Methods in Anopheles Research - MR4

Chapter 6 : Dissection Techniques6.9 A. gambiae s.l. Salivary Gland Chromosome PreparationPage 1 of 46.9 An. gambiae s.l. Salivary Gland Chromosome PreparationAnthony Cornel 1IntroductionChromosome preparations from larval salivary glands first require a clean dissection of the salivaryglands from L4 larvae. Fourth stage larvae of appropriate age are at about two to three hours beforepupal trumpets can be seen developing in the thorax under the cuticle. Thereafter, histolysis of thesalivary glands renders them useless for this purpose.Solutions• Modified Carnoy’s fixative (three parts pure (100%) ethanol and one part glacial acetic acid).Ethanol absorbs water from the atmosphere so ensure that the ethanol is pure, as water infixative compromises quality of spreads.• Propionic acid - prepare 5% and 50% in water.• 2% lacto-aceto orcein - prepare this solution by adding slowly 2% by weight of synthetic orceinpowder to a solution of 1 part glacial acetic acid and 1 part pure lactic acid under constant stirring(use a magnetic stirrer). Remove un-dissolved orcein particulates by filtering the solution throughWhatman 3MM paper. The solution can be stored indefinitely at room temperature.• Ethanol – 70%, 90% and 100% in water.Materials• 0.13- 0.17 mm thick cover glasses of dimensions 22L X 22W mm• Frosted 1mm thick microscope slides 75 x 25 mm in size.• Beaker• Aluminum foil• Forceps• Pasteur pipettes• Absorbent paper (Whatman 3MM paper))• Dissecting needles and minutien pins• Dissecting Microscope• 1.5 ml screw cap polypropylene vials• Phase contrast compound microscope with 10X, 40 or 60 X and 100X objective lenses.• Digital imaging systemProcedure1. Dissect salivary glands by placing a larva in a drop of 5% propionic acid on a microscope slide. Severthe abdomen from the thorax and insert a needle from the rear of the thorax along the mid dorsalsurface just underneath the cuticle up to just inside the head. Rub another needle over the inserted1 Mosquito Control Research Laboratory, Department of Entomology and Center for Vector borneDiseases, University of California at Davis, Parlier, CA 93648, E-mail: cornel@uckac.edu