CN102607907B - Paraffin section method for fern gametophytes - Google Patents
Paraffin section method for fern gametophytes Download PDFInfo
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- CN102607907B CN102607907B CN201210043067.4A CN201210043067A CN102607907B CN 102607907 B CN102607907 B CN 102607907B CN 201210043067 A CN201210043067 A CN 201210043067A CN 102607907 B CN102607907 B CN 102607907B
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- dimethylbenzene
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Abstract
The invention discloses a paraffin section method for fern gametophytes, which comprises the steps of material selection and fixation, washing, coloration and bluing, dehydration and hyalinizing, waxing and embedding, sectioning, patching, dewaxing and mounting, as well as microscopic examination and photographing, wherein improved FAA (formalin, acetic acid and alcohol) stationary liquid is adopted in the fixation, and the stationary liquid consists of formalin, acetic acid and 30% alcohol (formalin:acetic acid:30% alcohol = 1:1:18). The method is adopted, so that the disadvantage of much youngness of the fern gametophytes can be effectively overcome; the fixation can be well performed for cell divisions at all stages; from a paraffin section in a later stage, a plurality of mitotic sections can be observed, so that a whole development process can be reflected detailedly; therefore, the method has high development and application values.
Description
Technical field
The present invention relates to a kind of method of plant paraffin section, especially a kind of method of Garnetophytes development paraffin section.
Technical background
Up to now be not specifically applied to the technology of preparation pteridophyte section, conventionally prepare the anatomical slice of pteridophyte by conventional paraffin section technology.But conventional paraffin section technology can cause Garnetophytes development loss moisture, thereby gross distortion has changed cell interior shape, cannot normally observe gametophytic anatomical structure by conventional paraffin section technology.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of method of Garnetophytes development paraffin section, comprise and drawing materials and fixing, flushing, dyeing and oil blackeite, dehydration and transparent, waxdip and embedding, section, paster, dewaxing and mounting, microscopy and photograph, the wherein fixing middle fixing liquid of FAA that adopts improvement, it consists of formalin: glacial acetic acid: 30% ethanol=1: 1: 18, and to solve the very young tender problem of Garnetophytes development.
In described dehydration and transparent process, the processing stage of absolute alcohol and pure dimethylbenzene, the time is 5min, and other each phases-time is 10 minutes.
Dyeing adopts haematine bulk dyeing method.
In paraffin method, often use be FAA immobile liquid (formalin: glacial acetic acid: 70% ethanol=1: 1: 18) and Kano immobile liquid (absolute ethyl alcohol: glacial acetic acid=3: 1), the FAA immobile liquid that young tender materials'use 50% alcohol configures.Garnetophytes development individuality is less, gametophyte major part only have one layer of cells to form and water cut abundant, the immobile liquid of alcohol in high concentration and for a long time in dehydration, transparent process easily dehydration excessively shrink.
The present invention is directed to the very young tender feature of Garnetophytes development, inquired into the alcohol concentration in FAA immobile liquid and Kano immobile liquid, finally select the FAA (formalin: glacial acetic acid: 30% ethanol=1: 1: 18) of improvement as immobile liquid.Shorten dehydration and clearing time interval, i.e. 30% absolute ethyl alcohol+2/3, dimethylbenzene → 1/3, ethanol+1/2, dimethylbenzene → 1/2, ethanol+1/3, ethanol → 50% ethanol → 70% ethanol → 85% ethanol → 95% ethanol → absolute ethyl alcohol I → absolute ethyl alcohol II → 2/3 dimethylbenzene → pure dimethylbenzene I → pure dimethylbenzene II, every grade of 10min, wherein absolute alcohol and pure dimethylbenzene 5min.
In improvement FAA immobile liquid, can divide each period by good fixed cell, from the paraffin section in later stage, we can observe a large amount of mitotic sections, the whole growth course of reflection that can be detailed.In this experiment, adopt the paraffin method of improvement, dyeing adopts haematine bulk dyeing method, test period and later stage dyeing time are greatly shortened, and obtained good experimental result, for the tender material of children particularly fixing the and paraffin section of Garnetophytes development faster and better dicing method is provided.
Brief description of the drawings
The paraffin section that Fig. 1 adopts this method to prepare
A embryonic development B archegonium is grown
The paraffin section that Fig. 2 adopts classic method to prepare
A embryonic development B archegonium is grown
Embodiment
Below introduce in detail the method that the present invention adopts.
