590 LABORATORY EXERCISES 



Rhdich-Weigcrl Anil in Methyl ViolcL 



Alcoholic solution of methyl violet (saturated) 1 1 



Absolute alcohol ic 



Anilin water i oo 



This preparation does not keep well. 



Gram's Stain. — This is a method of differential bleaching after a stain. The 

 cover-glass preparations, or sections, are passed from absolute alcohol into Ehrlich's 

 anilin gentian violet, or into a water> solution of methyl violet, where they remain 

 one to three minutes, except tubercle bacilli preparations, which remain commonly 

 twelve to twenty-four hours (Gram). They are then placed for one to three minutes 

 (occasionally five minutes) in iodine potassium iodide water (iodine crystals, potassic 

 iodide 2 gr., water 300 c.c), with or without first washing lightly in alcohol. In 

 this way they remain one to three minutes. They are then placed in absolute alcohol 

 until sufliciently bleached, after which they are cleared in clove oil and mounted 

 in Canada balsam. By this method the stain is removed from some kinds of bacteria 

 and not from others. Too much confidence must not be placed in this method, since 

 in some cases the removal, or non-removal of the stain from the organism depends 

 on the length of exposure to iodine water. It would be better, therefore, to expose 

 all for the same period, e.g., two minutes. 



DelafieWs Hematoxylin. — To 100 c.c. of a saturated solution of ammonia alum 

 add, drop by drop, a solution of i gram of haematoxylin dissolved in 6 c.c. of absolute 

 alcohol. Expose to air and light for one week. FUter. 



Add 25 c.c. of glycerin and 25 c.c. of methyl alcohol. Allow to stand uhtU the 

 color is sufficiently dark. Filter, and keep in a tightly stoppered bottle. The 

 addition of the glycerin and methyl alcohol will precipitate some of the ammonia 

 alum in the form of small crystals. The last filtering should take place four or five 

 hours after the addition of the glycerin and methyl alcohol. 



The solution should stand for at least two months before it is ready for using. 

 This "ripening" is brought about by the oxidation of the haematoxylin into haematin, 

 a reaction which may be secured in a few minutes by a judicious application of per- 

 oxide of hydrogen (see Chamberlain, Methods in Plant Histology, p. 34). 



Safranin Gentian Violet. — Stain two to three days in safranin (dissolve 0.5 gram 

 safranin in 50 c.c. absolute alcohol, and after four days add 10 c.c. distilled water); 

 rinse quickly in water; stain one to three hours in a 2 per cent, aqueous solution 

 of gentian violet, wash quickly in water. Transfer from stain to absolute alcohol, 

 clear in clove oil and mount in balsam. 



Other useful stains in mycologic work are Fuchsin and Methyl Green, Fuchsin 

 and Methylene Blue, Eosin Water, Erythrosin and Acid Fuchsin. For the prepara- 

 tion of these and directions for using consult Chamberlain, Methods in Plant His- 

 tology, and other books on microscopic technique. 



Neisser's -Stain. — To differentiate between diphtheiia bacilli and pseudo- 

 diphtheria bacilli. 



1. Cultivate the organisms on fresh Loeffier's blood-serum at 34° to 35°C. for 

 ten to twenty hours. 



2. Stain with acid methylene blue three seconds. 



