A rapid preparation method for rumen ciliate ammonia silver staining samples
By optimizing the ammonia silver staining method, the double addition reaction of methyl green-formalin-normal saline solution and 35% formaldehyde aqueous solution was solved, and the problem of unstable staining of rumen ciliates in the prior art was achieved, and efficient, stable staining and long-term preservation of cervical subfamilies and large phenomenaceae were achieved.
Patent Information
- Application Number
- CN202310137751.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-02-17
- Publication Date
- 2025-07-08
- Estimated Expiration
- 2043-02-17
AI Technical Summary
The existing ammonia silver staining method is not effective when staining rumen ciliates, especially for the cilia pattern staining of the cephalosa subfamily and the large tetrama subfamily. Inaccurate reagent sampling and improper temperature control lead to unstable experimental results.
The rumen fluid was fixed with methyl green-formalin-normal saline solution, combined with the double addition reaction of 35% formaldehyde aqueous solution and Rio-Hortega solution, and the reaction temperature in the dark was controlled to be 20-25℃. By accurately measuring the dosage of each reagent and centrifuging treatment, the silver chromatin suspension was prepared and stored at 4℃ to prolong the staining effect.
It has achieved efficient and stable staining of the rumen ciliates of the erectile dysfunction and the large erectile dysfunction subfamily. The dyeing effect is beautiful and easy to observe, and it does not require permanent production materials, so that the samples can be preserved for a long time.
Smart Images
Figure CN116296700B_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biotechnology, and particularly relates to a method for rapidly preparing a sample of rumen ciliate ammonia silver staining. Background Art
[0002] Rumen ciliates are a type of protozoa that mainly live in the rumen of ruminants such as cattle and sheep and have a mutualistic symbiosis with the host. They are an important part of rumen microorganisms and play an important role in maintaining the stability of the rumen ecosystem and maintaining the health and high production performance of the host animals. The entire surface of the rumen ciliate, all the kinetosomes, the corresponding cilia (organs), and the fibers (or microfilaments) and microtubules under the pellicle connected to them are collectively called the ciliary pattern. Because it has a specific two-dimensional or three-dimensional structural pattern in different species, it needs to be stained appropriately before it can be displayed, and it is one of the most important bases in species identification and higher-level taxonomic classification.
[0003] The ammonia silver method, also known as the silver carbonate method or the Fernandez-Galiano method, is a simple, rapid and highly effective silver staining method, which is applied to the staining of ciliate ciliary patterns, silver line systems on the body surface and nuclear organs. At present, the ammonia silver staining method is mainly applicable to small rumen ciliates in the Entodiniinae and small Diplodiniinae, and has poor staining effects on the ciliary patterns of large rumen ciliates in the Ophryoscolecinae and large Diplodiniinae. In addition, in current research, the reaction reagents in the ammonia silver method are added by the number of drops of a dropper, and the reagents cannot be accurately quantified, and the temperature of the staining reaction environment is not clearly defined, which all lead to unstable experimental results. Therefore, for rumen ciliates, it is necessary to improve and optimize the ammonia silver staining method to stain and prepare slides quickly and stably.
[0004] Therefore, how to provide a method for efficiently, stably and high-quality preparing a sample of rumen ciliate ammonia silver staining, which is applicable to the ammonia silver staining of more species of rumen ciliates such as Ophryoscolecinae and large Diplodiniinae, and can preserve the ammonia silver staining sample for a long time, is an urgent problem to be solved by those skilled in the art. Summary of the Invention
[0005] The purpose of the present invention is to provide a method for rapidly and stably preparing a sample of rumen ciliate ammonia silver staining, which is applicable to the ammonia silver staining of more species of rumen ciliates such as Ophryoscolecinae and large Diplodiniinae, can efficiently and high-quality prepare a sample of rumen ciliate ammonia silver staining, optimize the slide preparation steps, and can preserve the ammonia silver staining sample for a long time.
[0006] To achieve the above object, the present invention provides a rapid preparation method for a rumen ciliate ammonia silver staining sample, comprising the following steps: preparing a methyl green-formalin-saline solution, a Rio-Hortega solution, a 35% aqueous formaldehyde solution, and a 4% bacterial peptone solution; collecting rumen fluid, fixing the rumen fluid with the methyl green-formalin-saline solution, and storing the fixed rumen fluid in a light-tight manner at 4°C to obtain a fixed sample solution; taking 5-10 mL of the fixed sample solution, centrifuging it, discarding the supernatant, and retaining the precipitate to obtain enriched ciliates; adding 3 mL of distilled water, 80-100 μL of the enriched ciliates, 130-140 μL of pyridine, and 305-330 μL of the bacterial peptone solution to a centrifuge tube and mixing them evenly, and then placing the centrifuge tube in the dark for reaction for 20 min; after the reaction is completed, adding 140-160 μL of the aqueous formaldehyde solution to the centrifuge tube and mixing them evenly, then adding 1255-1400 μL of the Rio-Hortega solution to the centrifuge tube and mixing them evenly, and then placing the centrifuge tube in the dark for reaction for 30 min; after the reaction is completed, placing the centrifuge tube in a 70°C water bath and oscillating it for 30 min, then taking out the centrifuge tube and placing it in cold water, adding 245-290 μL of the Rio-Hortega solution to the centrifuge tube and mixing them evenly, placing the evenly mixed centrifuge tube in a 70°C water bath and oscillating it for 15 min, then taking out the centrifuge tube and placing it in cold water; taking out the centrifuge tube from the cold water, allowing the centrifuge tube to stand or centrifuging it, and then sucking out the supernatant in the centrifuge tube and storing it separately as a separately stored solution; adding distilled water to the centrifuge tube, then allowing the centrifuge tube to stand and discarding the supernatant in the centrifuge tube, adding the separately stored solution to the centrifuge tube to obtain a silver-stained ciliate suspension.
