CN106053177A - Specimen making method for observing anatomic structure of lamina of tillandsia - Google Patents
Specimen making method for observing anatomic structure of lamina of tillandsia Download PDFInfo
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- CN106053177A CN106053177A CN201610612203.5A CN201610612203A CN106053177A CN 106053177 A CN106053177 A CN 106053177A CN 201610612203 A CN201610612203 A CN 201610612203A CN 106053177 A CN106053177 A CN 106053177A
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- ethanol
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- wax
- dimethylbenzene
- tillandsia
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/2813—Producing thin layers of samples on a substrate, e.g. smearing, spinning-on
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/36—Embedding or analogous mounting of samples
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
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Abstract
The invention discloses a specimen making method for observing an anatomic structure of a lamina of tillandsia. The specimen making method for observing the anatomic structure of the lamina of the tillandsia comprises the following steps of (1), selecting the lamina; (2), fixing a material; (3), softening the material; (4), dehydrating and transparentizing the material; (5), sealing the material with wax; (6), infiltrating the material with wax; (7), embedding the material; (8), trimming and slicing the material; (9), sticking the material; (10), dyeing the material; (11), sealing and observing the material. In comparison with the prior art, according to the specimen making method for observing the anatomic structure of the lamina of the tillandsia, a softening process is improved; a new softening reagent combination is used; aiming at the lamina of the tillandsia, an ideal softening effect can be obtained. In addition, an embedding process is innovated; an operation method is simple and convenient; a complete wax stone can be obtained; an original embedding operating procedure is simplified. The problem that a clear observation material of the anatomic structure of the lamina of the tillandsia cannot be obtained in the prior art is solved.
Description
Technical field
The present invention relates to a kind of Slide processing for observing Tillandsia natomical leaf structure, belong to leaf blade specimen
Manufacture technology field.
Background technology
Tillandsia (Tillandsia), in originating in, the tropical and subtropical region of South America, belongs to pineapple family
(Bromeliaceae) herbaceos perennial, wherein based on pineapple family Tillandsia.Sight and valency due to Tillandsia
Value is more unique, and China at the beginning of 21 century has started the introduction to these species, and Tillandsia is the most gradually protected at landscape art, environment
The aspect such as protect to show up prominently.
Moisture required for growth of Tillandsia and nutrient substance are both from air, it is not necessary to by means of soil, and blade
Exposing the most most parts as it, will certainly become absorption moisture and the major organs of nutrient substance, structure is merit
The basis of energy, so the structure existence that gas raw Fructus Ananadis comosi blade interior is the most special.Tillandsia is of a great variety, in order to air phoenix
Pears more reasonably utilize, and the blade construction characteristic of different cultivars is badly in need of further investigation.
But more conventional plant is very different the blade construction of Tillandsia, the research method of common blade anatomical structure
It is difficult to be suitable for it.
Summary of the invention
Goal of the invention: in order to solve above-mentioned technical problem, the present invention provides a kind of for observing Tillandsia Leaf Anatomic
The Slide processing of structure.
Technical scheme: in order to realize foregoing invention purpose, the invention discloses a kind of for observing Tillandsia blade solution
Cut open the Slide processing of structure, comprise the following steps:
(1) blade is chosen: select, without pest and disease damage, individual plant that upgrowth situation is good, to choose healthy leaves in the middle part of it；
(2) material is fixed: blade is cut into rectangular piece, puts in FAA fixative fixing after rinsing with clear water, fixing
24-36 hour；
(3) material softening: first put into by step (2) gained sample in 50% glycerol+50% ethanol 1:1 solution, soaks 20-
30 days, then place in the mixed liquor of hydrogen peroxide and propanoic acid, took out sample every 30 minutes-1 hour, repeat 6-10 time,
