Bacterial colony specimen manufacturing method

Abstract

The invention relates to the technical field of specimen preparation, in particular to a bacterial colony specimen preparation method which comprises the following steps: (1) performing bacterial colony culture on a plate culture medium, and performing bacterial colony sterilization when the bacterial colony is cultured to a logarithmic phase or a stable phase; (2) pouring epoxy resin type crystal drop glue onto the sterilized bacterial colony, and removing the plate culture medium and the bacterial colony cultured by the plate culture medium after solidification to obtain a bacterial colony specimen made of an epoxy resin material; (3) placing the bacterial colony specimen in a rollover enclosure frame, fixing the bacterial colony specimen in the middle of a rollover enclosure frame bottom plate, pouring food-grade silica gel to cover the bacterial colony specimen, and demolding after curing to obtain a bacterial colony silica gel mold; and (4) pouring the epoxy resin type crystal drop glue into a bacterial colony silica gel mold, solidifying, and demolding to obtain a bacterial colony specimen in the same form. According to the method, the morphological characteristics of bacterial colonies are re-carved by epoxy resin type crystal glue dripping, bacterial colony specimens which can be stored for a long time are formed, and batch production of the bacterial colony specimens is realized.

Description

Technical Field

[0001] The present invention relates to the technical field of specimen preparation, and in particular to a method for preparing a bacterial colony specimen. Background Art

[0002] Microorganisms, a group of tiny organisms that are difficult to identify with the naked eye, are widely distributed in all corners of nature, such as soil, water, air, and inside and outside living organisms. They are rich in species and numerous in number, closely linked to human life, and play an indispensable role in many fields such as food fermentation, medical pharmaceuticals, and environmental protection. The colonies formed by microorganisms, with their unique morphology and texture, carry key information such as microbial species and growth characteristics. They are extremely valuable both in microbial identification and in ecological research. For example, in clinical diagnosis, by observing the morphology of the colonies, it is possible to preliminarily determine the type of pathogen, providing a basis for subsequent precise treatment.

[0003] Although the colonies cultured on the flat culture medium can be observed with the naked eye, obtaining specimens that can be preserved for a long time and fully present the characteristics of the colonies is crucial for microbial research, teaching and popular science. In the field of microbial specimen preparation, there are many mature methods for preparing large fungal specimens. For example, Chinese patent CN105325404A discloses a method for preparing large fungal specimens, which includes liquid nitrogen quick freezing, vacuum drying, etc., and requires specific equipment such as vacuum dryers and liquid nitrogen and other materials, which limits its application. In addition, small-sized fungi lack outer layer protection and are easily damaged by squeezing; Chinese patent CN104996397A discloses a method for preparing large fungal specimens, which includes multiple steps such as cleaning, preservation, dehydration, plasticization, and embedding. The process is complicated, and the liquid used for the pre-treatment of the preservative contains harmful components such as formaldehyde, which is not conducive to promotion. Moreover, these microbial specimens have the disadvantage that they cannot be reproduced in batches. Each production requires repeating the complex process, which consumes a lot of time and resources and is difficult to meet the growing needs of teaching and scientific research. Summary of the Invention

[0004] In order to solve the technical problem that the existing technology cannot reproduce microbial specimens of the same morphology in batches, the present invention provides a method for preparing colony specimens.

[0005] The technical solutions of the present invention are as follows: A method for preparing a colony specimen comprises the following steps: (1) culturing a colony on a plate culture medium, and sterilizing the colony when the colony reaches the logarithmic phase or the stable phase; (2) pouring epoxy resin type crystal glue onto the sterilized colony, and removing the plate culture medium and the cultured colony after solidification to obtain a colony specimen made of epoxy resin; (3) placing the colony specimen in a mold frame and fixing it in the middle of the bottom plate of the mold frame with the side having the colony morphology pattern facing upward, pouring food-grade silica gel to cover the colony specimen, and demolding the colony specimen after solidification to obtain a colony silica gel mold; (4) pouring epoxy resin type crystal glue into the colony silica gel mold, and obtaining a colony specimen of the same morphology after solidification and demolding.

[0006] Furthermore, the colonies include bacterial colonies and fungal colonies; bacteria include aerobic bacteria, anaerobic bacteria, facultative anaerobic bacteria and microaerophilic bacteria; fungi include yeasts and molds; aerobic bacteria include Escherichia coli and Bacillus subtilis, and anaerobic bacteria are Bifidobacterium suis; yeasts include Pichia pastoris and Saccharomyces cerevisiae.

[0007] Furthermore, before colony culture is performed on a plate culture medium, activation of the strain and determination of the growth cycle are sequentially performed.

[0008] Furthermore, in step (1) colony culture, the bacterial solution is dipped into an inoculating loop and streaked on a plate culture medium, preferably using a Z-shaped three-zone streak method, and then cultured in an incubator.

[0009] Furthermore, in step (1), the colony is sterilized by selecting a plate culture medium with obvious single colonies and transferring it to a sterile clean bench for ultraviolet sterilization for 0.5-1 hour.