[0007] As a further technical solution of the present invention: after obtaining the silver-stained ciliate suspension, the preparation method further comprises: storing the obtained silver-stained ciliate suspension in a 4°C refrigerator for microscopic examination and observation. If the stained ciliates in the silver-stained ciliate suspension fade, placing the silver-stained ciliate suspension in a 70°C water bath and oscillating it until it shows color, then stopping the water bath, taking out the silver-stained ciliate suspension for slide preparation and microscopic examination.
[0008] As a further technical solution of the present invention: preparing the methyl green-formalin-saline solution comprises: weighing 8 g of sodium chloride and 0.6 g of methyl green, dissolving the sodium chloride and the methyl green in 100 mL of the aqueous formaldehyde solution to obtain a mixed solution, making the volume of the mixed solution up to 1 L with distilled water, and then storing it in a light-tight manner to obtain the methyl green-formalin-saline solution.
[0009] A further technical solution of the present invention is as follows: preparing the Rio-Hortega solution includes: preparing a silver nitrate solution with a concentration of 10% and a sodium carbonate solution with a concentration of 5%. Take 50 mL of the silver nitrate solution and 150 mL of the sodium carbonate solution, mix them well, and a white precipitate will be produced. Dropwise add ammonia water to the white precipitate until the white precipitate dissolves, then add ultrapure water to dilute to 750 ml, and store it sealed to obtain the Rio-Hortega solution.
[0010] A further technical solution of the present invention is as follows: during the process of fixing the rumen fluid with the methyl green-formalin-saline solution, the volume ratio of the rumen fluid to the methyl green-formalin-saline solution is 1:4.
[0011] A further technical solution of the present invention is as follows: during the process of adding 3 mL of distilled water, 80 - 100 μl of the enriched worms, 130 - 140 μl of pyridine, and 305 - 330 μl of the bacterial peptone solution into a centrifuge tube and mixing them well, and then placing the centrifuge tube in the dark for reaction, the ambient temperature of the reaction in the dark is 20 - 25 °C; during the process of adding 1255 - 1400 μl of the Rio-Hortega solution to the centrifuge tube and mixing them well after mixing, and then placing the centrifuge tube in the dark for reaction, the ambient temperature of the reaction in the dark is 20 - 25 °C.
[0012] A further technical solution of the present invention is as follows: during the process of adding 3 mL of distilled water, 80 - 100 μl of the enriched worms, 130 - 140 μl of pyridine, and 305 - 330 μl of the bacterial peptone solution into a centrifuge tube and mixing them well, add 130 - 140 μl of the pyridine into the centrifuge tube and mix them well in a fume hood.
[0013] A further technical solution of the present invention is as follows: after preparing the bacterial peptone solution with a concentration of 4%, filter and sterilize the prepared bacterial peptone solution with a 0.22 μm sterile needle filter in a laminar flow hood, and store the filtered and sterilized bacterial peptone solution at 4 °C for standby; during the process of adding 3 mL of distilled water, 80 - 100 μl of the enriched worms, 130 - 140 μl of pyridine, and 305 - 330 μl of the bacterial peptone solution into a centrifuge tube and mixing them well, add 305 - 330 μl of the bacterial peptone solution into the centrifuge tube in a laminar flow hood.
[0014] As a further technical solution of the present invention: during the process of adding distilled water to the centrifuge tube and then discarding the supernatant in the centrifuge tube after standing the centrifuge tube, the operations of adding distilled water to the centrifuge tube and then discarding the supernatant in the centrifuge tube after standing the centrifuge tube are repeated until the impurities are removed.
[0015] As a further technical solution of the present invention: during the process of centrifuging 5-10 mL of the fixed sample solution, the rotation speed of the centrifugation is 2000 rpm and the centrifugation time is 1-3 min; when taking out the centrifuge tube from cold water, if the centrifuge tube is centrifuged, the rotation speed of the centrifugation is 2000 rpm and the centrifugation time is 1-3 min.