Flowing water rinses；
(4) material dehydration, transparent: through the material softened, utilize ethanol solution and the dimethylbenzene of variable concentrations, by setting
Determine program to be dehydrated；
(5) material sealing wax: room temperature cooling again after being melted completely by white paraffin wax, so repeats 30-40 time, until paraffin is complete
Entirely become milk yellow；Then the milk yellow wax stone boiled is melted stand-by, material is put into container, then it is complete to pour paraffin into
Flood end；
(6) material oozes wax: be placed in 24-48h in the electro-heating standing-temperature cultivator of 37 DEG C after sealing wax, by electrothermostat liter
Temperature, to 60 DEG C of 0.5h, is then removed the mixture of 1/3 volume paraffin and dimethylbenzene, is added equal-volume pure wax 1-2h, then go
1/2 volume paraffin and the mixture of dimethylbenzene, add equal-volume pure wax 1-2h, the purest wax I 1-2h, pure wax II 1-2h,
Embed after 0.5h；
(7) material embedding: make the armed rectangular paper box of both sides band with the paper that hardness is moderate and smooth, will process
Carton put into by good material, respectively places a block of material near two armrest sides in a carton；The milk yellow paraffin melted is fallen
Entering in carton, pouring volume accounts for the 2/3 of carton, then tweezers burn-out on alcohol burner, drives the bubble in paraffin away, will with tweezers
Carton put into by material, rapidly material marshalling, when paraffin starts to solidify, is put in cold water by carton rapidly, and
Overturning carton in water immediately, bottom-up, wax stone can completely depart from carton, completes embedding；
(8) material repaiies block, section: accomplished by paraffin mass trapezoidal, is cut by paraffin unnecessary for surrounding materials with blade, makes to cut
Face is the least, but is not expressed to material, device materials, adjusts thickness marker, makes indication scale just be suitable for wanted thickness,
Obtain wax band；
(9) material bonding die
One droplet alite paste is coated on microscope slide, smoothens with finger, put down step (8) gained wax band gently microscope slide
On, and drip 3% formalin, make wax band launch, the microscope slide with wax band is placed on temperature platform on, make surface dry, will
Microscope slide is placed in 2-5d in 37 DEG C of electro-heating standing-temperature cultivators, accelerates it and is dried；
(10) dyeing material: use sarranine-fast green counterstaining to dye；
(11) material mounting is observed: the coverslip after processing is middle rapidly to be taken out, and drips a 1-2 and drips neutral gum mounting, covers
Coverslip, it is to avoid have bubble to produce, after drying overnight, to obtain final product in 37 DEG C of electro-heating standing-temperature cultivators, available observation by light microscope
And take pictures.
As preferably, described Tillandsia is overlord, torsion Ye Tielan, trichroism ferrum is blue, tie up road orange red Na, bronze gas is spent, this is vertical
Ke Te, Bei Ji, Harris, Usnea or line leaf ferrum are blue.
Preferred as another kind, described in step (2), FAA fixative volume is 20 times of fixing organization, and it is configured that
70% or 50% ethanol 90mL: glacial acetic acid: formaldehyde=18:1:1.
Preferred as another kind, described in step (3) in the mixed liquor of hydrogen peroxide and propanoic acid, hydrogen peroxide and propanoic acid
Volume ratio is 1:1 or 2:1.
Preferred as another kind, described in step (4), setting program is as follows:
Using the ethanol of 30% successively, 70% ethanol (adds the sarranine of 0.5g) in the ethanol of every 100ml 70%, 85% second
Alcohol (1-2h), 95% ethanol (1-2h), dehydrated alcohol (1-2h), dehydrated alcohol (0.5-1h), 3/4 ethanol+1/4 dimethylbenzene (1-
2h), 1/2 ethanol+1/2 dimethylbenzene (1-2h), 1/4 ethanol+3/4 dimethylbenzene (1-2h), dimethylbenzene (1-2h), dimethylbenzene (0.5-
1h) it is dehydrated.
Preferred as another kind, described in step (10), dyeing course is as follows:
Dimethylbenzene I (20-30min), dimethylbenzene II (20-30min), 1/2 dimethylbenzene+1/2 ethanol (5-10min), 100%
Ethanol (5-10min), 95% ethanol (5-10min), 85% ethanol (5-10min), 70% ethanol (5_10min), 50% ethanol
(5-10min), 30% ethanol (5-10min), 1% sarranine (12h), 50% ethanol (5-10min), 70% ethanol (5-10min),
85% ethanol (5-10min), 95% ethanol (5-10min), 0.1% fast green (1-3min), 95% ethanol (5-10min), 100%
Ethanol I (5-10min), 100% ethanol II (5-10min), 1/2 dimethylbenzene+1/2 ethanol (5-10min), dimethylbenzene I (20-
30min), dimethylbenzene II (20-30min).
