CN113398306A

CN113398306A - Rapid biological indicator for sterilization effect monitoring and use method

Info

Publication number: CN113398306A
Application number: CN202110745210.3A
Authority: CN
Inventors: 田友昊; 韩宝福; 王文波; 朱本仁; 王衍龙
Original assignee: Shinva Medical Instrument Co Ltd
Current assignee: Shinva Medical Instrument Co Ltd
Priority date: 2021-06-30
Filing date: 2021-06-30
Publication date: 2021-09-17
Anticipated expiration: 2041-06-30
Also published as: CN113398306B

Abstract

The invention belongs to the technical field of medical equipment, and particularly relates to a rapid biological indicator for monitoring a sterilization effect and a using method thereof. The biological indicator is a self-contained structure and comprises a bottle body, a bacterial sheet dyed with test microorganisms, a nylon membrane, a bearing piece and a glass tube sealed with recovery culture solution, wherein the upper end of the bottle body is provided with a bottle cap. When the device is used, after the glass tube sealed with the recovery culture solution is damaged, the recovery culture solution is contacted with the test microorganism on the bacterial sheet dyed with the test microorganism, the fluorescence reaction is rapidly excited, the generated fluorescence product is captured, and whether the microorganism survives or not is judged according to the strength of the fluorescence intensity. The invention can realize that the biological monitoring result is judged within 30min after the recovery culture solution is fully contacted with the test microorganism, can solve the problems of false positive and false negative in the actual use process of medical institutions, and can be used for monitoring the pressure steam sterilization and hydrogen peroxide low-temperature plasma sterilization effects of the medical institutions.

Description

Rapid biological indicator for sterilization effect monitoring and use method

Technical Field

The invention belongs to the technical field of medical equipment, and particularly relates to a rapid biological indicator for monitoring a sterilization effect and a using method thereof.

Background

The high health requirements of people promote the great progress of medical health and the rapid development of various works in hospitals. It is therefore becoming more and more noticed by medical institutions that in order to improve medical quality and guarantee service quality, the management of supply rooms must be strengthened. The main services whose importance is reflected in the disinfection supply center (room) include: the recycling, decontamination, sterilization, article supply, research, development, improvement and maintenance of medical instruments and the like are not related to medical treatment, nursing, inspection, infection monitoring and other work. The guarantee of the quality of the supply of the sterile goods is the core of the work of a disinfection supply center (room), and is an important link for reducing the occurrence of nosocomial infection and guaranteeing the medical quality of hospitals. The introduction of third party disinfection supply centers is a future trend. According to the national standard requirements for sterilization of medical instruments, standard operations must include three modes of physical monitoring, chemical monitoring and biological monitoring. The three kinds of monitoring all meet the standard requirements, and the sterilization effect is qualified.

Hospital infection control traditionally uses a method of culturing biological indicators and detecting whether the pH value changes, and the principle is as follows: bacteria grow after the spores are contacted with the culture solution, acidic or alkaline substances are discharged during the growth of the bacteria, and the substances change the pH value of the culture solution to change the color of an acid-base indicator contained in the culture solution. If the sterilization is successful and all the bacterial spores die, no bacteria grow after the bacterial spores are contacted with the culture solution, and the pH value of the culture solution is not changed, so that the color of the culture solution is not changed, and the original color is still kept, thereby judging the success of the sterilization. If sterilization fails, bacterial spores survive, the pH value of the culture solution changes, and the color of the culture solution changes, so that the sterilization failure is judged.

The above conventional biological indicators have a problem that the sterilization monitoring time is too long, and it often takes 48 hours to obtain the biological monitoring result. Is not beneficial to the turnover of the apparatus, greatly reduces the efficiency of the hospital hand, and additionally increases the cost. Sometimes, biological monitoring results are not obtained, and sterilized instruments are used in clinic, so that a great risk is brought to infection control in hospitals.

CN201180052944.8 biological sterilization indicator and methods of using the same, the read time of the biological sterilization indicator 100 (i.e., the time used to determine the effectiveness of a sterilization process) can be, in some embodiments, less than 8 hours, in some embodiments, less than 1 hour, in some embodiments, less than 30 minutes, in some embodiments, less than 15 minutes, in some embodiments, less than 5 minutes, and in some embodiments, less than 1 minute.

