CN107739729B

CN107739729B - Portable outdoor spore separator and spore separation and collection method

Info

Publication number: CN107739729B
Application number: CN201710778285.5A
Authority: CN
Inventors: 陈春; 胡亚强; 彭锐
Original assignee: China Jiliang University
Current assignee: China Jiliang University
Priority date: 2017-01-20
Filing date: 2017-09-01
Publication date: 2023-09-12
Anticipated expiration: 2037-09-01
Also published as: CN107739729A; CN107674828B; CN107674828A; CN207330932U

Abstract

The invention provides a brand new portable outdoor spore separator and a separation method, wherein the spore separator at least comprises a disinfection groove, an upper cover disc and a collecting hole arranged in the separator, the upper cover disc covers the disinfection groove, the upper opening of the disinfection groove is a liquid volatilizing hole, a sample storage groove is arranged on the upper cover disc, and the sample storage groove is positioned above the volatilizing hole or the collecting hole. The separator and the separation method can separate fungus spores under the aseptic condition, avoid the equipment requirement that the traditional method needs to operate in an ultra-clean workbench, are portable outdoor spore separators which are convenient to carry, small and easy to operate, and bring convenience for collecting pathogenic fungi outdoors.

Description

Portable outdoor spore separator and spore separation and collection method

Technical Field

The invention relates to the field of separation and collection of strains, in particular to a spore separation and collection device and a spore separation and collection method suitable for a non-clean environment.

Background

In the research process of fungi, the separation and the expansion culture of strains are very important. The strain separation is to separate target fungi from mixed microorganisms, and then purify and culture the separated target fungi in a proper environment. To avoid contamination, the separation work is required to be performed under aseptic conditions. Currently, isolation culture is performed in a sterile room, in a sterile box, or on a sterile bench (ultra clean bench). The aseptic chamber and the aseptic box are subjected to spray dust removal and sterilization by using medicines or ultraviolet irradiation, wherein the common sterilization medicines are spray of 70% alcohol, 2% coal phenol soap solution, 5% carbolic acid solution and the like. If the ultraviolet lamp is used for irradiation, 20-30 minutes are needed.

Taking the fungus from the order of the Pest, for example, the fungus from the order of the Pest (Entomophthlides) belongs to the subfamily of Pest (Entomophthlycotina), and the fungus is widely distributed and reported all over the world. The fungus of the order entomomycetes is mostly an obligate pathogen of terrestrial arthropods, wherein insects are mainly used, and a few species infest lower invertebrates such as nematodes. Many entomomycetes are able to exert a pest controlling effect by inducing a high intensity of epidemics in insect populations. The investigation of the fungus of the order entomomycetes often requires that the infected aphid cadavers are brought back to a laboratory for separation and identification, and in addition, the fungus of the order entomomycetes can eject conidia in a short time, and the conidia are not easy to preserve. The aseptic operation in the open air can not be ensured, so the traditional method of separating by depending on a laboratory ultra-clean workbench can not be applied to outdoor collection, which brings great obstacle to deep research of fungus of the order entomomycete.

Disclosure of Invention

Based on the problems, the invention provides the separator which is simple and compact in structure, convenient to carry and easy to operate and can be used for aseptically collecting fungus spores outdoors.

The invention provides a portable outdoor spore separator which comprises a disinfection tank, an upper cover disc and a collecting hole arranged in the separator, wherein the upper cover disc can cover the disinfection tank, an upper opening of the disinfection tank is a liquid volatilizing hole, a sample storage groove is arranged on the upper cover disc, and the sample storage groove can be positioned above the volatilizing hole or the collecting hole.

Further, still include the lower valve disc that sets up between disinfection groove and upper cover dish, the collecting hole sets up in the valve disc down, still is provided with the sterilization hole that runs through the valve disc down on the valve disc down, and the stock groove can be located sterilization hole's top and with volatilize hole intercommunication or sterilization hole be located collecting hole top.

Further, a middle valve disc is arranged between the lower valve disc and the upper cover disc, a pore canal penetrating the middle valve disc is arranged on the middle valve disc, and the sample storage groove can be communicated with the pore canal and the sterilization hole and is positioned above the volatilization hole, or the sample storage groove is communicated with the pore canal and is positioned above the collection hole.

Preferably, the sterilizing groove, the lower valve disc, the middle valve disc and the upper cover disc are cylinders, the circular surface equal division radiation part of the lower valve disc cylinder is provided with a sterilizing hole and a collecting hole, the circular surface equal division radiation part of the middle valve disc cylinder is provided with a pore canal, the circular surface equal division radiation part of the upper cover disc cylinder is provided with a sample storage groove, the distances from the circle centers of the sterilizing hole and the collecting hole to the center point of the circular surface of the lower valve disc, the distances from the circle centers of the pore canal to the center point of the circular surface of the middle valve disc, and the distances from the circle centers of the sample storage groove to the circle center of the circular surface of the upper cover disc are equal.

The number of the sterilizing holes and the collecting holes of the lower valve disc is at least three, the sterilizing holes and the collecting holes are distributed at intervals, the number of the pore channels of the middle valve disc is at least three, and the number of the sample storage grooves of the upper cover disc is at least six.

Further, the valve further comprises a stud bolt, a central hole is formed in the center of the cylindrical round surface of the lower valve disc, the central valve disc and the upper cover disc, and the stud bolt is inserted into the central hole to assemble the lower valve disc, the central valve disc and the upper cover disc together.

Further, the cylinders of the lower valve disc, the middle valve disc and the upper cover disc are machined into concave-convex structures which can be matched with each other.

Further, the device also comprises a positioning hole and a positioning pin for judging whether the rotation angles of the lower valve disc, the middle valve disc and the upper cover disc are in place, and the positioning hole is arranged at the corresponding positions of the three discs.

Further, marking scores are provided at the disc edges of the lower, middle and upper cover discs, respectively.

In order to facilitate the replacement of liquid in the disinfection tank, the upper part of the disinfection tank is provided with a liquid injection port, and the lower part is provided with a liquid discharge port.

An O-shaped ring can be embedded on the inner side wall of the collecting hole.

The spore separator can also include a timer and/or a temperature controller.

The sterilization tank can serve as a base for the spore separator. In another form, the spore separator further includes a collection tank that is independent of and removably assembled with the sterilization tank and serves as a base for the separator.

Further, a slide rail is provided on one side wall of each of the sterilizing tank and the collecting tank, and a slide groove is provided on the opposite side wall thereof.

