THE MITOCHONDRIAL CONSTITUENTS OF PROTOPLASM. 63 



Working on the same principle, a number of stains can be made up for mito- 

 chondria (Cowdry, 1913a, p. 485). Note also Bensley's neutral gentian method. 



(7) Bensley's (1916, p. 47) brazilin-wasserblau method: 



Fixation: 



(1) Fix in Zenker's fluid, plus less acetic acid, 10 per cent formalin, 24 hours. 



(2) Wash, dehydrate, clear, embed, and section (p. 59). 



Staining: 



(1) Pass down to water (p. 59). 



(2) Iodize with Lugol's solution, 30 seconds. 



(3) Stain in following solution several hours: Phosphotungstic acid, 1 gm.; aq. dest., 100 c.c; braziUn, 0.05 gm. 



The brazilin is first dissolved in a small quantity of distilled water by the aid of heat and added to the 

 phosphotungstic-acid solution. Ripening may be accelerated by the addition of 0.4 c.c. of hydrogen pero.x- 

 ide, or of a few drops of a solution of soluble molybdic acid. The solution deteriorates with age and 

 should not be used after 3 days. 



(4) Rinse in aq. dest. and place for 1 to 5 minutes in phosphotungstic acid, 1 gm.; wasserblau, 0.2 gm.; aq. dest., 



100 c.c. 



(5) Wash rapidly in water, dehydrate in absolute alcohol, clear in toluol, and mount in balsam. 



(8) Meves's (1905, p. 102) new Victoria green method: This method is intended 

 for red blood-cells which are simply stained in the fresh condition by the addition 

 of a 4 per cent iodic-acid solution to which a small quantity of new Victoria green 

 (malachite green) has been added. 



(9) The methods of silver reduction employed by many ItaUan investigators 

 are essentially modifications of the original method of Golgi. They undoubtedly 

 reveal mitochondria in most cases, but one would hesitate to attribute any high 

 degree of specificity to them. For details see Veratti (1909, p. 34), Pensa (1910, 

 p. 326), Rina Monti (1915, p. 21), and Cajal (1915, p. 3). 



For still other mitochondrial methods see Sjovall (1906, p. 563), Rubaschkin 

 (1910, p. 407), Koltzoff (1906, p. 384), Kingsbury (1911, p. 317), Rchultze (1911a, 

 p. 465), Maximow (1916a, p. 462), and others. 



In unskilled hands the experimental error in some of these methods of technique 

 is sometimes very great, but it is a mistake to regard the methods as difficult. 



In the examination of living cells it is of course essential that the medium should 

 be as nearly as possible isotonic. The presence or absence of Brownian movement 

 is a valuable criterion of the condition of the cells. Where it is very marked the 

 material should be discarded, because it indicates that an unusually large amount 

 of water has entered into the cells. Each particle, instead of being held in place 

 by the balanced action of many bombarding molecules, is subject to the action of 

 only a few, now on one side and now on the other, causing it to jump from place 

 to place. The smaller the particle the less chance there is of molecules on the 

 opposite side compensating. Large granules, on the other hand, offer a greater 

 surface, are more likely to be bombarded from all sides, and are thereby held in 

 place and do not show so much tendency toward exhibiting Brownian movement. 



One of the most common and annoying sources of error is mechanical manipu- 

 lation of the fresh tissue before fixation. A mere crushing of the tissue with the 

 forceps will bring about the most astonishing and perplexing modifications in the 

 mitochondria. Allowing a surface film of the tissue to dry in air, as it stands on 

 the autopsy table, is another common blunder which changes the whole appearance 

 of the mitochondria. Osmotic changes in the tissues before fixation must also be 