In some embodiments, the active enzyme source may be (1) a purified isolated enzyme derived from a suitable microorganism; (2) a microorganism in which the enzyme is native or added by genetic engineering; and/or (3) a microorganism, wherein the enzyme has been added during sporulation or growth so that the enzyme binds or associates with the microorganism, e.g., the enzyme is added to the spore during sporulation so as to bind into the spore. In some embodiments, microorganisms useful as a source of enzymes include bacteria or fungi in a spore or vegetative state. In some embodiments, the enzyme source comprises bacillus, clostridium, neurospora, candida, or a combination of such microorganisms.

alpha-D-glucosidase has been identified in spores of Bacillus stearothermophilus such as "ATCC 8005" and "ATCC 7953" commercially available from the American Type Culture Collection, Rockville, Md. beta-D-glucosidase has been found in Bacillus subtilis (commercially available, for example, as ATCC9372 from the American type culture Collection).

In some embodiments, spore viability can be determined by using enzyme activity. For example, in Material et al, entitled "Rapid Method for Determining effectiveness of a differentiation Cycle and Rapid Read-out biological indicationor "(rapid method of determining the effectiveness of a sterilization cycle and rapid reading of biological indicators) U.S. patent No.5,073,488, which is incorporated herein by reference, identifies enzymes for specific types of spores in which the enzymes have particularly useful properties that can be used to determine the effectiveness of a sterilization process. Such characteristics may include the following: (1) when subjected to a temperature sufficient to convert 1 × 10⁶Reduced by about 6 log units (i.e., to about zero, as measured in the absence of test microorganism outgrowth) of the total number of test microorganisms, the enzyme has a residual activity equal to the "background" as measured by reaction with the enzyme substrate system; and (2) when subjected to conditions sufficient only to convert 1 × 10⁶The enzyme has an enzymatic activity greater than the "background" as measured by reaction with an enzyme substrate system under sterilization conditions in which the total number of test microorganisms is reduced by at least 1 log unit but less than 6 log units. An enzyme substrate system may include a substrate or mixture of substrates that are acted upon by an enzyme to produce a detectable enzyme-modified product, as evidenced by a detectable change.

Disclosure of Invention

The technical problem to be solved by the invention is as follows: the defects of the prior art are overcome, the rapid biological indicator for monitoring the sterilization effect and the using method thereof are provided, the biological monitoring result can be judged within 30min after the recovery culture solution is fully contacted with the test microorganism, the problems of false positive and false negative in the actual use process of a medical institution can be solved, and the rapid biological indicator can be used for monitoring the sterilization effect of pressure steam sterilization and hydrogen peroxide low-temperature plasma in the medical institution.

The invention relates to a rapid biological indicator for monitoring sterilization effect, which is a self-contained biological indicator and comprises a bottle body, wherein a bacterial sheet dyed with test microorganisms is vertically placed in a lower clamping groove at the bottom of the bottle body, a nylon membrane, a bearing piece and a glass tube sealed with recovery culture solution are sequentially arranged on the upper part of the bacterial sheet dyed with the test microorganisms, and a bottle cap is arranged at the upper end of the bottle body.

The lower part of the bearing piece is matched with a bacterium sheet dyed with test microorganisms and is provided with an upper clamping groove, and the upper part of the bearing piece is matched with a glass tube sealed with recovery culture solution and is provided with a lower placing groove.

The bottle cap is provided with a plurality of through holes, and the bottle cap is internally provided with an upper placing groove matched with the glass tube sealed with the recovery culture solution.

The outer wall of the bottle body or the bottle cap is pasted with a label with a special color-changing medicine strip for a corresponding sterilization mode, and whether the medicine strip is sterilized or not can be judged according to the color change of the medicine strip.

The nutrient components of the recovery culture solution in the glass tube sealed with the recovery culture solution comprise: 4-methylumbelliferone-alpha-D-glucopyranoside, tryptone, IPTG, soybean peptone, L-alanine, L-glycine and L-aspartic acid, can rapidly recover the bacillus stearothermophilus spores, enable the bacillus stearothermophilus spores to grow and reproduce to generate sufficient alpha-glucosidase, and have the color indication effect of pH change caused by microbial reproduction and metabolism.

