CN207330932U - The outer spore separator of household portable - Google Patents

Abstract

The utility model proposes a brand-new portable outdoor spore separator and a separation method. The spore separator at least includes a disinfection tank, an upper cover plate and a collection hole arranged in the separator, and the upper cover plate is covered on the disinfection tank. The upper opening of the disinfection tank is a liquid volatilization hole, the upper cover plate is provided with a sample storage tank, and the sample storage tank is located above the volatilization hole or above the collection hole. The separator and separation method described in the utility model can separate fungal spores under aseptic conditions, avoiding the equipment requirements that traditional methods need to be operated in an ultra-clean workbench, and is a portable outdoor spore separator that is easy to carry, compact and easy to operate , to bring convenience for outdoor collection of pathogenic fungi.

Description

Portable Outdoor Spore Separator

technical field

The utility model relates to the field of separation and collection of strains, in particular to a spore separation collector suitable for unclean environments.

Background technique

In the research process of fungi, the isolation and expansion of strains are very important. Strain isolation is to separate the target fungi from the mixed microorganisms, and then purify and cultivate the isolated target bacteria in a suitable environment. In order to avoid contamination, the separation work is required to be carried out under sterile conditions. At present, isolation culture needs to be carried out in a sterile room, a sterile box or a sterile workbench (ultra-clean workbench). Sterile rooms and aseptic boxes should be dedusted by spraying and disinfected with drugs or ultraviolet radiation. Commonly used disinfectants are sprays such as 70% alcohol, 2% creol soap, and 5% carbolic acid. It takes 20-30 minutes to irradiate with ultraviolet light.

Taking Entomophthorales as an example, Entomophthorales belong to the subphylum Entomophthoromycotina and are widely distributed. There are reports about Entomophthorales all over the world. The fungi of the order Entomomycetes are mostly obligate pathogens of terrestrial arthropods, mainly insects, and a few species infect nematodes and other low-level invertebrates. Many entomogenous fungi are capable of controlling pests by causing high-intensity epidemics in insect populations. However, the investigation of Entomomycetes fungi often requires the infected aphid corpses to be brought back to the laboratory for isolation and identification. In addition, Entomomycetes fungi will eject conidia in a relatively short period of time, and conidia are not easy to preserve. Aseptic operation cannot be guaranteed outdoors, so the current traditional method of relying on laboratory ultra-clean workbench separation cannot be applied to outdoor collection, which brings great obstacles to the in-depth study of Entomomycetes fungi.

Utility model content

The utility model is based on the problems mentioned above, and proposes a separator that is simple in structure, small and exquisite, easy to carry, easy to operate and can aseptically collect fungal spores outdoors.

The utility model provides a portable outdoor spore separator, which includes a disinfection tank, an upper cover plate and a collection hole arranged in the separator, the upper cover plate can be covered on the disinfection tank, and the upper opening of the disinfection tank is a liquid volatilization hole , There is a sample storage tank on the upper cover plate, and the sample storage tank can be located above the volatilization hole or above the collection hole.

Further, it also includes a lower valve plate arranged between the disinfection tank and the upper cover plate, the collection hole is arranged on the lower valve plate, and the lower valve plate is also provided with a sterilization hole passing through the lower valve plate, and the sample storage tank can be located at the sterilizer. The top of the bacteria hole is connected with the volatilization hole or the sterilization hole is located above the collection hole.

Further, a middle valve disc is arranged between the lower valve disc and the upper cover disc, and there is a hole through the middle valve disc, and the sample storage tank can communicate with the hole and the sterilization hole and be located above the volatilization hole, or store The sample tank communicates with the channel and is located above the collecting hole.

Preferably, the disinfection tank, the lower valve disc, the middle valve disc and the upper cover disc are cylinders, and the circular surface of the lower valve disc cylinder is provided with sterilization holes and collection holes at the circular surface of the lower valve disc cylinder. There is a hole at the equally divided radiation part, and a sample storage tank is set at the equal parted radiation part of the cylinder of the upper cover plate. The distance between the center point of the disc disc, the distance from the center of the sample storage tank to the center of the disc disc of the upper cover, the above three distances are equal.

The number of further lower valve disc sterilization holes and collection holes is at least three, and the sterilization holes and collection holes are arranged at intervals, the number of holes in the middle valve disc is at least three, and the number of sample storage slots on the upper cover disc At least six.

Further, it also includes a stud bolt, and a central hole is opened in the center of the cylindrical circular surface of the lower valve disc, the middle valve disc and the upper cover disc, and the stud bolt is inserted into the center hole to connect the lower valve disc, the middle valve disc Assemble together with the upper cover plate.

Further, the cylinders of the lower valve disc, the middle valve disc and the upper cover disc are processed into concavo-convex structures that can cooperate with each other.

Further, it also includes positioning holes and positioning pins 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 holes are set at the corresponding positions of the above three discs.

Further, marking marks are provided on the disc edges of the lower valve disc, the middle valve disc and the upper cover disc.

In order to facilitate the replacement of the 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-ring can also be embedded on the inner side wall of the collecting hole.

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

The disinfection tank can be used as the base of the spore separator. In another form, the spore separator also includes a collection tank, which is independent from the disinfection tank and assembled together in a detachable manner, and serves as a separator base.

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

The utility model provides a method for collecting spores by using a portable spore separator, comprising the following steps:

Put volatile disinfectant in the disinfection tank, and sterilize the upper cover plate; set viscose material in the sample storage tank of the upper cover plate, and stick the susceptible organisms in the sample storage tank; cover the upper cover plate On the disinfection tank, make the sample storage tank above the volatilization hole of the disinfection tank, and sterilize; after the sterilization, make the sample storage tank above the collection hole, and place a sterile medium collection tube in the collection hole Under certain conditions, the fungal spores in the susceptible organisms are ejected into the collection tube; after the collection is completed, the collection tube in the collection hole is taken out for use.