[0016] Beneficial effects: The present invention provides a method for rapidly and stably preparing a rumen ciliate ammoniacal silver staining sample. The steps are simple, the preparation period is simplified, it is more efficient and convenient, and the staining effect is more stable. When performing ammoniacal silver staining, by setting the concentration of the formaldehyde aqueous solution to 35% (w / v) and reacting by adding a certain amount of Rio-Hortega solution in two portions, the formaldehyde reduces the silver ions in the Rio-Hortega solution to metallic silver under alkaline conditions and deposits it on the protein surface of the ciliate to form staining, enhancing the staining effect. It is applicable to ammoniacal silver staining of more types of rumen ciliates such as Ophryoscolecinae and large Diplodininae. After staining, the ciliates are white or light yellow and the ciliary pattern is significantly dark brown. Compared with the traditional method, the stained ciliates and ciliary pattern of the present invention are more beautiful and easier to observe; by precisely quantifying the dosage range of each reagent and the reaction time, etc., the staining effect can be made more stable; when washing the silver-stained ciliates, the supernatant in the centrifuge tube is aspirated and stored separately as a storage solution, and the storage solution is re-added after washing the silver-stained ciliates to retain the reaction system, facilitating the long-term preservation of the silver-stained ciliate suspension without the need for materials and reagents for permanent specimen preparation. Description of the Drawings
[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present specification or the prior art, the following will briefly introduce the drawings required to be used in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.
[0018] Figure 1 It is a test flow chart of a method for rapidly and stably preparing a rumen ciliate ammoniacal silver staining sample in an embodiment of the present invention;
[0019] Figure 2 It is an ammoniacal silver staining effect diagram of rumen ciliates of the genus Ophryoscolex obtained in Example 2 of the present invention (scale bar = 50 μm);
[0020] Figure 3 This is the ammoniacal silver staining effect diagram of rumen ciliate of the genus Eudiplodinium obtained in Example 2 of the present invention (scale bar = 50 μm);
[0021] Figure 4 This is the ammoniacal silver staining effect diagram of rumen ciliate of the genus Eudiplodinium obtained in Example 3 of the present invention (scale bar = 50 μm);
[0022] Figure 5 This is the ammoniacal silver staining effect diagram of rumen ciliate of the genus Polyplastron obtained in Example 3 of the present invention (scale bar = 50 μm); Detailed implementation manners
[0023] Next, the technical solutions in the present invention will be clearly and completely described in conjunction with the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art belong to the scope of protection of the present invention.
[0024] Meanwhile, throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as having the meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the technical field to which the present invention belongs. In case of contradiction, this specification shall prevail.
[0025] Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment, etc. used in the present invention can be obtained through market purchases or can be prepared by existing methods.
[0026] Example 1
[0027] Such as Figure 1As shown in the figure, the first embodiment provides a rapid preparation method for a rumen ciliate ammonia silver staining sample, including the following steps: preparing solutions, preparing a methyl green-formalin-saline solution, a Rio-Hortega solution, a formaldehyde aqueous solution with a concentration of 35% (w / v), and a bacteriological peptone solution with a concentration of 4% (w / v); fixing the ciliate, collecting rumen fluid, fixing the rumen fluid with the methyl green-formalin-saline solution, and storing the fixed rumen fluid in the dark and sealed at 4°C to obtain a fixed sample solution; enriching the ciliate, taking 5-10 mL of the fixed sample solution, centrifuging it, discarding the supernatant, and retaining the precipitate to obtain enriched ciliates; ammonia silver staining, adding 3 mL of distilled water, 80-100 μL of the enriched ciliates, 130-140 μL of pyridine, and 305-330 μL of the bacteriological peptone solution into a centrifuge tube and mixing them evenly. After mixing, place the centrifuge tube in the dark for reaction for 20 min; after the reaction is completed, add 140-160 μL of the formaldehyde aqueous solution into the centrifuge tube and mix evenly. After mixing, add 1255-1400 μL of the Rio-Hortega solution into the centrifuge tube and mix evenly. After mixing, place the centrifuge tube in the dark for reaction for 30 min; after the reaction is completed, place the centrifuge tube in a 70°C water bath and oscillate for 30 min, then take out the centrifuge tube and place it in cold water. Add 245-290 μL of the Rio-Hortega solution into the centrifuge tube and mix evenly. After mixing, place the centrifuge tube in a 70°C water bath and oscillate for 15 min, then take out the centrifuge tube and place it in cold water; washing the silver-stained ciliates, taking out the centrifuge tube from the cold water, letting the centrifuge tube stand still or centrifuging it, then sucking out the supernatant in the centrifuge tube and storing it separately as a stored solution; adding distilled water into the centrifuge tube, then letting the centrifuge tube stand still and discarding the supernatant in the centrifuge tube, adding the stored solution into the centrifuge tube to obtain a silver-stained ciliate suspension.
[0028] Specifically, the present invention provides a method for rapidly and stably preparing a sample of rumen ciliate ammonia silver staining. The steps are simple, which simplifies the preparation cycle, is more efficient and convenient, and the staining effect is more stable. During ammonia silver staining, by setting the concentration of the formaldehyde aqueous solution to 35% (w / v) and reacting by adding a certain amount of Rio-Hortega solution in two portions, the formaldehyde reduces the silver ions in the Rio-Hortega solution to metallic silver under alkaline conditions and deposits it on the protein surface of the ciliate to form a stain, enhancing the staining effect. It is applicable to the ammonia silver staining of more types of rumen ciliates such as Ophryoscolecinae and large Diplodininae. After staining, the ciliate body is white or light yellow while the ciliary pattern is significantly dark brown. Compared with the traditional method, the ciliate body and ciliary pattern after staining in the present invention are more beautiful and easier to observe. By precisely quantifying the dosage range of each reagent and the reaction time, etc., the staining effect can be made more stable. When washing the silver-stained ciliate, the supernatant in the centrifuge tube is aspirated and stored separately as a storage solution. After washing the silver-stained ciliate, the storage solution is re-added to retain the reaction system, facilitating the long-term preservation of the silver-stained ciliate suspension without the need for materials and reagents for permanent preparation.
