Method for verifying dry recovery rate of smearing rod for environmental monitoring

Abstract

In order to solve the problem that a method for verifying the recovery rate of a smearing rod for environmental monitoring in the prior art cannot accurately reflect the capacity of the smearing rod for capturing microorganisms in real environmental monitoring, and the like, the invention provides a method for verifying the dry recovery rate of the smearing rod for environmental monitoring, which comprises the following steps: adjusting the concentration of a bacterial suspension by adopting a fatality rate; drying the surface of the stainless steel flat plate for detection coated with the bacterial suspension under set conditions; wiping and sampling the bacterial suspension in the grids of the dried stainless steel flat plate for detection by using a to-be-detected smearing rod, and sampling in the grids which are not wiped on the same stainless steel flat plate for detection wiped by the to-be-detected smearing rod by using a contact flat plate; the higher the recovery rate is, the higher the capture capability of the to-be-detected smearing rod on the microorganisms in the environment is, and the better the quality is. The device and the method have the beneficial technical effects that the capture capability of the smearing rod on microorganisms is reflected more truly, and the verification result is more real and reliable.

Description

Technical Field

[0001] The present invention relates to a dry recovery rate verification technology for a smear stick for environmental monitoring, and in particular to a dry recovery rate verification method for a smear stick for environmental monitoring. Background Art

[0002] In order to ensure the safety and reliability of the sterile drug production environment, according to the requirements of relevant regulations, it is necessary to monitor the microorganisms in the clean area of ​​the sterile drug production environment to ensure that the production process is in a sterile environment. Usually, sedimentation bacteria, airborne bacteria, contact plates, gloves, clean clothes and surface sampling are used to monitor the microorganisms in the environment, that is, microbial sampling is carried out in the clean area of ​​the production environment, and the sampled microorganisms are transferred to the plate for cultivation, and then the number of colonies grown on the plate is compared with the standards specified in the relevant regulations to determine whether the clean area of ​​the production environment meets the requirements of the relevant regulations. Among them, the plate refers to a plate containing nutrient agar; the relevant regulations refer to the Chinese Pharmaceutical GMP Appendix Sterile Drugs (2010 Edition) and the European GMP Appendix 1 Sterile Drug Production (2022 Edition).

[0003] Usually, contact plates are used for sampling of microbial detection of gloves and clean clothes, while smear sticks are used for sampling of microbial detection of various irregular surfaces (for example, convex and concave surfaces or inner surfaces of gaps). The structure or appearance of the smear stick is similar to that of a medical cotton swab, but the requirements are much higher than those of a medical cotton swab. During specific sampling, the head of the smear stick is immersed in the liquid in the smear stick protective tube and moistened, taken out of the tube, and the head of the smear stick is contacted to the irregular surface to be detected and moved back and forth in an S-shaped trajectory. Appropriate pressure is required and continued for a set time. The area of ​​the wiping area is about 25 square centimeters. Then, the head of the smear stick is moved back and forth in an S-shaped trajectory on the agar medium plate at a set angle (about 30º to 45º) to ensure that the entire smear stick head is in contact with the agar medium. Then, the agar medium plate is cultured, and the number of colonies is detected after the culture is completed, and compared with the standards specified in relevant regulations to determine whether the microorganisms on the irregular surface being detected exceed the standard.

[0004] Obviously, the smear stick as a sampling tool is critical to the capture ability of microorganisms in the clean area environment. Sampling with a smear stick is one of the important test methods for pharmaceutical companies to monitor the clean area environment. According to the requirements of relevant regulations, the capture ability of the smear stick needs to be verified. The existing environmental monitoring recovery rate verification method for smear sticks usually adopts a wet recovery rate verification method, that is, a bacterial suspension of a set bacterial species and concentration is prepared, the bacterial suspension is added to the contact surface (such as the surface of a stainless steel plate), and the bacterial suspension is dispersed with an L-shaped sterile coating stick; in the wet state of the bacterial suspension, the designated area of ​​the contact surface is wiped with the smear stick to be verified, and then the microorganisms on the smear stick are transferred to the agar medium plate, which is the test plate; at the same time, the contact surface that is the same as the smear stick sampling is sampled at least 3 times in a row using a contact plate, which is the control plate. The test plate and the control plate are cultured at 30℃-35℃ for no more than 72 hours; the colonies of the test plate and the control plate are counted respectively; the recovery rate of the smear stick to be verified is calculated by the following formula (1) : (1) In the formula, is the recovery rate of the smear stick to be verified; It is the average number of colonies on the test plate, in cfu; The average number of colonies on the control plate, in cfu. Usually, the recovery rate of the smear stick to be verified The higher the value, the higher the ability of the smear stick to capture microorganisms in the environment and the better the quality.

