Meiotic Preparations Research Articles

A method for meiotic preparations of mammalian ova.

Superovulation is induced in the female rodent with a priming dose of PMS followed by an ovulating dose of HCG. At proper time intervals thereafter, follicular or ductal ova can be obtained in large numbers, freed of coronal cells while in a 0.7 % sodium citrate solution and fixed in 50 % acetic acid. Freezing with dry ice and methanol permits removal of the cover slip after mild squashing and the dried preparation can then be hydrolysed with in HCL and stained with Giemsa stain. Preparations of good quality which permit examination of number and morphology of the chromosomes of ova in first or second meiotic metaphase can thus be obtained.

AN AIR-DRYING METHOD FOR MEIOTIC PREPARATIONS FROM MAMMALIAN TESTES.

A suspension is made in isotonic (2.2%) sodium citrate solution from the contents of the tubules from a whole testis or a testicular biopsy specimen. The germinal cells are sedimented by centrifuging, leaving most of the sperm in the supernatant fluid, which is discarded. The cells are resuspended in hypo-tonic (1%) sodium citrate solution and left to stand at room temperature for 12 minutes, after which they are sedimented again and fixed as a concentrated suspension in a mixture of 3 parts absolute ethyl alcohol to 1 part glacial acetic acid plus a trace of chloroform. Two quick changes into fresh fixative follow. Air-dried preparations are made from the final fixed suspension and stained in lactic-acetic-orcein. The method is suitable for stages of male meiosis in which the chromosomes are condensed. Its principle advantage is the separation of the clumps of spermatogonia and spermatocytes into individual cells which are randomly dispersed over the preparations. Compared with squash techniques, the air-drying method gives improved spreading of the chromosomes and less cell breakage.

The Use of Lacto-Propionic Orcein in Rapid Squash Methods for Chromosome Preparations

A solution of 2 gm of natural orcein dissolved in 100 ml of a mixture of equal parts of lactic and propionic acids, and diluted to 45% with water, proved more effective than other stain fixatives for meiotic preparations from fresh pollen mother cells. When used after 5 min fixation in modified Carnoy's fixative (alcohol, acetic acid, chloroform, formalin; 10:2:2:1) and 5 min maceration in 1 N HC1 at 60° C, the same stain proved the most suitable for the rapid preparation of root-tip chromosomes for counting and for studying detailed morphology.