[0010] Furthermore, in step (2), epoxy resin-type crystal glue is poured onto the sterilized colony, first placed in a refrigerator for refrigeration, and then allowed to stand in a cool and ventilated place. After solidification, the plate culture medium and the cultured colonies are removed, and the specimens are cleaned, dried, and polished to obtain epoxy resin colony specimens. The refrigeration temperature is 0-4°C, the refrigeration time is 6-8 hours, and the specimens are allowed to stand in a cool and ventilated place for 24 hours.

[0011] Furthermore, in step (3), the mold frame is assembled from mold building blocks, and a plastic sheet or a disposable square culture dish is fixed at the bottom as the bottom plate of the mold frame.

[0012] Furthermore, in step (3), the colony specimen is placed in the mold frame and fixed in the middle of the bottom plate of the mold frame, with the side with the colony morphology pattern facing up, and food-grade silica gel is poured to cover the colony specimen. Then, the specimen is placed in the refrigerator and cooled for 30-40 minutes, and then aired in a ventilated and cool place for 4-8 hours. After curing, the specimen is demoulded to obtain a colony silica gel mold.

[0013] Furthermore, in step (4), the epoxy resin crystal glue is poured into the bacterial colony silicone mold, first placed in a refrigerator for 6-8 hours, then taken out, and then placed in a cool and ventilated place for 24 hours. After solidification and demolding, a bacterial colony specimen of the same morphology is obtained.

[0014] Furthermore, the colony specimen is treated with a special epoxy resin coloring dye to obtain a stained colony specimen.

[0015] The beneficial effects of the present invention are: The method for preparing colony specimens provided by the present invention has significant advantages in many aspects. In terms of colony feature preservation and reproduction, by using epoxy resin type crystal glue, the morphological characteristics of microbial colonies can be accurately reproduced, and the shape, texture, etc. of the colony can be clearly preserved to form specimens that can be preserved for a long time, meeting the needs of microbial research, teaching and popular science work for long-term preservation of specimens. In terms of specimen safety processing, without affecting the colony morphology, the biosafety risks that may be brought by living microorganisms are avoided. In terms of specimen production efficiency and cost control, food-grade silicone is used to make colony molds, which greatly simplifies the production process and breaks through the limitation that traditional methods cannot reproduce the same morphological colony specimens in batches. Mass production can be achieved with one molding, saving time and resources, and meeting the growing needs of teaching and scientific research. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0017] Figure 1 It is a silicone mold of E. coli colonies.

[0018] Figure 2 It is a specimen of Escherichia coli colony.

[0019] Figure 3 This is a stained Escherichia coli colony specimen.

[0020] Figure 4 It is a colony specimen of Bifidobacterium suis.

[0021] Figure 5 It is a colony specimen of Bacillus subtilis.

[0022] Figure 6 This is a stained Pichia pastoris colony specimen.

[0023] Figure 7 This is a stained specimen of the Saccharomyces cerevisiae colony. DETAILED DESCRIPTION

[0024] In order to enable those skilled in the art to better understand the technical solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts should fall within the scope of protection of the present invention.

[0025] The growth cycle of microorganisms includes four stages: lag phase, logarithmic phase, stationary phase and decay phase. The optimal growth cycle in the following embodiments belongs to the logarithmic phase.

[0026] Example 1 A method for preparing a colony specimen using Escherichia coli ( Escherichia coli ) as an example, including the following steps: Step 1: Activation of E. coli: Thaw the E. coli stored at -80°C in an ice box and activate in a test tube. Specifically, inoculate 1 mL of thawed E. coli inoculum into 10 mL of LB broth medium, incubate in a shaker at 37°C and 200 rpm for 24 hours, and activate for two generations to obtain E. coli culture liquid.

[0027] Step 2. Growth cycle determination: Add 30 μL of E. coli culture and 270 μL of LB broth to a 96-well plate used for growth curve analysis as the experimental group. Set up three experimental groups and one control group (the control group is a 96-well plate used for growth curve analysis with 300 μL of LB broth added). Then, place the plate in a BIOSCREEN C PRO fully automatic growth curve analyzer, and incubate, shake, and measure (measure the growth cycle of E. coli) simultaneously. The optimal growth cycle of E. coli is measured to be 18-24 hours. The oscillation condition is linear oscillation using the normal speed and low amplitude mode of the BIOSCREEN C PRO fully automatic growth curve analyzer, and the measurement condition is at 37° C. and detection at 600 nm absorbance.

[0028] Step 3: Colony culture: Use an inoculating loop to dip the E. coli liquid and make three Z-shaped lines on a 9-cm diameter plate culture medium (using LB broth solid culture medium). Perform three parallel experiments, then place the three streaked plate culture media in an incubator and culture for 24 hours until colonies grow.

[0029] It should be noted that the E. coli liquid in step 3 is prepared according to the preparation method of the E. coli liquid in step 1 after obtaining the growth cycle data of E. coli.

[0030] Step 4. Colony disinfection: Select a plate culture medium with obvious three-zone lines and single colonies and transfer it to a sterile clean bench. Disinfect it with ultraviolet light for half an hour to ensure colony disinfection and set aside.