Technique effect: relative to prior art, the inventive method improves softening process, employs new softening reagent set
Close, preferable bating effect can be obtained for Tillandsia blade.Additionally, innovated embedding process, operational approach is easy, can obtain
Obtain wax stone completely, simplify original embedding operation sequence.Solve prior art and cannot obtain Tillandsia blade clearly
The problem that anatomical structure observes material
Accompanying drawing explanation
Fig. 1: for overlord's natomical leaf structure；
Fig. 2: for turning round leaf ferrum orchid natomical leaf structure.
Detailed description of the invention
Embodiment 1
1, blade is chosen
Choose without pest and disease damage, the good individual plant of upgrowth situation, choose healthy leaves in the middle part of plant.Selection kind is overlord.
2, material is fixed:
Different cultivars leaf blade size is different, chooses a kind of specification depending on leaf blade size, blade is cut into 3mm × 5mm, 4mm ×
The rectangular piece of 6mm, 5mm × 7mm or 6mm × 8mm, the material clear water cut puts in FAA fixative fixing after rinsing,
Fixative volume is 20 times of fixing organization.FAA fixative configures: 70% or 50% ethanol 90mL: glacial acetic acid: formaldehyde=18:
1:1, fixes 30 hours.
3, material softening:
1) sample is put in 50% glycerol+50% ethanol (95%) 1:1 solution, soak 25 days.
2) material is put in the mixed liquor of hydrogen peroxide and propanoic acid (1:1), took out with tweezers every 30 minutes-1 hour
Sample, optic lobe sheet material soft or hard, repeat 6-10 time, after flowing water rinses about 10 minutes, use blade trial cut, if soft.
4, material dehydration, transparent:
First rinse well with tap water before the material dehydration softened, then proceed to dewatering of ethanol → 70% of 30%
(sarranine pre-dyed overnight) (adds the sarranine of 0.5g) → 85% ethanol (1-2h) → 95% second to ethanol in the ethanol of every 100ml 70%
Alcohol (1-2h) → dehydrated alcohol (1-2h) → dehydrated alcohol (0.5-1h) → 3/4 ethanol+1/4 dimethylbenzene (1-2h) → 1/2 ethanol+
1/2 dimethylbenzene (1-2h) → 1/4 ethanol+3/4 dimethylbenzene (1-2h) → dimethylbenzene (1-2h) → dimethylbenzene (0.5-1h).
5, material sealing wax
1) wax is endured
White paraffin wax is put into Porcelain Jar heat on microwave oven, treat that paraffin melts room temperature cooling completely.So repeat 30-
40 times, until paraffin becomes milk yellow completely.
2) sealing wax
By the yellow wax stone that boils as in the electrothermostat of 60 DEG C, melt stand-by.Material is put into container, falls
Enter paraffin and flood end completely.
6, material oozes wax
36h in the electro-heating standing-temperature cultivator of 37 DEG C it is placed in after sealing wax.Electrothermostat is warming up to 60 DEG C about
0.5h, removes the mixture of 1/3 volume paraffin and dimethylbenzene, add the pure wax of equal-volume (1～2h) → go 1/2 volume paraffin with
The mixture of dimethylbenzene, adds the pure wax of equal-volume (1～2h) → pure wax I (1～2h) → pure wax II (1～2h).Carry out after 0.5h
Embedding.
7, material embedding
Box processed:
3cm × 5cm, the armed rectangular paper of both sides band is made with the paper (such as journal surface) that hardness is moderate and smooth
Box.And it is standby to prepare a basin cold water, tweezers, a basin cold water, an alcohol burner.
Embedding:
The material handled well is put into carton, in a carton, respectively places a block of material near two armrest sides.By thawing well
Yellow paraffin pour in carton, pouring volume accounts for the 2/3 of carton.Then tweezers burn-out on alcohol burner, drive away in paraffin
Bubble.With tweezers, material is put into carton, rapidly material marshalling.When paraffin starts to solidify, rapidly carton is put
Entering in cold water, and overturn carton in water immediately, bottom-up, wax stone can completely depart from carton, completes embedding.
8, material repaiies block, section
Paraffin mass is accomplished trapezoidal, with blade, paraffin unnecessary for surrounding materials is cut, make tangent plane the least, but not
It is expressed to material.Device materials, adjusts thickness marker, makes indication scale just be suitable for wanted thickness.Wax stone hardness and section speed
Degree all can affect chipping qualities.