The invention provides a way of collecting spores by using a portable spore separator, comprising the following steps:

volatile disinfectant is filled in the disinfection tank, and the upper cover plate is sterilized; an adhesive material is arranged in the sample storage groove of the upper cover disc, and a disease-sensitive organism is adhered in the sample storage groove; covering the upper cover disc on the sterilizing groove, so that the sample storage groove is positioned above the volatilizing hole of the sterilizing groove, and sterilizing; after sterilization, the sample storage groove is positioned above the collecting hole, a sterilized culture medium collecting pipe is arranged in the collecting hole, and under proper conditions, fungus spores in the infected organism are ejected into the collecting pipe; and after the collection is finished, taking out the collecting pipe in the collecting hole for standby.

The present invention also provides a collection method using a portable spore separator having a lower disc comprising the steps of: volatile disinfectant is filled in the disinfection tank, and the lower valve disc and the upper cover disc are sterilized; an adhesive material is arranged in the sample storage groove of the upper cover disc, and a disease-sensitive organism is adhered in the sample storage groove; placing a collecting pipe containing sterile culture medium in a collecting hole of a lower valve disc, rapidly covering an upper cover disc, and enabling a sample storage groove of the upper cover disc to be communicated with a sterilizing hole of the lower valve disc for sterilization; after sterilization, the sample storage groove is communicated with the collecting hole of the lower valve disc, and under proper conditions, fungus spores in the infected organism are ejected into the collecting pipe; and after the collection is finished, taking out the collecting pipe in the collecting hole for standby.

The present invention also provides a collection method using a portable spore separator having a lower disc, a middle disc and an upper cover disc, comprising the steps of: volatile disinfectant is filled in the disinfection groove, and the lower valve disc, the middle valve disc and the upper cover disc are sterilized; an adhesive material is arranged in the sample storage groove of the upper cover disc, and a disease-sensitive organism is adhered in the sample storage groove; placing a collecting pipe containing sterile culture medium in a collecting hole of a lower valve disc, rapidly covering an upper cover disc, and communicating a sample storage groove of the upper cover disc, a pore canal of a middle valve disc and a sterilization hole of the lower valve disc, so as to sterilize; after sterilization, the middle valve disc and the upper cover disc rotate together for a certain angle to enable the sample storage groove, the pore canal and the collecting hole of the lower valve disc to be communicated, and under proper conditions, fungus spores in the infected organism are ejected into the collecting pipe; after the collection is finished, the upper cover disc and the middle valve disc rotate for a certain angle again, so that the collecting pipe is in a closed state, the upper cover disc and the middle valve disc are removed, and the collecting pipe is taken out for standby.

Further, the method also comprises the step of replacing the volatile sterilizing liquid in the sterilizing tank with sterile water after the sterilization of the organisms is finished.

Further, after the spore collection is completed, the sterile water in the disinfection tank is replaced with volatile disinfection solution.

The beneficial effects of the invention are as follows: in the outdoor spore separating and collecting process, the volatile sterilizing liquid stored in the sterilizing tank of the separator disclosed by the invention is utilized, so that the space inside and nearby the spore separator is ensured to be in a relatively sterile environment. The lower valve disc of the portable outdoor spore separator is provided with collecting holes and sterilizing holes in an radiating mode, wherein the collecting holes for placing the spore collecting pipes and the sterilizing holes for forming the fluid channels are distributed at intervals; the middle valve disc is provided with equally distributed channels, the equally distributed angle is twice as large as that of the lower valve disc, and the number of the channels is the same as that of the collecting holes and/or the sterilizing holes; by using the fact that the distribution angle of the holes of the lower valve disc in the disc is different from that of the holes of the middle valve disc, the formation of the fluid channel and the cutting-off of the fluid channel are realized by rotation, namely the function similar to a valve.

The spore separator changes the traditional method of separating by the original laboratory ultra-clean bench, and can efficiently and conveniently collect spores of pathogenic fungi from the disease-sensitive aphid corpse in a short time under the condition of guaranteeing sterility. The portable outdoor spore separator is small and exquisite, occupies no space and is easy to carry; the structure is simple, and the operation method is simple and convenient; the separation effect is better, avoids the resource waste caused by excessive investment and idle equipment, has strong practicability and is convenient to popularize.

Drawings

FIG. 1 is a portable outdoor spore separator including a sterilization tank, a lower valve disc, a middle valve disc, and an upper cover disc.

Fig. 2 is an exploded view of the portable outdoor spore separator of fig. 1.

Fig. 3 includes a schematic view of a disinfection tank with a fill port and a drain port.

FIG. 4 is a perspective view of the lower disc of FIG. 1.

FIG. 5 is a schematic front view of the lower disc of FIG. 1.

Fig. 6 is a cross-sectional view in the direction of fig. 5 A-A.

Fig. 7 is a perspective view of the middle valve disc of fig. 1.

FIG. 8 is a front view of the mid-valve disc of FIG. 1.

Fig. 9 is a cross-sectional view taken in the direction of fig. 8 A-A.

Fig. 10 is a perspective view of the upper cover tray of fig. 1.

Fig. 11 is a front view of the upper cover tray of fig. 1.

Fig. 12 is a cross-sectional view taken in the direction of fig. 11 A-A.

FIG. 13 is the daily average number of elastosis in the cryopreservation tube and spore separator. When the same lowercase letters appear, no significant difference is indicated, and different lowercase letters indicate significant differences; the appearance of different capital letters indicates extremely significant differences, and the same capital letters indicate electrodeless significant differences.

FIG. 14 shows the average total spore yield per aphid for the cryopreservation tube and spore separator.

FIG. 15 is a diagram of an experimental analysis of the sterility of the separator.

FIG. 16 is a comparison of sterility of a conventional process versus a spore separator process.

FIG. 17 is a portable spore separator including a sterilization well, a lower valve disc, and an upper cover disc.

FIG. 18 is a portable spore separator including a sterilization well and an upper tray.

A schematic view of a collecting pipe placing frame is arranged in the sterilizing groove in fig. 19.

FIG. 20 is a schematic view of the collection tube placed in the rack of FIG. 19.

Fig. 21 is a schematic view of the sterilization tank and collection tank connected by a slide rail and chute.

Fig. 22 shows a lower valve disc with two sterilization holes and two collection grooves, a middle valve disc and an upper cover disc cooperating therewith.

