BONE, TEETH 435 



Mall {op. cit.) clears formalin embryos in 10 per cent, potash 

 for about a month or longer. Formalin renders the connective 

 tissues very tough, and this strong KOH solution is necessary. 

 Refer also to § 809. 



925. Dawson's Method of Staining the Skeleton of Cleared 

 Specimens with Alizarin Red S (Sodium Alizarin Monosulphonate). 

 Alden B. Dawson states {in Uteris) that the methods commonly 

 used for obtaining a differential staining of developing bone in 

 specimens already cleared or to be cleared (Lundvall, Spalteholz, 

 Batson) have involved the staining of the entire specimen in a 

 rather concentrated solution of alizarin, followed by differential 

 decoloration. The alizarin is ordinarily made up as an alcoholic 

 solution, usually in 95 per cent, alcohol. Such a solution stains 

 the soft parts as well as the skeleton and must be followed by 

 a decolourising fluid. Decoloration is accomplished, either by 

 using an acid-alcohol, such as a i per cent, solution of sulphuric 

 acid in 95 per cent, alcohol, or by allowing the specimen to stand 

 in strong sunlight until the stain has been removed from the 

 tissues surrounding the bones. 



Decoloration by means of any acid solution is objectionable 

 in a study of ossification on account of the danger of decalcifying 

 minute ossification centres. Decolourisation in strong sunlight, 

 while not open to the objection just raised for the acid method, 

 also has some drawbacks. In continuous cloudy weather, it is 

 obvious, no progress can be made and, in addition to this, many 

 specimens prove very refractory to the sunlight method and the 

 process of decoloration may in some cases take days, weeks or 

 even months. 



Dawson's new method has the advantage of introducing no 

 factors of error due to possible decalcification and takes less 

 time than bleaching in strong sunlight. 



Legs of rats from one to thirty days of age have been prepared 

 by this modified method. The animals are fixed in toto in 95 per 

 cent, alcohol for from forty-eight to seventy-two hours, depending 

 on the age of the animal. Prolonged fixation in the alcohol 

 seems to render the tissue less liable to macerate in the potash 

 solutions used later. 



The tissue is then placed in a 1 per cent, solution of potassium 

 hydroxide. Mall's (1902) modification of Schultze's (1897) method, 

 for from twenty-four to seventy-two hours or until the bones are 

 clearly visible through the muscle. The specimens are then 

 placed in a dilute solution of alizarin and potash, 1 part alizarin 

 to 10,000 parts of 1 per cent, potassium hydroxide. They are 

 allowed to remain in this solution until the bones are stained the 

 desired colour. If the dye is absorbed from the solution before 

 the maximum intensity is obtained the specimen can be trans- 

 ferred to a fresh solution of stain. If clearing in the initial potash 