[0031] Step 5. Preparation of epoxy resin specimens: Select high-hardness and high-transparency epoxy resin type crystal glue (including glue A and glue B, Ruijuan brand), stir for 5 minutes at a weight ratio of glue A: glue B of 3:1, and mix evenly to obtain a mixed colloid. That is, follow the instructions of the epoxy resin type crystal glue to obtain a mixed colloid. Pour 25g of the mixed colloid onto the disinfected colony, place it in a refrigerator at 4°C for 6 hours, then take it out and let it stand in a cool and ventilated place for 24 hours. Then take out the solidified epoxy resin type crystal glue, remove the residual LB broth solid culture medium and Escherichia coli (LB broth solid culture medium and Escherichia coli are uniformly recovered and discarded into hazardous waste bins after high-temperature sterilization), wash, dry, and polish the edges to obtain the Escherichia coli colony specimen.

[0032] Step 6. Prepare the silicone mold: Mix two components of food-grade silicone (Huagong brand) with a hardness of 10-30 degrees in a 1:1 ratio. Stir at a constant speed for 2 minutes to obtain the mixed silicone. Set aside, avoiding bubbles during stirring. Prepare the mold frame. The frame is composed of 14 mold building blocks, 3.3 cm long, 1.5 cm wide, and 1.1 cm high. The inner diameter is 9.6 cm, the outer diameter is 12.7 cm, and the height is 2.1 cm. A plastic sheet is fixed to the bottom of the mold frame as a base to prevent glue leakage.

[0033] Step 7. Silicone mold: Fix the E. coli colony specimen prepared in step 5 in the middle of the bottom plate of the mold frame (the smooth side of the E. coli colony specimen faces down, and the side with E. coli morphological lines faces up), pour in 100g of mixed silica gel, and ensure that the E. coli colony specimen is completely covered by the mixed silica gel.

[0034] Step 8, mold curing and demoulding: put the mold frame containing E. coli colony specimen and mixed silica gel into a 4℃ refrigerator, cool it down and store it for 30 minutes to quickly defoam, then dry it in a ventilated and cool place for 4-8 hours (high temperature curing at about 30℃ for 4 hours, low temperature curing at about 15℃ for 8 hours), cure and demould to obtain the E. coli colony silica gel mold (see Figure 1 ).

[0035] Step 9. Batch production: When batch producing E. coli colony specimens, take the mixed colloid in step 5 and pour it into the E. coli colony silica gel mold, place it in a refrigerator at 4℃ for 6 hours, then take it out and let it stand in a cool and ventilated place for 24 hours. Remove the mold and take out the solidified E. coli colony specimens (see Figure 2 ), and repeat the operation multiple times to achieve batch production of Escherichia coli colony specimens of the same morphology.

[0036] Step 10: Specimen staining: Take 1ml of epoxy resin special color dye (Fengxu brand red dye) and add 100ml of 75% alcohol and mix evenly. Put the E. coli colony specimen into the solution and make it completely submerged. Soak for 2 minutes (not overtime), take out and rinse with water and dry. The stained E. coli colony specimen is obtained (see Figure 3 ).

[0037] Throughout the entire production process, strict laboratory safety regulations must be adhered to. The mixing and curing process of epoxy resin crystal glue and food-grade silicone should be performed in a well-ventilated environment to avoid direct skin contact or inhalation of volatile gases. The prepared E. coli colony specimens should be stored in a dry, dark environment, away from high temperature and humidity to prevent deformation or deterioration. Regularly check the integrity of the specimens to ensure their long-term stability. If the E. coli colony specimens are to be used for teaching or display, they can be placed in a transparent display box and labeled with detailed instructions (such as the strain name, culture conditions, and production date). For scientific research communication, the specimens can be digitized using high-definition photography or 3D scanning technology to facilitate online sharing and discussion.

[0038] Example 2 A method for preparing a colony specimen using Bifidobacterium suis ( Bifidobacterium suis ) as an example, including the following steps: Step 1, activation of Bifidobacterium suis: thaw Bifidobacterium suis stored at -80°C in an ice box and activate in an anaerobic tube. Specifically, inoculate 1 mL of Bifidobacterium suis inoculum into 10 mL of MRS broth medium, culture in a shaker at 37°C and 200 rpm for 24 hours, and activate for two generations to obtain Bifidobacterium suis bacterial liquid.

[0039] Step 2, growth cycle determination: 20 μL of Bifidobacterium suis bacterial liquid and 180 μL of MRS broth medium were added to a 96-well plate dedicated to the growth curve, and the plate was sealed with 100 μL of paraffin oil as the experimental group. A total of 3 experimental groups and 1 control group were set up (the control group was a 96-well plate dedicated to the growth curve with 200 μL of MRS broth medium and 100 μL of paraffin oil added). The plate was then placed in a BIOSCREEN C PRO fully automatic growth curve analyzer, and culture and measurement (measuring the growth cycle of Bifidobacterium suis) were performed simultaneously. The optimal growth cycle of Bifidobacterium suis was measured to be 24-28 hours; The measurement conditions were 37°C and detection at 600 nm absorbance.

[0040] Step 3. Colony culture: Use an inoculation loop to dip the bacterial liquid of Bifidobacterium suis and make three Z-shaped lines on a 9 cm diameter plate culture medium (using MRS broth solid culture medium). Perform three parallel experiments, and then place the three streaked plate culture media in an incubator and culture for 24 hours until colonies grow.