[0029] In some possible embodiments, after obtaining the silver-stained ciliate suspension, the preparation method further includes: storing the obtained silver-stained ciliate suspension in a 4°C refrigerator for microscopic examination. If the stained ciliates in the silver-stained ciliate suspension fade, the silver-stained ciliate suspension is placed in a 70°C water bath and oscillated until it becomes colored, then the water bath is stopped, and the silver-stained ciliate suspension is taken out for slide preparation and microscopic examination.
[0030] Among them, during the oscillation process, the silver-stained ciliate suspension is aspirated onto a glass slide, and then the glass slide is placed under a microscope to observe the coloring effect. When the coloring effect is good, the water bath is stopped.
[0031] This is because after the water bath staining by the traditional method, if long-term storage is required, permanent slide preparation is necessary: adding distilled water and centrifuging to wash away the reaction liquid, then using protein gelatin to fix the stained ciliate to the glass slide, and then performing slide preparation processes such as fixation - dehydration - clearing - mounting. There are disadvantages such as increased material and reagent costs, long time consumption, more cumbersome steps, and risks of over-coloring and ciliate deformation during the process. Therefore, in the present invention, the silver-stained ciliate suspension retaining the reaction system liquid is stored in a 4°C refrigerator for microscopic examination. If the stained ciliates in the silver-stained ciliate suspension fade, the silver-stained ciliate suspension is placed in a 70°C water bath and oscillated until it becomes colored, then the water bath is stopped, and the silver-stained ciliate suspension is taken out for slide preparation and microscopic examination. When storing the silver-stained ciliate suspension, when the silver-stained ciliate suspension fades, it can be repeated to extend the good staining effect of the silver-stained ciliate suspension, achieving the purpose of long-term storage of the ammonia silver staining sample without the need for permanent slide preparation operations.
[0032] In some possible embodiments, preparing the methyl green-formalin-saline solution includes: weighing 8 g of sodium chloride and 0.6 g of methyl green, dissolving the sodium chloride and the methyl green in 100 mL of the aqueous formaldehyde solution to obtain a mixed solution, and then making up the volume of the mixed solution to 1 L with distilled water, and storing it sealed and away from light to obtain the methyl green-formalin-saline solution.
[0033] Those skilled in the art can understand that the mixed fixing solution of the methyl green-formalin-saline solution is prepared by sodium chloride, methyl green and an aqueous formaldehyde solution with a concentration of 35% (w / v). Among them, the aqueous formaldehyde solution, that is, the formalin-saline fixing solution, has the advantages of strong bactericidal ability, strong antiseptic property, large penetration power, fast fixing speed, etc., and can play a role in preservation after fixation. By adding the methyl green staining agent, the cytoplasm and nucleus of the worm body can be stained for subsequent ammoniacal silver staining operation.
[0034] In some possible embodiments, preparing the Rio-Hortega solution includes: preparing a silver nitrate solution with a concentration of 10% (w / v) and a sodium carbonate solution with a concentration of 5% (w / v), taking 50 mL of the silver nitrate solution and 150 mL of the sodium carbonate solution, fully mixing them to produce a white precipitate; adding ammonia water dropwise to the white precipitate until the white precipitate dissolves, and then adding ultrapure water to dilute it to 750 ml, and storing it sealed to obtain the Rio-Hortega solution.
[0035] Those skilled in the art can understand that the Rio-Hortega solution prepared by the above method can be stored for a long time and can be used for ammoniacal silver staining of the enriched worm bodies.
[0036] In some possible embodiments, during the process of fixing the rumen fluid with the methyl green-formalin-saline solution, the volume ratio of the rumen fluid to the methyl green-formalin-saline solution is 1:4.
[0037] This is because when the volume ratio of the rumen fluid to the methyl green-formalin-saline solution is 1:4, the morphological structure of the worm cells and tissues in the rumen fluid can be best maintained during the fixation of the rumen fluid.
[0038] In some possible embodiments, when 3 mL of distilled water, 80 - 100 μl of the enriched worms, 130 - 140 μl of pyridine, and 305 - 330 μl of the bacterial peptone solution are added to a centrifuge tube and mixed evenly, during the process of placing the centrifuge tube in the dark for reaction, the ambient temperature of the reaction in the dark is 20 - 25 °C; after mixing evenly, when 1255 - 1400 μl of the Rio - Hortega solution is added to the centrifuge tube and mixed evenly, during the process of placing the centrifuge tube in the dark for reaction, the ambient temperature of the reaction in the dark is 20 - 25 °C.
[0039] This is because, by controlling the ambient temperature of the reaction in the dark to be 20 - 25 °C, the staining effect of rumen ciliates can be made more stable at a suitable ambient temperature.
[0040] In some possible embodiments, during the process of adding 3 mL of distilled water, 80 - 100 μl of the enriched worms, 130 - 140 μl of pyridine, and 305 - 330 μl of the bacterial peptone solution to a centrifuge tube and mixing evenly, 130 - 140 μl of the pyridine is added to the centrifuge tube in a fume hood and mixed evenly.
