28 W. H. E M I G 



obtained by cotmterstaining with direct blue dyes such as Niagara 

 Sky Blue. 



27. Orange G, Acid Orange G is an acid dye, light 3 to 4; -J 

 gram in 100 ml. of distilled water and 1 ml. of 1 per cent sulfuric 

 acid. The fastness of Orange G to washing is greatly increased 

 by the presence of acids. If the material is treated with -J per 

 cent potassium dichromate, the stain reacts more rapidly; potas- 

 sium dichromate cannot be added to the dye. Best results occur 

 on tissues fixed in a solution containing picric acid; with other 

 types of fixatives Orange II is better. 



Orange G is used extensively on animal tissues as a counter- 

 stain in conjunction with iron Haematoxylin. Because the iron 

 alum present in the tissues as a mordant for Haematoxylin also 

 serves as a chemical assistant for Orange G, the stain need not 

 contain sulfuric acid. Since the orange tends to obscure a portion 

 of the finer details of cell structure, many technicians prefer iron 

 Haematoxylin without a counterstain. 



Mallory's connective tissue stain includes Orange G, Aniline 

 Blue, and Acid Fuchsine. On many sections, Aniline Blue fails to 

 differentiate well with Orange G; the colors may be striking but 

 they usually lack clear definition. The deficiencies inherent in 

 Mallory's triple stain are also found in sections stained with Ehr- 

 lich's Orange G, Methyl Green, and Acid Fuchsine. 



A more satisfactory combination of stains is a mixture of nine 

 parts Orange G and one part Acid Fuchsine for two minutes which 

 is followed by Aniline Blue for 15 seconds on most sections of 

 animal tissues. As an alternative procedure try Orange II and 

 Acid Fuchsine followed by Niagara Sky Blue. 



Flemming's triple stain is frequently used on all kinds of plant 

 tissues. Saf ranine is applied first ; the excess of red is removed in 

 acid-alcohol. A brief stain in Gentian Violet is followed by dehy- 

 dration and then Orange G in clove oil is added. These three dyes 

 may be applied in a simpler manner. Stain the plant tissue in 

 Orange G, or preferably Orange II, and then apply the counter- 

 stains in a mixture of three parts Saf ranine and one part Methyl 

 Violet, page 21. 