FIG. 23 is a portable outdoor spore separator with a timer on the upper cover tray.

FIG. 24 is a lower valve disc with an O-ring disposed in the collection port.

Detailed Description

The present invention will be described in detail with reference to the accompanying drawings. These specific examples are intended to be limiting and do not preclude other embodiments of the invention by those of ordinary skill in the art combining the present invention with the present invention.

Example 1 Structure of Portable spore separator

The portable outdoor spore separator as shown in fig. 1 to 12 comprises a sterilizing tank 1, a lower valve disc 2, a middle valve disc 3 and an upper cover disc 4, and is detachably assembled together from bottom to top.

The sterilizing tank 1 is used for containing volatile sterilizing liquid or sterile water, and is positioned at the bottom of the portable outdoor spore separator as a supporting device of the separator.

The lower valve disc 2 is a cylinder and is provided with through holes of a certain diameter at six equally divided radiation positions on the circular surface 21 of the cylinder. The three through holes are sterilization holes 22, and the sterilization holes are used as volatile sterilization liquid volatilization channels for sterilizing collected biological samples (such as aphid bodies) and fluid channels formed by assembling three plates of a lower valve plate, a middle valve plate and an upper cover plate which are arranged on a sterilization groove. The other three through holes of the lower valve disc are collecting holes 23 for placing and fixing the collecting tube 9 with sterile medium. In this embodiment, the sterilization holes 22 and the collection holes 23 are arranged at intervals.

The middle valve disc 3 is a cylinder, a hole channel 32 with a certain diameter is arranged on the trisection radiation position on the circular surface 31 of the cylinder, and the distance between the center of the hole channel 32 and the center point of the circular surface of the middle valve disc is the same as the distance between the center points of the sterilization hole and the collection hole of the lower valve disc and the center point of the circular surface of the lower valve disc. The middle valve disc plays a role of sealing and isolating.

The upper cover plate 4 is a cylinder, a groove 42 is arranged on the lower surface of the circular surface 41 of the cylinder, and a sample storage groove 43 is arranged on the circular surface of the bottom of the groove at the position of trisection of radiation distribution and used for storing collected organisms. The distance between the center of the slotted hole of the sample storage groove 43 and the center of the circular surface of the upper cover disc is the same as the distance between the center of the sterilizing hole and the collecting hole of the lower valve disc and the center of the circular surface of the lower valve disc. In one embodiment of the manufacture of the upper disc, a small cylinder of slightly smaller diameter and height than the cylinder is first hollowed out in the upper disc cylinder to form the recess 42. Three small circular grooves with certain diameters and depths are processed on the circular surface of the bottom of the groove at the position of trisecting the radiation distribution, and the small circular grooves are used as sample storage grooves 43 for storing biological samples. In this embodiment, the wells 43 do not extend through the upper tray. In a preferred embodiment, the sample storage well of the upper cover tray is provided with an adhesive layer.

In order to increase the speed of spore counting, in another embodiment, a sample storage groove 43 is provided on the circular surface of the bottom of the groove 42 of the upper cover plate 4 at a position of six equal parts of radiation distribution for storing the collected organisms. Three of the sample storage tanks are used for spore collection, and the other three sample storage tanks are used for spore counting, so that samples in six sample storage tanks can be used for counting, and the counting speed is increased by 1 time.

The materials of the sterilizing tank 1, the lower valve disc 2, the middle valve disc 3 and the upper cover disc 4 are selected from stainless steel, high-temperature sterilizable plastic materials and the like. Preferably stainless steel, such as the lower disc 2, the middle disc 3 and the upper cover disc 4 are all machined from a cylindrical stainless steel material having a certain height and diameter.

When the portable outdoor spore separator is used, the disinfection tank 1, the lower valve disc 2, the middle valve disc 3 and the upper cover disc 4 are assembled together from bottom to top. When the three passages 32 of the middle valve disc 3 are communicated with the sterilizing holes 22 of the lower valve disc 2 to form a fluid channel, the volatile sterilizing liquid in the sterilizing tank sterilizes the biological sample (such as aphid cadaver) placed in the sample storage groove 43 of the upper cover disc 4, the fluid channel and the upper cover and other devices through the fluid channel. The sterilization hole 22 of the lower valve disc 2 is not communicated with the middle valve disc pore canal 32, but the collection hole 23 of the collecting pipe placed on the lower valve disc is communicated with the middle valve disc pore canal 32, at the moment, the collection hole 23 is communicated with the pore canal 32 and the upper sample storage groove 43, and fungi carried by the biological sample are ejected out of spores under the action of gravity and smoothly fall into the collecting pipe placed in the sterile culture medium in the lower valve disc collection hole 23.

The sterilizing chamber 1, the lower valve disc 2, the middle valve disc 3 and the upper cover disc 4 of the outdoor spore separator are detachably assembled with each other, for example, by screwing, buckling of the buckling piece 52, etc. In one embodiment, the sterilization slot is threadably engaged with the lower valve disc.

In this embodiment, the centers of the circular cylindrical surfaces of the lower valve disc 2, the middle valve disc 3 and the upper valve disc 4 are respectively drilled with a central hole 51 with a certain diameter and threads, and a stud bolt 5 can be inserted into the central hole, and the function of the stud bolt is to make the upper valve disc, the middle valve disc and the lower valve disc be assembled into a whole. The length of the stud bolt is determined by the thickness of the upper cover disc, the middle valve disc and the lower valve disc when the upper cover disc, the middle valve disc and the lower valve disc are assembled together, and the diameter of the stud bolt is matched with the diameter of the central hole of each disc. In a preferred embodiment, the stud has a thread of a certain length only at the beginning end.

In order to allow the lower disc 2, the middle disc 3 and the upper disc 4 to be tightly fitted to each other, in a preferred embodiment, the cylinders of the three discs are machined with mating embossments. For example, the lower valve disc 2 comprises a protrusion 25; the middle valve disc 3 comprises a concave part 34 and a convex part 35; the upper cover disc 4 comprises a recess 44. When the three disks are assembled together, the boss 25 of the lower disk fits into the recess 34 of the middle disk 3, and the boss 35 of the middle disk fits into the recess 44 of the upper cover disk.

