In real life
1. Understand the general principle and method of organelle separation through chloroplast separation of plant cells.
2. Observe the spontaneous fluorescence and secondary fluorescence of chloroplasts, and be familiar with the use of fluorescence microscope.
Empirical principle
The common method of separating organelles is to homogenize tissues and suspend them in isotonic medium for differential centrifugation. Chloroplast separation should be carried out in isotonic solution (0.35mol/L sodium chloride or 0.4mol/L sucrose solution) to avoid chloroplast damage caused by osmotic pressure change. Centrifuge the homogenate at the speed of 1000 rpm for 2 minutes to remove tissue residues and intact cells. Then centrifuge at 3000r/5min for 5min to obtain precipitated chloroplasts (mixed with partial nuclei). The separation process is preferably carried out at 0~5℃; If it is at room temperature, it should be separated and observed quickly.
When using fluorescence microscope to detect fluorescent substances, it will be affected by many factors such as temperature, light and quencher. Therefore, we should seize the time in fluorescence observation and take pictures immediately if necessary. In addition, when making fluorescent microscopic specimens, it is best to use non-fluorescent glass slides, cover sheets and non-fluorescent oil.
Practical test product
I. Equipment
1. main equipment: general centrifuge, tissue masher, coarse balance, fluorescence microscope.
2. Small equipment: 2 500ml beakers, 1 250ml measuring cylinder, 20 droppers, 20 centrifugal tubes with 10ml scale, 5 test racks, some gauze, 4 non-fluorescent glass slides and 4 covers.
Second, the material is fresh spinach.
Three, reagent 0.35mol/L sodium chloride solution, 0.0 1% acridine orange.
Experience method
Isolation and observation of chloroplasts from 1.
1. Select fresh tender spinach leaves, clean and dry them, remove stems and veins, weigh 30g in 150ml NaCl solution, and put them into a tissue masher.
2. Homogenize at low speed (5,000 rpm) with a tissue masher for 3-5 minutes.
3. Filter the homogenate with 6 layers of gauze in a 500ml beaker.
4. Take 4 ml of filtrate and centrifuge for 2 minutes at 1000 rpm. Discard the sediment.
5. Centrifuge the supernatant at 3000rpm for 5min. Discard the supernatant and precipitate chloroplasts (mixed with some nuclei).
6. Suspend the precipitate with 0.35 mol/ml solution,
7. Take a drop of chloroplast suspension on the glass slide, add a cover glass, and observe it under ordinary optical microscope and fluorescence microscope.
(1) was observed under a common optical microscope.
(2) Observe the direct fluorescence of chloroplasts under fluorescence microscope.
(3) Observing the indirect fluorescence of chloroplasts under fluorescence microscope: Drop a drop of chloroplast suspension on a non-fluorescent glass slide, add a drop of 0.0 1% acridine orange fluorescent dye, cover the glass slide and observe it under fluorescence microscope.
2. Observation of spinach leaves by hand slicing.
Cut the fresh spinach leaves into inclined planes with a shaving blade, put them on a glass slide, drop 1~2 drops of 0.35mol/L NaCl solution, cover the slices, gently press them and observe them under a microscope.
(1) was observed under a common optical microscope.
(2) Observe its direct fluorescence under a fluorescence microscope.
(3) Observation of indirect fluorescence: Drop 1 ~ 2 drops of 0.05438+0% acridine orange dye solution on the hand slice, dye 1min, wash off the residual solution, cover the slice and observe the indirect fluorescence under a fluorescence microscope.
Real fruit
Isolation and observation of chloroplasts from 1.
1. Under the ordinary optical microscope, we can see that the chloroplast is green and olive-shaped. Under the high-power microscope, we can see that the chloroplast contains dark green particles, that is, basal particles.
2. Take Olympus fluorescence microscope as an example. Under the conditions that B (blue) is used to excite the filter, B dichroic mirror is used, and O530 (orange) is used to break the filter, chloroplasts emit fiery red fluorescence.
3. After acridine orange staining, chloroplasts can emit orange-red fluorescence, and some of their nuclei emit green fluorescence.
2. Observation of spinach leaves by hand slicing.
1. Epidermal cells of three kinds of cells (1) can be seen under ordinary light microscope: squamous cells with serrated edges; (2) guard cells: paired kidney-shaped cells, which form stomata; (3) The mesophyll cells are oblong cells arranged in a grid. Chloroplast is green olive-shaped, and green grana can be seen under high magnification.
2. Under the fluorescence microscope, the chloroplast emits red fluorescence, but its fluorescence intensity is weaker than that of the free chloroplast, and the stomata emit green fluorescence. The red chloroplasts in the two guard cells are arranged in a circle around the stomata. The number of chloroplasts in epidermal cells is less than that in mesophyll cells.
3. After acridine orange staining, chloroplasts emit orange-red fluorescence, nuclei emit green fluorescence, and stomata remain green.
Zuo Ye
1. Under the ordinary optical microscope, the long axis and short axis of chloroplasts were measured by visual micrometer and desk micrometer, and 5~ 10 chloroplasts were measured respectively, and the average values were obtained.
