Methods in Anopheles Research - MR4

Chapter 6 : Dissection Techniques6.8 A. gambiae s.l. Ovarian Polytene Chromosome PreparationPage 5 of 611. To make the chromosome squashes, remove the ovaries from the vials with a pair of forceps andplace them into a drop of modified Carnoy’s (approximately 25 µl) on a dust- and grease-freemicroscope slide. Quickly separate half the ovules or follicles from one ovary, and return the rest ofthe ovaries to the vial to use as back-up for later preparations. You have to do this quickly before themodified Carnoy’s solution dries out on the slide as no spreads can be recovered from dry ovules.12. Drop about 50 µl of 50% propionic acidonto the ovules and leave them for about 3n memABminutes until they have cleared andswollen to about twice their original size.Consult Figure 6.8.4 for further guidanceovulento visualize appropriately aged ovules.Once again do not let the ovules dry out.ovule13. Under a dissecting microscope carefullyovuleseparate the ovules from each other andCDgently squeeze each ovule (follicle) out ofmemits surrounding membrane. Removeovulenconnecting tissues, trachea andmembranes by sliding them away from theovules and draw up the waste tissues withthe tip of tightly rolled up absorbent paper.Figure 6.8.4. Examples of ovules after 5 minutes in 5% 14. From my experience, staining thepropionic acid that were dissected at various stages of chromosomes is unnecessary particularlydevelopment. A – ovule at early Christopher’s II stage in these days of improved microscopewhich is too young for harvesting, Note for comparison optics. Staining with 2% lacto-aceto orceinthe edge of an ovule of appropriate age and size in the is optional and this step should be donetop right hand corner; B and C – ovules at Christopher’s after the ovules have been separated andIII stage of development which are at the correct age cleaned (after completion of step 13). Doand size for producing good chromosome squashes. In this by adding a drop of the stain to theimage B the ovule to the right is too old. In both B and C ovules in the 50% propionic acid. Createthe ovules have popped out of their surroundingan even-staining mixture around the ovulesmembranes (mem) and the nuclei of nurse cells are by stirring gently with a needle. Leave thisenlarged (n); C- ovule too old. All images taken at 100 for about 4 minutes for the ovule cells toX mag. under phase contrast.absorb the stain. Gently absorb the excessstain with a piece of tightly rolled upabsorbent paper and add a drop of 50% propionic acid to the ovules which by now should have apale pink hue to them. Proceed to step 15.15. Place a further drop of 25µl of 50% propionic acid onto the clean ovules.16. Wipe a cover-glass with a clean sheet of lint free paper – lens cleaning tissue or KimWipe®(Kimberley-Clark- Roswell GA) and place on top of the ovules. Ensure the cover-glass is dust andgrease free.17. Carefully set the microscope slide on a piece of absorbent paper on a flat surface.18. Various people use different tapping techniques to make chromosome squashes, but essentially allbegin by tapping quite firmly to break open the nuclear membranes and spreading out thechromosomes followed by harder tapping or pressing to flatten the chromosomes. I prefer to firstgently tap the cover-glass with a mattress needle about 8 to 10 times (end bent downwards asdepicted in Figure 6.8.2). While tapping with the needle the cover-glass will shift around a little whichhelps to shear the nuclear membranes and spread the chromosome arms out. View the slide at 100Xmagnification under phase contrast in a compound microscope to determine if more tapping to spreadchromosomes is required.