Methods in Anopheles Research - MR4

Chapter 3 : Specific Anopheles Techniques3.6 Determination of Lipid, Glycogen, and Sugars in MosquitoesPage 2 of 4c. Store at 4°C. Stable for several weeks.Standards1. Lipid: 100 mg per 100 mL of a commercial vegetable oil (e.g. soy bean oil) in chloroforma) In triplicate, add 50, 100, 200 and 400 μl of solution to glass tube.b) Place in heating block at 90-110 o C to evaporate the solvent.c) Add 0.2 mL of sulfuric acid and heat for 10 min at 90-110 o Cd) Add vanillin reagent to 5 mL level and mix.e) Remove from heating block and allow to cool.f) Allow reddish color to develop; this will take approximately 5 min and will be stable up to30 min.g) Determine OD at 625 nm and plot μg lipid vs. OD for calibration line.2. Sugar and glycogen: 100 mg per 100 mL of anhydrous glucose in deionized water.a) In triplicate, add 25, 50, 100, 150 and 200 μl of glucose solution to glass tubeb) Add anthrone reagent to 5 mL level and mix.c) Heat for 17 minutes at 90-110 o C.d) Remove from heating block and allow to cool.e) Determine OD at 625 nm and plot μg glucose vs. OD for calibration standard.Extraction of Lipid, Glycogen and Sugar Fractions from Mosquito1. Add mosquito to glass centrifuge tube.2. Add 0.2 mL sodium sulfate solution.3. Homogenize mosquito in solution until no identifiable parts remain (glass rod or other utensil).4. Wash glass rod into centrifuge tube with two x 0.8 mL volumes of chloroform/methanol solution.5. Centrifuge (3000 rpm, 1 min).6. Transfer supernatant to clean centrifuge tube. Retain pellet for glycogen analysis.7. Add 0.6 mL DI water to supernatant. Mix.8. Centrifuge (3000 rpm, 1 min).9. Separate top fraction (water/methanol) for sugar analysis.10. Bottom portion (chloroform) holds the portion for lipid analysis.Lipid Analysis1. Place portion for lipid analysis in a tube with a marking at the 5 mL level.2. Place in heating block at 90-110 o C to evaporate the solvent.3. Add 0.2 mL of sulfuric acid and heat for 10 min at 90-110 o C4. Add vanillin reagent to 5 mL level and mix.5. Remove from heating block and allow to cool.6. Allow reddish color to develop; this will take approximately 5 min and will be stable up to 30 min.7. Determine OD at 625 nmSugar Analysis1. Place portion for sugar analysis in a tube with a marking at the 5 mL level.2. Place in heating block at 90-110 o C to evaporate the solvent down to 0.1-0.2 mL.3. Add anthrone reagent to 5 mL level and mix.4. Heat for 17 minutes at 90-110 o C.5. Remove from heating block and allow to cool.6. Determine OD at 625 nmGlycogen Analysis1. Add anthrone reagent to 5 mL level and mix.2. Heat for 17 minutes at 90-110 o C.3. Remove from heating block and allow to cool.4. Determine OD at 625 nm.