Source of content: Microbiology Lab.
Operation method
Plant cell structure and plant freehand section observation test
Principle
1. understand the diversity of plant cell morphology; simple staining techniques. 2. understand the structure of plant cells and plant freehand sectioning techniques. 3. identify and characterize common posterior inclusions in plant cells.
Materials and Instruments
Onions Green chili peppers Red chili peppers Potato tubers Duckweed Spinach leaves Hawthorn Tomatoes Wheat grains Castor Lentil flowers Apples Seeds Roots Move I. Plant Cell Structure For more product details, please visit Aladdin Scientific website.
Methyl blue, alkaline violet, resorcinol, alcohol, hydrofluoric acid, distilled water.
Microscope Dissecting glass Blade Slide Coverslip Tweezers Absorbent paper Petri dish Surface dish Staining dish Straw Straws Alcohol lamp Gauze Sateen cloth Brush Brush
1. Observation of plant cell morphology
(1) Wash the slides Wash the coverslips and slides with water and dry them with gauze.
(2) Take materials Place the slide flat on the lab bench, add a drop of distilled water to its center, and use tweezers or a pipette to take plant tissue isolation materials in the distilled water.
(3) Add a coverslip Put one side of the coverslip in contact with the edge of the drop of water on the slide first, and then slowly lower the other side to squeeze out the air under the coverslip to avoid air bubbles.
(4) Under the microscope, cells of various shapes are visible.
2. Observation of cell wall, nucleus, cytoplasm and vesicles
Cut a narrow strip (0.5 cm wide) of onion scale leaves (or onion scale leaves) longitudinally, and then use a razor blade to make two light cuts in the direction perpendicular to the narrow strip at the inner epidermis of the scale leaves (just cut through the epidermis) with a distance of about 0.5 cm between the two cuts. The piece of epidermis between the two cuts was gently torn off with forceps. When mounting the slides, first put a drop of water on a clean slide, then put the onion epidermis and cover the coverslip. Under low magnification, many long rows of colorless, transparent cells can be seen, with a round nucleus in the cytoplasm of the cells. Remove the mount from the microscope and stain it with iodine solution. Observe with high magnification to see the cell wall and the nucleus stained yellowish brown, the cytoplasm stained yellowish and the vesicles in the cytoplasm.
3. Observation of mitochondria
Tear a small piece of the inner epidermis of a duckweed leaf or an onion leaf, put it on a slide, add a drop of 0.25% alkaline violet, rinse it for 1-2 minutes to remove the stain, and then observe it under a high magnification microscope with aqueous film. You can see some tiny particles stained purple, which have Brownian motion, and these particles are mitochondria.
4. Observation of plastids in plant cells
(1) Chloroplast
Take the cucurbit moss anthophyllum or black algae on a slide, scrape off a small part of the leaf with a knife, make a mount, and observe it under a low-power microscope, which shows that the shape of the leaf of cucurbit moss is nearly rectangular, and each cell contains many granular chloroplasts. Changing to a high magnification microscope for magnified observation, it can be clearly seen that the chloroplasts within its cells are discus-shaped.
(2) White Body
Take the younger leaves of purple duckweed, tear the epidermis along the leaf veins, and observe the preparation, many granular white bodies can be seen, distributed around the nucleus of the cell.
(3) Colored bodies
Take the ripe tomato (or red pepper) fruit, with a needle from under the pericarp to pick a small piece of pulp made of pressed film, observed with a high magnification lens, can be seen in the cytoplasm of the thin-walled cells of their pulp with orange-red colored bodies.
5. Observation of the inclusions of plant cells
(1) Starch granules
Take a potato tuber to do freehand section, first in the low magnification observation, can see its thin-walled cells contain many white particles that starch granules. Changed to high magnification observation, can see the starch grain whorl, and can find with two umbilical starch grain.
(2) Storage protein
Take castor seeds, peel off the seed coat, and use the fat endosperm to make freehand slices. First put the slices into a petri dish containing pure alcohol and wash for a few minutes, so that the fat in the slices is dissolved in alcohol. Then the slices were taken out to make mounts, and when observed under a high magnification microscope, the paste powder grains within the endosperm cells could be seen, which were composed of protein crystals stored in the vesicles, globulin bodies, and amorphous gelatinous material that filled the vesicles. If a drop of iodine-potassium iodide reagent is added to the section, the proteins appear yellow.
(3) Oil drop
The endosperm of castor bean is sliced freehand and stained with Sudan III solution. When observed under low magnification, oil droplets (fat droplets) stained red can be seen inside the cells.
