MICROSCOPICAL EXAMINATION OF BACTERIA. 97 



they must be treated with a half-saturated solution of carbonate of 

 potash. In either case the sections are next washed with distilled 

 water, and passed through 60 per cent, alcohol into absolute alcohol. 

 When almost decolorised, spread out the section carefully on a 

 platinum lifter and transfer it to clove-oil, or stain with picro-carmine 

 solution (Weigert's) for half an hour, wash in water, alcohol, and 

 then treat with clove-oil. After the final treatment with clove- oil, 

 transfer with the platinum lifter to a clean glass slide. Dry the 

 preparation by pressure with a piece of filter-paper folded several 

 times, and preserve in Canada balsam, dissolved in xylol. 



Gram's Method. In the method of Gram sections are stained 

 for ten minutes in a capsule containing aniline-gentian- violet solution. 

 Great care must be taken not to injure the sections. If there is 

 any difficulty in finding them, it is best to carefully pour off the 

 stain and fill up the capsule with water. The sections are then readily 

 visible, and can be taken up on the end of a glass rod and placed 

 in the iodine and iodide of potassium solution, where they remain for 

 two or three minutes, until stained uniformly brown and resembling 

 in appearance a boiled tea-leaf. They are then placed in absolute 

 alcohol, and washed by carefully moving the sections in the liquid 

 with a glass rod. When completely decolorised they are spread out 

 on a lifter, and transferred to clove-oil until completely clarified. 

 Each is transferred with a lifter to a slide, and the clove-oil is 

 run off and then completely removed by gently pressing two or 

 three layers of filter-paper upon the section. Finally, the section 

 is mounted in Canada balsam. 



The process of decolorisation may be hastened by transferring the 

 section from alcohol to clove-oil, and back again to alcohol, repeating 

 this two or three times. 



On examination the tissue appears colourless, or slightly tinged 

 yellow from too long immersion in the iodine solution, while the 

 micro-organisms are stained blue or blue-black. 



Double staining is obtained by transferring the sections after 

 decolorisation to eosin, Bismarck brown, or vesuvin. They are left 

 in a watery solution for two or three minutes, then again washed in 

 alcohol, before clarifying in clove-oil and mounting in balsam. 



Another instructive method is to place the decolorised sections 

 in picro-carminate of ammonia for three or four minutes, and then 

 treat with alcohol and clove-oil. 



A similar result is obtained by placing the sections in Orth's 

 solution (picro-lithium carmine), transferring to acidulated alcohol, 

 and then passing through clove-oil and mounting in balsam. 



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