The recovery culture solution in the glass tube sealed with the recovery culture solution also comprises a pH color indicator, wherein the pH color indicator is preferably bromopotassium phenol purple, and the color reaction from yellow to purple occurs along with the change of the pH value, so that the effect of observing the survival of microorganisms is considered.

The recovery culture solution is sealed in the glass tube in a high-temperature melt-sealing mode, and a sterile treatment means is adopted, so that the nutrient components in the recovery culture solution cannot be consumed by conventional microorganisms and fluorescent products cannot be generated in advance in the preparation process and the long-time melt-sealing process of the culture solution. If the fluorescent product is generated in advance without the sterilization treatment, a false positive condition can occur in the actual use process.

The test microorganism is used for pressure steam sterilization and hydrogen peroxide low-temperature plasma sterilization to form bacillus stearothermophilus spores, and timed manganese ion induction is added in a culture process to generate a strain with high spore rate and high enzyme activity.

When the top-speed biological indicator for monitoring the sterilization effect is used, a glass tube sealed with a recovery culture solution is damaged, the recovery culture solution is contacted with a test microorganism on a bacterial sheet dyed with the test microorganism, a fluorescence reaction is rapidly excited to occur, a generated fluorescence product is captured, and whether the microorganism survives or not is judged according to the strength of fluorescence intensity.

Adopt automatic cracker to have the glass pipe damage of resumeing the culture solution with sealing in the biological indicator of extremely fast type, automatic cracker includes gear motor, the guide bar, the crushing pole, the backup pad, place the broken hole of the biological indicator of extremely fast type and drive the vibrating motor of broken hole vibration, gear motor is connected with the guide bar transmission, the articulated backup pad that is fixed in of crushing pole, the one end of crushing pole is passed through the crushing pole connecting piece and is connected with the guide bar, the upper end in broken hole is located to the other end cooperation extremely fast type biological indicator of crushing pole.

The top-speed biological indicator is placed in the crushing hole, after the switch is started, the speed reduction motor drives the guide rod to lift up and press down the crushing rod, the glass tube sealed with the recovery culture solution is automatically crushed, and the recovery culture solution is fully contacted with the test microorganisms through oscillation of the vibrating motor.

The fluorescence reaction is to recover 4-methylumbelliferone-alpha-D-glucopyranoside in the culture solution and alpha-glucosidase generated by bacillus stearothermophilus spores in the recovery process, and the fluorescence reaction is rapidly carried out under the condition of 55-65 ℃ (preferably 58 ℃). The fluorescent product 4-methylumbelliferone is produced, and the survival condition of the microorganism is judged according to the fluorescence intensity which is achieved within 30 min. Survival is indicated if the fluorescence intensity exceeds the threshold, and death is indicated if the fluorescence intensity is below the threshold. While the biological indicators of different fluorescence intensity continue to be incubated at 55-65 deg.C (preferably 58 deg.C) to observe the color change caused by the change in pH; the fluorescence measurement result achieved within 30min can be 100% consistent with the actual survival condition of the microorganism, and the aim of shortening the culture time is achieved.

Compared with the prior art, the invention has the following beneficial effects:

1. the biological monitoring time can be shortened to be within 30 min.

2. The invention solves the problems of false negative and false positive in the actual use process.

3. The monitoring results of the invention clearly shorten the time and are unified in terms of consistency with the results of actual microorganism survival.

Drawings

FIG. 1 is a schematic view of an exploded structure of a biological indicator according to the present invention;

FIG. 2 is a perspective view of a biological indicator of the invention;

FIG. 3 is a cross-sectional view of the biological indicator of the invention in the A direction;

FIG. 4 is a cross-sectional view of the biological indicator of the invention in the orientation B;

FIG. 5 is a schematic view of an automatic crusher according to the present invention;

in the figure: 1. a bottle cap; 101. a through hole; 102. an upper placing groove; 2. a glass tube sealed with a recovery culture solution; 3. a carrier; 301. a lower placing groove; 302. an upper clamping groove; 4. a nylon membrane; 5. a bacterial pellet contaminated with a test microorganism; 6. a bottle body; 601. a lower clamping groove; 7. a label; 8. a reduction motor; 9. a guide bar; 10. a breaking bar; 11. a breaker bar attachment; 12. a support plate; 13. breaking the holes; 14. a vibration motor.

Detailed Description

The invention is further described below with reference to the accompanying drawings and examples.