(4) Crystallization
Take begonia petioles (or geranium stems) to do cross-section freehand section, observed under the microscope, can be seen in the basic tissue cells often have single crystals or clusters of crystals.
(5) Anthocyanin
Lentil petals laid flat on a slide, with a razor blade to scrape off the lower epidermis and part of the thin-walled tissue, the remaining part of the mount, observed under the microscope, visible in its thin-walled cells within the cytosol was red, which is the cytosol in the anthocyanins show the color.
6. Intercellular connectivity observation
With a razor blade along the surface of the peel of the fresh via chili pepper in a parallel direction, cut a thin slice (or put the inside of the peel of the pepper face up on the table, scrape off the hypertrophic material with a fast knife, so that it is very thin), plus iodine solution stained tablets for observation. Under high magnification, it can be seen that the epidermis is composed of irregular groups of cells, cells with yellowish cytoplasm. The cell wall is very thick, dark yellow in color, with small pores on the wall (striated pores), with cytoplasm passing through the pores.
7. Changes in the cell wall
(1) Cell wall lignification
Take the old stems of broad beans and other herbaceous plants and make transverse freehand sections, put them on slides, first add a drop of hydrochloric acid to saturate the cells, wait for 2-3 minutes, remove the excess hydrochloric acid, then add a drop of mesitylene trisphenol solution (50% alcoholic solution), and finally add a coverslip and observe under a microscope.
It can be seen that some thick-walled cell groups of cell walls with red, which is in the acidic environment of lignin and resorcinol red reaction.
(2) Cell wall keratinization
Take a broad bean, tomato or sunflower and make a transverse freehand section. Add a drop of Sudan III alcohol reagent and stain the section for microscopic observation.
Can see the stem of the outermost epidermal cells of the outer wall with orange-red. This is the stem epidermal cells deposited on the outer wall of the keratin (fat-like substances) and Sudan Ⅲ reaction results.
(3) Cell wall embolization
Take the potato tuber and cut it into rectangles of about 1 cm thickness, then make a freehand transverse section along the surface of the short diameter of the rectangles, and then stain the sections with Sudan Ⅲ alcohol staining and observe them by microscopic examination.
It can be seen that the cell wall of several layers of cells on the surface of the orange-red, which is the result of the reaction between the cell wall plug plasmalogens and Sudan Ⅲ.
Second, plant freehand section
1. Selection of materials
Generally choose normal development, soft and hard moderate plant organs or tissues as materials.
The fresh material taken should be put into water in time to avoid wilting during freehand sectioning.
If the material is too hard, it can be softened with 1.5% hydrofluoric acid or an equal mixture of glycerin and 70% alcohol; if the material is too soft, it can be sliced by sandwiching the material with a potato tuber or carrot root as a support.
For some plants, the leaves can be rolled into a tube for slicing or made into a simple "cutter" with three double-sided blades for slicing.
The size of the material to be sliced is usually no more than 5 mm in diameter and 15-25 mm in length.
2. Slicing
(1) First, cut the material to be sliced into pieces of appropriate size with a razor blade, and flatten the cut surface, and then dip the material and the blade into water to moisten it (and then moisten it with water every time you cut a piece).
(2) Hold the material with the thumb, forefinger and middle finger of your left hand. To prevent cutting your fingers while slicing, keep the top end of the material (the cut surface) slightly higher than your index finger and your thumb slightly lower than your index finger. Pinch the end of the blade with the thumb and index finger of the right hand and place it on top of the index finger of the right hand, with the blade parallel to the cut surface of the material and with the blade placed slightly below the cross-section of the material on the left front side of the material.
(3) with uniform force and smooth movement to make the knife blade from the left front to the right rear oblique slide pull cutting, pay attention to not straight cut, non-stop in the middle of the pull cutting speed should be fast, do not push forward and drag the back (sawing) cutting, the left hand forefinger slightly downward movement, so that the material slightly rise, so as to mobilize the thickness of each piece of slicing. Slicing process of the right hand does not move, just move the right arm, move the role of arm force without using wrist force.
(4) slices should be thin, flat and intact, the slices will be cut with a brush dipped in water from the blade gently moved into a petri dish of water or directly submerge the blade in water to rinse down the slices.
3. Provisional slide preparation.
In the center of a clean slide drop of water, with tweezers in the Petri dish to select thin and transparent, intact slices placed in the drop of water, take a clean cover slice from the left side of the drop of water slowly and diagonally under the cover to avoid the emergence of air bubbles, so that made the clinical fashion slides.