[0041] Those skilled in the art can understand that since pyridine has a foul smell and strong irritation, by adding 130 - 140 μl of the pyridine to the centrifuge tube in a fume hood and mixing evenly, it can avoid the human body inhaling pyridine and thus causing irritation to the respiratory tract and endangering the human body.
[0042] In some possible embodiments, after preparing the bacterial peptone solution with a concentration of 4%, the prepared bacterial peptone solution is filtered and sterilized using a 0.22 μm sterile needle filter in a laminar flow hood, and the filtered and sterilized bacterial peptone solution is stored at 4 °C for standby; during the process of adding 3 mL of distilled water, 80 - 100 μl of the enriched worms, 130 - 140 μl of pyridine, and 305 - 330 μl of the bacterial peptone solution to a centrifuge tube and mixing evenly, 305 - 330 μl of the bacterial peptone solution is added to the centrifuge tube in a laminar flow hood.
[0043] This is because, by filtering and sterilizing the prepared bacterial peptone solution using a 0.22 μm sterile needle filter in a laminar flow hood, storing the filtered and sterilized bacterial peptone solution at 4 °C for standby, and adding 305 - 330 μl of the bacterial peptone solution to the centrifuge tube in a laminar flow hood, the bacterial peptone solution can be added to the reaction system in a sterile environment, avoiding bacteria from contaminating the sample and affecting the color development effect.
[0044] In some possible embodiments, during the process of adding distilled water into the centrifuge tube, allowing the centrifuge tube to stand still, and then discarding the supernatant in the centrifuge tube, the operations of adding distilled water into the centrifuge tube, allowing the centrifuge tube to stand still, and then discarding the supernatant in the centrifuge tube are repeated until impurities are removed.
[0045] Those skilled in the art can understand that by repeating the operations of adding distilled water into the centrifuge tube, allowing the centrifuge tube to stand still, and then discarding the supernatant in the centrifuge tube multiple times, the black precipitate and impurities generated by the reaction can be washed away, thereby improving the color development effect and facilitating observation.
[0046] In some possible embodiments, during the process of centrifuging 5 - 10 mL of the fixed sample solution, the rotation speed of the centrifugation is 2000 rpm, and the centrifugation time is 1 - 3 min; when taking out the centrifuge tube from cold water, if centrifuging the centrifuge tube, the rotation speed of the centrifugation is 2000 rpm, and the centrifugation time is 1 - 3 min.
[0047] Those skilled in the art can understand that during the process of centrifuging 5 - 10 mL of the fixed sample solution, with the rotation speed of the centrifugation being 2000 rpm and the centrifugation time being 1 - 3 min, the worm bodies in the fixed sample solution can be separated by centrifugation; when taking out the centrifuge tube from cold water, if centrifuging the centrifuge tube, with the rotation speed of the centrifugation being 2000 rpm and the centrifugation time being 1 - 3 min, the silver-stained worm bodies can be separated for subsequent cleaning of the silver-stained worm bodies.
[0048] In order to further elaborate on the technical solution of this application to support the technical problem to be solved by this application, the following provides a specific example of a method for rapidly preparing a sample of rumen ciliate ammonia silver staining, such as Examples 2 - 3. It should be noted that the rumen ciliates in the following Examples 2 - 3 were all collected from Chinese Holstein cows artificially raised in a ranch in Jiangxia District, Wuhan City, Hubei Province. A special rumen fluid collection device was used to collect rumen fluid, and it was immediately filtered on-site to fix the rumen fluid sample.
[0049] Example 2
[0050] Taking the rumen ciliate of the family Ophryoscolecidae as an example, this example provides a method for rapidly preparing a sample of rumen ciliate ammonia silver staining, including the following steps:
[0051] Prepare solutions, including preparing methyl green-formalin-saline solution, Rio-Hortega solution, 35% (w / v) aqueous formaldehyde solution, and 4% (w / v) bacterial peptone solution; preparing the methyl green-formalin-saline solution includes: weighing 8 g of sodium chloride and 0.6 g of methyl green, dissolving the sodium chloride and the methyl green in 100 mL of the aqueous formaldehyde solution to obtain a mixed solution, diluting the mixed solution to 1 L with distilled water, and then storing it sealed and protected from light to obtain the methyl green-formalin-saline solution; preparing the Rio-Hortega solution includes: preparing a 10% (w / v) silver nitrate solution and a 5% (w / v) sodium carbonate solution, taking 50 mL of the silver nitrate solution and 150 mL of the sodium carbonate solution, fully mixing them to produce a white precipitate; adding ammonia water dropwise to the white precipitate until the white precipitate dissolves, and then adding ultrapure water to dilute to 750 ml and storing it sealed; the solution preparation also includes filtering and sterilizing the prepared bacterial peptone solution with a 0.22 μm sterile needle filter in a laminar flow cabinet, and storing the filtered and sterilized bacterial peptone solution at 4 °C for standby;
[0052] Fix the protozoa, collect rumen fluid, fix the rumen fluid with the methyl green-formalin-saline solution, and store the fixed rumen fluid sealed and protected from light at 4 °C to obtain a fixed sample solution; the volume ratio of the rumen fluid to the methyl green-formalin-saline solution is 1:4;
[0053] Enrich the protozoa, select the fixed sample solution with a higher abundance of rumen ciliates in the family Ophryoscolecidae from the obtained fixed sample solution; after centrifuging 6 mL of the fixed sample solution, discard the supernatant and retain the precipitate to obtain enriched protozoa; the rotation speed of the centrifugation is 2000 rpm and the centrifugation time is 2 min;