When the sterilization hole 22 of the lower valve disc is communicated with the pore canal 32 of the middle valve disc and the upper cover disc to form a fluid passage, the separator is in a sterilization state; when the collection hole 23 of the lower valve disc of the spore separator communicates with the hole 32 of the middle valve disc and the stock groove 43 of the upper cover disc to form a fluid passage, the separator is in a state of collecting spores. The spore separator can realize the spore collection work under the aseptic condition after the holes of the lower valve disc, the middle valve disc and the upper cover disc of the spore separator are sterilized outdoors. In the embodiment, the upper cover disc, the middle valve disc and the lower valve disc realize the conversion of the disinfection state or the collection state of the separator in a rotary fit mode. In order to enable an operator to intuitively and effectively control the rotation angles of the three discs, the separator also comprises a positioning device, and the angle of each rotation is a fixed value, so that smooth communication between the sterilizing holes or the collecting holes, the pore canal and the sample storage groove is ensured. The positioning device is selected from but not limited to the following modes: the cooperation of the positioning hole 6 and the positioning pin 7, marking the notch indication 8, etc.

In the embodiment of the matching of the positioning holes and the positioning pins, the corresponding positions of the upper cover disc, the middle valve disc and the lower valve disc are respectively provided with the positioning holes 6, and whether the positioning pins 7 can be completely inserted into the positioning holes 6 on the three discs is utilized to judge whether the rotation angles of the three discs are in place or not. As shown in fig. 1 and 2, at least two positioning holes 6 are formed in the lower valve disc, namely a disinfection state positioning hole and a spore collection state positioning hole, and at least one positioning hole is formed in each of the upper cover disc and the middle valve disc. The positioning holes of the upper cover disc and the middle valve disc are aligned with the positioning holes of the lower valve disc in a disinfection state, the positioning pins can be smoothly inserted into the three positioning holes, and at the moment, the disinfection holes of the lower valve disc, the pore canal of the middle valve disc and the sample storage groove of the upper cover disc are completely aligned to form a fluid channel, so that disinfection can be implemented; the locating holes of the upper cover disc and the middle valve disc are aligned with the locating holes of the spore collecting state of the lower valve disc, the locating pins can be smoothly inserted into the three locating holes, and at the moment, the collecting holes of the lower valve disc are aligned with the pore canal of the middle valve disc and the sample storage groove of the upper cover disc, so that spores can be collected.

In the example of the marking scores, at least two marking scores 8 are provided on the disc edges of the lower disc, and at least one marking score 8 is provided on the disc edges of the upper cover disc and the middle disc. When the mark nicks of the upper cover disc and the middle valve disc are aligned with the mark nicks of the lower valve disc in the disinfection state, the disinfection holes of the lower valve disc are communicated with the pore canal of the middle valve disc and the upper cover disc to form a fluid channel, and the separator is in the disinfection state; the other marking notch of the lower valve disc is a marking notch in a collecting state, and when the marking notch of the upper cover disc and the middle valve disc are aligned with the disinfection state marking notch of the lower valve disc, the collecting hole of the lower valve disc is communicated with the pore canal of the middle valve disc and the sample storage groove of the upper cover disc to form a fluid channel, and the separator is in a spore collecting state.

In a more preferred embodiment, the separator includes a locating hole and a locating pin, with a marking score provided on the corresponding disc edge at the locating hole. On the one hand, the combination of the positioning hole and the positioning pin ensures the accuracy of the rotation angle of the three coils, and on the other hand, the mark nicks can easily enable experimenters to observe the direction and the angle which need to be rotated. In a preferred embodiment, the score of the mark may also be marked with a sterilization or collection letter to alert the experimenter of the current status of the separator.

Example 2 method of collecting spores Using the Portable spore separator of example 1

The collection of spores of fungi of the order Pest mould is described in detail. All operations are performed with a mask and disposable rubber gloves.

The lower valve disc 2, the middle valve disc 3, the upper cover disc 4 and the like are sterilized first with 75% alcohol or other sterilizing fluid.

A quantity of volatile sterilizing liquid is filled into the sterilizing tank 1, and the aphid is obtained by a sterilized sampling rod (for example, the aphid is obtained by a writing brush which is sprayed with 75% alcohol), and the aphid (aphid cadaver) is fixed in a sample storage tank 43 at the top interval in the upper cover plate. The mode of fixing the aphids with the disease can be realized by means of double-sided adhesive tape or an adhesive layer of a sample storage groove.

The lower valve disc 2 is mounted on the sterilizing tank 1, after a few minutes of sterilization, a spore collection tube (not shown) containing sterile medium (e.g., sterile egg yolk medium) is placed in three collection holes 23 spaced apart, the sterilized middle valve disc 3 is quickly assembled, and the sterilization holes 22 of the lower valve disc 2 are kept in communication with the openings 32 in the middle valve disc 3, i.e., the collection tube is in a closed state. Finally, the upper cover plate 4 is arranged on the middle valve plate, and the sample storage groove filled with the aphid cadaver is communicated with the sterilizing hole of the lower valve plate and the pore canal of the middle valve plate for sterilization for 1min. The spore collection tube can be a test tube or a concave bottom container, etc.

After the sterilization, the upper cover plate 4 and the middle valve plate 3 are kept to synchronously rotate and rotate for 60 degrees together, and at the moment, the aphid storage groove 43 stuck on the upper cover plate is communicated with the middle valve plate pore canal 32 and the collecting pipe in the lower valve plate collecting hole 23. The separator was allowed to stand overnight in a constant temperature incubator at 20℃until the fungal elastase was allowed to stand, which under the force of gravity would fall into a collection tube containing sterile medium.

After the incubator is placed overnight, the upper cover disc 4 and the middle valve disc 3 are synchronously rotated for 60 degrees, at the moment, the sample storage groove of the upper cover disc is communicated with the sterilization hole of the lower valve disc and the pore canal of the middle valve disc, then the upper cover disc and the middle valve disc are sequentially removed, the collecting pipe in the lower valve disc is taken out, and spores in the collecting pipe are harvested for later use.

The operation has reached the aim of collecting fungal spores in a non-ultra-clean bench. The function of the sterilizing tank in the whole operation is to make the space of the separator be a relatively sterile environment by utilizing the volatile sterilizing liquid therein, and the sterilizing tank has the best of the sterile environment provided around the flame of the alcohol lamp.