The utility model also provides a collection method using a portable spore separator with a lower valve disc, which includes the following steps: put volatile disinfectant in the disinfection tank, and sterilize the lower valve disc and the upper cover disc; Set the viscose material in the sample storage tank of the cover plate, and stick the susceptible organisms in the sample storage tank; place the collection tube containing the sterile medium in the collection hole of the lower valve plate, quickly cover the upper cover plate, Make the sample storage tank of the upper cover plate communicate with the sterilization hole of the lower valve disc, and sterilize; The fungal spores are ejected into the collection tube; after the collection is completed, take out the collection tube in the collection hole for use.

The utility model also provides a collection method using a portable spore separator with a lower valve disc, a middle valve disc and an upper cover disc. The valve disc and the upper cover disc are sterilized; the viscose material is set in the sample storage tank of the upper cover disc to stick the susceptible organisms in the sample storage tank; the collection tube containing the sterile medium is placed in the lower valve disc Quickly cover the upper cover plate in the collection hole of the upper cover plate, so that the sample storage tank of the upper cover plate, the hole channel of the middle valve plate and the sterilization hole of the lower valve plate are connected to perform sterilization; after the sterilization is completed, the middle valve plate and the upper cover The discs rotate at a certain angle together to connect the sample storage tank, the channel and the collection hole of the lower valve disc. Under suitable conditions, the fungal spores in the susceptible organisms are ejected into the collection tube; after the collection is completed, the upper cover disc and the middle valve disc Rotate again at a certain angle so that the collection pipe is in a closed state, remove the upper cover plate and the middle valve plate at the same time, take out the collection pipe, and set it aside.

Further, it also includes replacing the volatile disinfectant in the disinfection tank with sterile water after the organism is sterilized.

Further, after the spores are collected, the sterile water in the disinfection tank is replaced with a volatile disinfectant.

The beneficial effects of the utility model are: in the process of separating and collecting spores outdoors, the volatile disinfectant stored in the disinfection tank of the separator described in the utility model is used to ensure that the space inside and near the spore separator is one relatively sterile environment. The lower valve disc of the portable outdoor spore separator is equipped with collection holes and sterilization holes in equal parts, in which the collection holes for placing spore collection tubes and the sterilization holes forming fluid channels are arranged at intervals; the middle valve disc is equally divided into radiation distribution The holes in the lower valve disc have an equally divided angle twice that of the lower valve disc, and the number of channels is the same as the collection holes and/or sterilization holes; the distribution angle of the holes in the lower valve disc in the disc is different from that of the middle valve disc. The rotation realizes the formation of the fluid channel and the cutting off of the fluid channel, which is similar to the role of a valve.

The spore separator changes the traditional method of separating the original laboratory ultra-clean workbench, and realizes the efficient and convenient collection of pathogenic fungi from the corpses of susceptible aphids in a short period of time under the condition of ensuring sterility. spore. Moreover, the portable outdoor spore separator is small and does not take up space, and is easy to carry; the structure is simple, the operation method is simple; the separation effect is good, and the waste of resources caused by excessive investment and idle equipment is avoided. It is practical and easy to promote.

Description of drawings

Fig. 1 is the portable outdoor spore separator comprising disinfection tank, lower valve disc, middle valve disc and upper cover disc.

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

Fig. 3 is a schematic diagram of a disinfection tank including a liquid injection port and a liquid discharge port.

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

Fig. 5 is a schematic front view of the lower valve disc shown in Fig. 1 .

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

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

Fig. 8 The front view of the middle valve disc described in Fig. 1 .

Fig. 9 is a cross-sectional view along the direction A-A of Fig. 8 .

FIG. 10 is a perspective view of the upper cover plate shown in FIG. 1 .

Fig. 11 is a front view of the upper cover plate in Fig. 1 .

Fig. 12 is a sectional view along the direction of Fig. 11A-A.

Figure 13 shows the daily average shellfish counts for cryovials and spore separators. When the two have the same lowercase letters, it means there is no significant difference, and different lowercase letters mean there is a significant difference; when there are different uppercase letters, it means there is a very significant difference, and the same uppercase letters mean there is no significant difference.

Figure 14 shows the average total sporulation per aphid in the cryopreservation tube and spore separator.

Figure 15 is an experimental diagram of the sterility analysis of the separator.

Figure 16 is a comparison of the sterility rate of the traditional method and the spore separator method.

Figure 17 is a portable spore separator comprising a disinfection tank, a lower valve disc and an upper cover disc.

Fig. 18 is a portable spore separator including a disinfection tank and an upper cover tray.

Fig. 19 is a schematic diagram of a collection tube placement rack arranged in the disinfection tank.

Fig. 20 is a schematic diagram of putting the collection tube into the rack shown in Fig. 19 .

Figure 21 is a schematic diagram of the connection between the disinfection tank and the collection tank through slide rails and chute.

Fig. 22 has the lower valve disc of two sterilization holes and two collecting tanks, the middle valve disc and the upper cover disc matched therewith.

Fig. 23 is the portable outdoor spore separator that is provided with timer on the loam cake.

Figure 24 is a lower valve disc with an O-ring in the collecting hole.

Detailed ways

Below in conjunction with specific accompanying drawing, the utility model is described in detail. These specific embodiments are only limited enumerations without violating the spirit of the utility model, and do not exclude other specific implementations produced by those skilled in the art by combining the prior art with the utility model.

Embodiment 1 A kind of structure of portable spore separator

The portable outdoor spore separator as shown in Figures 1 to 12 includes a disinfection tank 1, a lower valve disc 2, a middle valve disc 3 and an upper cover disc 4, which are detachably assembled together from bottom to top.