[0005] However, in the actual production clean area, the surface of the clean area that the smear stick contacts is mostly dry. The number of microorganisms that survive on the surface of objects under dry conditions and the ability of the smear stick to capture microorganisms are completely different from those under wet conditions, and there may be a big difference between the two. Therefore, the test results under wet conditions cannot accurately reflect the ability of the smear stick to capture microorganisms in real-world environmental monitoring, that is, they cannot accurately reflect the quality of the smear stick.

[0006] Obviously, the existing environmental monitoring smear stick recovery rate verification method has problems such as not being able to accurately reflect the ability of the smear stick to capture microorganisms in real environmental monitoring. Summary of the invention

[0007] In order to solve the problems that the recovery rate verification method of the prior art smear stick for environmental monitoring cannot accurately reflect the ability of the smear stick to capture microorganisms in actual environmental monitoring, the present invention proposes a dry recovery rate verification method for a smear stick for environmental monitoring.

[0008] The present invention discloses a dry recovery rate verification method for a smear stick for environmental monitoring, which uses lethality to adjust the concentration of a bacterial suspension prepared with test bacteria; the bacterial suspension is spread on the surface of a stainless steel plate and dried under set conditions; the stainless steel plate is wiped and sampled with a smear stick to be verified and the collected microorganisms are transferred to a culture plate to obtain a test plate; at the same time, a contact plate is used to sample the same stainless steel plate to obtain a control plate; the test plate and the control plate are cultured under the same conditions and their respective colony counts are detected; the recovery rate is calculated, and the higher the recovery rate, the higher the capture ability of the smear stick to be verified for microorganisms in the environment; wherein the lethality refers to the ratio of the number of dead colonies of the bacterial suspension spread on the surface of the stainless steel plate until it is dried to the number of spread colonies; the recovery rate is the ratio of the number of colonies on the test plate to the number of colonies on the control plate.

[0009] Preferably, the test bacterial species for preparing the bacterial suspension are: Staphylococcus aureus, Bacillus subtilis, Aspergillus niger, Staphylococcus epidermidis and Micrococcus luteus.

[0010] Preferably, the setting of drying conditions includes, in a biological safety cabinet, a temperature of 10° C. to 30° C., a wind speed of 0.36 m / s to 0.54 m / s; and standing for 20 to 50 minutes.

[0011] Preferably, the culture plate refers to a prefabricated TSA L&P80 plate, i.e., a plate of tryptic soy agar containing Tween 80 and lecithin; the contact plate refers to a prefabricated TSA L&P80 contact plate, i.e., a contact plate of tryptic soy agar containing Tween 80 and lecithin.

[0012] Preferably, the lethality rate is obtained by the following steps: S101, to set the concentration Prepare bacterial suspension, and at the same time, add part of the concentration Set the dilution multiple of bacterial suspension The concentration obtained is of bacterial suspension; S102, the concentration is The bacterial suspension is coated on the surface of the stainless steel plate, and the stainless steel plate is dried under set conditions; S103, sampling the surface of the dried stainless steel plate using a contact plate to obtain a test plate; S104, the concentration is The bacterial suspension is spread on the surface of the culture plate to obtain a reference plate; S105, culturing the test plate and the reference plate; S106. Count the colonies on the test plate and the reference plate, and calculate the lethality rate using the following formula (2): : (2) In the formula, is the fatality rate, in %; is the number of colonies on the test plate, in cfu; is the colony count of the reference plate, in cfu; To set the dilution multiple.