Maintaining the ambient humidity throughout the separator will be more conducive to spore culture. Therefore, in a preferred scheme, after the sample storage groove filled with aphid corpse is communicated with the sterilizing hole of the lower valve disc and the pore canal of the middle valve disc, and sterilization is carried out for 1min, the upper cover disc 4 and the middle valve disc 3 synchronously rotate together for 60 degrees, and at the moment, the sample storage groove 43 stuck with aphid on the upper cover disc is communicated with the pore canal 32 of the middle valve disc and the collecting pipe in the collecting hole 23 of the lower valve disc. The volatile sterilizing liquid in the sterilizing tank is then replaced by sterile water to maintain the ambient humidity of the whole device during constant temperature culture. After the separator is placed in the constant temperature incubator overnight, sterile water in the disinfection tank 1 is replaced by volatile disinfection solution, then the upper cover disc 4 and the middle valve disc 3 are synchronously rotated for 60 degrees, the upper cover disc and the middle valve disc are sequentially removed, and the collecting pipe is taken out to harvest spores. The replacement of the sterilizing liquid and the sterile water in the sterilizing tank can be directly completed from the upper opening of the sterilizing tank through liquid suction, liquid injection and other modes. In the embodiment of the disinfection tank shown in fig. 3, the upper part of the disinfection tank is provided with a liquid filling port 11, the lower part is provided with a liquid discharging port 12, and the liquid filling port and the liquid discharging port are sealed by an openable sealing member 13. In a specific replacement method, after the components of the separator are sterilized by the sterilizing liquid in the sterilizing tank, the sealing member 13 at the liquid outlet 12 is opened to discharge the volatile sterilizing liquid in the sterilizing tank, and then the liquid outlet is closed again by the sealing member. Then, the sealing member 13 of the pouring port 11 is opened, and sterile water is poured from the pouring port as needed, and after the pouring is completed, the pouring port is closed again by the sealing member.

The sterile water in the disinfection tank provides a moist environment for the fungal elastores, with a relatively uniform change in daily elastore number without sudden extreme decline. In the field of aphids, it is not possible to replace the culture medium with new one at intervals to collect spores, so the performance of the separator according to the invention is in fact that the possibility of separating to pure fungi is prolonged in the time dimension. Thus, the spore separator of the invention not only ensures the tightness not to be disturbed by the outside, but also provides a humidity requirement suitable for fungus growth.

Example 3 comparative experiment of spore production quantity

The comparison test of the spore production quantity index adopts two collection modes of the portable spore separator collection and the freezing tube collection, which are described in the embodiment 1, and the spore production quantity in the two collection modes is counted. And carrying out differential analysis on the spore numbers in the two modes by a statistical method, so as to judge the feasibility of the outdoor spore separator collection mode. 30 infected aphids are selected as test objects, and statistical analysis is carried out, namely, 30 infected aphids with approximate sizes are randomly selected for each of two modes to test.

Using the mounting of the spore separator described in example 1 and the trays of example 2, the collected aphids were secured in the sample storage well in the upper tray of the separator using double sided tape, the well bottom container was placed in the collection well of the lower tray of the separator, and 250. Mu.L of 0.5% SDS solution was added to the well bottom container in advance to make the aphids correspond one to one with the well bottom container. Then, the separators were incubated in an illumination incubator (20 ℃,12L: 12D) with concave containers replaced every 24 hours until the aphis felt to be no longer sporulated. The spore liquid in each well was counted under observation under an optical microscope using a blood cell counting plate method for 7 spores in 0.1. Mu.L liquid samples. The blood cell counting area has 25 grids, only 5 grids are selected randomly for counting each time, and the liquid volume of the 5 grids is 0.02ul, so that the number of spores in the concave bottom container needs to be multiplied by a coefficient k=5×10 when calculating ⁴ . Will eject spores every dayThe sum of the numbers of the aphids can be used for obtaining the total spore yield of the aphids.

The freezing tube collection mode is as follows: a part of the middle of the frozen storage tube body is cut off, the cover part is close to the bottom, 200 mu L of 0.5% SDS solution is added to the bottom, and the collected aphid is fixed in the center of the cover by double-sided adhesive tape, so that the insect body is close to the reagent after being covered and cannot contact with the surface of the reagent solution, and spores ejected from the aphid can be collected as much as possible. Then, all the spore collection cryopreservation tubes are cultured in an illumination incubator (20 ℃,12L: 12D), and the bottom cryopreservation tubes are replaced every 24 hours until the aphids with the disease do not play spores any more. The spore liquid in each of the cryopreservation tubes was counted for 7 spores in 0.1. Mu.L of liquid sample by observation under an optical microscope using a blood cell counting plate method. The blood cell counting area has 25 grids, only 5 grids are selected randomly for counting each time, and the liquid volume of the 5 grids is 0.02 mu L, so that the number of spores in the freezing tube needs to be multiplied by a coefficient k=5×10 when the number of spores in the freezing tube is calculated ⁴ . And adding up the quantity of the catapulting spores every day to obtain the total spore yield of the aphid.

The results of statistical analysis of the data from consecutive counts of aphids with a sample size of 30 are shown in fig. 13 and 14.

FIG. 13 is a graph showing that there is no significant difference in the number of spores collected in the first, second, third and fourth day of the time series of spores by t-test of the average number of spores produced by 30 samples from each day of the time series of bullet spores in the freezing tube and separator, and that there is a significant difference in the number of spores collected in the freezing tube and separator at the fourth and fifth days of the time series of bullet spores. This property of the separator has gained time for the field harvesting of pathogenic fungi, which is of great practical value.

FIG. 14 shows the total sporozoite amounts accumulated daily, the fifth day being the total amount of all sample elastosis amounts. The difference between the numbers of spores collected in the two ways is not large, as can be seen from fig. 14, the number of spores per day in the first and second days is larger than that in the separator, but there is no significant difference between them from a statistical point of view, the number of spores in the separator is also no significant difference between them from the third day, but there is a very significant difference between them from the fourth and fifth days, and the final accumulated result is that the total number of spores collected in the separator is larger than that in the freeze tube. The freezing tube mode is used for collecting a little more spore production in the early stage of spores, the total number is less, the separator method is used for collecting a little less spore production in the early stage of spores, the total amount of spore production is more, and the separator has better collection performance from the result of the total amount of elastosis. The results in connection with fig. 13 and 14 show that the separator method of the present invention is superior to the conventional freezing tube method.

EXAMPLE 4 separator sterility verification

The sterility evaluation index is mainly used for judging the feasibility of the separator collection mode by carrying out statistical analysis on the number of sterility samples in the collection result under the two collection modes of the separator collection mode and the traditional collection mode. 30 aphids with a disease susceptibility were selected as subjects.

