Meiotic Preparations from Testis
Muriel T. Davisson*
The Jackson Laboratory
*Slightly modified from method of E.P. Evans, Oxford, England.
(Evans, E.P., Breckon, G., and Ford, C.E. Cytogenetics 1964; 3:289-294)
- 2.2% Na citrate in distilled water to 37°C
- 1.1% Na citrate -made by diluting 2.2% 1:1 with warm distilled water
- Fixative = 3:1 Methanol:glacial acetic acid
- Stain - 2 ml Fisher Giemsa (#SG28-475) in 48 ml Fisher KPO4 -NaOH buffer (pH 7). (#SB108-20)
- Dissecting tools, including curved forceps for teasing
- 9" Pasteur pipets and small dish for washing tubules
- Conical centrifuge tubes and Coplin jars for staining
- Remove testis and wash in 2.2% trisodium citrate (Na citrate).
- Remove tunica from tubules and wash tubules in at least 3 changes of 2.2% Na citrate or until there are no more fat globules on the surface of the saline.
- Tease tubules apart until pieces are small enough to get into a Pasteur pipet. Pipet 100 times (~1 min.) with 9" Pasteur pipet.
- Tilt dish and allow tubule fragments to settle. This can be from 10 - 15 min. depending upon the number of samples being processed. Transfer supernatant cell suspension to 12- or 15-ml conical centrifuge tube (we use plastic disposable).
- Centrifuge for 5 min. on clinical bench centrifuge (approximately 200 x g).
- Remove supernatant and add 4 ml 1.1% Na citrate to resuspend pellet. Incubate cells at room temperature for 15 min.
- Centrifuge as in step 5 and remove hypotonic Na citrate.
- Flick pellet "violently" into suspension and add fixative directly onto cells, drop-by drop, flicking continually to mix well. Total fixative should be about 3 mls.
- Centrifuge immediately as before, remove fixative and add fresh fixative as before. Allow to sit at least 5 min. at room temperature. Cells can be stored for several days in fixative in the refrigerator at this point. Always change fixative (step 8) just before making slides.
- Change fixative once or twice more and make air dried slides. For standard slides stain 6 min. in 4% Fisher Giemsa. Rinse in cold tap water 3 times, distilled water 3 times.
- Slide Preparation 1. Immerse pre-cleaned slides in fixative at least 15 min. prior to use. Wipe slides dry with a Kimwipe or other lint-free tissue. 2. Drop small drops of cell suspension onto slide surface with a Pasteur pipet and allow it to spread. If too much suspension is used it will bubble at the edges. 3. As soon as the drop begins to contract and Newton's rings are visible (rainbow colors around the edge of the drop), blow on the slide surface to accelerate drying. Slides may also be dried by using tubing connected to an air supply or by spraying lightly with Dust-off Plus. 4. Repeat steps 2 and 3 until sufficient sample is on the slide. Monitor cell concentration by phase microscopy.