[0054] For ammoniacal silver staining, add 3 mL of distilled water, 80 μL of the enriched worms, 135 μL of pyridine, and 320 μL of the bacterial peptone solution to a 15-mL centrifuge tube and gently shake to mix. Among them, add 135 μL of the pyridine to the centrifuge tube in a fume hood and mix; add 320 μL of the bacterial peptone solution to the centrifuge tube in a laminar flow hood; after mixing, place the centrifuge tube in the dark for reaction for 20 min. The environmental temperature of the reaction in the dark is 25 °C, and the solution is bluish-green at this time; after the reaction is completed, add 150 μL of the formaldehyde aqueous solution to the centrifuge tube and gently shake to mix. After mixing, add 1400 μL of Rio-Hortega solution to the centrifuge tube and gently shake to mix. At this time, the solution is milky white and the worms are bluish-green. After mixing, place the centrifuge tube in the dark for reaction for 30 min. The environmental temperature of the reaction in the dark is 25 °C, and the solution is grayish-black after the reaction. After the reaction is completed, place the centrifuge tube in a 70 °C water bath and continuously shake for 30 min, then take out the centrifuge tube and place it in cold water. Add 270 μL of Rio-Hortega solution to the centrifuge tube and gently shake to mix. Place the mixed centrifuge tube in a 70 °C water bath again and continuously shake for 15 min, then take out the centrifuge tube and place it in cold water. At this time, the solution is grayish-black;
[0055] Washing of silver-stained worms: Take out the centrifuge tube from cold water. After allowing the centrifuge tube to stand for 10 min, suck out the supernatant in the centrifuge tube and save it separately as a saved solution; add an appropriate amount of distilled water to the centrifuge tube to resuspend the precipitate, and then briefly allow the centrifuge tube to stand and discard the supernatant in the centrifuge tube. Repeat the operations of adding distilled water to the centrifuge tube and then allowing the centrifuge tube to stand and discarding the supernatant in the centrifuge tube multiple times until impurities are removed; add the saved solution to the centrifuge tube to obtain a silver-stained worm suspension;
[0056] Preparing slides for microscopic examination: Take 20 μL of the silver-stained worm suspension and place it on a glass slide. After covering the glass slide with a coverslip, place the glass slide under the microscope to observe the ciliary pattern of the worms in the silver-stained worm suspension and take pictures.
[0057] The morphology of the ammoniacal silver-stained samples of rumen ciliates of the genera Ophryoscolex and Prostomatea in the subfamily Ophryoscolecinae prepared in this example observed under a microscope is as Figure 2 、 Figure 3 shown. From Figure 2 、 Figure 3It can be seen that the ciliary pattern of the worm body is very vividly displayed. After staining, the worm body is light yellow and white, while the ciliary pattern is significantly dark brown. The contrast between the two is obvious. Compared with the traditional method, both the worm body and the ciliary pattern are deeply stained after staining, which is more beautiful and easier to observe. It should be noted that in ammoniacal silver staining, after adding 35% formaldehyde, only by following the steps of adding a certain amount of Rio-Hortega solution in two portions for reaction according to this application can the excellent staining effect shown in the attached drawings be obtained.
[0058] Example 3
[0059] In this example, taking large rumen ciliates of the family Diplotriichidae as an example, a rapid preparation method for an ammoniacal silver staining sample of rumen ciliates is provided, including the following steps:
[0060] Prepare solutions, prepare methyl green-formalin-saline solution, Rio-Hortega solution, formaldehyde aqueous solution with a concentration of 35% (w / v), and bacteriological peptone solution with a concentration of 4% (w / v); preparing the methyl green-formalin-saline solution includes: weighing 8 g of sodium chloride and 0.6 g of methyl green, dissolving the sodium chloride and the methyl green in 100 mL of the formaldehyde aqueous solution to obtain a mixed solution, fixing the volume of the mixed solution to 1 L with distilled water, and storing it sealed and protected from light to obtain the methyl green-formalin-saline solution; preparing the Rio-Hortega solution includes: preparing a silver nitrate solution with a concentration of 10% (w / v) and a sodium carbonate solution with a concentration of 5% (w / v), taking 50 mL of the silver nitrate solution and 150 mL of the sodium carbonate solution, fully mixing them to produce a white precipitate; adding ammonia water dropwise to the white precipitate until the white precipitate dissolves, and then adding ultrapure water to dilute to 750 ml, and storing it sealed; the preparation of the solution also includes filtering and sterilizing the prepared bacteriological peptone solution with a 0.22 μm sterile needle filter in a laminar flow hood, and storing the filtered and sterilized bacteriological peptone solution at 4 °C for standby;
[0061] Fix the worm body, collect rumen fluid, fix the rumen fluid with the methyl green-formalin-saline solution, and store the fixed rumen fluid sealed and protected from light at 4 °C to obtain a fixed sample solution; the volume ratio of the rumen fluid to the methyl green-formalin-saline solution is 1:4;
[0062] Enrich the worm body, select the fixed sample solution with a higher abundance of large rumen ciliates of the family Diplotriichidae from the obtained fixed sample solution; after centrifuging 6 mL of the fixed sample solution, discard the supernatant and retain the precipitate to obtain enriched worm bodies; the rotation speed of the centrifugation is 2000 rpm, and the centrifugation time is 3 min;
