REAGENTS AND PROCESSES 537 



with a digestive fluid made by mixing i part of pepsin-glycerine with 3 parts of 

 water acidified with 0.2 per cent, of chemically pure hydrochloric acid. 



Double Staining. There are certain stains which have a peculiar affinity for 

 the lignified and suberized or cutinized membranes; others which color the cellulose 

 membranes without affecting the modified membranes, and still others which stain 

 all membranes, but with different degrees of intensity. The latter are known as 

 diffuse stains, and are able to differentiate the tissues when used singly. Thus, 

 safranin will stain the lignified membranes a cherry-red and the unlignified mem- 

 branes a brownish-red. Double staining by the use of two .stains, one of which has 

 an affinity for the modified membranes and the other for the cellulose membranes, 

 gives excellent results in differentiating the tissues. In general, the sections should 

 first be treated with those stains which color the lignified membranes, and then, 

 after rinsing in water or acidulated alcohol, as the case may require, the sections 

 are to come into a stain which has a particular affinity for the cellulose walls. 

 Fuchsin is an excellent stain for the lignified membranes, and hasmatoxylin, aniline 

 blue, methyl blue, and Berlin blue are good stains for the cellulose membranes. 

 Microtome sections should be left in aqueous solution of fuchsin for a quarter of an 

 hour or longer; then they should be washed in a mixture of I part of concentrated 

 alcoholic solution of picric acid and 2 parts of water. Then the sections should be 

 placed for about an hour in one of the blue stains above named, and thereafter 

 washed in strong alcohol, transferred to xylol, and thence mounted in Canada 

 balsam. The lignified membranes will be stained red and the cellulose membranes 

 blue. The cutinized and suberized membranes may be stained together with the 

 lignified membranes in the following manner: Ammonia is added to an alcoholic 

 solution of fuchsin until the solution attains a straw-yellow color; then the sections 

 are placed in this and treated thereafter as described for the simple fuchsin 

 solution. 



A mixture of fuchsin and iodine green produces an excellent differentiation. 

 One volume of a concentrated aqueous solution of fuchsin is mixed with 9 volumes 

 of a o.l per cent, aqueous solution of iodine green. The sections remain in this 

 solution for about eight minutes; then they are washed in a mixture of 100 mils of 

 absolute alcohol, I mil of glacial acetic acid, and o.i Gm. of iodine; then transferred 

 to xylol, and thence they are mounted in Canada balsam. The sections should be 

 left longer in the stain if a double stain is not achieved in eight minutes. The 

 proper time for a given material can soon be determined by experiment. In gen- 

 eral, those sections stain best which have been fixed in a fixative containing chro- 

 mic acid or corrosive sublimate. Material which has been fixed in alcohol should, 

 just before staining, be placed for about a day in a I per cent, solution of chromic 

 acid, and then washed out in water for some hours. When permanent mounts are 

 made, staining is best carried out with sections already fixed to the slide. 



The iron-haematoxylin method of staining imparts different intensities of gray 

 or blue to the different tissues and cell-contents, and is one of the simplest and best 

 differentiating stains. It is particularly useful for staining the dividing nucleus. 

 For staining sections from which photomicrographs are to be made, it is unsur- 

 passed. The method of procedure is as follows: The sections mounted on the 

 slide are placed in a 4 per cent, solution of ferric ammonium sulphate for an hour 

 or so. They are then washed in water and placed in a 0.5 per cent, aqueous solu- 

 tion of haematoxylin for an hour or more. The hsematoxylin solution should be 

 several weeks old. The sections are again washed in water, and placed in 1.5 per 

 cent, solution of ferric ammonium sulphate and left there until examination with 

 the microscope shows that the desired intensity of color is achieved. They are 

 then washed in water, dehydrated in alcohol, cleared in xylol, and mounted in 

 Canada balsam. 



