Worm Breeder's Gazette 3(1): 13
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In the meeting, some persons were interested by my stained gonads slides. This technical note describes how I proceed to get them. DISSECTED WORMS Fixative = sublimate acetic : 95 ml sublimate (dissolve 10 g of HgCl2 in 100 ml boiling water-let decant one night) and 5 ml acetic acid. When dessication of dissected worms is completed, deposit a fixative drop on the frottis, then immerse the slide in sublimate acetic for 10 mn. Rinse in water and let one (or more) night in iodine-iodized alcohol (100 ml 70% ethanol, 2g I2, 4g KI). Rinse in a 0.25 % sodium hyposulfite solution (5 mn), then in running water (10 mn). Immerse in HCl for 12 mn in a 56 C water-bath, stop hydrolysis by a room temperature water rinse, immerse in Schiff reagent for 2 hours and rinse in running water (15 mn). Stain finally by a fast passing over in : - 1% light green solution, rinse and dehydrate in 1 x 70, 1 x 95, 2 x 100 alcohols, then 2 x Toluene and mount. - picro indigo-carmine (0.25 g indigo-carmine in 100 ml of picric acid aqueous saturated solution). Rinse directly in 100 ethanol ( twice). I prefer use P.I.C. to light green, because the green staining disappears from cytoplasm in 2 to 3 months ENTIRE WORMS (for sections) Fixative = Zenker fluid - stock solution = HgCl2 : 5 g; K2Cr2O7 = 2. 5 g; Na2SO4 = 1g; distilled water 100 ml - Before use, add 5 ml acetic acid. Background staining = Heidenhain's blue - Dissolve in 100 ml hot 0.5 g aniline blue and 2 g orange G - When cold, add 8 ml acetic acid - For use, dilute 2 or 3 times. Worms are washed several times in saline water (NaCl 8%) then fixed by Zenker fluid for 1 hour. Rinse alcohol, wash in 0.25 % sodium hyposulfite solution (15 mn), and in distilled water (2 x 15 mn). Worms are double-embedded, in 1.3 % agar and in filtered paraffin, after 4 baths in paraffin (2 x 30 mn, 2 x I hour). Do the Feulgen reaction as before on hydrated sections, then, rinse in distilled water, transfer the slides in 5 % phosphotungstic acid for 10 mn, rinse in distilled water, stain by Heidenhain's blue for 30 sec to 2 mn (depending dye solution concentration). Dehydrate directly in 95% ethanol, then toluene (twice) and mount. Another background colorings can be use : light green or picro indigo carmine .