Plasmodium vivax morphology observation experiment
Plasmodium vivax morphology observation experiment
This experiment is from the official website of Parasitology, School of Medicine, Shandong University.
Operation method
Plasmodium vivax morphology observation experiment
Principle
There are four species of human malaria parasites, namely Plasmodium vivax, Plasmodium malariae, Plasmodium falciparum and Plasmodium ovale. Plasmodium vivax and P. falciparum are more common, while Plasmodium vivax and P. ovale are less common and rare. Plasmodium requires two hosts to complete its life history. It undergoes cleavage proliferation in the human body, including extra-erythrocytic and intra-erythrocytic stages, and gametogenic and sporotogenic reproduction in the mosquito. First, Plasmodium vivax Thin Blood Smears of Plasmodium vivax ) Plasmodium vivax patients blood smear, Rui Yi Ji staining, microscopic observation. First, under low magnification to determine the plane of the blood film, blood smear thin and uniform or red blood cells in a monolayer uniformly arranged parts (usually for the blood film near the end), after refueling, turn the oil microscope observation. Look for Plasmodium in the erythrocytes, after Ruiji staining, the cytoplasm of Plasmodium stained blue, the nucleus stained red, malarial pigment (malaria pigment) is yellowish brown. Note the difference with other cells in the blood film, dye residue, etc. 1. Ring form (Ring form) The parasitized erythrocytes in this stage are not yet changed. The cytoplasm of the malaria parasite is ring-shaped, and the red nucleus is dotted, on one side of the ring, much like a jeweled ring. The size of the annulus accounts for about l/3~l/4 of the diameter of the erythrocyte.2. Large trophozoite (Large trophozoite) The annulus further develops in the form of the parasitized erythrocyte, which is swollen and lighter in color, and there are many red dots on the erythrocyte, i.e., Schuffner's dots. The cytoplasm of the protozoa increased with pseudopods protruding. The red nucleus is also significantly enlarged, and there are yellow-brown malaria pigment particles on the cytoplasm. 3. Schizont has two forms: immature and mature. At this time the cytoplasm is more dense, the pseudopods have disappeared, the nucleus first split, and then the cytoplasm splits, immature schizont (unmature schizont) of the nucleus has not yet split to a certain number, the cytoplasm has not yet been separated. Mature schizont (mature schizont) of the nucleus has been divided to a certain number that is 12 ~ 24 (average 16), the cytoplasm around the nucleus of each cell to form a schizont. At this time, the yellow-brown malaria pigment particles are concentrated in the center or one side of the body.4. Gametocyte After three to five generations of proliferation of schizont, a part of the schizont invades the erythrocyte and develops into gametocyte. At this time the parasitized erythrocytes are enlarged. Both male and female gametocytes are round, and the malaria pigment granules are evenly distributed in the cytoplasm. Female gametocytes (macrogametocyte) have a dense nucleus and cytoplasm, and after staining, the cytoplasm is blue, and the nucleus is red on one side, i.e., the nucleus is small, dense, and on one side. In microgametocyte, the nucleus and cytoplasm were looser, and the nucleus was larger and located near the center, i.e., the nucleus was large, loose and in the center. Fig. 1: Normal red cell; Figs. 2-6: Small trophozoites Young trophozoites (ring stage parasites); Figs. 7-18: Large trophozoite Trophozoites; Figs. 19-27: Schizonts. (19-22 unmature schizont; 23-27 mature schizont) Figs. 28 and 29: female gametophytes Macrogametocytes (female); Fig. 30: male gametophytes Microgametocyte (male). Thin Blood Smears of Plasmodium falciparum (P. falciparum) were observed in the same way as P. vivax specimens. The annulus is smaller than that of Plasmodium falciparum, accounting for about 1/6 of the red blood cells. The rings are small, and there is often a nucleus in one ring, and some rings are attached to the edge of the erythrocyte, especially in the shape of a bird of prey. Two or more Plasmodium falciparum annuli are often seen in one erythrocyte. The large trophozoites and schizonts of P. falciparum are generally not easily detected in the surrounding blood. The erythrocytes on which the gametocytes are parasitized are often swollen and missing. Female gametocytes are crescent-shaped with pointed ends. The male gametophyte is salami shaped with rounded ends. The nuclei are all in the center of the body and the nucleus is surrounded by malarial pigment granules. Fig. 2, 1: Normal red cell; Figs. 2-18: Trophozoites (among these, Figs. 2-10 correspond to ring-stage trophozoites); Figs. 19-26: Schizonts ( Fig. 19-26: Schizonts (Fig. 26 is a ruptured schizont); Figs. 27, 28: female gametophytes macrogametocytes (female); Figs. 29, 30: male gametophytes microgametocytes (male).
