Aceto-orcein squash

Stage 5 Late gametogony. Characterised by the maturation of the male and female genitalia. This stage is recognised by the presence of spermatozoa which can be readily identified by preparing routine aceto-orcein squashes (796). 

Protocol 1.1 Aceto-Orcein Squashes of Larval Brains, 5... 

Depending on the experimental purpose, aceto-orcein squashes can be prepared by three different experimental regimes, which are summarized in Protocol 1.1. First, dissected brains can be squashed in aceto-orcein without colchicine treatment and hypotonic shock (i.e.. Protocol 1.1, with the omission of steps 3 and 4). This procedure allows observation of all phases of mitosis and permits evaluation of the mitotic index and the frequency of anaphases (Gatti and Baker 1989). However, chromosome morphology is poorly defined in these preparations. 

Unstained brain chromosomes can be prepared according to the same experimental regimes described above for aceto-orcein squashes. The procedure for this type of preparation is given in Protocol 1.2. 

Although well-sealed aceto-orcein squashes remain in good condition for 1-2 months, there are cases in which permanent chromosome preparations are needed. This can be... 

Leave the brains in the aceto-orcein drops for 1-2 minutes and then lower a very clean, dust-free nonsiliconized slide onto the coverslip surface tension will cause the coverslip to adhere to the slide. Invert the sandwich and squash between two or three sheets of blotting paper. 

Note Squashing should be performed in two steps First exert a gentle pressure to remove the excess of aceto-orcein and then squash very hard. The gentle presquashing prevents coverslip sliding and the consequent damage to the preparation. 

The salivary gland chromosome method of detecting newly induced rearrangements has been described in detail by Bauer et al, (1938), who used it to study the pattern and frequency of rearrangements induced in sperm as a function of X-ray dosage. Some improvements in techniques have occurred since the work of these investigators principally, these are (1) the use of siliconed coverslips to improve the spreading of the chromosomes as the nuclei are squashed and (2) the use of aceto-lactic-orcein... 

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