A new basic metachromatic dye, 1:9-DimethylMethylene Blue (1)
With pectin and potassium chondroitin sulphate the first two dyes show more absorption at what may be called the non-metachromatic peak, but a marked shift to the metachromatic peak was found with 1:9-Dimethyl Methylene Blue.
The chemical reactions involved in the preparation of the dye are shown. The reaction mechanism requires that one position meta to the dialkyl amino group should be unsubstituted to form the thioether (-S-) bond. Since in NN-dimethyl-m-toluidine one of the two positions is occupied, only one formulation of the resultant thiazine is possible.
1,9-Dimethyl Methylene Blue solution was prepared as below. 6.0 g of sodium sulphide (analytical reagent grade Na~S.9H20) was dissolved in 25 ml of distilled water, 40.5 g of ferric chloride was dissolved separately in 150 ml of N HCL. The solution of the amine hydrochloride was stirred mechanically and alternate additions of 2 ml of the sodium sulphide solution followed by 12 ml of the ferric chloride solution were made until all of the solutions had been added. Stirring was continued for 1 hr after the final addition. The purple precipitate of the crude dye was filtered off on a Buchner funnel using Whatman No. 542 paper.
Staining method
1. Paraffin sections of fixed tissue (fixed in formalin, Bouin’s fluid, etc.) were dewaxed with xylene and passed through aqueous alcohols to water. Unfixed cryostat sections may also be used; they sometimes give more striking results.
2. The buffered stain solution was poured on the slide and allowed to act for 5 main.
3. The stained section was washed well with distilled water.
4. The slide was passed rapidly through 25%, 50%, 75% and 100% dioxan.
5. The section was rinsed with chloroform, cleared in xylene or benzene, and mounted in D.P.X. or XAM.
Colour plate (I) shows a section of rat femur stained with 1:9-Dimethyl Methylene Blue according to the method outlined above. Colour plate (2) shows a similar section stained with Toluidine Blue according to the method of Hess and Hollander (1947), except that the material was fixed in Bouin’s fluid and mounted in XAM. Similar sections stained with Methylene Blue and with Azur B are shown in Colour plate (3) and (4). Slides stained with 1:9-Dimethyl Methylene Blue were found to give a consistently more intense metachromasia than any of the other three dyes tested. It is a particularly good stain for the demonstration of the acid mucosaccharides in cartilage.
