Methods in Anopheles Research - MR4

Chapter 6 : Dissection Techniques6.8 A. gambiae s.l. Ovarian Polytene Chromosome PreparationPage 6 of 619. Once the first round of tapping has been completed, remove excess propionic acid by gently pressingabsorbent paper over the cover-glass edges. Place a new dry piece of absorbent paper over thecover-glass, and press firmly downwards with a thumb directly over the cover-glass. Do not move thecover-glass laterally while thumb pressing as the chromosomes will roll and be unrecognizable. Scanthe slide at 100X magnification to get an overall perspective if the chromosomes are flat enough. Ifthey are not sufficiently flat then give them a further thumb press. Chromosome spreads can also beflattened by holding the slide at about 70C for 5 to 10 seconds on a slide warmer. Don’t overheat thechromosomes as the banding will fade. View again at 100X magnification and select the bestchromosome compliment to examine at higher power.20. On occasions you may wish to view the spreads under oil at 600 to 1,000X magnification and thendecide to go back to a lower magnification that does not require oil. The oil can be absorbed relativelyeasily from the surface of the cover-glass with tightly rolled up absorbent paper. Oil cannot beremoved effectively from an unsiliconized cover-glass which is another reason why I recommend youroutinely coat cover-glasses (refer to step 1).21. On occasions the slide will begin to dry up (air creeps in between the cover-glass and microscopeslide) while you are examining the spreads under the microscope. Simply add a small drop of 50%propionic acid to the edge of the cover-glass and the acid will, by capillary action, draw under thecover-glass.22. If results are required immediately, chromosomes can be prepared from freshly dissected ovaries byfirst dissecting them out of the female mosquito in 5% propionic and then transferring them into adrop of 50% propionic acid on a clean microscope slide. Continuing with the process as describedfrom steps 11 to 20.23. Interpretable chromosome spreads, but of lesser quality, can be extracted from ovaries preserved forless than 1 hour in modified Carnoy’s solution. This comes in useful in the field when on the spotdecisions are required to know which An. gambiae s.l. member or chromosomal forms of An.gambiae occur in an area. Use the same procedure as described from steps 11 to 20.24. Permanent records of chromosome preparations can be kept either by taking photographs or bymaking permanent mounts. Digital cameras attached to the microscope and computer makecapturing images much easier than the past when emulsion films were used. If permanentpreparations are still required then proceed to the next step.25. Remove the cover-glass by holding the microscope slide in one corner with a forceps and holding inliquid nitrogen until the bubbling stops. Take it out of the liquid nitrogen and immediately place themicroscope slide on a flat surface and briskly pry the cover-glass off with a razor blade form onecorner. Immediately begin dehydrating the preparations placing the slides into an ethanol series of70%, 90% and 100% for 5 minutes each at 4C. Air dry the slide and add a drop of mounting mediumused for histology onto the chromosomes and cover with a cover-glass (does not have to besiliconized). Leave to dry and ring the cover-glasses with sealant according to recommendedmounting medium instructions.ReferencesClements AN (1992) The Biology of Mosquitoes: Development, Nutrition and Reproduction. Chapman &Hall, LondonColuzzi M, Sabatini A, della Torre A, Di Deco MA, Petrarca V (2002) A polytene chromosome analysis ofthe Anopheles gambiae species complex. Science 298:1415-1418Holt RA et al. (2002) The genome sequence of the malaria mosquito Anopheles gambiae. Science298:129-149