Disinfection tank 1 is used to hold volatile disinfection and sterilization liquid or sterile water, and is located at the bottom of the separator as a supporting device of the portable outdoor spore separator.

The lower valve disc 2 is a cylinder and is provided with a through hole of a certain diameter at the position of the sextile radiation on the circular surface 21 of the cylinder. Three of the through holes are sterilizing holes 22, and the sterilizing holes can be used as a volatilization passage for the volatile sterilizing liquid to collect biological samples (such as aphid corpses) and the lower valve disc and the middle valve disc installed on the disinfection tank. The fluid channels formed by assembling the three plates with the upper cover plate are sterilized. The other three through holes of the lower valve disc are collection holes 23, which are used to place and fix the collection pipe 9 that is equipped 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 and is provided with a hole 32 of a certain diameter at the trisecting radiation position on the cylindrical surface 31, and the distance between the center of the hole 32 and the center point of the middle valve disc disc is the same as that of the lower valve disc. The distance between the center point of the sterilization hole and the collection hole is the same as the center point of the lower valve disc circular surface. The middle valve disc plays the role of sealing and isolation.

The upper cover plate 4 is a cylinder and is provided with a groove 42 on the lower surface of the cylinder circular surface 41, and a sample storage groove 43 is provided at the circular surface at the bottom of the groove to divide the radiation distribution position into three equal parts and is used for storing collected samples. organism. The distance between the center of the hole of the sample storage tank 43 and the center point of the upper cover disc is the same as the distance between the center of the circle of the lower valve disc's sterilization hole and the collection hole and the center point of the lower valve disc disc. In an embodiment of manufacturing the upper cover plate, firstly, a small cylinder with a diameter and height slightly smaller than the cylinder is dug out in the upper cover plate cylinder to form a groove 42 . Three small circular grooves with a certain diameter and depth are processed at the positions where the radiation distribution is trisected on the circular surface at the bottom of the groove as sample storage grooves 43 for storing biological samples. In this embodiment, the sample storage groove 43 does not penetrate through the upper cover plate. In a preferred embodiment, the sample storage tank of the upper cover tray has an adhesive layer.

In order to increase the speed of spore counting, in another solution, a sample storage tank 43 is provided at the position of the radiation distribution on the circular surface at the bottom of the groove 42 of the upper cover plate 4 to store the collected organisms. Three of the spaced sample storage tanks are used for spore collection, and the other three spaced sample storage tanks are used for spore counting, so that the samples in the six sample storage tanks can be used for counting, and the counting speed is doubled.

The materials of the disinfection 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 and non-deformable plastic materials and the like. Stainless steel is preferred. For example, the lower valve disc 2, the middle valve disc 3 and the upper cover disc 4 are all processed on the basis of a cylindrical stainless steel material with 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 holes 32 of the middle valve disc 3 communicate with the sterilization holes 22 of the lower valve disc 2 to form a fluid channel, the volatile sterilizing liquid in the disinfection tank is placed in the sample storage tank 43 of the upper cover plate 4 through the fluid channel pair Sterilize the biological samples (such as aphid corpses), the fluid channel itself and the upper cover and other devices. The sterilization hole 22 of the lower valve disc 2 is not connected with the middle valve disc hole 32, but the collection hole 23 where the collection pipe is placed on the lower valve disc is connected with the middle valve disc hole 32. The three of the sample tanks 43 are connected, and under the action of gravity, the fungi carried by the biological sample eject spores and smoothly fall into the collection tube that is placed in the collection hole 23 of the lower valve disc and is equipped with a sterile medium.

The disinfection tank 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 together, for example, by means of screw threading, buckle 52 fastening and the like. In one embodiment, the disinfection tank and the lower valve disc are combined together by threading.

In this embodiment, the central hole 51 with a certain diameter and a thread is drilled in the center of the cylindrical surface of the lower valve disc 2, the middle valve disc 3 and the upper cover disc 4, and the stud bolt 5 can be inserted in the central hole. The effect of the stud bolt is to assemble the upper cover plate, the middle valve plate and the lower valve plate into a whole. The length of the stud is determined by the thickness of the upper cover plate, the middle valve disc and the lower valve disc when they are assembled together, and the diameter of the stud is adapted to the diameter of the center hole of each disc. In a preferred embodiment, the stud bolt has a length of thread only at the beginning end.

In order to allow the parts of the lower valve disc 2, the middle valve disc 3 and the upper cover disc 4 to be tightly embedded with each other, in a more optimal embodiment, each cylinder of the above three discs is processed with concavities and convexities that can cooperate with each other. structure. For example, the lower valve disc 2 includes a protrusion 25 ; the middle valve disc 3 includes a recess 34 and a protrusion 35 ; the upper cover disc 4 includes a recess 44 . When the three discs are assembled together, the convex portion 25 of the lower valve disc fits into the concave portion 34 of the middle valve disc 3 , and the convex portion 35 of the middle valve disc fits into the concave portion 44 of the upper cover disc.

When the sterilization hole 22 of the lower valve disc communicates with the hole 32 of the middle valve disc and the upper cover plate to form a fluid channel, the separator is in a sterilized state; When the sample storage tank 43 of the upper cover plate is connected to form a fluid channel, the separator is in the state of collecting spores. That is to say, the spore separator described in the utility model can realize the spore collection in an aseptic state after the holes of the lower valve disc, the middle valve disc and the upper cover disc of the separator are sterilized outdoors. In this embodiment, the upper cover plate, the middle valve plate and the lower valve plate are rotated to realize the conversion of the sanitation state or the collection state of the separator. In order to allow the operator to control the rotation angles of the three discs more intuitively and effectively, the separator also includes a positioning device to ensure that the angle of each rotation is a fixed value, thereby ensuring that the sterilization hole or collection hole and the hole channel and the sample storage There is a smooth communication between the slots. The positioning device is selected but not limited to the following methods: the cooperation of the positioning hole 6 and the positioning pin 7, the mark and the score indication 8, etc.

