Infecting mosquitoes with microfilaremic blood.

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Materials

• Glass slides
• Glass cover slips
• Defibrinated sheep's blood (Hemostat Laboratories DSB100)
• Bleach
• Glass or electric blood feeders
• Parafilm
• Ring stand (glass feeders only)

Preparing blood for blood feeding

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All pipette tips, slides, serological pipettes, tubes, and other materials that come into contact with blood must be treated with bleach prior to disposal. Prepare bleach solution in the bleach-specific pitcher in the insectary (labeled with yellow tape) by adding ~1/2 cup of bleach to 1 L of tap water.

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Remove the sucrose pads at least 2 hours before feeding for LVP and AaSD mosquito species.

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Starting with microfilaremic blood

1. Invert the tube of infected blood several times to mix the microfilariae (mf). If the microfilariae are shipped in multiple glass vials from the same subject, combine all vials into a single 50 mL conical tube and mix by inversion.

2. Add 20 μL of infected blood to 50 μL of dH₂O on a glass slide and stir with the pipette tip to mix.

3. Add a cover slip to the slide. Beginning at an angle and slowly lowering it will reduce bubbles in the middle of the slide.

4. Return the mf to the incubator while counting.

5. Place the slide on a compound microscope and count the mf. Start at a corner of the cover slip and count the mf in the fluid outside the coverslip. Once the perimeter has been counted, count the mf underneath the cover slip using an S-pattern.

   

   Microfilariae counting pattern

6. Repeat steps 2–5 three times. Take the average of the three mf counts and calculate the mf concentration in the microfilaremic blood stock.