The culture of new aphid pestilential fungi under ordinary and conventional conditions is very difficult, since new aphid pestilential fungi are a specific entomopathogenic fungus which has high demands on living environment and nutrition, we use Sa's medium and add yolk and milk to it for isolation and in vitro culture.

Traditional collection mode:

1. alcohol wiping is carried out on the table top of the ultra-clean workbench, and then ultraviolet sterilization is carried out for 30min;

2. cutting small double-sided adhesive tapes, and adhering the small double-sided adhesive tapes to the central position on the inner surface of the dish cover of the culture dish;

3. taking one end of aphid with 75% alcohol sterilized writing brush, sticking on double-sided adhesive tape, holding the dish cover, placing above sterilizing tank containing sterilizing liquid for sterilizing, and placing the dish cover on culture dish containing yolk culture medium after 1 min;

placing overnight in an incubator at 4.20 ℃, replacing the dish cover adhered with the disease-causing aphid cadaver with a new sterile dish cover, and continuously placing in the incubator for several days;

5. After several days of subsequent observation, the medium was checked for contamination and the results recorded.

The spore separator of the invention is collected by the following steps:

1. on the table top of the experiment table, the sterilizing tank is filled with volatile sterilizing liquid, and 75% alcohol is used for sterilizing the lower valve disc, the middle valve disc and the upper cover disc in the separator;

2. sticking double-sided adhesive tapes in three spaced sample storage grooves in the upper cover plate, taking aphids with a writing brush, and sticking the aphids with the disease on the double-sided adhesive tapes;

3. the egg yolk culture medium which is arranged in advance and is contained in the concave bottom container is placed in the collecting hole of the lower valve disc at intervals, the middle valve disc is quickly covered to enable the concave bottom container to be in a closed state (a sterile space is created by the volatile disinfectant in the bottom disinfection groove);

4. covering the upper cover disc to enable the holes of the lower valve disc and the middle valve disc to be communicated and enable the holes to face the aphid cadaver of the upper cover disc;

5. after sterilization for 1min, the middle valve disc and the upper cover disc rotate together for 60 degrees, and at the moment, the aphid is in a communication state with the concave bottom container of the lower valve disc;

6. sterilizing for 1min;

7. placing the separator in an incubator at 20 ℃, and rotating the middle valve disc and the upper cover disc together for 60 degrees after overnight placement, wherein the concave bottom container is in a closed state;

8. taking out the separator, placing on a laboratory bench, removing the upper cover disc and the middle valve disc, rapidly taking down the concave container, placing the concave container into a disposable culture dish, and sealing the culture dish by using a sealing film;

9. Placing the culture dish in an incubator at 20 ℃ for culture;

10. the medium was checked for contamination during the incubation and recorded.

Identification of sterility. The definition of sterility should include two parts: one is that the culture medium is separated into single colonies, and the other is that the culture medium is kept sterile, namely no mixed bacteria grow on the culture medium, because in the practical situation, whether aphids are infected or not is judged according to the experience of a sampler, namely that the aphids infected by people are possibly not infected, then the target fungus spores cannot be obtained at the moment, and spores cannot be separated, and if the mixed bacteria do not grow on the separation culture medium, the sterility in the separation operation is ensured by the separator.

The experimental results are shown in fig. 15: a, a sample of a culture medium unchanged in a traditional separation mode, b, a sample of a single colony collected in the traditional separation mode, and c, a sample of contaminated bacteria in the traditional separation mode; d2 and d3 in the graph d are samples of the medium unchanged in the separation mode by the separator method, e is the case of separating a single colony, f and d1 are the cases of staining the medium itself. The diagram ab in the conventional method is regarded as sterile and the diagrams d2, d3 and e in the separator method are regarded as sterile.

Statistical analysis and evaluation of sterility:

the test subjects were selected from 30 aphids with a sample size, and statistical analysis was performed, i.e. 30 aphids with a sample size were randomly selected for each of the two separation methods.

In the experiment, three batches are developed in the traditional separation method and the separation method of the spore separator, wherein the sterility rate of the traditional method is 96.67% in the first batch, and the sterility rate of the separator method is 76.67%; in the second batch, the sterility rate of the traditional method is 66.67%, and the sterility rate of the separator method is 58.33%; in the third batch, the sterility rate of the conventional method was 69.44% and the sterility rate of the separator method was 94.44%. Two sets of data in the same batch were obtained under the same conditions. As shown in fig. 16, the sterility rates in the first, second and third batches are listed in the form of a bar graph, and t-test analysis was performed on these three sets of data, so that there was no significant difference in sterility performance between the two separation methods, i.e., the spore separator method of the present invention was not different from the conventional method in sterility performance between fungi separated in an ultra clean bench.

Example 5 Another portable spore separator according to the invention

The portable outdoor spore separator, as shown in fig. 17, includes a sterilization tank 1, a lower valve disc 2, and an upper cover disc 4. Through holes are arranged at symmetrical positions of the lower valve disc, wherein one hole is a sterilization hole, and the other hole is a collection hole. A sample storage groove is formed in one side of the bottom of the upper cover disc, and the sample storage groove can be opposite to the through hole of the lower valve disc when the sample storage groove is used. The sterilization groove 1, the lower valve disc 2 and the upper cover disc 4 are detachably assembled with each other. The assembly method may be the method exemplified in example 1.

When the sample storage groove of the upper cover disc is communicated with the sterilization hole of the lower valve disc to form a fluid channel, the volatile sterilization liquid can sterilize the aphid corpse, the fluid channel, the upper cover device and the like which are positioned at the sample storage groove through the fluid channel; the collection hole of the lower valve disc is communicated with the collection groove of the upper cover disc, and spores sprung by fungi fall into the collection tube filled with sterile culture medium under the action of gravity.

The specific collecting step comprises the steps of installing a lower valve disc on a sterilizing groove, placing a collecting pipe filled with sterile culture medium in a collecting hole after a few minutes of sterilization, removing a pipe cover, rapidly assembling an upper cover disc, enabling a sample storage groove filled with aphid bodies and the sterilizing hole of the lower valve disc to be communicated, and sterilizing for 1min. The upper cover disk rotates relative to the lower valve disk, the sample storage groove of the upper cover disk is communicated with the collecting hole of the lower valve disk, and spores are collected in a constant-temperature environment. After the spores are collected, the upper cover disc is separated from the lower valve disc, and the aseptic tube cover is covered for recycling the header for later use. The liquid in the disinfection tank is used interchangeably between volatile disinfection liquid and sterile water during collection as required.