In the embodiment where the positioning hole and the positioning pin are matched, positioning holes 6 are provided at the corresponding positions of the upper cover plate, the middle valve plate and the lower valve plate. Whether the positioning pin 7 can be completely inserted into the positioning holes 6 on the three plates To judge whether the rotation angle of the three discs is in place. As shown in Figures 1 and 2, at least two positioning holes 6 are provided on the lower valve disc, which are respectively the positioning holes in the disinfection state and the positioning holes in the spore collection state, and at least one positioning hole is respectively provided on the upper cover disc and the middle valve disc. The positioning holes of the upper cover plate and the middle valve plate are aligned with the sterilized positioning holes of the lower valve plate, and the positioning pins can be inserted into the three positioning holes smoothly. The inventory sample tank is completely aligned to form a fluid channel, which can be disinfected; the positioning holes of the upper cover plate and the middle valve plate are aligned with the spore collection state positioning holes of the lower valve plate, and the positioning pins can be smoothly inserted into the three positioning holes. The collection hole of the valve disc is aligned with the hole channel of the middle valve disc and the sample storage groove of the upper cover disc, so that spores can be collected.

In the embodiment of marking marks, at least two marking marks 8 are provided on the edge of the lower valve disc, and at least one marking mark 8 is provided on the edges of the upper cover disc and the middle valve disc. A marking mark on the lower valve disc is the marking mark of the disinfection state. When the marking marks on the upper cover plate and the middle valve disc are aligned with the marking marks on the lower valve disc, the sterilization hole of the lower valve disc is aligned with the marking mark of the middle valve disc. When the hole of the valve disc and the upper cover plate are connected to form a fluid channel, the separator is in a sterile state; the other marking mark on the lower valve disc is the marking mark of the collection state. When the disinfection status marks on the lower valve disc are aligned, and when the collection hole of the lower valve disc communicates with the channel of the middle valve disc and the sample storage tank of the upper cover disc to form a fluid channel, the separator is in the state of collecting spores.

In a more preferred example, the separator includes a positioning hole and a positioning pin, and marking marks are provided on the edge of the disk corresponding to the positioning hole. On the one hand, the combination of positioning holes and positioning pins ensures the accuracy of the rotation angles of the three disks. On the other hand, the marking marks make it easier for the experimenter to observe the direction and angle of rotation. In a preferred solution, the words sterilized or collected can also be marked on the marked notch to remind the experimenter of the current state of the separator.

Embodiment 2 Utilizes the method for collecting spores by the portable spore separator described in embodiment 1

Take the collection of Entomycetes fungal spores as an example for specific illustration. All operations must be performed while wearing masks and disposable rubber gloves.

Use 75% alcohol or other sterilizing solution to firstly carry out disinfection treatment to components such as the lower valve disc 2, the middle valve disc 3, and the upper cover disc 4.

A certain amount of volatile sterilizing liquid is packed into disinfection tank 1, obtain susceptible aphids with a sterilized sampling stick (for example, get the susceptible aphids with a writing brush that has been sprayed with 75% alcohol), and the susceptible aphids (aphids) body) is fixed in the sample storage groove 43 at the top spacer in the upper cover tray. The method of immobilizing the susceptible aphids can be achieved by means of double-sided tape or the adhesive layer that comes with the sample storage tank.

The lower valve disc 2 is installed on the disinfection tank 1. After disinfection for several minutes, the spore collection tubes (not shown) that are equipped with sterile medium (such as sterile egg yolk medium) are placed in the three collecting tubes arranged at intervals. In the hole 23, quickly assemble the sterilized middle valve disc 3, keep the sterilization hole 22 of the lower valve disc 2 communicated with the hole 32 on the middle valve disc 3, that is, the collecting pipe is in a closed state. Finally, the upper cover plate 4 is installed on the middle valve plate, and the sample storage tank equipped with aphid corpses is connected with the sterilization hole of the lower valve plate and the channel of the middle valve plate, and sterilized for 1 min. The spore collection tube can be a test tube or a concave bottom container or the like.

After the above-mentioned sterilization is completed, keep the upper cover disc 4 and the middle valve disc 3 to rotate synchronously, and rotate 60 degrees together. The collection pipes in the collection holes 23 communicate. The separator was placed overnight in a constant temperature incubator at 20°C, waiting for the fungal shell spores, which would fall into the collection tube containing the sterile medium under the action of gravity.

After placing overnight in the incubator, the upper cover disc 4 and the middle valve disc 3 are rotated 60 degrees synchronously. At this time, the sample storage tank of the upper cover disc is connected with the sterilization hole of the lower valve disc and the channel of the middle valve disc, and then Remove the upper cover plate and the middle valve plate in turn, take out the collection tube in the lower valve plate, and harvest the spores in the collection tube for later use.

So far, the purpose of collecting fungal spores in non-clean workbenches has been achieved. The role of the disinfection tank in the whole operation is to use the volatile disinfectant in it to make the space where the separator is located a relatively sterile environment, which is similar to the sterile environment provided around the flame of the alcohol lamp.