Materials and Instruments
Infected mice; slides Sterilized cotton balls Ethanol Scissors Crayons Straws Distilled water Petri dishes Kiehl's or Rachel's stains Buffers Move 1. Thin blood film production method Caveat 1. The slide should be clean and free of grease. The volume of blood is moderate and pushed at a uniform rate to prevent the blood film from being too thick, too thin, or appearing as streaks of transverse lines. Blood film in the drying process to avoid dust or fly desorption. 2. Thick and thin dish membrane preparation in a slide, should pay attention to the thick blood film hemolysis before the thin blood film must be fixed with methanol, in order to avoid contact with water and make the thin blood film on the red blood cell lysis. Thick hemofilm hemolysis time should not be too long, do not oscillate, in order to prevent the blood film off. 3. The dye solution is a methanol solution, do not mix with water droplets, otherwise precipitation occurs, hindering the staining, so the dye solution should not be used again when precipitation is found. 4. Do not add too many drops of dye, otherwise the dye residue will stick to the blood film and cannot be washed away, thus affecting the examination. After adding water, it must be fully mixed with the dye, otherwise uneven staining will occur. When rinsing the blood film, the dye should be washed away directly with running water to avoid the dye sticking to the blood film.Microscopic view of the thick blood film method in the red end stage of Plasmodium vivax. For more product details, please visit Aladdin Scientific website.
(1) Blood collection: clinically, the patient's earlobe or fingertip blood is taken; in this experiment, the tail tip blood of mice infected with Plasmodium falciparum is used.
(2) Methods of operation: Cut off the tail tip of the mouse and take a clean slide, hold both ends of the slide in the left hand, and choose another slide with smooth and flat edges as a slide. Use the center of one end of the slide to take 1 small drop of blood (about a grain of rice) from the tail end of the mouse, place it in the middle of the slide, keep the angle between the slide and the slide at an angle of 30 to 40 degrees, and then push the drop of blood forward at an even speed after unfolding it on the edge of the slide, i.e., to form a tongue-like blood film.
2. Thick blood film (thick blood film) production method
(1) Blood collection is the same as thin blood film method.
(2) Method of operation: The thick blood film can be placed at the other end of the thin blood film. Take 2 or 3 drops of blood from the rat's tail with the corner of the push piece, and then form a blood film with a diameter of about 1 cm and a uniform thickness in one direction from the inside out. A line was drawn with a crayon to separate the thick and thin blood film. After sufficient drying, drops of distilled water were added to the thick blood film to dissolve the blood, the water was poured off, and the thin blood film was stained together after drying.
3. Fixation and staining
(1) Rachel's staining: first use a crayon to draw a line on both ends of the blood film to prevent spillage of the staining solution. Rachel's stain is methanol solution, the blood film does not need to be pre-fixed, this staining method is fast, suitable for clinical examination, but it is easy to fade, the preservation time is not long.
Add a few drops of Rachel's stain to cover the blood film, about 1 to 2 min after the blood film is fixed by the methanol in the stain, and then add an equal amount of buffer or distilled water, gently shake the slide, so that the stain and the diluent mix well, 3 to 5 min after the buffer or tap water from the end of the slide rinsing, drying microscopic examination.