Example 6 Another portable spore separator according to the invention

The portable outdoor spore separator as shown in fig. 18 to 20 comprises a sterilizing tank 1 in which a rack 10 is provided to house a collection tube containing a sterile medium, and an upper cover tray 4 in which a sample storage tank is provided on the inner surface. When the upper cover plate is assembled on the sterilizing tank, the sample storage tank has at least two positions relative to the sterilizing tank 1: a sterilizing position of the sample storage tank opposite to the sterilizing liquid in the sterilizing tank, and a spore collecting position of the sample storage tank opposite to the spore collecting pipe. More specifically, the cross section of disinfection groove and upper cover dish is rectangle, and the stock groove is located one side of upper cover dish, and rack 10 is connected with the inner wall of disinfection groove and divide into disinfection position and two parts of collecting position with the disinfection groove, has seted up the hole in the middle of the rack, and the spore collecting pipe inserts in this hole.

When the sample storage groove of the upper cover disc is communicated with the sterilizing position of the sterilizing groove, the volatile gas of the sterilizing liquid realizes the sterilization of aphid corpse, upper cover device and the like in the sample storage groove of the upper cover disc; after the disinfection is finished, the upper cover disc is rotated for 180 degrees, and the tube cover of the collecting tube is rapidly removed to enable the sample storage groove to be communicated with the collecting tube of the disinfection groove, and the fungus elastospore falls into the collecting tube filled with the sterile culture medium under the action of gravity. After the spores are collected, the upper cover disc is removed, the aseptic tube cover is covered on the recovery header again, and the collection tube with the spores is collected for standby. The liquid in the disinfection tank is used interchangeably between volatile disinfection liquid and sterile water during collection as required.

Example 7

The separator comprises a lower valve disc, a middle valve disc, an upper cover disc, a sterilizing tank 1 and a collecting tank 17, wherein the sterilizing tank and the collecting tank are mutually independent and assembled cavities, and the sterilizing tank and the collecting tank which are assembled together are used as a base of the separator as shown in figure 21. The lower valve disc is assembled on the disinfection tank and the collection tank which are connected together in a buckling manner.

In this embodiment, a slide rail 15 is provided on one side wall of each of the sterilizing tank and the collecting tank, and a slide groove 16 is provided on the other side wall thereof, and the sterilizing tank and the collecting tank are mutually engaged and sleeved together through the slide rail and the slide groove. In a more preferred design, the chute is arranged inside the side wall, i.e. the chute outer vertical surface 61 is not protruded outside the side wall, the outer side wall 51 of the slide rail is in contact with the outer vertical surface of the chute, and when the disinfection tank and the collecting tank are connected through the slide rail and the chute, the disinfection tank and the collecting tank are closely attached, so that the use space can be saved more.

The separator described in this example may employ the sterilization and spore collection methods outlined in the previous examples. The culture solution is preloaded in the collecting tank, and the opening of the collecting tank is sealed by a sealing film. When sterilization is completed, the sealing film is torn to align the sample storage well of the upper cover tray with the collection well to collect spores.

When the base of the spore separator adopts the mode of the disinfection tank and the collection tank in the embodiment, the upper cover disc can also be directly combined with the base, or the upper cover disc and the lower valve disc can be combined and assembled on the base.

Example 8

The sample storage groove 43 of the upper cover plate of the present embodiment is designed as a through hole, and the rest of the structure and the sterilization collection mode are the same as those of the previous embodiment.

In the biological sample collection step, the rubberized fabric with a slightly larger area than the sample storage groove of the upper cover plate is sheared, a writing brush sterilized by 75% alcohol is used for taking the aphids with the disease and transferring the aphids to the central position of the rubber surface of the rubberized fabric, and then the rubberized fabric with the aphids is adhered to the sample storage groove on the upper surface of the upper cover plate to cover the sample storage groove. The penetrating design of the sample storage groove makes the aphid storing operation on the upper cover plate easier.

Example 9

The spore separator of the invention is also provided with a timer 55 which can be transported along with the separator, so that no matter where the separator is transported or an experimenter is replaced, the timer can directly obtain time information from the timer of the separator, and after each step of container sterilization and/or spore collection is finished, the timer can send out a prompt tone to remind the experimenter. In a preferred embodiment, the timer can display and set the sterilization start time and/or sterilization end count down, the spore collection start time and/or spore collection end count down, and the like. In the embodiment shown in fig. 23, the timer is mounted on the upper cover disc.

Example 10

The spore separator can also be provided with a temperature controller to control the temperature of the separator to an optimum temperature for spore collection and growth. In the embodiment shown in fig. 18, the bottom of the stainless steel sterilizing tank is provided with a temperature controller 53, which can be adjusted to 20 c or other suitable temperature to keep the separator in a constant temperature culture state. In a preferred embodiment, insulation is provided outside the disinfection tank 1, the lower valve disc 2, the middle valve disc 3 and the upper cover disc 4 to reduce the heat exchange of the separator with the environment.

Example 11

The number and arrangement of the sterilizing holes and the collecting holes of the lower valve disc, the number and arrangement of the tunnels of the middle valve disc and the number and arrangement of the sample storage slots of the upper cover disc are not limited to those in the above embodiments, but may be, for example, 2 sterilizing holes and collecting holes of the lower valve disc respectively and are arranged at intervals in the quarter radiation positions, and the number of tunnels of the middle valve disc or the sample storage slots of the upper cover disc corresponding to the sterilizing holes and the collecting holes of the lower valve disc respectively is 2. The sterilization holes and collection holes of the lower valve disc shown in fig. 22 are respectively 2 and symmetrically arranged at the quarter radiation positions, and the number of the corresponding pore channels of the middle valve disc or the sample storage grooves of the upper cover disc is respectively 2. The number of the collecting pore channels and the sample storage grooves of the sterilizing hole can be 1 to N according to the requirement, and N is a number larger than 1.

The shapes of the lower valve disc, the middle valve disc and the upper cover disc are not limited to the circular or square shapes exemplified in the above embodiments, but may be, for example, elliptical, polygonal, etc.