Maintaining the ambient humidity throughout the separator will be more conducive to the cultivation of spores. Therefore, in a preferred solution, the sample storage tank containing aphid corpses is connected with the sterilization hole of the lower valve disc and the hole of the middle valve disc. Rotate 60 degrees together, now the sample storage tank 43 that is stuck with aphids on the upper cover plate communicates with the collection pipe in the middle valve plate hole 32 and the lower valve plate collection hole 23. Afterwards, the volatile disinfectant in the disinfection tank is replaced with sterile water to maintain the ambient humidity of the entire device during constant temperature cultivation. After the separator is placed in the constant temperature incubator overnight, replace the sterile water in the disinfection tank 1 with a volatile disinfectant, then rotate the upper cover plate 4 and the middle valve plate 3 synchronously by 60 degrees, and remove the upper cover plate in turn , in the valve disc, remove the collection tube, and harvest the spores. The replacement of the disinfectant solution and sterile water in the disinfection tank can be completed directly from the upper opening of the disinfection tank by suction and injection. In the embodiment of the disinfection tank as shown in Figure 3, the upper part of the disinfection tank is provided with a liquid injection port 11, and the lower part is provided with a liquid discharge port 12, and both the liquid injection port and the liquid discharge port are sealed by an openable seal 13. In a specific replacement method, after the disinfectant in the disinfection tank sterilizes the components of the separator, the seal 13 at the liquid discharge port 12 is opened to discharge the volatile disinfectant in the disinfection tank, and then the seal is used to seal the drain again mouth. Then open the sealing member 13 of the liquid injection port 11, inject aseptic water from the liquid injection port as required, and seal the liquid injection port with the sealing member again after the liquid injection is completed.

The sterile water in the disinfection tank provides a certain humidity environment for the fungal shell spores, and the daily number of shell spores changes relatively uniformly without a sudden extreme drop. When collecting susceptible aphids in the wild, it is impossible to replace new medium every once in a while to collect spores. Therefore, the performance of the separator described in this utility model actually prolongs the possibility of separating pure fungi from the time dimension. sex. In this way, the spore separator of the utility model not only ensures the airtightness not disturbed by the outside world, but also provides a humidity requirement suitable for the growth of fungi.

The comparative experiment of embodiment 3 sporulation quantity

The comparative test of the sporulation number index adopts the two collection methods of portable spore separator collection and cryopreservation tube collection described in Example 1, and counts the sporulation number in the two collection methods. Statistical methods are used to analyze the difference in the number of spores in the two modes, and then determine the feasibility of the collection mode of the outdoor spore separator described in the utility model. Select 30 susceptible aphids as test objects for statistical analysis, that is, for each of the two methods, randomly select 30 susceptible aphids of similar size for the test.

Utilize the spore separator described in Example 1 and the installation method of each plate of Example 2, the susceptible aphids collected are fixed in the sample storage tank in the upper cover plate of the separator with double-sided tape, and the concave bottom container is placed On the collection hole of the lower valve plate of the separator, add 250 μL of 0.5% SDS solution to the concave bottom container in advance, so that the susceptible aphids correspond to the concave bottom container one by one. Then, the separator was placed in a light incubator (20°C, 12L:12D) for cultivation, and the concave-bottomed container was replaced every 24 hours until the susceptible aphids were no longer shellfish. The spore liquid in each concave bottom container was observed under an optical microscope by the method of a hemocytometer and the spores in 7 0.1 μL liquid samples were counted. There are 25 grids in the blood cell counting area, and only 5 grids are randomly selected for counting each time. The liquid volume of 5 grids is 0.02ul, so when calculating the number of spores in the concave bottom container, a coefficient k=5*10 ⁴ needs to be multiplied. Add up the number of ejected spores every day to get the total sporulation of the aphid.

The collection method of the cryopreservation tube is: cut off a part of the cryopreservation tube body so that the cover part is closer to the bottom, add 200 μL of 0.5% SDS solution to the bottom, and fix the collected susceptible aphids on the center of the cover with double-sided tape. After the cover is covered, the worm body and the reagent fit closer together and cannot be in contact with the surface of the reagent solution, so that as many spores ejected from the aphids as possible can be collected. Then, culture all the spore collection cryopreservation tubes in a light incubator (20°C, 12L:12D), and replace the bottom cryopreservation tubes every 24 hours until the susceptible aphids no longer have shellfish. The spore liquid in each cryopreservation tube was observed under an optical microscope by the method of a hemocytometer and the spores in seven 0.1 μL liquid samples were counted. There are 25 grids in the blood cell counting area, and only 5 grids are randomly selected for counting each time. The liquid volume of the 5 grids is 0.02 μL, so when calculating the number of spores in the cryopreservation tube, a coefficient k=5*10 ⁴ needs to be multiplied. Add up the number of ejected spores every day to get the total sporulation of the aphid.

According to the continuous counting of susceptible aphids with a sample size of 30, the results of statistical analysis of the data are shown in Figure 13 and Figure 14.

Figure 13 is obtained by carrying out the t test on the average spore number of 30 samples in the daily cryopreservation tube and separator of the shellfish time series, as can be seen from Figure 13, the first day, the second day, and the third day There was no significant difference in the number of spores collected in the cryopreservation tube and the separator, but there was a very significant difference in the number of spores collected in the cryopreservation tube and the separator on the fourth day and the fifth day. This performance of the separator wins time for the field collection of pathogenic fungi, which is of great practical application value.

Figure 14 shows the cumulative spore production per day, and the fifth day is the total amount of spore production for all samples. The difference between the number of sporulations collected by the two methods is not large. It can be seen from Figure 14 that in the first and second days, the number of shell spores per day, although the number in the cryopreservation tube is more than that in the separator, but There is no significant difference between the two from a statistical point of view, and there is no significant difference in the number of spores between the two on the third day, but starting from the third day, the number of spores in the separator is more than that in the cryopreservation tube. There was a very significant difference between the two in five days, and the final cumulative result was that the total shellfish quantity collected in the separator was more than that in the cryopreservation tube. The spores collected by the cryopreservation tube were slightly more sporulated in the early stage, but the total number was small. The spores collected by the separator method were slightly less sporulated in the early stage, but the total amount of sporulation was more. From the results of the total number of spores, it also confirmed that Separators have better collection performance. Combining the results of Fig. 13 and Fig. 14 shows that the separator method described in the utility model is better than the traditional cryopreservation tube method.