[0013] Preferably, the concentration of the bacterial suspension is When the turbidity of Staphylococcus aureus is 0.90-1.10, select 10 -4 ~10 -5 ; When the turbidity of Bacillus subtilis is 2.0-2.5, select 10 -4 ~10 -5 When the turbidity of Aspergillus niger is 3.3-3.5, select 10 -4 ~10 -5 ; When the turbidity of Staphylococcus epidermidis is 1.10-1.25, select 10 -4 ~10 -5 ; When the turbidity of Micrococcus luteus is 0.9-1.0, select 10 -4 ~10 -5 ; Wherein, the solvent of the bacterial suspension is 0.9% sterile sodium chloride solution or sterile sodium chloride solution containing polysorbate; the unit of the turbidity is McF, i.e. McFarland turbidity; the unit of the bacterial suspension concentration is volume percentage v / v %.

[0014] Preferably, the dilution factor 1 to 12 times.

[0015] Preferably, the recovery rate is obtained by the following steps: S201, using the following formula (3) to adjust the concentration of the bacterial suspension, and according to the adjusted concentration Prepare bacterial suspension; (3) In the formula, is the fatality rate, in %; The set concentration of the bacterial suspension prepared to obtain the lethality rate is expressed in v / v%; is the adjusted bacterial suspension concentration, unit is v / v %; S202, the concentration is The bacterial suspension is spread on the squares on the surface of the stainless steel plate; S203, drying the stainless steel plate under set conditions; S204, swabbing the stainless steel plate grid with the smear stick to be verified to collect samples, and transferring the microorganisms collected by the smear stick to be verified to the culture plate to obtain a test plate; S205, sampling other squares on the same stainless steel plate using the contact plate to obtain a control plate; S206, culturing the test plate and the control plate under the same conditions; S207, count the number of colonies on the test plate and the control plate; and calculate the recovery rate by the following formula (4): : (4) In the formula, is the recovery rate; is the number of colonies on the test plate, in cfu; is the colony count of the control plate, in cfu.

[0016] The beneficial technical effect of the dry recovery rate verification method of the smear stick for environmental monitoring of the present invention is that it more truly reflects the capture ability of the smear stick on microorganisms, and the verification result is more real and reliable. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Attached Figure 1 A block diagram of the lethality detection of the smear stick dry recovery verification method for environmental monitoring of the present invention; Attached Figure 2 The figure is a block diagram of the recovery rate detection of the smear stick dry recovery rate verification method for environmental monitoring of the present invention.

[0018] The following is a further description of the dry recovery rate verification method of the smear stick for environmental monitoring of the present invention in conjunction with the accompanying drawings and specific implementation methods. DETAILED DESCRIPTION

[0019] The "coating" mentioned in the present invention refers to dispersing the bacterial suspension on the surface of a stainless steel plate; including marking a number of squares on the surface of the stainless steel plate, washing with injection water and disinfecting with sterile 70% ethanol; dripping the bacterial suspension into each square on the surface of the stainless steel plate; each stainless steel plate can only add a bacterial suspension of one strain, and each square adds the same set amount of bacterial suspension; using an L-shaped sterile coating rod to disperse the bacterial suspension, starting from the upper left square to the lower right square for coating; the dispersed bacterial suspension occupies the middle of each square.

[0020] The "transfer" mentioned in the present invention refers to moving the collected test bacteria back and forth on the culture plate in an S-shaped trajectory at a set angle (about 30º to 45º), ensuring that the entire smear stick head is in contact with the culture medium on the culture plate, thereby migrating the collected test bacteria to the surface of the culture plate.

[0021] The "culture" mentioned in the present invention refers to placing a plate with or possibly with test bacteria in an incubator, and allowing the test bacteria to grow for a set time under set temperature and humidity conditions. The plate includes a prefabricated TSA L&P80 plate, i.e., a plate of tryptic soy agar containing Tween 80 and lecithin; or, a prefabricated TSA L&P80 contact plate, i.e., a contact plate of tryptic soy agar containing Tween 80 and lecithin. The temperature, humidity and culture time vary depending on the test bacteria; for example, for Staphylococcus aureus, Bacillus subtilis, Staphylococcus epidermidis and Micrococcus luteus, the suitable temperature is 30°C to 35°C, and the culture should not exceed 72 hours; for Aspergillus niger, the suitable temperature is 20°C to 25°C, and the culture should not exceed 120 hours.