The spore collection tube may have machining errors, and some collection tubes may have diameters slightly larger or slightly smaller than the design dimensions, so that they cannot be placed into or stand up in the collection hole smoothly. Therefore, in a preferred embodiment, the O-ring 56 is also embedded on the inner side wall of the collecting hole 23 as shown in FIG. 24, the O-ring has certain elasticity, the spore collecting pipe is inserted into the collecting hole with the O-ring and can be blocked by the O-ring, particularly, the diameter of the spore collecting pipe is deviated, and the collecting pipe slightly larger than the normal collecting pipe can be smoothly inserted into the collecting groove by extruding the O-ring by utilizing the elasticity of the O-ring.

The comparison can be carried out through experiments of spore production quantity statistics: the portable outdoor spore separator has better collection performance than the spore collection mode of the traditional freezing tube method, and researches show that the separator device has better collection stability on spores and more total collected spores. Although the traditional spore collection method allows aphids and culture fluid to be almost in close contact, which ensures that as much as possible of the spores that are bounced by fungi are collected, the collection tube is too closed, which is detrimental to the growth of the fungi and further affects the spores. The upper cover disc of the spore separator is a certain distance from the collecting pipe, so that an environment which is more favorable for fungal growth is provided for the closed space.

From a statistical analysis of the sterility, it can be derived that: the sterility of the portable outdoor spore separator disclosed by the invention is not remarkably different from that of the traditional method, but the traditional mode has great operation limitation, separation experiments need to be carried out by means of sterile equipment such as an ultra-clean workbench, and the separator device is simple and compact in structure, convenient to carry and easy to operate, can be used outdoors anytime and anywhere, greatly improves the working efficiency and is wider in application. Meanwhile, the separator device has a plurality of functions which are not possessed by the traditional collection method, and the separator device can realize a plurality of functions of sterilization, collection culture and the like. The separator device has great practical application value, openness, popularity and low cost, has great market prospect, and can provide key equipment support for many industries such as biological control and the like.

Claims

1. The portable outdoor spore separator comprises a sterilizing groove, an upper cover disc and a collecting hole arranged in the separator, wherein the upper cover disc covers the sterilizing groove.

2. The portable outdoor spore separator as recited in claim 1, further comprising a lower valve disc disposed between the sterilization well and the upper cover disc, the collection well being disposed in the lower valve disc, the lower valve disc further having a sterilization well, the collection well and sterilization well being disposed through the lower valve disc, the storage well being in communication with the evaporation well when the storage well is disposed above the evaporation well.

3. The portable outdoor spore separator as recited in claim 2 further comprising a middle valve disc disposed between the lower valve disc and the upper cover disc, the middle valve disc having a passage extending therethrough, the reservoir being in communication with the passage and the sterilization well and being positioned above the volatilization well, or the reservoir being in communication with the passage and being positioned above the collection well.

4. The portable outdoor spore separator as recited in claim 3, wherein the sterilization tank, the lower valve disc, the middle valve disc and the upper cover disc are cylinders, a sterilization hole and a collection hole are formed in a circular surface equal division radiation position of the lower valve disc cylinder, a hole channel is formed in a circular surface equal division radiation position of the middle valve disc cylinder, a sample storage tank is formed in a circular surface equal division radiation position of the upper cover disc cylinder, and distances from a circle center of the sterilization hole and the collection hole to a center point of a circular surface of the lower valve disc, a distance from a circle center of the hole channel to a center point of a circular surface of the middle valve disc, and a distance from a circle center of the sample storage tank to a circle center of a circular surface of the upper cover disc are equal.

5. The portable outdoor spore separator as defined in claim 4, further comprising: the valve further comprises a stud bolt, wherein a central hole is formed in the central position of the cylindrical round surface of the lower valve disc, the central valve disc and the upper cover disc, and the stud bolt is inserted into the central hole to assemble the lower valve disc, the central valve disc and the upper cover disc together.

6. The portable outdoor spore separator as claimed in one of claims 1 to 5, characterised in that: a timer is also installed.

7. The portable outdoor spore separator as claimed in one of claims 1 to 5, characterised in that: a temperature controller is arranged at the bottom of the sterilizing tank.

8. The method for collecting spores using the portable spore separator of claim 1, comprising the steps of:

(1) Volatile disinfectant is filled in the disinfection tank, and the upper cover plate is sterilized;

(2) An adhesive material is arranged in the sample storage groove of the upper cover disc, and a disease-sensitive organism is adhered in the sample storage groove;

(3) Covering the upper cover disc on the sterilizing groove, so that the sample storage groove is positioned above the volatilizing hole of the sterilizing groove, and sterilizing;

(4) After sterilization, the sample storage groove is positioned above the collecting hole, a collecting pipe containing a sterilization culture medium is arranged in the collecting hole, and under proper conditions, fungus spores in the infected organism are ejected into the collecting pipe;

(5) And after the collection is finished, taking out the collecting pipe in the collecting hole for standby.

9. A method of collecting spores using the portable spore separator of claim 2, comprising the steps of:

(1) Volatile disinfectant is filled in the disinfection tank, and the lower valve disc and the upper cover disc are sterilized;

(3) Placing a collecting pipe containing sterile culture medium in a collecting hole of a lower valve disc, rapidly covering an upper cover disc, and enabling a sample storage groove of the upper cover disc to be communicated with a sterilizing hole of the lower valve disc for sterilization;

(4) After sterilization, the sample storage groove is communicated with the collecting hole of the lower valve disc, and under proper conditions, fungus spores in the infected organism are ejected into the collecting pipe;

10. A method of collecting spores using the portable spore separator of claim 3, comprising the steps of:

(1) Volatile disinfectant is filled in the disinfection tank, and the lower valve disc, the middle valve disc and the upper cover disc are sterilized;

(3) Placing a collecting pipe containing sterile culture medium in a collecting hole of a lower valve disc, rapidly covering an upper cover disc, and communicating a sample storage groove of the upper cover disc, a pore canal of a middle valve disc and a sterilization hole of the lower valve disc, so as to sterilize;

(4) After sterilization, the middle valve disc and the upper cover disc rotate together to be communicated with the sample storage groove, the pore canal and the collecting hole, and under proper conditions, fungus spores in the infected organism are ejected into the collecting pipe;

(5) After the collection is finished, the upper cover disc and the middle valve disc rotate for a certain angle again, so that the collecting pipe is in a closed state, the upper cover disc and the middle valve disc are removed, and the collecting pipe is taken out for standby.

11. The method according to any one of claims 8 to 10, further comprising the step of replacing the volatile sterilizing liquid in the sterilizing tank with sterile water between step (3) and step (4).