[0041] It should be noted that the Bifidobacterium suis bacterial solution in step 3 is prepared according to the preparation method of the Bifidobacterium suis bacterial solution in step 1 after obtaining the growth cycle data of Bifidobacterium suis.

[0042] Step 4. Colony disinfection: Select a plate culture medium with obvious three-zone lines and single colonies and transfer it to a sterile clean bench. Disinfect it with ultraviolet light for half an hour to ensure colony disinfection and set aside.

[0043] Step 5. Preparation of epoxy resin specimens: Select high hardness and high transparency epoxy resin crystal glue (including glue A and glue B, Ruijuan brand), stir for 5 minutes at a weight ratio of glue A: glue B of 3:1, and mix evenly to obtain a mixed colloid, that is, follow the instructions of the epoxy resin crystal glue to obtain a mixed colloid, pour 25g of the mixed colloid on the sterilized colony, place it in a 4℃ refrigerator for 6 hours, take it out, and let it stand in a cool and ventilated place for 24 hours, then take out the solidified epoxy resin crystal glue, remove the residual MRS broth solid culture medium and porcine Bifidobacterium (MRS broth solid culture medium and porcine Bifidobacterium are uniformly recovered and discarded into hazardous waste bins after high-temperature sterilization), wash, dry, and polish the edges to obtain a porcine Bifidobacterium colony specimen (see Figure 4 ).

[0044] Step 6. Prepare the silicone mold: Mix two components of food-grade silicone (Huagong brand) with a hardness of 10-30 degrees in a 1:1 ratio. Stir at a constant speed for 2 minutes to obtain the mixed silicone. Set aside, avoiding bubbles during stirring. Prepare the mold frame. The frame is composed of 14 mold building blocks, 3.3 cm long, 1.5 cm wide, and 1.1 cm high. The inner diameter is 9.6 cm, the outer diameter is 12.7 cm, and the height is 2.1 cm. Use a 10×10 cm plastic disposable square Petri dish as the bottom of the mold frame to prevent glue leakage.

[0045] Step 7, silicone mold: fix the porcine Bifidobacterium colony specimen prepared in step 5 in the middle of the bottom plate of the mold frame (the smooth side of the porcine Bifidobacterium colony specimen faces down, and the side with the porcine Bifidobacterium colony morphological lines faces up), pour in 100g of mixed silica gel, and ensure that the porcine Bifidobacterium colony specimen is completely covered by the mixed silica gel.

[0046] Step 8. Molding, curing and demolding: Place the mold frame containing the porcine Bifidobacterium colony specimen and mixed silica gel into a 4°C refrigerator, cool it down and store it for 30 minutes to quickly defoam, then air it in a ventilated and cool place for 4-8 hours (high temperature curing at about 30°C for 4 hours, low temperature curing at about 15°C for 8 hours), and cure and demold to obtain a porcine Bifidobacterium colony silica gel mold.

[0047] Step 9, batch production: When batch-producing Bifidobacterium suis colony specimens, take the mixed colloid in step 5 and pour it into a Bifidobacterium suis colony silicone mold, place it in a refrigerator at 4°C for 6 hours, then take it out and let it stand in a cool and ventilated place for 24 hours. De-mold and take out the solidified Bifidobacterium suis colony specimens. Repeat the operation multiple times to achieve batch production of Bifidobacterium suis colony specimens of the same morphology.

[0048] Throughout the entire production process, laboratory safety regulations must be strictly adhered to. The mixing and curing process of epoxy resin crystal glue and food-grade silicone should be carried out in a well-ventilated environment to avoid direct contact with skin or inhalation of volatile gases. The completed Bifidobacterium suis colony specimens should be stored in a dry, dark environment, away from high temperature and humidity to prevent deformation or deterioration of the specimens. Regularly check the integrity of the specimens to ensure their long-term storage stability. If the Bifidobacterium suis colony specimens are to be used for teaching or display, they can be placed in a transparent display box and labeled with detailed instructions (such as the strain name, culture conditions, and production date). For scientific research communication, the specimens can be digitized using high-definition photography or 3D scanning technology to facilitate online sharing and discussion.

[0049] Example 3 A method for preparing a colony specimen using Bacillus subtilis ( Bacillus subtilis ) as an example, including the following steps: Step 1: Activation of Bacillus subtilis: Thaw Bacillus subtilis stored at -80°C in an ice box and activate in a test tube. Specifically, inoculate 1 mL of Bacillus subtilis inoculum into 10 mL of LB broth, incubate in a shaker at 37°C and 200 rpm for 24 hours, and activate for two generations to obtain a Bacillus subtilis culture solution.

[0050] Step 2: Growth cycle determination: 30 μL of Bacillus subtilis culture and 270 μL of LB broth were added to a 96-well plate used for growth curve analysis as experimental groups. Three experimental groups and one control group were set up (the control group consisted of 300 μL of LB broth added to a 96-well plate used for growth curve analysis). The plates were then placed in a BIOSCREEN C PRO fully automatic growth curve analyzer for simultaneous cultivation, shaking, and measurement (to measure the growth cycle of Bacillus subtilis). The optimal growth cycle of Bacillus subtilis was found to be 21-24 hours. The oscillation condition is linear oscillation using the normal speed and low amplitude mode of the BIOSCREEN C PRO fully automatic growth curve analyzer, and the measurement condition is at 37° C. and detection at 600 nm absorbance.