2. Under the fluorescence microscope, when observing the autofluorescence of chloroplasts, does the color of chloroplasts change when the filter system is changed?
Experiment 5: Preparation and Observation of Plant Chromosome Specimens
Experimental purpose
Learn the preparation technology of plant chromosome specimens, understand the basic principle of Fulgen reaction, learn its operation method and tabletting method, and initially grasp the main characteristics of each stage of cell division.
Nucleic acid is the most important part of an organism. Nucleic acids can be divided into two categories, namely deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Their distribution and chemical properties in cells are different. DNA is mainly distributed in chromatin or chromosome during mitosis. RNA is distributed in cytoplasm and nucleoli. However, chromatin and chromosome also contain a small amount of RNA, and nucleolus also contains a small amount of DNA. In addition to RNA, there are organelles such as mitochondria and chloroplasts.
Experimental principle
The principle of Fulgen reaction is that after DNA is hydrolyzed by weak acid (1mol/L), the bond between purine and deoxyribose is opened, and a free aldehyde group is formed at one end of deoxyribose. These aldehyde groups combine with Schiff reagent in situ to form compounds containing quinone group (). Aldehyde groups are chromophores, so they are colored, so wherever there is DNA, they are purple-red.
experimental materials
Barley, rye or wheat seeds
Experimental content
Preparation of Plant Chromosome Specimens and DNA Display Method —— Fulgen Reaction
pressed disc method
Observation of mitotic phase of cells
experimental procedure
(1) Preparation of Plant Chromosome Specimens
1. Put the plant seeds on wet filter paper and germinate at 20℃. When the radicle grows to 1 ~ 2 cm, cut off the root tip part with a length of 0.5cm.
2. Pretreatment: Immerse the cut root tips in 0. 1% colchicine solution and treat for 3-4 hours at room temperature.
3. Fixation, hydrolysis and dyeing:
Camouflage fixative
Fixed to 10-30 minutes? 95% alcohol 10 minutes? 70% alcohol 10 minutes? Distilled water → (control) 5% trichloroacetic acid? 90℃15min
1mol/l HCl/distilled water? 1mol/L HCl？ 60 oC 8 minutes? Schiff reagent for 40 -60 minutes? Sulfite water (1, 2,3)? Change tap water for five minutes three times? Rinse the dyed root tips and hang materials with good dyeing effect? Distilled water? Tablet? Microscopic observation
(II) Tablet pressing method
The operation steps of tabletting are as follows: put the root tip on the glass slide, cut off the root tip (0.3cm) with a blade, cut the root tip longitudinally, add a drop of water, divide the root tip longitudinally with a dissecting needle, keep 1-2, add a cover glass, and tap the cover glass gently with a pencil tip to separate the cells and flatten them into clouds.
(3) Observation of Vicia faba (or onion) root tip slices.
Firstly, under the low-power microscope, the cells in the meristem area and the elongated tissue area of the root tip are distinguished. Then observe the morphology of the nucleus under a high-power microscope, and pay attention to whether there are mitotic cells on your film. If yes, you can find out several stages of cell division and their characteristics (see appendix)
Several problems that should be paid attention to when doing Fulgan reaction
Selection of stationary liquid: Canoy stationary liquid is usually used. Other fixatives can be used, such as Fleming fixative and Champy fixative, but Bouin fixative cannot be used.
Hydrolysis time: Hydrolysis time must be appropriate, not too long or insufficient, otherwise it will affect the experimental results. For materials fixed with Canoy stationary solution, the hydrolysis time is generally between 8-65438 0.5 minutes. The length of hydrolysis time depends on different materials and different fixatives.
Quality of Schiff reagent: The quality of Schiff reagent is an important factor in the Fulgen reaction. Attention should be paid to whether the color of the reagent is normal and whether there is the smell of SO2.
Importance of detergent: When rinsing, it is best to prepare sulfurous acid water temporarily before each experiment to keep the concentration of SO2.
Experimental control group: Be sure to make correct photos to illustrate the authenticity of the experimental results.
Use tweezers to take the apical growth area during the operation, and don't clamp the root crown.
During opium, root meristem cells should keep their original distribution.
utilize
Briefly describe the principle of Fulgen reaction.
What should be paid attention to in the process of making a good Furgen reaction membrane?
Draw a picture of cell division.
Experiment 6 Preparation of Plant Protoplasts
Experimental purpose
1. Learn the separation and preparation techniques of plant protoplasts and observe the morphology of protoplasts.
2. Learn the technology of plant protoplast fusion, and observe the morphology and changes of cells fused by different methods.
Experimental supplies
Microscope, mirror cleaning paper, scissors, tweezers, saucer, four straws, straight funnel, 300-mesh nylon net, 10ml centrifuge tube, glass slide and cover.
Experimental principle
The term protoplast was first proposed by Hanstein in 1880. Specifically, the protoplast of edible fungi refers to the bare cell structure left after the cell wall is completely eliminated.
After the petal cells of plants were treated with cellulase and ionolytic enzyme, the cellulose and pectin components were destroyed, which made protoplasts leave the cell wall and enter the culture medium.