[0022] The present invention uses a dry recovery rate verification method for environmental monitoring with a smear stick, and uses a lethality rate to adjust the concentration of the bacterial suspension prepared by the test bacteria; the bacterial suspension is applied to the surface of a stainless steel plate and dried under set conditions; the smear stick to be verified is used to wipe the stainless steel plate for sampling and the collected microorganisms are transferred to the culture plate to obtain a test plate; at the same time, a contact plate is used to sample on the same stainless steel plate to obtain a control plate; the test plate and the control plate are cultured under the same conditions and the respective colony counts are detected; the recovery rate is calculated, and the higher the recovery rate, the higher the capture ability of the smear stick to be verified for microorganisms in the environment; wherein the lethality rate refers to the ratio of the number of dead colonies of the bacterial suspension applied to the surface of the stainless steel plate to the number of coated colonies after drying; the recovery rate is the ratio of the number of colonies of the test plate to the number of colonies of the control plate. The method of the present invention samples the stainless steel surface after drying, so that the sampling is closer to the actual sterile environment. Since part of the test bacteria will die during the drying process of the bacterial suspension, and the sampling mechanism of the smear stick under wet conditions and dry conditions is different, therefore, sampling on the dried stainless steel surface can better reflect the capture ability of the smear stick to microorganisms in a real environment. The concentration of the bacterial suspension is adjusted by the lethality, so that the number of residual colonies after the bacterial suspension coated on the surface of the stainless steel plate is able to meet the sampling requirements of the smear stick and meet the requirements of relevant specifications. Therefore, the dry recovery rate verification method for environmental monitoring smear sticks of the present invention can more accurately reflect the capture ability of the smear stick to microorganisms in a real environment, and the verification result is more real and reliable. Since there are currently no relevant quality standards and testing standards for smear sticks, it seems more scientific and accurate to characterize the capture ability of the smear stick with the recovery rate.

[0023] As one of the preferred technical solutions, the test strains for preparing the bacterial suspension are: Staphylococcus aureus, Bacillus subtilis, Aspergillus niger, Staphylococcus epidermidis and Micrococcus luteus. The above strains are common strains in environmental monitoring and are easy to obtain, and are suitable for verifying the capture ability of the smear stick.

[0024] As one of the preferred technical solutions, the drying conditions are set to include, in a biological safety cabinet, a temperature of 10°C to 30°C, a wind speed of 0.36m / s to 0.54m / s, and standing for 20 to 50 minutes. In fact, the actual drying time of each bacterial species and bacterial suspension of different concentrations is different, but it is based on drying until the surface of the stainless steel plate is dry.

[0025] As one of the preferred technical solutions, the culture plate refers to a prefabricated TSA L&P80 plate, i.e., a plate of tryptic soy agar containing Tween 80 and lecithin; the contact plate refers to a prefabricated TSA L&P80 contact plate, i.e., a contact plate of tryptic soy agar containing Tween 80 and lecithin. Both of the above plates are standard microbial culture plates containing microbial culture media, which are very suitable for the growth or cultivation of various test bacteria used in the method of the present invention.

[0026] As one of the preferred technical solutions, the lethality rate is obtained by the following steps: S101, to set the concentration Prepare bacterial suspension, and at the same time, add part of the concentration Set the dilution multiple for the bacterial suspension The concentration obtained is of bacterial suspension; S102, the concentration is The bacterial suspension is coated on the surface of the stainless steel plate, and the stainless steel plate is dried under set conditions; S103, sampling the surface of the dried stainless steel plate using a contact plate to obtain a test plate; S104, the concentration The bacterial suspension is spread on the surface of the culture plate to obtain a reference plate; S105, culturing the test plate and the reference plate; S106. Count the colonies on the test plate and the reference plate, and calculate the lethality rate using the following formula (2): : (2) In the formula, is the fatality rate, in %; is the number of colonies on the test plate, in cfu; is the colony count of the reference plate, in cfu; To set the dilution multiple. Since the colony count of the test bacteria in the bacterial suspension is difficult to calculate and the error is large, the number of colonies coated on the surface of the stainless steel plate is determined by actual culture measurement, that is, the test bacteria are directly coated on the culture plate for culture, and then the number of colonies on the culture plate is counted, and the number of colonies on the culture plate is multiplied by the dilution multiple of the bacterial suspension. As the number of coated colonies on the surface of the stainless steel plate. At the same time, in order to count conveniently and accurately, the bacterial suspension was changed from the set concentration Dilution setting multiple Finally, spread it on the culture plate to ensure that the colony count after culture is more convenient and accurate.

