Methods in Anopheles Research - MR4

5. Place eggs in a new Petri dish containingpurified water while you “test crack” a sampleof the embryos. This step is necessary toensure that the exochorion was removedduring the bleaching step. If this layer is notremoved, the fixatives will not permeate theembryo and fixation will fail.a. Aliquot approx. 25 test embryos into ascintillation vial. Remove the waterwith a pipette and add 5 ml heptane.b. Incubate at room temperature for 5minutes while occasionally swirlinggently.c. Add 5 ml of methanol and vigorouslyswirl once to mix. Place scintillationvial on its side under the stereoscopeand watch for cracking (Figure3.1.2.2). It is normal for the embryosto seep out of their chorion. If theembryos crack, discard these testChapter 3 : Specific Anopheles Techniques3.1 Embryonic Techniques3.1.2 Anopheles Embryo FixationPage 2 of 4eggs and proceed with the protocol. If they do not, longer bleaching is needed.6. Rinse embryos into a new scintillation vial with deionized water. Remove as much water aspossible using a Pasteur pipette.7. Add 5 ml heptane and shake 3-4 times gently by hand to mix. Remove as much remaining wateras possible. Add 5 ml formaldehyde. Shake on rotary platform for 25 minutes on a medium speedsetting. Eggs will accumulate between layers as shown inFigure 3.1.2.3.8. Remove formaldehyde phase only (leaving heptanephase) using a fresh pipette. Replace with a large volumeof deionized water. Briefly shake 3-4 times gently by handand remove only the water phase (leaving heptanephase). Add 10ml of fresh deionized water.9. Shake on platform an additional 30 minutes on a mediumspeed setting.10. Remove only water phase (leaving heptane phase). Fillvial to the top with boiling deionized water. Incubate for 30seconds.11. Remove hot water phase (leaving heptane phase) andreplace with ice-cold deionized water.12. Place vial on ice for 10 minutes.13. Remove water phase using a glass pipette.14. Remove as much of the heptane phase as possible.Figure 3.1.2.3. Nondamagedeggs willaccumulate at theinterface between twolayers.Figure 3.1.2.2. Examples of eggs crackingproperly. Cracking is an indication that theprocess is proceeding correctly, and you cancontinue. Discard these test eggs.15. Add 5ml of fresh heptane. Remove as much water aspossible.16. Add 5ml methanol and swirl vigorously once, placescintillation vial on its side under stereo scope to watch forcracking, a sign the fixation has been successful to this