Acetic orcein

Stain slides with 2% lactic-acetic-orcein by adding a drop of stain to the slide and then placing a cover glass onto it. After some 2 min, gently press the cover glass and the slide within a bibulous pad to remove excess of fluid. Slides prepared this way do not dry up for at least a month. 

In preliminary experiments it may often be more important that a rough estimation of the effectiveness of the chemical can be quickly obtained than to have good permanent preparations for a detailed analysis. In such cases, the acetic-orcein method of Tjio and Levan (1950) is useful. 

Staining procedure When dry, invert into a drop of 1.0% acetic orcein (0.5 g orcein in 45 ml glacial acetic acid reflux % hr, add 55 ml distilled water while warm, reflux % hr, let stand 24 hr, filter, and filter fresh before using) on a clean slide, suck out excess out excess stain with filter paper, and seal with Kroenig s cement. If permanent slides are desired treat dry coverslip preparation in following sequence 1.0% acetic orcein (30 min), 45% acetic acid (dip until free of excess stain), tertiary butyl alcohol equal equal xylene (1 min), xylene (1 min), xylene (1 min), and invert wet into Permount (thinned with xylene) on clean slide. 

Staining can be achieved by standard procedures utilizing acetic-orcein, Giemsa stain, or the Feulgen reaction. 

Staining may be achieved by acetic-orcein, lactic-acetic-orcein, or 2% toluidine blue. 

Small pieces of tubules (1-2 cm length) are placed on a slide and stained with a drop of lactic-acetic-orcein. 

The preparations can be stored or stained immediately with lactic-acetic-orcein, toluidine, or Unna blue. Toluidine blue is generally preferred for immediate examination of nonpermanent preparations. Lactic-acetic-orcein and Unna blue are used for finer examination and for permanent preparations. 

A 2% solution of natural orcein (Gurr s) in 60% glacial acetic acid is prepared by dissolving the orcein in boiling glacial acetic acid with stirring. Cool to 55 °C. Add distilled water to make the acid 60%. Cool to room temperature. Filter twice through Whatman filter paper No. 1. 

Pure crystallised orcein is insoluble in water, etber, and benzene soluble in alcohol, acetone, and acetic acid. Pure orcein possesses nearly two hundred times the tinctorial power of orchil extract. 

Dissolve 2 g of orcein (natural) in 45 ml of hot glacial acetic acid. When the solution is cooled, add 7.5 ml of distilled water and 47.5 ml of lactic acid. Filter and store (4°C) in a brown bottle. Filter the stain periodically. Long storage will result in precipitation of the stain. 

Boil synthetic orcein powder (Gurr, BDH Laboratory Supplies, Poole, Dorset BH15 ITD England Phone -1-44 1202 660444 Fax -1-44 1202 666856 Web site http //www.bdh.com) in 45% acetic acid for 45 minutes in a reflux condenser. We usually prepare 5% orcein, which is subsequently diluted to 2% with 45% acetic acid. 

Orcein (3%), dissolved in 60% acetic acid (for preparation of 2% aceto-orcein, see p. 100) Ringer s Solution... 

Lactic acid 3% orcein in 60% acetic acid (1 1 ratio)... 

Stain in 3% orcein dissolved in 60% acetic acid for 5 minutes. 

Cut testes under dissecting scope to release testis content. Add a drop of Lacto-Aceto-Orcein (1 1 lactic acid to 3% orcein in 60% acetic acid). 

Perform steps 1-3 of Protocol 5.10 but use 45% acetic acid instead of 2% aceto-orcein, and use siliconized coverslips. 

Staining Solution of Lactic Acid, Acetic Acid, and Orcein... 

The final concentrations of acetic acid and orcein are 50% and 2%, respectively. Although differing concentrations of lactic acid (between 16% and 30%) are used, the lactic acid concentration suggested above by Lefevre (1976) is preferred. 

Place a drop ( 25 ll) of staining solution containing lactic acid, acetic acid, and orcein on a siliconized coverslip. After the glands are fixed sufficiently, transfer them to the drop of staining solution. Allow the glands to sit in the staining solution for several minutes. 

Acetic acid/orcein/carmine (Ashburner 1989, p. 332) or 4% Giemsa... 

Stain chromosomes in acetic acid/orcein/carmine or in Giemsa. 

Prepare squashes of salivary glands from female third-instar larvae (raised on a rich diet under uncrowded conditions). Use lactic-acetic acid without orcein or other DNA dyes (see Pimpinelli et al., this volume [Protocol 1.2]). 

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