Embodiment 4 separator sterility verification

The sterility evaluation index mainly judges the feasibility of the separator collection method through the statistical analysis of the number of sterile samples in the collection results under the two collection methods of the separator collection method and the traditional collection method. 30 susceptible aphids were selected as test subjects.

It is very difficult to cultivate P. neoaphidii under ordinary conditions, because P. neoaphidii is an obligate entomopathogenic fungus, and this obligate pathogenic fungus has high requirements for living environment and nutrition, so we adopt Sabouraud culture base and add egg yolk and milk for isolation and in vitro culture.

Traditional collection methods:

1. Wipe the surface of the ultra-clean workbench with alcohol, and then sterilize it with ultraviolet light for 30 minutes;

2. Cut out a small piece of double-sided tape and paste it on the center of the inner surface of the petri dish cover;

3. Use a writing brush sterilized with 75% alcohol to take a susceptible aphid, stick it on the double-sided tape, and then hold the dish cover and place it above the disinfection tank filled with disinfectant for disinfection. After 1 minute, Place the dish lid on the petri dish filled with egg yolk culture medium;

4. After overnight storage in the incubator at 20°C, replace the lid of the dish with the dead aphid with a new sterile lid, and continue to place it in the incubator for several days;

5. After several days of continuous observation, check whether the medium is contaminated with bacteria, and record the results.

The collection mode of the spore separator described in the utility model:

1. On the desktop of the test bench, the disinfection tank contains volatile disinfectant, and sterilizes the lower valve disc, middle valve disc and upper cover disc in the separator with 75% alcohol;

2. Paste double-sided tape on the three interval sample storage slots in the upper cover plate, pick up the susceptible aphids with a brush, and stick the susceptible aphids on the double-sided tape;

3. Place the pre-configured egg yolk culture medium in the concave bottom container at intervals in the collection hole of the lower valve plate, quickly cover the middle valve plate to keep the concave bottom container in a closed state (the volatile disinfectant in the bottom disinfection tank creates a sterile space);

4. Cover the upper cover plate so that the holes of the lower valve plate and the middle valve plate are connected and the holes are facing the aphid corpse of the upper cover plate;

5. After sterilizing for 1 minute, the middle valve disc and the upper cover disc rotate 60° together, and at this time, the susceptible aphids are in a state of communication with the concave bottom container of the lower valve disc;

6. Sterilize for 1 minute;

7. Put the separator in an incubator at 20°C, and after standing overnight, the middle valve disc and the upper cover disc rotate 60° together, and the concave bottom container is in a closed state at this time;

8. Take out the separator and put it on the test bench, remove the upper cover plate and the middle valve plate, quickly remove the concave bottom container and put it into a disposable petri dish, and seal the petri dish with parafilm;

9. Place the culture dish in an incubator at 20°C for cultivation;

10. Check the contamination of the medium during the cultivation process and make records.

Instructions for identification of sterility. The definition of sterility should include two parts: one is that the medium is isolated to a single colony, and the other is that the medium remains sterile, that is, no bacteria grow on the medium, because in actual situations it is determined whether aphids are susceptible Depending on the experience of the sampler, that is, the aphids that people think are susceptible may not actually be susceptible. Then at this time, the spores of the target fungus cannot be obtained and the spores cannot be isolated. Explain that the separator ensures the sterility in the separation operation.

The experimental results are shown in Figure 15: a is a sample with no change in the culture medium under the traditional separation method, b is a sample of a single colony collected under the traditional separation method, and c is a sample contaminated with bacteria under the traditional separation method; d The d2 and d3 in the figure are the samples with no change in the culture medium under the separation method of the separator, the case in the e picture is a single colony isolated, and the f picture and the d1 picture are the situation of the culture medium itself being contaminated. Figures ab in the traditional method are considered aseptic conditions, and figures d2, d3 and e in the separator method are considered as sterile conditions.

Statistical analysis and evaluation of sterility performance:

A sample size of 30 susceptible aphids was selected as the test object for statistical analysis, that is, 30 susceptible aphids were randomly selected for each of the two separation methods.

The traditional separation method and the spore separator separation method described in the utility model have all carried out three batches in this experiment, and in the first batch, the sterility rate of the traditional method is 96.67%, and the sterility rate of the separator method is 76.67%. %; in the second batch, the sterility rate of the traditional method was 66.67%, and the sterility rate of the separator method was 58.33%; in the third batch, the sterility rate of the traditional 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 Figure 16, the sterility rates in the first, second, and third batches were listed in the form of histograms, and the t-test analysis was carried out with these three sets of data, and the result was that the two separation methods were not significant The difference is that in terms of aseptic performance, there is no difference in the aseptic performance of the spore separator method described in the utility model and the traditional method in separating fungi in the ultra-clean bench.

Embodiment 5 Another portable spore separator described in the utility model

The portable outdoor spore separator as shown in FIG. 17 includes a disinfection tank 1 , a lower valve disc 2 and an upper cover disc 4 . There are through holes at the symmetrical positions of the lower valve disc, one of which is a sterilization hole and the other is a collection hole. One side of the bottom of the upper cover plate is provided with a sample storage tank, and the sample storage tank can be opposite to the through hole of the lower valve disc during use. The disinfection tank 1, the lower valve disc 2 and the upper cover disc 4 are disassembled and assembled together. The assembly method can adopt the method listed in Example 1.

When the sample storage tank of the upper cover plate is connected with the sterilization hole of the lower valve disc to form a fluid channel, the volatile sterilizing liquid passes through the fluid channel to realize the sterilization of the aphid corpses placed at the sample storage tank, the fluid channel itself and the upper cover. Sterilization of devices, etc.; the collection hole of the lower valve plate is connected with the collection tank of the upper cover plate, and the spores ejected by the fungus fall into the collection tube containing the sterile medium under the action of gravity.