[0051] Step 3: Colony culture: Dip an inoculating loop into the Bacillus subtilis culture medium and make three Z-shaped streaks on a 9-cm diameter plate (using LB broth solid culture medium). Perform three parallel experiments, then place the three streaked plate culture media in an incubator and culture for 24 hours until colonies grow.

[0052] It should be noted that the Bacillus subtilis bacterial solution in step 3 is prepared according to the preparation method of the Bacillus subtilis bacterial solution in step 1 after obtaining the growth cycle data of Bacillus subtilis.

[0053] Step 4. Colony disinfection: Select a plate culture medium with obvious three-zone lines and single colonies and transfer it to a sterile clean bench. Disinfect it with ultraviolet light for half an hour to ensure colony disinfection and set aside.

[0054] Step 5. Preparation of epoxy resin specimens: Select high hardness and high transparency epoxy resin crystal glue (including glue A and glue B, Ruijuan brand), stir for 5 minutes at a weight ratio of glue A: glue B of 3:1, and mix evenly to obtain a mixed colloid, that is, follow the instructions of the epoxy resin crystal glue to obtain a mixed colloid, pour 25g of the mixed colloid on the sterilized colony, place it in a 4℃ refrigerator for 6 hours, then take it out, and let it stand in a cool and ventilated place for 24 hours, then take out the solidified epoxy resin crystal glue, remove the residual LB broth solid culture medium and Bacillus subtilis (LB broth solid culture medium and Bacillus subtilis are uniformly recovered and discarded into hazardous waste bins after high-temperature sterilization), wash, dry, and polish the edges to obtain a Bacillus subtilis colony specimen (see Figure 5 ).

[0055] Step 6. Prepare the silicone mold: Mix two components of food-grade silicone (Huagong brand) with a hardness of 10-30 degrees in a 1:1 ratio. Stir at a constant speed for 2 minutes to obtain the mixed silicone. Set aside, avoiding bubbles during stirring. Prepare the mold frame. The frame is composed of 14 mold building blocks, 3.3 cm long, 1.5 cm wide, and 1.1 cm high. The inner diameter is 9.6 cm, the outer diameter is 12.7 cm, and the height is 2.1 cm. A plastic sheet is fixed to the bottom of the mold frame as a base to prevent glue leakage.

[0056] Step 7, silicone mold: fix the Bacillus subtilis colony specimen prepared in step 5 in the middle of the bottom plate of the mold frame (the smooth side of the Bacillus subtilis colony specimen faces down, and the side with Bacillus subtilis colony morphology lines faces up), pour in 100g of mixed silica gel, and ensure that the Bacillus subtilis colony specimen is completely covered by the mixed silica gel.

[0057] Step 8. Mold curing and demolding: Place the mold frame containing the Bacillus subtilis colony specimen and mixed silica gel into a 4°C refrigerator, cool it down and store it for 30 minutes to quickly defoam, then air it in a ventilated and cool place for 4-8 hours (high temperature curing at about 30°C for 4 hours, low temperature curing at about 15°C for 8 hours), and cure and demold to obtain a Bacillus subtilis colony silica gel mold.

[0058] Step 9, mass production: When making batches of Bacillus subtilis colony specimens, take the mixed colloid in step 5 and pour it into a Bacillus subtilis colony silicone mold, place it in a refrigerator at 4°C for 6 hours, then take it out and let it stand in a cool and ventilated place for 24 hours. De-mold and take out the solidified Bacillus subtilis colony specimens. Repeat this operation multiple times to achieve mass production of Bacillus subtilis colony specimens of the same morphology.

[0059] Throughout the entire production process, laboratory safety regulations must be strictly adhered to. The mixing and curing process of epoxy resin crystal glue and food-grade silicone should be carried out in a well-ventilated environment to avoid direct contact with skin or inhalation of volatile gases. The completed Bifidobacterium suis colony specimens should be stored in a dry, light-proof environment, away from high temperature and humidity to prevent deformation or deterioration of the specimens. Regularly check the integrity of the specimens to ensure their long-term storage stability. If the Bifidobacterium suis colony specimens are to be used for teaching or display, they can be placed in a transparent display box and labeled with detailed instructions (such as the strain name, culture conditions, and production date). For scientific research communication, the specimens can be digitized using high-definition photography or 3D scanning technology to facilitate online sharing and discussion.

[0060] Example 4 A method for preparing a colony specimen using Pichia pastoris ( Komagataella pastoris ) as an example, including the following steps: Step 1: Activation of Pichia pastoris: Thaw the Pichia pastoris stored at -80°C in an ice box and activate it in a test tube. Specifically, inoculate 1 mL of the Pichia pastoris inoculum into 10 mL of YPD medium, and culture the culture in a shaker at 30°C and 200 rpm for 24 hours. Activate the culture for two generations to obtain a Pichia pastoris culture.