1, draw materials with fixing
For the first time when culture materials, determine spermatangium, archegonium, the embryonic development time range in each period according to development time, cultivate through repeatedly repeating, draw materials in a large number; Examine under a microscope and draw materials, as early as possible material being placed in fast to the FAA immobile liquid of improvement, and preserving in 4 DEG C of refrigerators, in improvement FAA immobile liquid, fixing 24h.
2, rinse
By the material fixing, rinse 1～2h at low discharge flowing water, 30% alcohol after punching, 50% alcohol, the each 30min of 70% alcohol.
3, dyeing and oil blackeite
Ai Shi brazilwood extract dyeing 2～5d, nucleus becomes reddish violet, and recycling tap water rinses, and makes nucleus become mazarine, and tenuigenin is dyed light blue, and the oil blackeite time is 3～5h; The material that oil blackeite is suitable is put into 30% alcohol and is carried out gradient dehydration.
4, dehydration and transparent
Adopt ethanol evaporation step by step, i.e. 30% ethanol → 50% ethanol → 70% ethanol → 85% ethanol → 95% ethanol → absolute ethyl alcohol I → absolute ethyl alcohol II, every grade of 10min, wherein absolute ethyl alcohol 5min; After dehydration through dimethylbenzene with transition, when in tissue while all being occupied by dimethylbenzene, light can see through, tissue presents pellucidity in various degree; 2/3 absolute ethyl alcohol+2/3, dimethylbenzene → 1/3, ethanol+1/2, dimethylbenzene → 1/2, ethanol+1/3 dimethylbenzene → pure dimethylbenzene I → pure dimethylbenzene II, every grade of 10min, wherein pure dimethylbenzene 5min.
5, waxdip and embedding
With paraffin substituted dimethyl benzene gradually, detailed process is: under normal temperature, the wax of cutting is considered to be worth doing, drop into gradually in the dimethylbenzene bottle that fills material and add wax bits to DEG C incubator of saturated → 37, being placed in constant temperature oven spends the night → bottle stoste is removed to fill into after 1/2 the 1/2 pure wax having melted, this process repeat 2～3 times → all change pure wax 2 times, every step 0.5～1h;
First build up little carton, then the paraffin of fusing is poured into wherein, rapidly material is moved in the carton of containing paraffin, after material being put on right position with tweezers or dissecting needle, in box, paraffin solidifies gradually.For accelerated solidification, in the time that paraffin surface condenses into milky, box is moved on cold platform and can become rapidly lump.
6, section
To fix and fixing paraffin mass platform wood is contained on the folder thing platform of microtome, slicer is fixed on cutter holder, and twisting promotes spiral, and paraffin mass and the edge of a knife are pressed close to, and adjust the angle between them, adjust thickness gauge, controlling slice thickness is 8 μ m, when section, right hand shake swiveling wheel handle, left hand is held writing brush wax band is mentioned, and moves on a piece of paper plate, prepares bonding die.
7, paster
On a clean microslide, be coated with skim alite paste (by egg white, glycerine modulation), drip again 1～2 distilled water, the wax band cutting open with blade in advance with pincet gripping, be placed on the water surface, set position, notice that the smooth one side of wax disk(-sc) light is affixed on microslide, and make it in one end of slide slightly partially, the other end is convenient to adhesive label, then by slide glass low-grade fever or be placed on pre-heated exhibition sheet platform (temperature remains on 40～45 DEG C) on spirit lamp, wax disk(-sc) is launched, then microslide is placed on and on exhibition sheet folder, finishes mark and be placed in 37 DEG C of incubators and dry 12h.
8, dewaxing and mounting
Dried section bubble 1～2h in dimethylbenzene is carried out to dewaxing treatment, take out afterwards; After dimethylbenzene volatilization Deng slice surface, drip canada balsam, get clean cover glass and drip to be sidelong from natural gum and build, avoid producing bubble; Labelled, be placed on fully de-benzene in baking oven, in the box of income section afterwards.
9, microscopy and photograph
Optical microphotograph Microscopic observation record, the lower photomicrograph of photomicrographic device (Olympus BH2～C5060WZ), adopts the result of this method acquisition as shown in Figure 1, adopts the result of conventional method acquisition as shown in Figure 2.。
Claims (2)
1. the method for a Garnetophytes development paraffin section, comprise and drawing materials and fixing, flushing, dyeing and oil blackeite, dehydration and transparent, waxdip and embedding, section, paster, dewaxing and mounting, microscopy and photograph, it is characterized in that the fixing fixing liquid of FAA that adopts improvement, it consists of formalin: glacial acetic acid: 30% ethanol=1:1:18; In described dehydration and transparent process, the processing stage of absolute alcohol and pure dimethylbenzene, the time is 5min, and other each phases-time is 10 minutes; Described dyeing adopts haematine bulk dyeing method.