[0063] For ammoniacal silver staining, add 3 mL of distilled water, 80 μL of the enriched worms, 140 μL of pyridine, and 320 μL of the bacterial peptone solution to a 15 mL centrifuge tube and gently shake to mix. Among them, add 140 μL of the pyridine to the centrifuge tube in a fume hood and mix; add 320 μL of the bacterial peptone solution to the centrifuge tube in a laminar flow hood; after mixing, place the centrifuge tube in the dark for reaction for 20 min. The ambient temperature for the reaction in the dark is 25 °C, and at this time the solution is bluish-green; after the reaction is completed, add 150 μL of the aqueous formaldehyde solution to the centrifuge tube and gently shake to mix. After mixing, add 1350 μL of Rio-Hortega solution to the centrifuge tube and gently shake to mix. At this time, the solution is milky white and the worms are bluish-green. After mixing, place the centrifuge tube in the dark for reaction for 30 min. The ambient temperature for the reaction in the dark is 25 °C, and the solution is grayish-black after the reaction. After the reaction is completed, place the centrifuge tube in a 70 °C water bath and continuously shake for 30 min, then take out the centrifuge tube and place it in cold water. Add 280 μL of Rio-Hortega solution to the centrifuge tube and gently shake to mix. Place the mixed centrifuge tube in a 70 °C water bath again and continuously shake for 15 min, then take out the centrifuge tube and place it in cold water. At this time, the solution is grayish-black;
[0064] Washing of silver-stained worms: Take out the centrifuge tube from cold water, centrifuge the centrifuge tube at 2000 rpm for 3 min, and suck out the supernatant in the centrifuge tube and save it separately as a saved solution; add an appropriate amount of distilled water to the centrifuge tube to resuspend the precipitate, and then briefly let the centrifuge tube stand and discard the supernatant in the centrifuge tube. Repeat the operations of adding distilled water to the centrifuge tube and then letting the centrifuge tube stand and discarding the supernatant in the centrifuge tube for multiple times until impurities are removed; add the saved solution to the centrifuge tube to obtain a silver-stained worm suspension;
[0065] Preparing slides and microscopic examination: Take 20 μL of the silver-stained worm suspension and place it on a glass slide. After covering the glass slide on the glass slide, place the glass slide under the microscope to observe the ciliary pattern of the worms in the silver-stained worm suspension and take pictures.
[0066] The morphological observations of the ammoniacal silver-stained samples of rumen ciliates of the genera Euplotidium and Polycalix in the subfamily Diplotriinae prepared in this example are as follows Figure 4 、 Figure 5 shown. It can be seen from Figure 4 、 Figure 5 that the ciliary pattern of the worms is very vividly displayed. After staining, the worms are light yellow and the ciliary pattern is significantly dark brown. The contrast between the two is obvious. Compared with the traditional method, both the worms and the ciliary pattern are darkly stained after staining, which is more beautiful and easier to observe.
[0067] Finally, it should also be noted that the endpoints and any values within the ranges disclosed in this document are not limited to the exact ranges or values. These ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed in this document.
[0068] The term "comprising", "including" or any other variant thereof is intended to cover non-exclusive inclusion, such that a process, method, article or apparatus comprising a series of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article or apparatus.
[0069] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications once they learn the basic creative concept. Therefore, the appended claims are intended to be construed as including the preferred embodiments as well as all changes and modifications falling within the scope of the present invention.
[0070] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these modifications and variations.
Claims
1. A rapid preparation method for a rumen ciliate ammonia silver staining sample, characterized in that, Comprising the following steps: Preparing a methyl green-formalin-saline solution, a Rio-Hortega solution, an aqueous formaldehyde solution with a concentration of 35%, and a bacterial peptone solution with a concentration of 4%; Collecting rumen fluid, fixing the rumen fluid with the methyl green-formalin-saline solution, and storing the fixed rumen fluid in the dark and sealed at 4°C to obtain a fixed sample solution; Taking 5 - 10 mL of the fixed sample solution, centrifuging it, discarding the supernatant, and retaining the precipitate to obtain enriched protozoa; Adding 3 mL of distilled water, 80 - 100 μL of the enriched protozoa, 130 - 140 μL of pyridine, and 305 - 330 μL of the bacterial peptone solution into a centrifuge tube and mixing well. After mixing, place the centrifuge tube in the dark for reaction for 20 min; after the reaction is completed, add 140 - 160 μL of the aqueous formaldehyde solution into the centrifuge tube and mix well. After mixing, add 1255 - 1400 μL of the Rio-Hortega solution into the centrifuge tube and mix well. After mixing, place the centrifuge tube in the dark for reaction for 30 min; after the reaction is completed, place the centrifuge tube in a 70°C water bath and oscillate for 30 min, then take out the centrifuge tube and place it in cold water. Add 245 - 290 μL of the Rio-Hortega solution into the centrifuge tube and mix well. Place the mixed centrifuge tube in a 70°C water bath and oscillate for 15 min, then take out the centrifuge tube and place it in cold water; Taking out the centrifuge tube from cold water, allowing the centrifuge tube to stand or centrifuging it, then sucking out the supernatant in the centrifuge tube and storing it separately as a separately stored solution; adding distilled water into the centrifuge tube, then allowing the centrifuge tube to stand and discarding the supernatant in the centrifuge tube, adding the separately stored solution into the centrifuge tube to obtain a silver-stained protozoa suspension.
