PLANT PARASITES. 153 



should be kept in tightly stoppered bottles, and from these the more dilute solutions 

 required foi staining may be prepared. For ordinary purposes one part of alcoholic 

 solution of fuchsin or methylene blue, added to twenty parts of water, will give a 

 staining solution of suitable strength. 



Special stains and modes of staining, such as are necessary for some forms of bac- 

 teriathe tubercle bacillus, for example will be described under the appropriate head- 

 ings. 



To Stain Bacteria in Fluids. A small drop of the fluid is placed on a clean cover 

 glass, spread a little with a needle, and dried by gentle heat. The cover glass is now 

 held with the forceps, specimen side up, and passed moderately rapidly three times 

 through the flame of an alcohol lamp or Bunsen burner. The material on the cover 

 should not be burned. This heating not only fixes the contents of the fluid firmly on 

 to the glass so that it will not easily soak off, but renders insoluble any albuminous 

 materials which may be mixed with the bacteria, and which might otherwise interfere 

 with subsequent examinations by forming granular precipitates. 



A drop of the aqueous staining fluid is now put on to the dried specimen on the 

 cover glass, and if this be held in the forceps and tilted slightly up and down a few 

 times so as to bring fresh portions of the staining fluid into contact with the bacteria, 

 the staining will usually be completed in two or three minutes. The stain is now 

 washed off with a jet of water from the wash bottle, and the specimen is either mounted 

 in a drop of water for temporary study, or the washing water is drained off and, after 

 drying in the air, it is mounted directly in balsam. It is well to use balsam which has 

 been softened, when this is necessary, with oil of cedar or xylol rather than with chloro- 

 form, since this is apt to decolorize the bacteria. Solid cultures of bacteria should be 

 mixed with a little water and spread on the cover glass before drying and staining. 



Gram's method (see below) is often useful and in some cases almost indispensable for 

 the differential staining of bacteria. 1 



'To Stain Bacteria in Tissues. The tissues should be well hardened in alcohol. 

 Thin sections are placed in the above-described aqueous coloring solutions, where they 

 may remain from five to fifteen minutes. In some cases a much longer staining is nec- 

 essary. Gentle warming (40 to 50 C.) will hasten the staining. The entire tissue as 

 well as the bacteria is in this way deeply colored. The sections are rinsed with distilled 

 water and then placed in alcohol. This, with varying degrees of rapidity with differ- 

 ent stains and tissues, gradually extracts the color from the tissue, most slowly from 

 the nuclei. The time required and the exact degree of decolorization to be sought for 

 must be learned by experience in different cases. Sometimes five, sometimes thirty 

 minutes are required, sometimes only a few seconds. It is often necessary, and the 

 decolorizing of the tissue is thereby hastened, to add a few drops of acetic acid to the 

 alcohol. When acetic acid is used it should be finally thoroughly washed out by alco- 

 hol. The specimens are now cleared up by oil of origanum and mounted in balsam. 

 In specimens prepared in the above way, the nuclei of cells usually retain to some ex- 

 tent a color similar to that of the bacteria, but their size and shape serve for the differ- 

 entiation. 



Grain's Method. This is a much more generally useful method of staining bacteria 

 in the tissues than that just given, although not in all cases applicable. The sections 

 are stained for from two to four minutes in anilin-gentian-violet solution, prepared by 

 adding 1 c.c. anilin oil to 20 c.c. distilled water, shaking thoroughly, and filtering 

 off the excess of anilin oil through a moistened paper filter. To the clear filtrate satu- 

 rated alcoholic solution of gentian violet is added in the proportion of .about 1 of the 

 dye to 10 of the anilin solution. 



From the staining solution the sections are transferred to a solution of iodin in 

 potassium iodid and water (I 1.0 KI 2.0 HO 300.0), remaining from one to three 

 minutes, when they are transferred to strong alcohol; this should be changed two or 

 three times so as to dehydrate the specimen, which at the same time will lose much of 

 its color. The section is cleared in oil of origanum to which a little eosin has been 



1 For the methods of staining spores see special works on bacteriology. 



