Methods in Anopheles Research - MR4

Chapter 6 : Dissection Techniques6.5 Dissecting Plasmodium-Infected Mosquitoes6.5.2 Salivary GlandsPage 1 of 26.5.2 Salivary GlandsMR4 StaffIntroductionDissection of the salivary glands should be done 10-18 dayspost-infection. The precise number of days that is best willvary with species. Some examples of variability are shown inTable 6.5.2.1 (WHO 1975). An alternate method by Lookerand Taylor-Robinson can be found in Methods in MalariaResearch (Looker and Taylor-Robinson 2004).Materials• Stereoscope• Dissecting needles• Microscope• Forceps• PBS or saline solution• Microscope slides• Giemsa stain• Cover slips• Chloroform, ethyl ether or ethylacetateSpeciesDays to sporozoitesP. vivax 8-10P. ovale 10-15P. falciparum 10-18P. malariae 15-21Table 6.5.2.1. Adapted from WHOManual on Practical Entomology, 1975.ProtocolDetecting the presence of sporozoites in the salivary glands.1. Anesthetize adult females (see 3.8 Mosquito Anesthesia).2. Place a drop of PBS on a clean microscope slide.3. Place a female on her side with her thorax in the PBS solution.4. While firmly holding the female with forceps or a needle, gently lay another needle across the necknear the head and slowly pull the head away (Figure 6.5.2.1).5. Gently detach the salivary glands from the head.6. If the salivary glands remain within the thorax, using the needle, apply gentle pressure to the thoraxtowards the mesonatum end and squeeze the glands out.7. At this stage you can either process the slides for staining or perform a brief inspection to determine ifsporozoites are present.Brief inspectionCover the dissected salivary glands with a cover slip and inspect the glands under a microscope for thepresence of the threadlike sporozoites. Gentle pressure on the glands will help rupture the tissues freeingthe sporozoites into solution.