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Microfilaria in methylene blue preparation

The above image is showing microfilaria in blood wet mount treated with supravital stain i.e. methylene blue preparation (40X objective).

Requirements for Blood wet mount

• New methylene blue
• Specimen: Blood
• Clean and grease-free slides and
• Cove slips(22- by 22-mm)
• Microscope
• Gloves

Procedure for blood  wet mount 

1. First, wear the groves.
2. Take a clean and grease-free slide.
3. Add one drop of blood and then also add a drop of  New methylene blue ( supravital stain).
4. Mix it properly and apply a coverslip over a uniform suspension without creating bubbles.
5. Examine the entire 22- by 22-mm coverslip systematically with the low power objective (10X ) and low light intensity.
6. If any suspicious objects encounter, examine with the high dry objective (40X) as shown above image and below video.

Keynotes

• Supravital dyes for red blood cells (RBCs) are as follows-

1. New methylene blue
2. Brilliant cresyl blue
3. Crystal violet
4. Methyl violet
5. Nile blue
6. Hoechst stain

• Supravital staining is a method of staining used in microscopy to examine living cells that have been removed from an organism. It differs from intravital staining, which is done by injecting or otherwise introducing the stain into the body. e.g. New methylene blue is a supravital stain whereas Gram stain is intravital.
• Microfilaria-The females of parasites are viviparously giving birth to larvae so-called microfilaria. This parasite includes four genera and species that are parasitic to humans. These filarial nematodes are as follows: 1. Wuchereria bancrofti 2. Brugia malayi 3. Oncocerca volvulus and 4. Loa loa but mainly two genera i.e. Wuchereria and Brugia are encountered in peripheral blood.
• Filariasis is an infectious disease caused by nematodes of the Filarioidea type e.g. Wuchereria bancrofti, Brugia malayi,  Oncocerca volvulus, and Loa loa. These are spread by blood-feeding insects such as black flies and mosquitoes. The filarial worms reside in the subcutaneous tissues, lymphatic system, or body cavities of humans.
• Periodicity-The microfilariae circulate in the bloodstream. They show a nocturnal periodicity in peripheral circulation; being seen in large numbers in peripheral blood only at night (between 10 pm and 4 am). This correlates with the night-biting habit of the vector mosquito.

Further Readings

1. Merkell and Voge’s medical parasitology
   9th edition.
2. Parasitology: 12th edition
   By K. D. Chatterjee
3. District laboratory practice in Tropical countries –Part-I.
   By Monica Chesbrough.
4. Isenberg clinical microbiology procedures Handbook
   2nd edition. Vol. 2
5. Atlas of Medical Helminthology and protozoology -4th edn  -P.L.  Chiodini, A.H. Moody, D.W. Manser
6. Medical Parasitology by Abhay R. Satoskar, Gary L. Simon, Peter J. Hotez and Moriya Tsuji
7. Atlas of Human Parasitology, Lawrence R Ash, Thomas C. Orihel, 3 rd ed, Publisher ASCP Press, Chicago.
8. Molecular Medical Parasitology. Editors: J. Joseph Marr, Timothy W. Nilsen, and Richard W. Komuniecki, Publisher Academic Press, an imprint of Elsevier Science.
9. Topley & Wilsons’ Principle of parasitology. Editors: M.T. Parker & L.H. Collier, 8 th ed 1990, Publisher Edward Arnold publication, London.
10. Illustrated Medical Microbiology 2nd ed, Satish Gupte