[0027] As one of the preferred technical solutions, the concentration of the bacterial suspension is When the turbidity of Staphylococcus aureus is 0.90-1.10, select 10 -4 ~10 -5 ; When the turbidity of Bacillus subtilis is 2.0-2.5, select 10 -4 ~10 -5 When the turbidity of Aspergillus niger is 3.3-3.5, select 10 -4 ~10 -5 ; When the turbidity of Staphylococcus epidermidis is 1.10-1.25, select 10 -4 ~10 -5 ; When the turbidity of Micrococcus luteus is 0.9-1.0, select 10 -4 ~10 -5 ; The solvent of the bacterial suspension is 0.9% sterile sodium chloride solution or sterile sodium chloride solution containing polysorbate; the unit of turbidity is Mcf, i.e. McFarland turbidity; the unit of bacterial suspension concentration is volume percentage v / v%. Since McFarland turbidity is used in the technical field to characterize the number of colonies in the bacterial suspension of each test strain, McFarland turbidity is used as a consideration indicator for bacterial suspension concentration when preparing bacterial suspension. Since the lethality of different test bacteria is different, although the concentration of the prepared bacterial suspension is 10 -4 ~10 -5 However, the turbidity of different test bacteria is different; its purpose is to ensure that the number of test bacteria after drying can meet the requirements of test verification.

[0028] As one of the preferred technical solutions, the dilution multiple The purpose of dilution is to prevent too many colonies on the plate after culture, which would affect the accuracy of counting.

[0029] As one of the preferred technical solutions, the recovery rate is obtained by the following steps: S201, using the following formula (3) to adjust the concentration of the bacterial suspension, and according to the adjusted concentration Prepare bacterial suspension; (3) In the formula, is the fatality rate, in %; The set concentration of the bacterial suspension prepared to obtain the lethality rate is expressed in v / v%; is the adjusted bacterial suspension concentration, unit is v / v %; S202, the concentration is The bacterial suspension is spread on the squares on the surface of the stainless steel plate; S203, drying the stainless steel plate under set conditions; S204, swabbing the stainless steel plate grid with the smear stick to be verified to collect samples, and transferring the microorganisms collected by the smear stick to be verified to the culture plate to obtain a test plate; S205, sampling other squares on the same stainless steel plate using the contact plate to obtain a control plate; S206, culturing the test plate and the control plate under the same conditions; S207, count the number of colonies on the test plate and the control plate; and calculate the recovery rate by the following formula (4): : (4) In the formula, is the recovery rate; is the number of colonies on the test plate, in cfu; is the colony count of the control plate, in units of cfu. Obviously, the control plate (i.e., the contact plate of tryptic soy agar containing Tween 80 and lecithin) has a much higher ability to capture microorganisms than the smear stick. The present invention uses the control plate as a reference to actually verify the ability of the smear stick to capture microorganisms. In essence, the control plate and the smear stick are compared under the same conditions to capture microorganisms, thereby verifying the capture ability of the smear stick, which has strong scientificity and practicality.

[0030] Example 1: Death of Staphylococcus aureus after drying on the surface of a stainless steel plate Starting with a Staphylococcus aureus suspension with a turbidity of 0.94Mcf, the suspension was diluted in stages at a ratio of 1:10 to 0.94Mcf×10 -6 Standby; In the biosafety cabinet, mark four squares on the surface of a stainless steel plate, clean the stainless steel surface with injection water, disinfect it with sterile 70% ethanol and dry it; -5The bacterial suspension was dripped into each square on the surface of the stainless steel plate, and 0.1 ml of the bacterial suspension was dripped into each square; the bacterial suspension was dispersed in the middle of the square using an L-shaped sterile coating rod; the stainless steel plate was placed at a temperature of 25±2℃ and a wind speed of 0.45m / s±0.09m / s for 35 minutes; samples were taken in four squares using a contact plate, and each square was continuously sampled using 3 contact plates to obtain a test plate 1 # ~12 # ; In addition, the concentration is 0.94Mcf×10 -6 The bacterial suspension was inoculated onto two culture plates at a volume of 0.1 ml / plate to obtain two reference plates; # ~12 # Place the two reference plates together in a biological incubator at 32.5±2.5℃ for 42 hours; # ~12 # The colony counts were performed on the reference plate, and the results are shown in Table 1 below: Table 1: Results of the test on the lethality of Staphylococcus aureus

