example be greater than or equal to 5 min, and for example may lie in the range 5 min to 15 min.

Thereafter, the injection of atomic nitrogen into the chamber40is interrupted.

A third suction stage A3is then performed during which the chamber40is evacuated. In this example, the pressure in the chamber40decreases progressively from the level value P1reached during the stage IA2down to the vacuum pressure Pv. The vacuum pressure Pv reached at the end of this third suction stage A3may be less than or equal to 1 mbar.

The conditioning C is then continued by performing a second stage ID2of injecting molecular nitrogen into the chamber40.

In this example, the pressure in the chamber40increases from the vacuum pressure Pv to a level value P1during the stage ID2. The pressure in the chamber40is then stabilized at this level value P1, which for example lies in the range 6 mbar to 10 mbar, during the second stage ID2of injecting molecular nitrogen. The figure shows that the level value for pressure that is reached during each of the molecular nitrogen injection stages is identical. However, it would not go beyond the ambit of the invention if that were not so. By way of example, the duration d22of the second injection stage ID2may be less than or equal to 5 min, and for example it may lie in the range 1 min to 5 min.

Thereafter, the injection of molecular nitrogen into the chamber40is interrupted.

A fourth suction stage A4is then performed during which the chamber40is evacuated. In this example, the pressure in the chamber40decreases progressively from the level value P1reached during the stage ID2down to the vacuum pressure Pv. The vacuum pressure Pv reached at the end of this fourth suction stage A4may for example be less than or equal to 1 mbar.

The conditioning C is then continued by performing a third stage IA3of injecting atomic nitrogen into the chamber40, during which a third non-zero concentration of atomic nitrogen is imposed in the chamber40. The third concentration may be identical to or different from the first concentration. The third concentration may be identical to or different from the second concentration.

In the example of conditioning C shown, the pressure in the chamber40begins by increasing from the vacuum pressure Pv to a level value P1during the stage IA3. The pressure in the chamber40is then stabilized at this level value P1during the stage IA3. This level value may lie in the range 6 mbar to 10 mbar. By way of example, the duration d13of the third injection stage IA3may be greater than or equal to 5 min, and for example it may lie in the range 5 min to 15 min.

Thereafter, the injection of atomic nitrogen into the chamber40is interrupted.

In the example shown, the third injection stage IA3constitutes the last stage of injecting atomic nitrogen in the conditioning C.

In this example, the conditioning C includes a transition stage PT that is performed after the last stage IA3of injecting atomic nitrogen and before the beginning of the sterilization step S1. The presence of this transition stage PT is optional.

The transition stage PT comprises a first additional suction stage A5during which the chamber40is evacuated. In this example, the pressure in the chamber40decreases progressively from the level value P1reached during the last injection stage IA3down to the vacuum pressure Pv. The vacuum pressure Pv reached at the end of this first additional suction stage A5may be less than or equal to 1 mbar.

Thereafter, during the transition stage PT, an additional stage ID3of injecting molecular nitrogen into the chamber40is performed. In this example, the pressure in the chamber40increases from the vacuum pressure Pv to a level value P1during the stage ID3. Thereafter, the pressure in the chamber40is stabilized at this level value P1during the additional stage ID3of injecting molecular nitrogen. This level value may lie in the range 6 mbar to 10 mbar. By way of example, the duration d23of the additional injection stage ID3may be less than or equal to 5 min, and for example may lie in the range 1 min to 5 min.

Thereafter, the injection of molecular nitrogen into the chamber40is interrupted.

A second additional suction stage A6is then performed during which the chamber40is evacuated. In this example, the pressure in the chamber40decreases progressively from the level value P1reached during the additional injection stage ID3down to the vacuum pressure Pv. The vacuum pressure Pv reached at the end of this second additional suction stage A6may be less than or equal to 1 mbar.

