Microfilariae are the larval stage of filarial parasites, which are of great medical significance and serve as the key basis for the diagnosis of filariasis. They do not reproduce directly themselves, but can be ingested by intermediate hosts (vector insects) during blood-sucking, and develop into infective larvae within the insects, thereby completing the transmission cycle. Microfilariae usually occur in the blood or skin of definitive hosts.
The presence of microfilariae in the peripheral blood of hosts exhibits a periodic variation characterized by fewer numbers during the day and greater numbers at night. During the daytime, they are mainly concentrated in the capillaries of deep tissues such as the lungs; at night (usually from 10:00 p.m. to 2:00 a.m. the next day), they appear in large quantities in the peripheral blood. This is exactly synchronized with the blood-sucking time of vector mosquitoes (at night), which greatly improves the transmission efficiency. Therefore, clinical blood collection for microfilariae examination must be conducted at night; otherwise, the detection rate will be extremely low.
Different species of filarial worms vary in the morphology of their microfilariae, their distribution in the human body, and the diseases they cause. Common human-infecting filarial worms include Wuchereria bancrofti, Onchocerca volvulus, and Brugia malayi, etc. The main morphological criteria for identifying different species of microfilariae are as follows:
Somatic nuclei: The worm body contains a large number of nuclei, known as somatic nuclei. Observing the morphology, size, and distribution of somatic nuclei is the key to identification.
Cephalic space: The nuclear-free region at the anterior-most end of the worm body. Measuring the length-width ratio of the cephalic space is of great significance.
Caudal nuclei: Some species (e.g., Wuchereria bancrofti microfilariae) have no somatic nuclei in the tail region, while others (e.g., Brugia malayi microfilariae) have two caudal nuclei in the tail.
Microfilariae can be examined by direct microscopic observation of fresh blood smears, but this method has a low detection rate and cannot determine the species, so it is mostly used for publicity and education purposes. In contrast, microscopic examination of stained blood smears not only allows clear visualization of the internal structures of microfilariae, but also facilitates species identification and specimen preservation.
Microfilariae are usually enveloped by a sheath. In stained smears, the sheath is generally non-staining, so the worm body appears to be located within a transparent envelope. Commonly used morphological staining methods include Wright's staining, Giemsa's staining, Delafield's hematoxylin staining, and borax methylene blue staining. Under the microscope, microfilariae stained with Giemsa's stain appear as filamentous, colorless and transparent organisms with a rounded anterior end and a tapering posterior end. After staining with borax methylene blue, the worm body shows bright coloration with a clearly defined sheath; the cephalic space, somatic nuclei, caudal nuclei, and nerve ring are distinctly visualized; the sheath stains pale red and the somatic nuclei stain blue.
This reagent is for research use only and is not intended for clinical diagnosis or any other purposes.
Self-prepared Materials:
1. Glass slides, ordinary light microscopes, coverslips
2. Wax pencils, methanol, distilled water, 1% hydrochloric acid-ethanol differentiation solution, eosin staining solution
Operating Procedures (For Reference Only):
1. Blood Collection Time: Optimal between 9:00 p.m. and 2:00 a.m. the next day. Blood Collection Sites: Earlobes, finger pulps, soles of infants’ feet, etc.
2. Preparation of Blood Films: The thick blood film should be slightly thinner than that used for Giemsa staining; a sub-thick blood film with a diameter of 1.2–1.5 cm can be prepared using 3 μl of blood. The thin blood film should not be overly thin; it can be spread at a 45° angle with a spreader to make a relatively thick thin blood film.
3. Fixation: After the blood films are completely air-dried, dip a glass rod into methanol or anhydrous alcohol and gently wipe the thin blood film to fix the cells. Before fixing the thick blood film, hemolysis must be performed first: add a few drops of water onto the thick blood film using a dropper; once the film turns grayish white, pour off the water, air-dry it, and then fix it with methanol or anhydrous alcohol. If both thin and thick blood films are prepared on the same glass slide, draw lines at both ends of the stained area of the thin film and a circle around the thick film with a wax pencil to prevent mutual interference during hemolysis and fixation.
4. Preparation of Delafield's Hematoxylin Working Staining Solution: Take an appropriate volume of microfilaria staining solution (Delafield's hematoxylin staining solution), add distilled water, and mix them at a ratio of 1:1 to 1:9 before staining.
5. Stain the blood films with Delafield's hematoxylin working solution for 10–20 minutes, followed by rinsing with water for 2–5 minutes.
6. (Self-prepared, Optional) Differentiate in 1% hydrochloric acid-ethanol solution for 30–60 seconds, then rinse with water for 5–10 minutes until the blood films turn blue. Counterstain with eosin staining solution for 30–60 seconds, followed by rinsing with water for 2–5 minutes.
7. Air-dry the slides and observe under the microscope.
Staining Results:
The somatic nuclei of microfilariae stain blue, and the worm bodies turn red after counterstaining with eosin.
Precautions:
1. Glass slides for preparing blood films shall be treated with chromic acid cleaning solution prior to use.
2. The thickness of blood films for staining must meet the required specifications.
3. The staining solution should be added in an amount sufficient to completely cover the blood films.
4. For your safety and health, please wear a lab coat and disposable gloves during the operation.
5. Please use the reagent as soon as possible after opening to avoid affecting subsequent experimental results.
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