[0031] Example 2: Lethality Detection of Bacillus subtilis (Spores) The bacterial suspension (vegetative body in liquid culture medium) after one day of culture was transferred to solid culture medium and cultured for 7 days. A single colony was picked and mixed with 0.9% sterile sodium chloride by oscillation, and then heated (80°C, 20 minutes) to activate the suspension to form subtilis spore suspension, i.e., Bacillus subtilis (spore body). The turbidity was adjusted to 3.56Mcf (as stock solution), and then diluted step by step at a ratio of 1:10 to 3.56Mcf×10 -4 The bacterial suspension was set aside for use. The test was carried out in the same manner as in Example 1, except that the drying conditions on the surface of the stainless steel plate were 25±2°C and a wind speed of 0.45m / s±0.09m / s for 34 minutes; the culture conditions were 32.5±2.5°C in a biological incubator for 43 hours; the results are shown in Table 2 below: Table 2: Results of the mortality test of Bacillus subtilis (Bacillus subtilis)

[0032] Example 3: Lethality Detection of Staphylococcus epidermidis Prepare a suspension of Staphylococcus epidermidis with a turbidity of 1.15Mcf and dilute it step by step at a rate of 1:10 to 1.15Mcf×10 -5Standby; the test was carried out in the same manner as in Example 1, except that the drying conditions on the surface of the stainless steel plate were 25±2°C and a wind speed of 0.45m / s±0.09m / s for 46 minutes; the culture conditions were 32.5±2.5°C in a biological incubator for 43 hours; the results are shown in Table 3 below: Table 3: Results of the test on the lethality of Staphylococcus epidermidis

[0033] Example 4: Lethality Detection of Micrococcus luteus Prepare a suspension of Micrococcus luteus with a turbidity of 1.07Mcf and dilute it step by step at a ratio of 1:10 to 1.07Mcf×10 -5 Standby; the test was carried out in the same manner as in Example 1, except that the drying conditions on the surface of the stainless steel plate were 25±2°C and 0.45m / s±0.09m / s for 30 minutes; the culture conditions were 32.5±2.5°C in a biological incubator for 46 hours; the results are shown in Table 4 below: Table 4: Results of Micrococcus luteus lethality test

[0034] Example 5: Mortality Detection of Aspergillus niger The Aspergillus niger spores were cultured on the solid medium SDA in an incubator at 22.5±2.5℃ for 7 days. The Aspergillus niger spores on the plate were washed with 0.9% sterile sodium chloride to prepare an Aspergillus niger spore suspension with a turbidity of 3.42Mcf. The suspension was diluted stepwise at 1:10 to 3.42Mcf×10 -5 Standby; the test was carried out in the same manner as in Example 1, except that the drying conditions on the surface of the stainless steel plate were 25±2°C and 0.45m / s±0.09m / s for 27 minutes; the culture conditions were 22.5±2.5°C in a biological incubator for 98 hours; the results are shown in Table 5 below: Table 5: Aspergillus niger lethality test results

[0035] Example 6: Detection of lethality of vegetative Bacillus subtilis suspension and comparison with Example 2 The suspension of Bacillus subtilis after one day of cultivation (vegetative in liquid culture medium, hereinafter referred to as vegetative Bacillus subtilis suspension) was used to prepare a suspension of Bacillus subtilis with a turbidity of 3.58 Mcf, which was diluted stepwise at a ratio of 1:10 to 3.58 Mcf × 10 -5Standby; the test was carried out in the same manner as in Example 1, except that the drying conditions on the surface of the stainless steel plate were 25±2°C and 0.45m / s±0.09m / s for 30 minutes; the culture conditions were 32.5±2.5°C in a biological incubator for 44 hours; the results of the lethality test and the comparison with Example 2 are shown in Table 6 below: Table 6: Results of the lethality test of vegetative Bacillus subtilis and spore-forming Bacillus subtilis (spores)