2. the method for paraffin section described in claim 1, is characterized in that concrete steps are as follows:
1) draw materials with fixing
For the first time when culture materials, determine that according to development time spermatangium, archegonium, embryonic development and sporangium grow the time range in each period, then cultivate through repeatedly repeating, draw materials in a large number; Examine under a microscope and draw materials, as early as possible material being placed in fast to the FAA immobile liquid of improvement, and preserving in 4 DEG C of refrigerators, in improvement FAA immobile liquid, fixing 24h;
2) rinse
By the material fixing, rinse 1～2h at low discharge flowing water, 30% alcohol after rinsing, 50% alcohol, 70% alcohol is respectively processed 30min;
3) dyeing and oil blackeite
Ai Shi brazilwood extract dyeing 2～5d, nucleus becomes reddish violet, and recycling tap water rinses, and makes nucleus become mazarine, and tenuigenin is dyed light blue, and the oil blackeite time is 3～5h; The material that oil blackeite is suitable is put into 30% alcohol and is carried out gradient dehydration;
4) dehydration is with transparent
Adopt ethanol evaporation step by step, i.e. 30% ethanol → 50% ethanol → 70% ethanol → 85% ethanol → 95% ethanol → absolute ethyl alcohol I → absolute ethyl alcohol II, every grade of 10min, wherein absolute ethyl alcohol 5min; After dehydration through dimethylbenzene with transition, when in tissue while all being occupied by dimethylbenzene, light can see through, tissue presents pellucidity in various degree; 2/3 absolute ethyl alcohol+2/3, dimethylbenzene → 1/3, ethanol+1/2, dimethylbenzene → 1/2, ethanol+1/3 dimethylbenzene → pure dimethylbenzene I → pure dimethylbenzene II, every grade of 10min, wherein pure dimethylbenzene 5min;
5) waxdip and embedding
With paraffin substituted dimethyl benzene gradually, detailed process is: under normal temperature, the wax of cutting is considered to be worth doing, drop into gradually in the dimethylbenzene bottle that fills material and add wax bits to DEG C incubator of saturated → 37, being placed in constant temperature oven spends the night → bottle stoste is removed to fill into after 1/2 the 1/2 pure wax having melted, this process repeat 2～3 times → all change pure wax 2 times, every step 0.5～1h;
First build up little carton, then the paraffin of fusing is poured into wherein, rapidly material is moved in the carton of containing paraffin, after material being put on right position with tweezers or dissecting needle, in box, paraffin solidifies gradually; For accelerated solidification, in the time that paraffin surface condenses into milky, box is moved on cold platform;
6) section
To fix and fixing paraffin mass platform wood is contained on the folder thing platform of microtome, slicer is fixed on cutter holder, and twisting promotes spiral, and paraffin mass and the edge of a knife are pressed close to, and adjust the angle between them, adjust thickness gauge, controlling slice thickness is 8 μ m, when section, right hand shake swiveling wheel handle, left hand is held writing brush wax band is mentioned, and moves on a piece of paper plate, prepares bonding die;
7) paster
On a clean microslide, be coated with skim alite paste, it is by egg white, glycerine modulation forms, drip again 1～2 distilled water, the wax band cutting open with blade in advance with pincet gripping, be placed on the water surface, set position, notice that the smooth one side of wax disk(-sc) light is affixed on microslide, and make it in one end of slide slightly partially, the other end is convenient to adhesive label, then by slide glass low-grade fever or be placed on pre-heated exhibition sheet platform on spirit lamp, temperature remains on 40～45 DEG C, wax disk(-sc) is launched, then microslide is placed on and on exhibition sheet folder, finishes mark and be placed in 37 DEG C of incubators and dry 12h,
8) dewaxing and mounting
Dried section bubble 1～2h in dimethylbenzene is carried out to dewaxing treatment, take out afterwards; After dimethylbenzene volatilization Deng slice surface, drip canada balsam, get clean cover glass and drip to be sidelong from natural gum and build, avoid producing bubble; Labelled, be placed on fully de-benzene in baking oven, in the box of income section afterwards;
9) microscopy and photograph
Optical microphotograph Microscopic observation record, photomicrograph under photomicrographic device.
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