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ICROSCOPICAL TECHNIQUE 47 



preparations, the presence of vacuoles, capsules, spores, the 

 ease with which the bacteria take the simple aqueous stains. 

 Gram's stain. Some stains are of value in that they not 

 only bring out the morphology of the organism, but also have 

 a diagnostic value in differentiating one species from another. 

 The one most frequently used for this purpose is the Gram 

 stain. The staining of the preparation is as follows : Prepare 

 and fix the preparation as previously described, and then stain 

 for one minute with aniline-water gentian violet. Wash the 

 preparation and then flood with iodine solution, leaving it 

 in contact with the preparation for two minutes. Flood with 

 95 per cent alcohol, repeating this operation until no more 

 color is given off. Wash, mount in water, and examine. 

 Organisms which retain the stain are- said to be Gram posi- 

 tive, all others Gram negative. 



Exercise. Stain preparations from the cultures furnished by 

 Gram's method. 



Flagella stain. The staining of the delicate flagella is a 

 difficult process. No one method is always successful. The 

 number and arrangement of the flagella are of diagnostic 

 value. The following method (Bunge's) is one of the best in 

 use : A mordant, which so alters the protoplasm of the cell 

 as to make it more readily stained, is prepared by mixing 

 25 cc. of a 5 per cent aqueous solution of ferric chloride with 

 75 cc. of a saturated aqueous solution of tannic acid. 



From an eighteen-hour to a twenty-four-hour culture of 

 the organism on agar remove a small amount of the growth 

 and place in a large drop of tap water. Do not mix, but 

 allow the organisms to diffuse themselves, as mixing with 

 the needle breaks off many of the flagella. After standing 

 a few moments remove a loopful to a perfectly clean cover 

 glass and allow the drop to spread of itself. Dry, and fix 