9, material bonding die
One droplet alite paste is coated on microscope slide that (microscope slide needs, in advance through 95% alcohol-pickled a few hours, to use cloth to wipe with
Dry), smoothen with finger, gained wax band put down gently on microscope slide, and drip 3% formalin, make wax band launch.Will be with
The microscope slide of wax band is placed on temperature platform, makes surface dry.Microscope slide is placed in 3d in 37 DEG C of electro-heating standing-temperature cultivators, accelerates it and do
Dry.
10, dyeing material
Using sarranine-fast green counterstaining, dyeing course is as follows: dimethylbenzene I (20-30min) → dimethylbenzene II (20-
30min) → 1/2 dimethylbenzene+1/2 ethanol (5-10min) → 100% ethanol (5-10min) → 95% ethanol (5-10min) →
85% ethanol (5-10min) → 70% ethanol (5_10min) → 50% ethanol (5-10min) → 30% ethanol (5-10min) →
1% sarranine (12h) → 50% ethanol (5-10min) → 70% ethanol (5-10min) → 85% ethanol (5-10min) → 95% second
Alcohol (5-10min) → 0.1% fast green (1-3min) → 95% ethanol (5-10min) → 100% ethanol I (5-10min) → 100%
Ethanol II (5-10min) → 1/2 dimethylbenzene+1/2 ethanol (5-10min) → dimethylbenzene I (20-30min) → dimethylbenzene II (20-
30min)。
11, material mounting is observed
Rapidly coverslip is taken out from dimethylbenzene (can not make dimethylbenzene dry for a long time after just mounting), drip a 1-2 and drip neutral tree
Glue mounting, covered, avoid bubble to produce as far as possible, after 37 DEG C of electro-heating standing-temperature cultivators are dried overnight, shown with optics
Micro mirror is observed and takes pictures.
Embodiment 2:
Select kind for turning round Ye Tielan.
During step 2 material is fixing, the set time is 24 hours；
In step 3 material softening, sample is put in 50% glycerol+50% ethanol (95%) 1:1 solution, soak 20 days；
The volume entering hydrogen peroxide and propanoic acid in mixed liquor is 2:1；
Step 6 material oozes in wax, is placed in 24h in the electro-heating standing-temperature cultivator of 37 DEG C after sealing wax；
In step 9 material bonding die, microscope slide is placed in 2d in 37 DEG C of electro-heating standing-temperature cultivators.
Embodiment 3:
Selecting kind is that trichroism ferrum is blue.
During step 2 material is fixing, the set time is 36 hours；
In step 3 material softening, sample is put in 50% glycerol+50% ethanol (95%) 1:1 solution, soak 30 days；
The volume entering hydrogen peroxide and propanoic acid in mixed liquor is 1:1；
Step 6 material oozes in wax, is placed in 48h in the electro-heating standing-temperature cultivator of 37 DEG C after sealing wax；
In step 9 material bonding die, microscope slide is placed in 5d in 37 DEG C of electro-heating standing-temperature cultivators.
Claims (6)
1. the Slide processing being used for observing Tillandsia natomical leaf structure, it is characterised in that comprise the following steps:
(1) blade is chosen: select, without pest and disease damage, individual plant that upgrowth situation is good, to choose healthy leaves in the middle part of it；
(2) material is fixed: blade is cut into rectangular piece, puts in FAA fixative fixing after rinsing with clear water, fixing 24-
36 hours；
(3) material softening: first put into by step (2) gained sample in 50% glycerol+50% ethanol 1:1 solution, soaks 20-30
My god, then place in the mixed liquor of hydrogen peroxide and propanoic acid, took out sample every 30 minutes-1 hour, repeat 6-10 time, stream
Water rinses；
(4) material dehydration, transparent: through the material softened, utilize ethanol solution and the dimethylbenzene of variable concentrations, by setting journey
Sequence is dehydrated；
(5) material sealing wax: room temperature cooling again after being melted completely by white paraffin wax, so repeats 30-40 time, until paraffin becomes completely
Become milk yellow；Then the milk yellow wax stone that boils is melted stand-by, material is put into container, then pour paraffin into and flood completely
End；
(6) material oozes wax: is placed in 24-48h in the electro-heating standing-temperature cultivator of 37 DEG C after sealing wax, is warming up to by electrothermostat
60 DEG C of 0.5h, then remove the mixture of 1/3 volume paraffin and dimethylbenzene, add equal-volume pure wax 1-2h, then remove 1/2 body
JISHI wax and the mixture of dimethylbenzene, add equal-volume pure wax 1-2h, the purest wax I 1-2h, pure wax II 1-2h, 0.5h