As shown in FIGS. 1-4, the rapid biological indicator for monitoring sterilization effect is a self-contained biological indicator, and the whole assembly process needs to be completed in a 10 ten thousand grade purification plant. Including body 6, the lower draw-in groove 601 of body 6 bottom is vertically placed in to the fungus piece 5 that has dyed the test microorganism, and the upper portion that has dyed the fungus piece 5 that has the test microorganism is equipped with nylon membrane 4 in proper order, holds carrier 3 and seals the glass pipe 2 that has the recovery culture solution, and the upper end of body 6 is equipped with bottle lid 1. The lower part of the bearing part 3 is matched with the bacterial sheet 5 dyed with the test microorganism to be provided with an upper clamping groove 302, and the upper part is matched with the glass tube 2 sealed with the recovery culture solution to be provided with a lower placing groove 301. The bottle cap 1 is provided with a plurality of through holes 101, and the bottle cap 1 is internally provided with an upper placing groove 102 matched with the glass tube 2 sealed with the recovery culture solution. The outer wall of the bottle body 6 is adhered with a label 7 with a special color-changing medicine strip for corresponding sterilization.

The bacterial sheet 5 dyed with the test microorganism is formed by dripping a certain amount of test microorganism on a carrier in a micro-titration mode, wherein the carrier is in an ultrasonic welding shapeAnd (4) obtaining. The drop dyeing amount is required to be 5 multiplied by 10⁵cfu/tablet-5X 10⁶cfu/tablet.

The glass tube 2 sealed with the recovery culture solution is sealed with the recovery culture solution prepared in a high-temperature sealing manner. Sterile distilled water is adopted when preparing a recovery culture solution, and the following components are added: 0.2 g/L4-methylumbelliferone-alpha-D-glucopyranoside, 3g/L tryptone, 1g/L IPTG, 7g/L soybean peptone, 1g/L L-alanine, 1.2g/L L-glycine, 1.5g/L L-aspartic acid, 6g/L sucrose and 1ml/L potassium bromide phenol purple, and immediately placing the mixture in an aseptic filling line for melt sealing after the preparation is finished. And (3) melting and sealing the glass tube, and performing sterilization treatment in a water bath sterilization mode under the sterilization condition of 121 ℃ for 30 min.

In the using process, an automatic crusher shown in figure 5 is adopted to damage a glass tube 2 sealed with recovery culture solution in the high-speed biological indicator, the automatic crusher comprises a speed reduction motor 8, a guide rod 9, a crushing rod 10, a supporting plate 12, a crushing hole 13 for placing the high-speed biological indicator and a vibration motor 14 for driving the crushing hole 13 to vibrate, the speed reduction motor 8 is in transmission connection with the guide rod 9, the crushing rod 10 is hinged and fixed on the supporting plate 12, one end of the crushing rod 10 is connected with the guide rod 9 through a crushing rod connecting piece 11, and the other end of the crushing rod 10 is matched with the high-speed biological indicator and arranged at the upper end of the crushing hole 13.

The top-speed biological indicator is placed in the crushing hole 13, after the switch is started, the speed reduction motor 8 drives the guide rod 9 to lift and press the crushing rod 10, the glass tube 2 sealed with the recovery culture solution is automatically crushed, and the recovery culture solution is fully contacted with the test microorganism through oscillation of the vibration motor 14.

The rapid biological indicator is placed in a special fluorescent capturing instrument for culturing after the recovery culture solution is fully contacted with the test microorganism. The instrument can provide a condition of 58 ℃, so that the bacillus stearothermophilus spores are revived under the action of nutrient substances of the recovery culture solution, and the alpha-glucosidase generated in the reviving process and the 4-methylumbelliferone-alpha-D-glucopyranoside in the recovery culture solution perform a fluorescence reaction. And (4) judging whether the microorganisms survive or not according to an analysis curve and a threshold value set by the fluorescence capture instrument for 30 min. Survival is indicated if the fluorescence intensity exceeds the threshold, and death is indicated if the fluorescence intensity is below the threshold.

The rapid biological indicator can be continuously cultured at 58 ℃ in a fluorescent capture instrument, and the color change caused by the change of the pH value is observed. The final color change of the biological indicator reaching the fluorescence threshold value is yellow, which represents that the fluorescence monitoring result is positive for 30min, and the final microbial culture is also a survival result; the final color change of the biological indicator which does not reach the fluorescence threshold value is purple, which represents that the fluorescence monitoring result is negative for 30min, and the final microbial culture is also a death result.