[0061] Step 2. Growth cycle determination: 30 μL of Pichia pastoris culture medium and 270 μL of YPD medium were added to a 96-well plate used for growth curve analysis as the experimental group. Three experimental groups and one control group were set up (the control group was prepared by adding 300 μL of YPD medium to a 96-well plate used for growth curve analysis). The plates were then placed in a BIOSCREEN C PRO fully automatic growth curve analyzer. Cultivation, shaking, and measurement (for measuring the growth cycle of Pichia pastoris) were performed simultaneously. The optimal growth cycle of Pichia pastoris was measured to be 24-28 hours. The oscillation condition is linear oscillation using the normal speed and low amplitude mode of the BIOSCREEN C PRO fully automatic growth curve analyzer, and the measurement condition is detection at 30° C. with absorbance at 600 nm.

[0062] Step 3: Colony culture: Dip the Pichia yeast liquid with an inoculating loop and make three Z-shaped lines on a 9-cm diameter plate culture medium (using YPD solid culture medium). Perform three parallel experiments, then place the three streaked plate culture media in an incubator and culture for 24 hours until colonies grow.

[0063] It should be noted that the Pichia pastoris solution in step 3 is prepared according to the preparation method of the Pichia pastoris solution in step 1 after obtaining the growth cycle data of Pichia pastoris.

[0064] Step 4. Colony disinfection: Select a plate culture medium with obvious three-zone lines and single colonies and transfer it to a sterile clean bench. Disinfect it with ultraviolet light for half an hour to ensure colony disinfection and set aside.

[0065] Step 5. Preparation of epoxy resin specimens: Select high-hardness and high-transparency epoxy resin type crystal glue (including glue A and glue B, Ruijuan brand), stir for 5 minutes at a weight ratio of glue A: glue B of 3:1, and mix evenly to obtain a mixed colloid, that is, follow the instructions of the epoxy resin type crystal glue to obtain a mixed colloid, pour 25g of the mixed colloid on the disinfected colony, place it in a refrigerator at 4℃ for 6 hours, then take it out, and let it stand in a cool and ventilated place for 24 hours, then take out the solidified epoxy resin type crystal glue, remove the residual YPD solid culture medium and Pichia pastoris (YPD solid culture medium and Pichia pastoris are uniformly recovered and discarded into hazardous waste bins after high-temperature sterilization), wash, dry, and polish the edges to obtain the Pichia pastoris colony specimen.

[0066] Step 6. Prepare the silicone mold: Mix two components of food-grade silicone (Huagong brand) with a hardness of 10-30 degrees in a 1:1 ratio. Stir at a constant speed for 2 minutes to obtain the mixed silicone. Set aside, avoiding bubbles during stirring. Prepare the mold frame. The frame is composed of 14 mold building blocks, 3.3 cm long, 1.5 cm wide, and 1.1 cm high. The inner diameter is 9.6 cm, the outer diameter is 12.7 cm, and the height is 2.1 cm. A plastic sheet is fixed to the bottom of the mold frame as a base to prevent glue leakage.

[0067] Step 7, silicone mold: fix the Pichia colony specimen prepared in step 5 in the middle of the bottom plate of the mold frame (the smooth side of the Pichia colony specimen faces down, and the side with Pichia morphological patterns faces up), pour in 100g of mixed silica gel, and ensure that the Pichia colony specimen is completely covered by the mixed silica gel.

[0068] Step 8. Molding, curing and demolding: Place the mold frame containing the Pichia pastoris colony specimen and mixed silicone into a 4°C refrigerator, cool it down and store it for 30 minutes to quickly defoam, then air it in a ventilated and cool place for 4-8 hours (high temperature curing at around 30°C for 4 hours, low temperature curing at around 15°C for 8 hours), and then cure and demold it to obtain a Pichia pastoris silicone mold.

[0069] Step 9, mass production: When batching Pichia pastoris colony specimens, take the mixed colloid in step 5 and pour it into a Pichia pastoris silicone mold, place it in a refrigerator at 4°C for 6 hours, then take it out and let it stand in a cool and ventilated place for 24 hours. Remove the mold and take out the solidified Pichia pastoris colony specimens. Repeat the operation multiple times to achieve batch production of Pichia pastoris colony specimens of the same morphology.

[0070] Step 10: Specimen staining: Take 1ml of epoxy resin special color dye (Fengxu brand red dye) and add 100ml of 75% alcohol and mix evenly. Put the Pichia pastoris colony specimen into the solution and make it completely submerged. Soak for 2 minutes (not overtime), take out and rinse with clean water and dry. The stained Pichia pastoris colony specimen is obtained (see Figure 6 ).

[0071] Strict laboratory safety regulations must be adhered to throughout the entire production process. The mixing and curing process of epoxy resin crystal glue and food-grade silicone should be performed in a well-ventilated environment to avoid direct skin contact or inhalation of volatile gases. The prepared Pichia colony specimens should be stored in a dry, dark environment, away from high temperature and humidity to prevent deformation or deterioration. Regularly check the integrity of the specimens to ensure their long-term storage stability. If the Pichia colony specimens are to be used for teaching or display, they can be placed in a transparent display box and labeled with detailed instructions (such as the strain name, culture conditions, and production date). For scientific research communication, the specimens can be digitized using high-definition photography or 3D scanning technology to facilitate online sharing and discussion.