After embed；
(7) material embedding: make the armed rectangular paper box of both sides band with the paper that hardness is moderate and smooth, by handle well
Carton put into by material, respectively places a block of material near two armrest sides in a carton；Pour the milk yellow paraffin melted into paper
In box, pouring volume accounts for the 2/3 of carton, then tweezers burn-out on alcohol burner, drives the bubble in paraffin away, with tweezers by material
Put into carton, rapidly material marshalling, when paraffin starts to solidify, rapidly carton is put in cold water, and in water
Overturning carton immediately, bottom-up, wax stone can completely depart from carton, completes embedding；
(8) material repaiies block, section: accomplished by paraffin mass trapezoidal, is cut by paraffin unnecessary for surrounding materials with blade, makes tangent plane to the greatest extent
May be little, but it is not expressed to material, device materials, adjust thickness marker, make indication scale just be suitable for wanted thickness, obtain wax
Band；
(9) material bonding die
One droplet alite paste is coated on microscope slide, smoothens with finger, step (8) gained wax band is put down gently on microscope slide, and
The formalin of dropping 3%, makes wax band launch, and is placed on by the microscope slide with wax band on temperature platform, makes surface dry, by microscope slide
It is placed in 2-5d in 37 DEG C of electro-heating standing-temperature cultivators, accelerates it and be dried；
(10) dyeing material: use sarranine-fast green counterstaining to dye；
(11) material mounting is observed: the coverslip after processing is middle rapidly to be taken out, and drips a 1-2 and drips neutral gum mounting, and close the lid glass
Sheet, it is to avoid have bubble to produce, after drying overnight, to obtain final product in 37 DEG C of electro-heating standing-temperature cultivators, and available observation by light microscope is also clapped
According to.
Slide processing for observing Tillandsia natomical leaf structure the most according to claim 1, its feature exists
In, described Tillandsia is overlord, torsion Ye Tielan, trichroism ferrum orchid, dimension road orange red Na, bronze gas flower, Si Chuikete, Bei Ji, Harry
This, Usnea or line leaf ferrum blue.
Slide processing for observing Tillandsia natomical leaf structure the most according to claim 1, its feature exists
In, FAA fixative volume described in step (2) is 20 times of fixing organization, and it is configured that 70% or 50% ethanol 90mL: ice
Acetic acid: formaldehyde=18:1:1.
Slide processing for observing Tillandsia natomical leaf structure the most according to claim 1, its feature exists
In, described in step (3) in the mixed liquor of hydrogen peroxide and propanoic acid, the volume ratio of hydrogen peroxide and propanoic acid is 1:1 or 2:1.
Slide processing for observing Tillandsia natomical leaf structure the most according to claim 1, its feature exists
In, described in step (4), setting program is as follows:
Using the ethanol of 30% successively, 70% ethanol (adds the sarranine of 0.5g) in the ethanol of every 100ml 70%, 85% ethanol (1-
2h), 95% ethanol (1-2h), dehydrated alcohol (1-2h), dehydrated alcohol (0.5-1h), 3/4 ethanol+1/4 dimethylbenzene (1-2h), 1/
2 ethanol+1/2 dimethylbenzene (1-2h), 1/4 ethanol+3/4 dimethylbenzene (1-2h), dimethylbenzene (1-2h), dimethylbenzene (0.5-1h) is carried out
Dehydration.
Slide processing for observing Tillandsia natomical leaf structure the most according to claim 1, its feature exists
In, described in step (10), dyeing course is as follows:
Dimethylbenzene I (20-30min), dimethylbenzene II (20-30min), 1/2 dimethylbenzene+1/2 ethanol (5-10min), 100% ethanol
(5-10min), 95% ethanol (5-10min), 85% ethanol (5-10min), 70% ethanol (5_10min), 50% ethanol (5-
10min), 30% ethanol (5-10min), 1% sarranine (12h), 50% ethanol (5-10min), 70% ethanol (5-10min),
85% ethanol (5-10min), 95% ethanol (5-10min), 0.1% fast green (1-3min), 95% ethanol (5-10min), 100%
Ethanol I (5-10min), 100% ethanol II (5-10min), 1/2 dimethylbenzene+1/2 ethanol (5-10min), dimethylbenzene I (20-
30min), dimethylbenzene II (20-30min).
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| CN106053177B (en) | 2018-10-23 |
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