Sporozoans lack distinct locomotor organelles and are parasitic throughout all developmental stages. Their life cycle is relatively complex, involving two reproductive modes: asexual schizogony and sporogony, as well as sexual gametogony. These processes can be completed either in a single host or separately in two different hosts. Sporozoans parasitic in humans mainly include Plasmodium, Toxoplasma gondii, Pneumocystis jirovecii, Cryptosporidium and so on.
Plasmodium is a parasitic sporozoan that primarily infects humans and various mammals, with a few species parasitizing birds and reptiles. To date, more than 130 species have been identified. The main species parasitic in humans include Plasmodium vivax, Plasmodium falciparum, Plasmodium malariae and Plasmodium ovale. The basic structure of Plasmodium consists of a nucleus, cytoplasm and cell membrane. After staining with Wright's or Giemsa's stain, the nucleus appears purplish red and the cytoplasm appears blue. Plasmodium develops successively in liver cells and red blood cells within the human body, and its pathogenicity occurs during the erythrocytic schizogony stage. The development of Plasmodium in red blood cells includes four stages: ring-form trophozoite, mature trophozoite, schizont and gametocyte. The distinct morphological characteristics of each stage can be observed after staining with Wright's or Giemsa's stain.
The etiological examination of Plasmodium is mainly divided into two methods: thick and thin blood film staining microscopy, and quantitative buffy coat (QBC) analysis. In thick blood films, the parasites are relatively concentrated, making them easy to detect. However, red blood cells tend to lyse during sample preparation, resulting in morphological changes of the parasites and thus making species identification difficult. In thin blood films, red blood cells are not easily damaged, and the parasites maintain intact and typical morphological structures, which facilitates species identification. Nevertheless, light infections are prone to missed detection due to the low density of parasites. Therefore, it is common practice to prepare both thick and thin blood films on a single slide, followed by staining with Wright's stain, Giemsa's stain or acridine orange to detect the presence of Plasmodium. The principle of QBC analysis is that red blood cells infected with Plasmodium are lighter than normal red blood cells but slightly heavier than white blood cells. After centrifugation and stratification, the infected red blood cells are concentrated in the upper part of the normal red blood cell layer and the lower part of the white blood cell layer. After adding acridine orange reagent, the results are observed under a fluorescence microscope. This method has much higher sensitivity than conventional microscopy, and is more simple and rapid, but it is relatively costly.
Aladdin Plasmodium Staining Solution (Acridine Orange Staining Solution) is mainly composed of acridine orange and phosphate. It is primarily used for staining thick and thin blood films with the fluorescent dye acridine orange. Through the effects of permeation, adsorption and combination, the dye binds to different components of parasites or tissue cells to form fluorescent specimens. Excited by a fluorescent light source, the specimens bound with fluorochrome emit fluorescence. The presence of Plasmodium can then be observed under a fluorescence microscope in a dark room. This method is simple and rapid, with a higher detection rate than conventional methods. This reagent is for research use only and is not suitable for clinical diagnosis or other purposes.
Product Components and Storage Conditions:
M1510409
| Component
| 100mL
| Storage
|
M1510409A
| Acridine Orange Stock Solution
| 1mL
| 4℃. Store in the dark.
|
M1510409B
| Phosphate Buffer Solution
| 100mL
| RT |
Self-prepared Materials:
1. Wax pencil, methanol, deionized water
2. Glass slides, fluorescence microscope, darkroom, anti-fluorescence quenching mounting medium
Operating Procedures (For Reference Only):
1. Prepare Blood Films: The thick blood film should be slightly thinner than that used for Giemsa staining; 3 μl of blood can be spread to form a sub-thick blood film with a diameter of 1.2–1.5 cm. The thin blood film should not be overly thin; it can be prepared by spreading the blood at a 45° angle with a spreader to form a relatively thick thin blood film.
2. Prepare Acridine Orange Staining Solution: Add 0.1 ml of acridine orange stock solution to 9.9 ml of phosphate buffer solution and mix well. The prepared staining solution can be stored at 4℃ for 10–15 days.
3. Before staining, draw a horizontal line between the thick and thin blood films with a wax pencil.
4. Fix the thin blood film with 0.5–1 ml of methanol, and rinse the thick blood film with clean water to dissolve the hemoglobin.
5. Pipette the acridine orange staining solution and drop it onto the blood films. Stain at room temperature in the dark for 30–120 seconds, then cover with a coverslip.
6. Observe the slides in a darkroom under a fluorescence microscope (excitation filter wavelength: 488 nm; barrier filter wavelength: 515 nm), then count and take photos. For samples that cannot be observed immediately, seal the slides with anti-fluorescence quenching mounting medium to delay fluorescence quenching.
Staining Results:
The nuclei of Plasmodium and white blood cells show yellow-green fluorescence, and the cytoplasm shows orange-red fluorescence.
Precautions:
1. Glass slides used for preparing blood films should be treated with chromic acid cleaning solution before use.
2. The thickness of the blood films for staining should meet the required specifications.
3. It is generally recommended to dilute the acridine orange stock solution immediately before use; the diluted staining solution should not be stored for an extended period.
4. The volume of the staining solution added should be sufficient to completely cover the blood films.
5. For your safety and health, please wear a lab coat and disposable gloves during operation.
6. Please use the reagent as soon as possible after opening to avoid affecting the results of subsequent experiments.
D2603001