Making slide specimens is of great significance for understanding the morphological structure of organisms. Slide specimens include temporary slide specimens (such as smearing, tabletting and temporary mounting) and permanent slide specimens (such as permanent mounting and slicing).
1. daubing method
Smear method is a method of making materials evenly coated on the glass slide.
Smear materials include unicellular organisms, microalgae, blood, bacterial culture solution, loose tissues of animals and plants, testicles, anthers, etc.
Attention should be paid when smearing: (1) The glass slide must be clean. (2) The slideway shall be smooth. (3) The coating should be uniform. Drop the smear liquid in the middle of the glass slide to the right and smear it evenly with a scalpel blade or toothpick. (4) The coating should be thin. Use the other slide as a pusher, gently push it from right to left along the surface of the slide with daubing liquid (the included angle between the two slides should be 30 ~ 45), and smear it into a uniform thin layer. (5) fixed. If necessary, it can be fixed by chemical fixative or drying method (bacteria). (6) dyeing. Methylene blue is used for bacteria and Wright's dye is used for blood. The dye solution shall cover all painted surfaces. (7) washing. Absorb or bake with absorbent paper. (8) sealing. Long-term preserved Canadian chewing gum tablets.
2. Tablet pressing method
The tabletting method is a tabletting method in which biomaterials are placed between a slide and a cover, and certain pressure is applied to disperse tissue cells.
The general process of tabletting method: (1) taking materials. In order to observe cell division, fresh tissue cells with vigorous cell division should be selected as materials. Such as root tip, stem tip meristem, bone marrow cells and anthers (pollen mother cells). Testicles (spermatocytes) and so on. (2) fixed. The material fixation can be determined as required, and it can be observed by tabletting immediately after taking the material, without separate fixation (synchronous with dyeing); If you don't check it immediately after taking the materials, you can fix it with a fixed solution (usually acetic alcohol fixed solution). Fix for 2 ~ 24 hours (depending on the material), then wash with 95% ethanol and store in 70% ethanol for later use. (3) isolation. Materials whose cells are difficult to disperse are treated with hydrolysis separation solution (such as 1N HCl or hydrochloric acid-alcohol semen), which is usually treated for 6 ~ 20min, and then washed with water after dissociation. (4) dyeing. There are many kinds of dyes. Observation chromosomes are often stained with fuchsin acetate staining solution. (5) tabletting. Place the material on the glass slide, add a drop of water or dye solution, cover the cover glass and press gently with your thumb. (6) observation. After tabletting, it can be observed under a microscope.
3. Loading method
Loading method is a method of sealing biological materials as a whole to make slide specimens, which can be used to make temporary or permanent loading.
Packaging materials include: microscopic organisms such as Chlamydomonas, Spirogyra, Amoeba, Hydra and leaf epidermis of plants; Insect wings, feet, mouthparts, human oral epithelial cells, etc.
It should be noted: (1) Keep the slides flat or put them on the platform. When dripping water, the amount of water should be appropriate to cover the cover glass. (2) unfold the material with a dissecting needle or tweezers without overlapping and flatten it on the same plane. (3) When putting the cover glass, slowly cover it on the water drop from one side to prevent bubbles. (4) When staining, drop a drop of staining solution on one side of the cover glass, and suck it out from the other side with absorbent paper to make the specimen under the cover glass evenly colored. After coloring, in the same way, drop a drop of water, suck out the dye solution and observe it under a microscope.
Second, the basic experimental technology of microscope
1. How to use low-power mirror (4X, 10X)
Microscope operation mainly includes two aspects: (1) photometric adjustment. Correctly aiming at light is the first condition for successfully observing objects. When aiming, first lift the condenser, open the aperture and turn the reflector. At this time, while looking at the field of vision in the mirror, adjust the condenser screw until the uniform and bright light is obtained in the field of vision. (2) adjustment of focal length. When focusing, slice the observation section first and aim the propeller at the center of the light hole. Then focus in two steps. First, fix the focal length with the coarse adjuster, look at the eyepiece with your left eye, and let the coarse adjuster rise slowly until you can see the specimen clearly. Then adjust the spinner and lift the lens barrel slightly until the image of the object is clearer.
2. How to use a high-power mirror (40x)
(1) Move the part of the object image found in the low-power mirror to the center of the field of view. (2) Turn the objective lens converter so that the 40X objective lens is aligned with the light hole. When turning, look at the objective lens from the side to prevent the lens from pressing the glass slide. (3) Adjust the fine-tuning screw to make the image clear.
3. How to use the oil mirror (100X)
(l) First, use the low-power lens to find the fine structure to be observed; for the high-power lens, move the structure to the center of the field of view. (2) Step down, drop a drop of aromatic asphalt on the slide specimen and change the oil mirror. Look at the oil mirror from the side, lift the mirror table and let the oil mirror soak in the aromatic tar drops. (3) Turn the fine-tuning screw to make the image clear and observe. (4) After observation, wipe off the aromatic tar on the oil lens and glass slide with lens paper, and then wipe the lens clean with a little xylene or ether/anhydrous ethanol.
4. A simple way to install the pointer
Unscrew the upper cover of the eyepiece (a lens), cut off a short piece of hair (the length of which is about equal to the radius of the eyepiece), clamp one end with tweezers, and dip the other end with a little neutral glue and stick it on the metal diaphragm (an iron ring) on the inner wall of the eyepiece. When it is slightly dry, tighten the upper cover to use.
Third, the counting method of single cells
It is often necessary to count bacteria, cells, unicellular algae, protozoa, blood cells and pollen in bacterial culture and cell culture in vitro, as well as in blood examination of environmental monitoring. The principle of cell counting method is to directly calculate the amount of a certain volume of cell fluid under the microscope, and use the obtained results to calculate the number of individual cells per milliliter of water. The specific method is as follows:
1. water drop counting method
Suck 1mL of water with a round, smooth and appropriate nozzle, and then determine how many drops of water this straw can drop (repeat until it is accurate). In this way, the volume of each drop of water dropped by the straw can be calculated. When sampling with a straw, if a single cell is active, it should be killed with iodine before counting; If the sample concentration is too large to count, dilute it to a suitable concentration by multiple; In addition, the sample must be mixed before sampling, and the sample should be taken immediately after mixing. After sampling and counting under the microscope, the average number of cells in a drop of water can be calculated according to the following formula.
1 ml number of cells in water = average count × number of drops per ml quantitative pipette × dilution multiple.
2. Blood cell counting plate counting method
The blood cell counting plate is a special thick glass slide with two counting rooms in the center. Each counting room is divided into 9 large squares (see the figure below), and the area of each large square with cover glass is 1mm2 and the depth is 0.1mm.. So the volume of each big square is 0.1mm. In addition, the central square is divided into 25 middle squares by double lines. Each middle square is divided into 16 small squares for cell counting.