Of course, the foregoing is only a preferred embodiment of the invention and should not be taken as limiting the scope of the embodiments of the invention. The present invention is not limited to the above examples, and equivalent changes and modifications made by those skilled in the art within the spirit and scope of the present invention should be construed as being included in the scope of the present invention.

Claims

1. An extremely rapid biological indicator for monitoring sterilization effect, characterized in that: the biological indicator is a self-contained biological indicator and comprises a bottle body (6), a bacterial sheet (5) dyed with test microorganisms is vertically placed in a lower clamping groove (601) at the bottom of the bottle body (6), a nylon membrane (4), a bearing piece (3) and a glass tube (2) sealed with recovery culture solution are sequentially arranged on the upper portion of the bacterial sheet (5) dyed with the test microorganisms, and a bottle cap (1) is arranged at the upper end of the bottle body (6).

2. The very rapid biological indicator for sterilization effect monitoring of claim 1, wherein: the lower part of the bearing piece (3) is matched with the bacterial sheet (5) dyed with the test microorganism to be provided with an upper clamping groove (302), and the upper part is matched with the glass tube (2) sealed with the recovery culture solution to be provided with a lower placing groove (301).

3. The very rapid biological indicator for sterilization effect monitoring of claim 1, wherein: a plurality of through holes (101) are arranged on the bottle cap (1), and an upper placing groove (102) is arranged in the bottle cap (1) and is matched with the glass tube (2) sealed with the recovery culture solution.

4. The very rapid biological indicator for sterilization effect monitoring of claim 1, wherein: the outer wall of the bottle body (6) or the bottle cap (1) is adhered with a label (7) with a special color-changing medicine strip for a corresponding sterilization mode.

5. The very rapid biological indicator for sterilization effect monitoring of claim 1, wherein: the nutrient components of the recovery culture solution in the glass tube (2) sealed with the recovery culture solution comprise: 4-methylumbelliferone-alpha-D-glucopyranoside, tryptone, IPTG, soy peptone, L-alanine, L-glycine and L-aspartic acid.

6. The very rapid biological indicator for sterilization effect monitoring according to claim 1 or 5, wherein: the recovery medium in the glass tube (2) sealed with the recovery medium also comprises a pH color indicator.

7. Use of the very rapid biological indicator for monitoring the effectiveness of sterilization according to any one of claims 1 to 6, wherein: when the rapid biological indicator for monitoring the sterilization effect is used, after a glass tube (2) sealed with recovery culture solution is damaged, the recovery culture solution is contacted with test microorganisms on a bacterial sheet (5) stained with the test microorganisms, the fluorescence reaction is rapidly excited, a generated fluorescence product is captured, and whether the microorganisms survive or not is judged according to the strength of fluorescence intensity.

8. Use of the very rapid biological indicator for monitoring the effectiveness of sterilization according to claim 7, characterized in that: adopt automatic cracker to have glass pipe (2) the damage of recovering the culture solution with sealing in the biological indicator of the utmost point speed type, automatic cracker includes gear motor (8), guide bar (9), broken pole (10), backup pad (12), place broken hole (13) of the biological indicator of the utmost point speed type and drive vibrating motor (14) of broken hole (13) vibration, gear motor (8) are connected with guide bar (9) transmission, broken pole (10) are articulated to be fixed in on backup pad (12), the one end of broken pole (10) is passed through broken pole connecting piece (11) and is connected with guide bar (9), the upper end in broken hole (13) is located to the biological indicator of the other end cooperation utmost point speed type of broken pole (10).

9. The use of the rapid biological indicator for sterilization effect monitoring according to claim 8, wherein: the top-speed biological indicator is placed in the crushing hole (13), after the switch is started, the speed reduction motor (8) drives the guide rod (9) to lift up and press down the crushing rod (10), the glass tube (2) sealed with the recovery culture solution is automatically squeezed and broken, and the recovery culture solution is fully contacted with the test microorganism through oscillation of the vibrating motor (14).

10. Use of the very rapid biological indicator for monitoring the effectiveness of sterilization according to claim 7, characterized in that: the temperature of the fluorescence reaction is 55-65 ℃.