The specific collection steps include installing the lower valve plate on the disinfection tank, placing the collection tube containing the sterile medium in the collection hole after disinfection for a few minutes, removing the tube cover and quickly assembling the upper cover plate, so that the aphid-filled The sample storage tank of the body is connected with the sterilization hole of the lower valve plate, and the sterilization is performed for 1 minute. Then the upper cover disc rotates relative to the lower valve disc until the sample storage tank of the upper cover disc communicates with the collection hole of the lower valve disc, and the spores are collected under a constant temperature environment. After the spores are collected, the upper cover plate is separated from the lower valve plate, and the sterile tube cover is put back into the collection tube for use. According to needs, the liquid in the disinfection tank is exchanged between volatile disinfectant and sterile water during the collection process.

Embodiment 6 Another portable spore separator described in the utility model

Portable outdoor spore separator as shown in Figure 18 to 20, comprises disinfection tank 1 and loam cake 4, is provided with placement rack 10 to place the collecting tube that aseptic medium is housed in the disinfection tank, the inner surface of loam cake There is a sample storage tank. After the upper cover plate is assembled on the disinfection tank, the sample storage tank has at least two positions relative to the disinfection tank 1: the disinfection position where the sample storage tank is opposite to the disinfectant in the disinfection tank, and the spores where the sample storage tank is opposite to the spore collection tube Collect bits. More specifically, the cross-sections of the disinfection tank and the upper cover plate are rectangular, the sample storage tank is located on one side of the upper cover plate, and the placement frame 10 is connected with the inner wall of the disinfection tank to divide the disinfection tank into two parts: a disinfection position and a collection position A hole is opened in the middle of the placing frame, and the spore collection tube is inserted into the hole.

When the sample storage tank of the cover plate is connected with the disinfection position of the disinfection tank, the volatile gas of the sterilization liquid can realize the sterilization of the aphid corpses and the cover device in the sample storage tank of the cover plate; The upper cover plate is rotated 180 degrees, and the tube cover of the collection tube is quickly removed to connect the sample storage tank with the collection tube of the disinfection tank, and the spores produced by the fungus fall into the collection tube containing the sterile medium under the action of gravity. After the spores are collected, remove the upper cover plate, cover the sterile tube cover back to the collection tube, and collect the collection tube with spores for use. According to needs, the liquid in the disinfection tank is exchanged between volatile disinfectant and sterile water during the collection process.

Example 7

The separator includes a lower valve disc, a middle valve disc, an upper cover disc, and a disinfection tank 1 and a collection tank 17. As shown in Figure 21, the disinfection tank and the collection tank are cavities that are independent of each other and can be assembled together. The disinfection tank and collection tank are used as the base of the separator. The lower valve disc is assembled on the connected disinfection tank and collection tank in a fastening manner.

In this embodiment, a slide rail 15 is designed on each side wall of the disinfection tank and the collection tank, and a slide groove 16 is arranged on the corresponding other side wall, and the disinfection tank and the collection tank pass through the slide rail and the slide groove. The ground is cooperating with each other and socketed together. In a more optimal design scheme, the chute is arranged inside the side wall, that is, the outer surface 61 of the chute does not protrude outside the side wall, and the outer side wall 51 of the slide rail contacts the outer surface of the chute, when the disinfection tank and the collection When the trough is connected with the chute through the slide rail, the disinfection trough and the collection trough are close to each other, which can save more space.

The separator described in this example can adopt the sterilization and spore collection methods listed in the preceding examples. The culture medium is pre-filled in the collection tank, and the opening of the collection tank is sealed with a sealing film. When the disinfection is completed, tear off the sealing film so that the sample storage tank of the upper cover plate is aligned with the collection tank to collect the spores.

When the base of the spore separator adopts the disinfection tank and collection tank described in the present embodiment, the upper cover plate can also be directly combined with the base, or the upper cover plate and the lower valve plate can be used in combination to assemble on the described base. on the base.

Example 8

The sample storage tank 43 of the upper cover tray in this embodiment is designed as a through hole, and the rest of the structure and sterilization collection method are the same as those of the previous embodiment.

In the collection step of biological samples, cut off the adhesive tape with a slightly larger area than the sample storage tank on the upper cover plate, use a 75% alcohol-sterilized writing brush to pick up the susceptible aphids and transfer the aphids to the center of the adhesive surface of the adhesive tape, Then stick the adhesive tape with aphids on the sample storage tank on the upper surface of the upper cover plate to cover the sample storage tank. The penetrating design of the sample storage tank makes it easier to store aphids in the upper cover tray.

Example 9

The spore separator described in the utility model is also equipped with a timer 55, and the timer can be transported together with the separator, so no matter where the separator is transported, or if an experimenter is changed, it can be directly from the Time information is obtained on the timer of the separator, and after each step such as container sterilization and/or spore collection is completed, the timer will emit a beep to remind the experimenter. In a preferred embodiment, the timer can simultaneously display and set the sterilization start time and/or the countdown to the end of sterilization, the start time of spore collection and/or the countdown to the end of spore collection and other items. In the embodiment shown in Figure 23, the timer is installed on the upper cover plate.

Example 10

A temperature controller can also be installed on the spore separator to control the temperature of the separator at the optimum temperature for spore collection and growth. In the embodiment shown in Figure 18, a temperature controller 53 is provided at the bottom of the stainless steel disinfection tank, and the temperature of the temperature controller can be adjusted to 20° C. or other suitable temperature to keep the separator in a constant temperature culture state. In a preferred solution, thermal insulation materials are 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 between the separator and the external environment.