[0036] Example 7: Verification of dry recovery of smear stick A and smear stick B using Micrococcus luteus According to the test in Example 4, the lethality of Micrococcus luteus on the stainless steel surface is 49%, so the concentration of the Micrococcus luteus suspension is adjusted to = ; Twelve squares were marked on the surface of a stainless steel plate. The stainless steel surface was first cleaned with injection water and then disinfected with sterile 70% ethanol and dried. -4 The bacterial suspension was dripped into each square on the surface of the stainless steel plate, and 0.1 ml of the bacterial suspension was dripped into each square; the bacterial suspension was dispersed in the middle of the square using an L-shaped sterile coating rod; the stainless steel plate was placed at a temperature of 25±2°C and a wind speed of 0.45m / s±0.09m / s for 34 minutes; a contact plate was used to sample four squares on the surface of the stainless steel plate, and three contact plates were used to continuously collect samples for each square, and a control plate 1 was obtained. # ~12 # ; Use four smear sticks A to take samples from the other four squares and transfer them to the surface of four culture plates to obtain test plate 1 # ~4 # ; Use four smear sticks B to take samples in the remaining four squares and transfer them to the surface of four culture plates to obtain test plate 5 # ~8 # ; Place control plate 1 # ~12 # and detection plate 1 # ~8 # Place them together in a biological incubator at 32.5±2.5℃ and culture for 42 hours; # ~12 # and detection plate 1 # ~8 # The number of colonies was counted, and the results are shown in Table 7 below: Table 7: Validation results of dry recovery of smear stick A and smear stick B using Micrococcus luteus

[0037] Example 8: Verification of dry recovery of smear stick A and smear stick B using Staphylococcus epidermidis According to the test in Example 3, the lethality of Staphylococcus epidermidis on the stainless steel surface is 71%; the concentration of the suspension of Micrococcus luteus is adjusted to = ; The test was carried out in the same manner as in Example 7, except that the drying conditions on the surface of the stainless steel plate were 25±2°C and a wind speed of 0.45m / s±0.09m / s for 46 minutes; the culture conditions were 32.5±2.5°C in a biological incubator for 45 hours; the results are shown in Table 8 below: Table 8: Validation results of dry recovery of smear stick A and smear stick B using Staphylococcus epidermidis

[0038] It can be seen from the above embodiments that, whether it is the detection of lethality or the detection of recovery rate, the dry recovery rate verification method of the smear stick for environmental monitoring of the present invention sets the detection methods and steps as much as possible in accordance with relevant laws and regulations, including the use of detection equipment and the selection of relevant reagents, etc. The relevant regulations include the Chinese Pharmaceutical GMP Appendix Sterile Drugs (2010 Edition) and the European GMP Appendix 1 Sterile Drug Production (2022 Edition), etc. It can be seen that the dry recovery rate verification method of the smear stick for environmental monitoring of the present invention can not only meet the requirements of relevant regulations to the greatest extent, but also provide the detection personnel with an operation method that is generally applicable to the actual situation of the clean area (dry surface); and it can also ensure the authenticity and reliability of the test results.

[0039] Obviously, the beneficial technical effect of the dry recovery rate verification method of the smear stick for environmental monitoring of the present invention is that it more truly reflects the capture ability of the smear stick on microorganisms, and the verification result is more real and reliable.

Claims

1. A method for verifying the dry recovery rate of a smear stick for environmental monitoring, characterized in that: The concentration of the bacterial suspension prepared with the test bacteria is adjusted using the lethality; the bacterial suspension is spread on the surface of a stainless steel plate and dried under set conditions; a sample is wiped on the stainless steel plate with a smear stick to be verified and the collected microorganisms are transferred to a culture plate to obtain a test plate; at the same time, a sample is taken on the same stainless steel plate with a contact plate to obtain a control plate; the test plate and the control plate are cultured under the same conditions and the number of colonies on each plate is detected; the recovery rate is calculated, and the higher the recovery rate, the higher the capture ability of the smear stick to be verified for microorganisms in the environment; wherein the lethality refers to the ratio of the number of dead colonies of the bacterial suspension applied to the surface of the stainless steel plate until it dries to the number of coated colonies; the recovery rate is the ratio of the number of colonies on the test plate to the number of colonies on the control plate.