2. The rapid preparation method of the rumen ciliate ammonia silver staining sample according to claim 1, characterized in that, After obtaining the silver-stained protozoa suspension, the preparation method further includes: Storing the obtained silver-stained protozoa suspension in a 4°C refrigerator for microscopic examination. If the stained protozoa in the silver-stained protozoa suspension fade, place the silver-stained protozoa suspension in a 70°C water bath and oscillate until it shows color, then stop the water bath, take out the silver-stained protozoa suspension and prepare a slide for microscopic examination.
3. The rapid preparation method of the rumen ciliate ammonia silver staining sample according to claim 2, characterized in that, Preparing the methyl green-formalin-saline solution includes: Weighing 8 g of sodium chloride and 0.6 g of methyl green, dissolving the sodium chloride and the methyl green in 100 mL of the aqueous formaldehyde solution to obtain a mixed solution, making the volume of the mixed solution up to 1 L with distilled water, and storing it in a sealed and light-proof manner to obtain the methyl green-formalin-saline solution.
4. The rapid preparation method of the rumen ciliate ammonia silver staining sample according to claim 3, wherein, Preparing the Rio-Hortega solution includes: Preparing a silver nitrate solution with a concentration of 10% and a sodium carbonate solution with a concentration of 5%. Taking 50 mL of the silver nitrate solution and 150 mL of the sodium carbonate solution, mixing them well to produce a white precipitate; adding ammonia water drop by drop to the white precipitate until the white precipitate dissolves, and then diluting it to 750 ml with ultrapure water and storing it in a sealed manner to obtain the Rio-Hortega solution.
5. The rapid preparation method of a rumen ciliate ammoniacal silver staining sample according to claim 4, wherein: During the process of fixing the rumen fluid with the methyl green-formalin-saline solution, the volume ratio of the rumen fluid to the methyl green-formalin-saline solution is 1:
4.
6. The rapid preparation method of the rumen ciliate ammonia silver staining sample according to claim 5, characterized in that: During the process of adding 3 mL of distilled water, 80 - 100 μL of the enriched ciliates, 130 - 140 μL of pyridine and 305 - 330 μL of the bacterial peptone solution into a centrifuge tube and mixing them evenly, and then placing the centrifuge tube in the dark for reaction, the ambient temperature for the reaction in the dark is 20 - 25 °C; During the process of adding 1255 - 1400 μL of the Rio-Hortega solution into the centrifuge tube after mixing evenly and mixing them evenly again, and then placing the centrifuge tube in the dark for reaction, the ambient temperature for the reaction in the dark is 20 - 25 °C.
7. The rapid preparation method of the rumen ciliate ammonia silver staining sample according to claim 6, characterized in that: During the process of adding 3 mL of distilled water, 80 - 100 μL of the enriched ciliates, 130 - 140 μL of pyridine and 305 - 330 μL of the bacterial peptone solution into a centrifuge tube and mixing them evenly, 130 - 140 μL of the pyridine is added into the centrifuge tube in a fume hood and mixed evenly.
8. The rapid preparation method of the rumen ciliate ammonia silver staining sample according to claim 7, characterized in that: After preparing the bacterial peptone solution with a concentration of 4%, the prepared bacterial peptone solution is filtered and sterilized using a 0.22 μm sterile needle filter in a laminar flow cabinet, and the filtered and sterilized bacterial peptone solution is stored at 4 °C for standby; During the process of adding 3 mL of distilled water, 80 - 100 μL of the enriched ciliates, 130 - 140 μL of pyridine and 305 - 330 μL of the bacterial peptone solution into a centrifuge tube and mixing them evenly, 305 - 330 μL of the bacterial peptone solution is added into the centrifuge tube in a laminar flow cabinet.
9. The rapid preparation method of the rumen ciliate ammonia silver staining sample according to claim 8, characterized in that: During the process of adding distilled water into the centrifuge tube, and then discarding the supernatant in the centrifuge tube after standing the centrifuge tube, the operation of adding distilled water into the centrifuge tube and then discarding the supernatant in the centrifuge tube after standing is repeated until impurities are removed.
10. The rapid preparation method of the rumen ciliate ammonia silver staining sample according to claim 9, characterized in that: During the process of centrifuging 5 - 10 mL of the fixed sample solution, the rotation speed of centrifugation is 2000 rpm, and the centrifugation time is 1 - 3 min; When taking out the centrifuge tube from cold water, if centrifuging the centrifuge tube, the rotation speed of centrifugation is 2000 rpm, and the centrifugation time is 1 - 3 min.