Example 11

The number and arrangement of the sterilization holes and collection holes of the lower valve disc, the number and arrangement of the holes of the middle valve disc and the number and arrangement of the sample storage tanks of the upper cover plate are not limited to the above embodiments As listed, it can also be, for example, that there are two sterilization holes and collection holes on the lower valve disc and are arranged at intervals in the quartering radiation positions, and the corresponding holes in the middle valve disc or the sample storage tanks in the upper cover disc The quantity is 2 each. As shown in Figure 22, there are 2 sterilization holes and collection holes in the lower valve disc, which are arranged symmetrically in the quartering radiation positions, and the corresponding number of holes in the middle valve disc or sample storage tanks in the upper cover disc are respectively 2. The number of sterilizing hole collection holes and sample storage tanks can be 1 to N according to needs, and N is a number greater than 1.

The shapes of the lower valve disc, the middle valve disc and the upper cover disc are not limited to the circles or squares listed in the above embodiments, and may also be elliptical, polygonal, etc. for example.

There may be processing errors in the spore collection tube, and the diameter of some collection tubes will be slightly larger or slightly smaller than the design size, so that they cannot be smoothly placed in the collection hole or stand upright in the collection hole. Therefore in a preferred embodiment, the inner wall of the collection hole 23 as shown in Figure 24 is also embedded with an O-ring 56, the O-ring has a certain elasticity, and the spore collection tube is inserted into the collection hole with the O-ring In the middle, it can be stuck by the O-ring, especially if the diameter of the spore collection tube deviates, the collection tube that is slightly larger than the normal collection tube can also use the elasticity of the O-ring to squeeze the O-ring and insert it into the collection tank smoothly.

It can be compared and drawn by the experiment of counting the number of spores: the collection performance of the portable outdoor spore separator described in the utility model is stronger than the spore collection method of the traditional cryopreservation tube method, and the research shows that the separator device has better stability for the collection of spores , and the total amount of spores collected is also more. Although the traditional spore collection method makes the aphids and the culture medium almost in close contact, which can ensure that as many spores as possible can be collected by the fungus, but the collection tube is too closed, which is not conducive to the growth of the fungus, and further affects the growth of the spores. . However, there is a certain distance between the upper cover plate and the collecting tube of the spore separator described in the utility model, which provides an environment more conducive to the growth of fungi in the confined space.

Can draw by the statistical analysis of sterility situation: the sterility of portable outdoor spore separator described in the utility model has no significant difference with the sterility of traditional method, but the operation limitation of traditional way is very big, needs Separation experiments are carried out with the help of sterile equipment such as ultra-clean workbenches. The separator device is simple in structure, small in size, easy to carry, easy to operate, and can be used outdoors anytime and anywhere, which greatly improves work efficiency and is more widely used. At the same time, the separator device also has many functions that traditional collection methods do not have. The separator device can take into account multiple functions such as sterilization, collection and cultivation. The separator device of the utility model has great practical application value, is open and popular, and has a low cost and great market prospects, and can provide key equipment support for many industries such as biological control.

Claims (8)

1. the outer spore separator of household portable, including disinfection trough, upper cover plate and the collection hole being arranged in separator, upper cover disk cover In on disinfection trough, it is characterised in that disinfection trough is equipped with evaporation hole, and upper cover plate is equipped with storage sample groove, and storage sample groove is in the separator at least With two positions, storage sample groove is located above evaporation hole or storage sample groove is positioned at collecting above hole, is between disinfection trough and upper cover plate Detachable connection.

2. the outer spore separator of household portable according to claim 1, it is characterised in that further include be arranged at disinfection trough and Lower valve disc between upper cover plate, collects hole and is located in lower valve disc, and lower valve disc also has sterilizing hole, collects hole and sterilizing Kong Jun runs through Lower valve disc is arranged at, when storage sample groove is located above evaporation hole, storage sample groove, evaporation hole, sterilizing hole are connected, disinfection trough, upper cover plate It is detachable connection between lower valve disc.

3. the outer spore separator of household portable according to claim 2, it is characterised in that further include be arranged at lower valve disc and Middle valve disc between upper cover plate, the duct of middle valve disc is provided through on middle valve disc, and storage sample groove can be mutual with duct and sterilizing hole Connect and be located above evaporation hole, or storage sample groove is with hole link and positioned at collection hole top, disinfection trough, upper cover plate, middle valve disc It is detachable connection between lower valve disc.

4. the outer spore separator of household portable according to claim 3, it is characterised in that disinfection trough, lower valve disc, middle valve disc With upper cover plate be cylinder, the disc of lower valve disc cylinder etc. divide at radiation be provided with sterilizing hole and collect hole, middle valve disc cylinder Disc of body etc., which divides, is provided with duct at radiation, disc of upper cover plate cylinder etc., which divides at radiation, is provided with storage sample groove, and sterilize hole With collect the center of circle in hole to the distance to middle valve disc disc central point of distance, the duct center of circle of lower valve disc disc central point, storage sample To the distance in the upper cover plate disc center of circle, the distance of above-mentioned three is equal in the groove center of circle.

5. the outer spore separator of household portable according to claim 4, it is characterised in that further include stud bolt, lower valve disc, The cylinder disc center of middle valve disc and upper cover plate offers centre bore, and the stud bolt is inserted into centre bore, by lower valve Disk, middle valve disc and upper cover plate three fit together.

6. spore separator outside the household portable according to one of claim 1 to 5, it is characterised in that disinfection trough includes note Liquid mouth and leakage fluid dram.

7. spore separator outside the household portable according to one of claim 1 to 5, it is characterised in that be also equipped with timing Device.

8. according to one of claim 1 to 5, the outer spore separator of household portable, it is characterised in that be also equipped with temperature Controller.