2. The dry recovery rate verification method for environmental monitoring using a smear stick according to claim 1, characterized in that: The test bacterial species for preparing the bacterial suspension are: Staphylococcus aureus, Bacillus subtilis, Aspergillus niger, Staphylococcus epidermidis and Micrococcus luteus.

3. The dry recovery rate verification method for environmental monitoring using a smear stick according to claim 1, characterized in that: The setting of drying conditions includes, in a biological safety cabinet, a temperature of 10° C. to 30° C., a wind speed of 0.36 m / s to 0.54 m / s; and standing for 20 to 50 minutes.

4. The dry recovery rate verification method for environmental monitoring using a smear stick according to claim 1, characterized in that: The culture plate refers to a pre-made TSA L&P80 plate, i.e., a plate of tryptic soy agar containing Tween 80 and lecithin; the contact plate refers to a pre-made TSA L&P80 contact plate, i.e., a contact plate of tryptic soy agar containing Tween 80 and lecithin.

5. The dry recovery rate verification method for environmental monitoring using a smear stick according to claim 1, characterized in that: The lethality rate is obtained by the following steps: S101, to set the concentration Prepare bacterial suspension, and at the same time, add part of the concentration Set the dilution multiple of bacterial suspension The concentration obtained is of bacterial suspension; S102, the concentration is The bacterial suspension is coated on the surface of the stainless steel plate, and the stainless steel plate is dried under set conditions; S103, sampling the surface of the dried stainless steel plate using a contact plate to obtain a test plate; S104, the concentration is The bacterial suspension is spread on the surface of the culture plate to obtain a reference plate; S105, culturing the test plate and the reference plate; S106. Count the colonies on the test plate and the reference plate, and calculate the lethality rate using the following formula (2): : (2) In the formula, is the fatality rate, in %; is the number of colonies on the test plate, in cfu; is the colony count of the reference plate, in cfu; To set the dilution multiple.

6. The dry recovery rate verification method for environmental monitoring with a smear stick according to claim 5, characterized in that: The concentration of the bacterial suspension When the turbidity of Staphylococcus aureus is 0.90-1.10, select 10 -4 ~10 -5 ; When the turbidity of Bacillus subtilis is 2.0-2.5, select 10 -4 ~10 -5 When the turbidity of Aspergillus niger is 3.3-3.5, select 10 -4 ~10 -5 ; When the turbidity of Staphylococcus epidermidis is 1.10-1.25, select 10 -4 ~10 -5 ; When the turbidity of Micrococcus luteus is 0.9-1.0, select 10 -4 ~10 -5 ; Wherein, the solvent of the bacterial suspension is 0.9% sterile sodium chloride solution or sterile sodium chloride solution containing polysorbate; the unit of the turbidity is McF, i.e. McFarland turbidity; the unit of the bacterial suspension concentration is volume percentage v / v %.

7. The dry recovery rate verification method for environmental monitoring using a smear stick according to claim 5, characterized in that: The dilution multiple 1 to 12 times.

8. The dry recovery rate verification method for environmental monitoring with a smear stick according to any one of claims 1 to 7, characterized in that: The recovery rate is obtained by the following steps: S201, using the following formula (3) to adjust the concentration of the bacterial suspension, and according to the adjusted concentration Prepare bacterial suspension; （3） In the formula, is the fatality rate, in %; The set concentration of the bacterial suspension prepared to obtain the lethality rate is expressed in v / v%; is the adjusted bacterial suspension concentration, in v / v%; S202, the concentration is The bacterial suspension is spread on the squares on the surface of the stainless steel plate; S203, drying the stainless steel plate under set conditions; S204, swabbing the stainless steel plate grid with the smear stick to be verified to collect samples, and transferring the microorganisms collected by the smear stick to be verified to the culture plate to obtain a test plate; S205, sampling other squares on the same stainless steel plate using the contact plate to obtain a control plate; S206, culturing the test plate and the control plate under the same conditions; S207, counting the number of colonies on the test plate and the control plate; The recovery rate is calculated by the following formula (4): : （4） In the formula, is the recovery rate; is the number of colonies on the test plate, in cfu; is the colony count of the control plate, in cfu.

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