The example of conditioning C shown has three stages IA1-IA3of injecting atomic nitrogen. Nevertheless, it would not go beyond the ambit of the invention when the conditioning comprises only two stages of injecting atomic nitrogen, or indeed when it comprises more than three stages of injecting atomic nitrogen.

The example of conditioning C shown includes a stage ID1-ID3of injecting molecular nitrogen that is performed between two suction stages. In the example shown, the durations of these stages ID1-ID3are less than the durations of the stages IA1-IA3. Between the stages of injecting atomic nitrogen, it is possible to perform a stage of injecting molecular nitrogen and at least one suction stage that may be performed before or after the stage of injecting molecular nitrogen. Nevertheless, it would not go beyond the ambit of the invention if the conditioning did not have such stages of injecting molecular nitrogen.

The method continues by performing a sterilization step S1using atomic nitrogen. It should be observed that it is possible to obtain partial destruction of the microorganisms present at the end of the conditioning C. In a variant, the conditioning C need not have any sterilizing effect.

The sterilization step S1performed after the conditioning C serves to obtain a sterile state. The duration of sterilization step S1may suffice to obtain an at least 6 log reduction in the number of microorganisms present relative to the start of the stage of conditioning C. In other words, under such circumstances, there remain fewer than one microorganism per million at the end of the sterilization step S1, compared with the number of microorganisms that were present at the beginning of the conditioning C. This reduction may be of at least 12 log.

In this example, the sterilization step S1comprises injecting IA10atomic nitrogen into the chamber40. During this injection IA10, a concentration of atomic nitrogen in the chamber40is imposed that is greater than any of the atomic nitrogen concentrations that were imposed during the injection stages IA1-IA3.

During the sterilization injection IA10, the pressure in the chamber40increases initially from the vacuum pressure Pv to a level value P2. The pressure in the chamber40is then stabilized at this level value P2during the sterilization injection IA10. As shown, the level value P2is higher than the level value P1reached during each of the injection stages IA1-IA3of the conditioning C. The level value P2may be greater than or equal to 10 mbar. The duration d10of the sterilization injection IA10may be greater than or equal to 75 min. In the example ofFIG. 2, atomic nitrogen is injected IA10continuously (without interruption) during the sterilization step S1. It would not go beyond the ambit of the invention if the procedure was otherwise, as is described below.

In the example ofFIG. 2, the pressure P2reached during the sterilization injection IA10is higher than the pressure P1reached during each of the stages IA1-IA3of injecting atomic nitrogen. The atomic nitrogen concentration imposed during the sterilization step S1is thus higher than the concentration imposed during the injection stages IA1-IA3. The pressure may be increased during the sterilization injection IA10by imposing a flow rate for injecting atomic nitrogen during this injection IA10that is higher than the atomic nitrogen injection flow rate imposed during each of the injection stages IA1-IA3. This increase in the injection flow rate may be obtained by increasing the flow rate of the air stream7, and thus of the dinitrogen stream16. In a variant, or in combination with this increase in flow rate, it is possible to increase the pressure during the injection IA10by reducing the intensity of suction by the vacuum pump48.

Nevertheless, it would not go beyond the ambit of the invention if the pressure reached during the sterilization step were not greater than the pressure reached during the stages of injecting atomic nitrogen during the conditioning. Specifically, it is possible to modulate the imposed concentration of atomic nitrogen by modifying the power of the microwave generator22used to form the plasma upstream from the chamber40. It is thus possible to increase the concentration of atomic nitrogen during the sterilization step by increasing that power but without increasing the pressure in the chamber.

By way of illustration, and regardless of the implementation under consideration, at least one of the following conditions may be satisfied:

the first, second, and optional third concentrations may each be less than or equal to 1013atoms per cubic centimeter (atom/cm3), e.g. lying in the range 1010atom/cm3to 1013atom/cm3; and/or

the atomic nitrogen concentration imposed in the chamber during the sterilization step may be greater than or equal to 1013atom/cm3, e.g. lying in the range 1013atom/cm3to 1016atom/cm3.