[0072] Example 5 A method for preparing a colony specimen using Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) as an example, including the following steps: Step 1: Activation of Saccharomyces cerevisiae: Thaw the Saccharomyces cerevisiae stored at -80°C in an ice box and activate it in a test tube. Specifically, inoculate 1 mL of Saccharomyces cerevisiae inoculum into 10 mL of YPD medium, and culture in a shaker at 30°C and 200 rpm for 24 hours. Activate two generations continuously to obtain Saccharomyces cerevisiae liquid.

[0073] Step 2. Growth cycle determination: Add 30 μL of Saccharomyces cerevisiae culture liquid and 270 μL of YPD medium to a 96-well plate dedicated to growth curves as the experimental group. Set up three experimental groups and one control group (the control group is a 96-well plate dedicated to growth curves with 300 μL of YPD medium added). Then place the plate in a BIOSCREEN C PRO fully automatic growth curve analyzer, and culture, shake, and measure (measure the growth cycle of Saccharomyces cerevisiae) simultaneously. The optimal growth cycle of Saccharomyces cerevisiae is measured to be 24-28 hours; The oscillation condition is linear oscillation using the normal speed and low amplitude mode of the BIOSCREEN C PRO fully automatic growth curve analyzer, and the measurement condition is detection at 30° C. with absorbance at 600 nm.

[0074] Step 3: Colony culture: Dip the brewer's yeast liquid with an inoculating loop and make three Z-shaped lines on a 9-cm diameter plate culture medium (using YPD solid culture medium). Perform three parallel experiments, and then place the three streaked plate culture media in an incubator and culture for 48 hours (exceeding its optimal growth period and belonging to the stable period).

[0075] It should be noted that the brewer's yeast liquid in step three is prepared according to the preparation method of the brewer's yeast liquid in step one after obtaining the growth cycle data of the brewer's yeast.

[0076] Step 4. Colony disinfection: Select a plate culture medium with obvious three-zone lines and single colonies and transfer it to a sterile clean bench. Disinfect it with ultraviolet light for half an hour to ensure colony disinfection and set aside.

[0077] Step 5. Preparation of epoxy resin specimens: Select high-hardness and high-transparency epoxy resin type crystal glue (including glue A and glue B, Ruijuan brand), stir for 5 minutes at a weight ratio of glue A: glue B of 3:1, and mix evenly to obtain a mixed colloid, that is, follow the instructions of the epoxy resin type crystal glue to obtain a mixed colloid, pour 25g of the mixed colloid on the disinfected colony, place it in a 4℃ refrigerator for 6 hours, then take it out, and let it stand in a cool and ventilated place for 24 hours, then take out the solidified epoxy resin type crystal glue, remove the residual YPD solid culture medium and brewer's yeast (YPD solid culture medium and brewer's yeast are uniformly recovered and discarded into hazardous waste bins after high-temperature sterilization), wash, dry, and polish the edges to obtain a brewer's yeast colony specimen.

[0078] Step 6. Prepare the silicone mold: Mix two components of food-grade silicone (Huagong brand) with a hardness of 10-30 degrees in a 1:1 ratio. Stir at a constant speed for 2 minutes to obtain the mixed silicone. Set aside, avoiding bubbles during stirring. Prepare the mold frame. The frame is composed of 14 mold building blocks, 3.3 cm long, 1.5 cm wide, and 1.1 cm high. The inner diameter is 9.6 cm, the outer diameter is 12.7 cm, and the height is 2.1 cm. A plastic sheet is fixed to the bottom of the mold frame as a base to prevent glue leakage.

[0079] Step 7. Silicone mold: Fix the brewer's yeast colony specimen prepared in step 5 in the middle of the bottom plate of the mold frame (the smooth side of the brewer's yeast colony specimen faces down, and the side with the brewer's yeast colony morphology pattern faces up), pour in 100g of mixed silica gel, and ensure that the brewer's yeast colony specimen is completely covered by the mixed silica gel.

[0080] Step 8. Molding, curing and demolding: Place the mold frame containing the brewer's yeast colony specimen and mixed silica gel into a 4°C refrigerator, cool it down and store it for 30 minutes to quickly defoam, then dry it in a ventilated and cool place for 4-8 hours (high temperature curing at about 30°C for 4 hours, low temperature curing at about 15°C for 8 hours), and cure and demold to obtain a brewer's yeast colony silica gel mold.

[0081] Step 9, mass production: When making batches of brewer's yeast colony specimens, take the mixed colloid in step 5 and pour it into a brewer's yeast colony silicone mold, place it in a 4°C refrigerator for 6 hours, then take it out and let it stand in a cool and ventilated place for 24 hours. De-mold and take out the solidified brewer's yeast colony specimens. Repeat the operation multiple times to achieve mass production of brewer's yeast colony specimens of the same morphology.