There are a lot of pigments in the vacuoles of plant petal cells, and different cytochromes are different, so we can identify the protoplasts of different cells and the fusion between homologous cells and heterogeneous cells by color range, without staining the cells.
PEG is a high molecular compound, which has negative polarity because of its ether bond, and forms hydrogen bonds with some positive groups such as water, protein and carbohydrates. When the PEG molecule is long enough, it can be used as a molecular bridge between the surfaces of adjacent protoplasts to make them adhere. PEG can also connect Ca2+ and other cations. Ca2+ can form a bridge between some negative polarity groups and PEG, thus promoting adhesion. In the washing process, PEG molecules attached to the protoplast membrane will be eluted, which will cause disorder and redistribution of charges, thus causing protoplast fusion. Washing with high Ca2+ and high pH will increase the fluidity of plasma membrane, thus greatly increasing the fusion frequency. Osmotic shock during washing may also play a role in fusion.
Experimental reagent
1. Washing liquid:
Mannitol 0.6 M
CaCl2？ 2h2o 8mm
NaH2PO4？ H2O 2 mm
Ph 5.6
experimental procedure
Preparation of enzyme solution 1. Cellulase (EAS-867) was dissolved in 0.5 ~ 0.7 mol/L mannitol, and 10 mmol/L calcium chloride solution was added as stabilizer. After the enzyme was dissolved, the protoplast was precipitated by a centrifuge at 4000 rpm, and after 20 minutes, the supernatant was taken. The enzyme solution was filtered by syringe filter, then filtered by 0.45 micron microporous membrane, packed in triangular bottles in aseptic boxes, and stored in refrigerator for later use.
2. Materials: Tobacco plants with seedling age of more than 60 days grown in greenhouse were taken from the middle and irradiated in the sun for 2 hours to make them wither. You can also do the same treatment with bean leaves planted in greenhouses, or put carrots harvested in the field at a low temperature above 0℃ for later use. The wilting leaves are soaked in 3% bleaching concentrate 15 ~ 20 minutes, then washed with sterile water, and the surface moisture is absorbed by sterile absorbent paper.
3. Enzymolysis Tear off the lower epidermis of the leaves with pointed tweezers and cut into small pieces. Cut off the epidermis and stele of carrot with a blade, and cut the cortex into small pieces. Put 65438 0 g of the above materials into a Petri dish containing 65438±00ml of enzyme solution, and cover it. Keep the temperature at 28 ~ 30℃ 1.5 ~ 3 hours.
4. After washing leaves or other tissues, round free protoplasts will appear, and there are undigested tissues, fragments, cells and broken protoplasts in the protoplast suspension. Filter the coarse impurities with filter paper or nylon cloth, then move the protoplast suspension into a centrifuge tube, rotate it with a hand centrifuge for 2 minutes, suck off the supernatant (see figure), and then add 0.5 ml of mannitol to wash it for 2-3 times, and finally get a relatively pure protoplast, which can be used for further culture or other experiments.
Experiment 7 Cell Fusion Method
Experimental purpose
1, and preliminarily master the method of PEG fusion of animal cells.
2. Understand cell fusion and its application.
Experimental principle
The phenomenon that two or more cells fuse into 1 cell is called cell fusion. The main methods of cell fusion are virus method, polyethylene glycol (PEG) method and electrofusion method.
Treating cells with PEG can change the properties of plasma membrane, leading to cell membrane fusion, cytoplasmic circulation and finally cell fusion.
Experimental equipment, materials and reagents
1, instrument:
Optical microscope, centrifuge, constant temperature water bath pot, CO2 incubator, ultra-clean table, inverted microscope, etc.
2. Materials
Fresh chicken red blood cells, sterile syringe, No.6 needle, graduated centrifuge tube, test tube, glass slide, cover glass.
3. Reagent
50% polyethylene glycol, Hanks solution, methanol, Giemsa dye solution, iodine, 75% ethanol.
experimental method
(1) Fresh chicken blood is made of 0.85% physiological saline 10% suspension.
(2) Weigh 0.5g PEG(MW = 4000), put it in a test tube, melt it on an alcohol lamp, and quickly add 0.5ml of preheated Hanks solution to make a 50% PEG solution. Put it in a water bath at 37℃ for later use.
(3) Put 1 ml of the above 10% chicken red blood cell suspension in a centrifuge tube, add 5 ml of Hanks solution and mix well, then centrifuge at 1 0,000 rpm for 5 minutes, carefully discard the supernatant, and break up the cell mass by finger elastic method.
(4) Take 0.5ml of the above 50% PEG solution, drop it into the chicken red blood cell suspension within 65438 0 minutes, and gently shake it while adding it. After adding all PEG, let it stand for about 65438 0 minutes. The whole process requires a water bath at 37℃.
(5) Slowly drop 9ml Hanks solution to stop the action of PEG, and let it stand in a water bath at 37℃ for 5 minutes.
(6) After centrifugation and discarding the supernatant, take a drop of fused cell suspension and cover it for microscopic examination.