FIG. 4shows an experimental result revealing the improved effectiveness of sterilization associated with performing a method as shown inFIG. 2.

In this graph, the ordinate axis represents colony forming units (cfu), and the abscissa axis represents treatment time. The strain used was ageobacillus stearothermophillusstrain. Curve A1relates to performing a sterilization step S1without conditioning C, while curve A2relates to performing a sterilization step S1with preliminary conditioning C.

In the test performed:

the pressure Pv was 0.3 mbar;

the durations of each of the suction stages A1-A6were 30 seconds (s); and

the temperature imposed during sterilization was less than 60° C.

It can be seen that there exists a saturation phenomenon from about 50 min when the conditioning C is not performed. This saturation means that there continues to remain some quantity of microorganisms that are not destroyed by the sterilization treatment, even if it is prolonged. When conditioning C is performed, curve A2shows that saturation is no longer encountered and that a sterile state (6 log reduction) can be obtained.

FIG. 3shows a variant sterilization step S2performed after the conditioning C. In this variant, the sterilization step S2comprises a plurality of successive injections IA20and IA21of atomic nitrogen. Two consecutive injections IA20and IA21of atomic nitrogen are separated by a sequence comprising:

a first suction A20or A21;

an injection ID20or ID21of molecular nitrogen that is performed after the first suction A20or A21; and

In this example, the pressure reached in the chamber during the injections IA20and IA21of concentration is higher than the pressure reached during the stages IA1-IA3of injecting atomic nitrogen. This thus imposes an atomic nitrogen concentration during the sterilization step S2that is greater than that imposed during the conditioning C. Each injection IA20or IA21of atomic nitrogen in this example includes a period of pressure stabilization to a level value P20or P21. In this example, each of the level values P20and P21is higher than the pressure P1reached during the stages IA1-IA3of injecting atomic nitrogen. In the example shown, the level value P21is also higher than the level value P20.

FIG. 5shows an experimental result revealing the improvement in sterilization effectiveness associated with performing a method as shown inFIG. 3.

In this graph, the ordinate axis represents colony forming units (cfu), and the abscissa axis represents treatment time. The strain used was ageobacillus stearothermophillusstrain. The curve B1relates to performing a sterilization step S2without conditioning C, and the curve B2to performing a sterilization step S2with preliminary conditioning C.

In the test performed:

the conditioning C was identical to that performed in the context of the test ofFIG. 4;

the duration of each of the steps IA20and IA21was 10 min;

the duration of each of the steps ID20and ID21was 2 min;

the durations of each of the suction stages A20and A21was 30 s;

the sterilization step S2was repeated a second time after raising the pressure to atmospheric pressure; and

the temperature imposed during sterilization was less than 60° C.

Curve B1shows that performing the sterilization step S2, even without conditioning C, already serves to obtain good results.

In addition, for the curve B2, the conditioning C was initially performed for a duration of 39 min followed by the sterilization step S2. Under such conditions, a 6 log reduction in the number of microorganisms was obtained in 130 min. Thus, the duration that serves to obtain a 1 log reduction in the number of microorganisms for the sterilization step S2in this context is evaluated as (130 min−39 min)/6, i.e. about 15.17 min. The duration of a complete sterilization cycle enabling a 12 log reduction to be obtained in the number of microorganisms under such conditions can then be evaluated as (15.17 min*12)+39 min, i.e. 221 min.

For curve B1, where conditioning was not performed, a 6 log reduction in the number of microorganisms was obtained in 140 min. The duration of a complete sterilization cycle making it possible to obtain a 12 log reduction can then be evaluated as (140 min*2), i.e. 280 min. Performing the conditioning C thus makes it possible to improve significantly the effectiveness of the sterilization step S2that is performed subsequently.

The term “lying in the range . . . to . . . ” should be understood as including the bounds.