[0082] Step 10: Specimen staining: Take 1ml of epoxy resin special color dye (Fengxu brand red dye) and add 100ml of 75% alcohol and mix evenly. Put the saccharomyces cerevisiae colony specimen into the solution and make it completely submerged. Soak for 2 minutes (not overtime), take out and rinse with water and dry. The stained saccharomyces cerevisiae colony specimen is obtained (see Figure 7 ).

[0083] Strict laboratory safety regulations must be adhered to throughout the entire production process. The mixing and curing process of epoxy resin crystal glue and food-grade silicone should be performed in a well-ventilated environment to avoid direct skin contact or inhalation of volatile gases. Completed Saccharomyces cerevisiae colony specimens should be stored in a dry, dark environment, away from high temperature and humidity to prevent deformation or deterioration. Regularly check the integrity of the specimens to ensure their long-term stability. If the Saccharomyces cerevisiae colony specimens are to be used for teaching or display, they can be placed in a transparent display box and labeled with detailed instructions (such as the strain name, culture conditions, and production date). For scientific research communication, specimens can be digitized using high-definition photography or 3D scanning technology to facilitate online sharing and discussion.

[0084] Although the present invention has been described in detail with reference to the accompanying drawings and in conjunction with preferred embodiments, the present invention is not limited thereto. Without departing from the spirit and essence of the present invention, persons of ordinary skill in the art may make various equivalent modifications or substitutions to the embodiments of the present invention, and such modifications or substitutions shall be within the scope of the present invention. Any changes or substitutions that can be easily conceived by persons skilled in the art within the technical scope disclosed in the present invention shall be within the scope of protection of the present invention.

Claims

1. A method for preparing a bacterial colony specimen, characterized in that: The method comprises the following steps: (1) culturing a colony on a plate culture medium, and sterilizing the colony when the colony reaches the logarithmic phase or the stable phase; (2) pouring an epoxy resin type crystal drop glue onto the sterilized colony, and removing the plate culture medium and the cultured colony after solidification to obtain a colony specimen made of epoxy resin; (3) placing the colony specimen in a mold frame and fixing it in the middle of the bottom plate of the mold frame with the side having the colony morphology pattern facing upward, pouring food-grade silica gel to cover the colony specimen, and demolding the colony specimen after solidification to obtain a colony silica gel mold; (4) pouring the epoxy resin type crystal drop glue into the colony silica gel mold, and obtaining a colony specimen of the same morphology after solidification and demolding.

2. The method for preparing a colony specimen according to claim 1, wherein: The colonies include bacterial colonies and fungal colonies; bacteria include aerobic bacteria, anaerobic bacteria, facultative anaerobic bacteria and microaerophilic bacteria; fungi include yeast and mold; aerobic bacteria include Escherichia coli and Bacillus subtilis, and anaerobic bacteria are Bifidobacterium suis; yeasts include Pichia pastoris and Saccharomyces cerevisiae.

3. A method for preparing a colony specimen according to any one of claims 1 to 2, characterized in that: Before culturing colonies on plate culture medium, the strain activation and growth cycle determination must be carried out in sequence.

4. A method for preparing a colony specimen according to any one of claims 1 to 2, characterized in that: Step (1) When culturing the colony, use an inoculation loop to dip the bacterial solution and streak the culture on the plate culture medium, and then culture it in an incubator.

5. A method for preparing a colony specimen according to any one of claims 1 to 2, characterized in that: In step (1), the colony is sterilized by selecting a plate culture medium with obvious single colonies and transferring it to a sterile clean bench for ultraviolet sterilization for 0.5-1 hour.

6. A method for preparing a colony specimen according to any one of claims 1 to 2, characterized in that: Step (2) Pour epoxy resin-type crystal glue onto the sterilized colony, first place it in a refrigerator for refrigeration, and then let it stand in a cool and ventilated place. After solidification, remove the plate culture medium and the cultured colonies, wash, dry, and polish the edges to obtain a colony specimen made of epoxy resin; the refrigeration temperature is 0-4°C, the refrigeration time is 6-8 hours, and the time of standing in a cool and ventilated place is 24 hours.

7. A method for preparing a colony specimen according to any one of claims 1 to 2, characterized in that: In step (3), the mold frame is assembled by mold building blocks, and a plastic sheet or a disposable square culture dish is fixed at the bottom as the bottom plate of the mold frame.

8. A method for preparing a colony specimen according to any one of claims 1 to 2, characterized in that: Step (3) Place the colony specimen in the mold frame and fix it in the middle of the bottom plate of the mold frame with the side with the colony morphology pattern facing up, pour food-grade silica gel to cover the colony specimen, then place it in the refrigerator, cool it down and store it for 30-40 minutes, then dry it in a ventilated and cool place for 4-8 hours, and demould it after curing to obtain a colony silica gel mold.

9. A method for preparing a colony specimen according to any one of claims 1 to 2, characterized in that: Step (4) Pour the epoxy resin crystal glue into the colony silicone mold, place it in the refrigerator for 6-8 hours, then take it out, and then place it in a cool and ventilated place for 24 hours. After solidification and demoulding, a colony specimen of the same morphology is obtained.

10. A method for preparing a colony specimen according to any one of claims 1 to 2, characterized in that: The colony specimen is treated with a special color-adjusting dye for epoxy resin to obtain a stained colony specimen.

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