94 MICROSCOPIC METHODS 



merits in which the freezing is accomplished by compressed 

 carbonic acid gas), but far finer results can be obtained by 

 embedding the tissue in solid paraffin and cutting with some of 

 the more delicate microtomes of which, for pathological purposes, 

 the small Cambridge rocker is by far the best. For bacterio- 

 logical purposes embedding in celloidin is not advisable, as the 

 celloidin takes on the aniline dyes which are used for staining 

 bacteria, and is apt thus to spoil the preparation, and besides, 

 thinner sections can be obtained by the paraffin method. 



The Fixation and Hardening of Tissues. — The following are 

 amongst the best methods for bacteriological purposes : — 



(a) Absolute alcohol may be used for the double purpose of fixing and 

 hardening. If the piece of tissue is not more than J inch in thickness, it 

 is sufficient to keep it in this reagent for a few hours. If the pieces 

 are thicker a longer exposure is necessary, and in such cases it is better 

 to change the alcohol at the end of the first twenty-four hours. The 

 tissue must be tough without being hard, and the necessary consistence, 

 as estimated by feeling with the fingers, can only be judged of after 

 some experience. If the tissues are not to be cut at once, they may be 

 preserved in 50 per cent, spirit. 



(b) Formalin solution. — This may be used as a 10 per cent, solution of 

 commercial formol-aldehyde in water. Small pieces of tissue are fixed 

 in this in twenty-four hours ; they are then placed in 50 per cent, spirit' 

 for a similar period, and then in pure spirit. 



(c) Formol-alcohol — formalin 1, absolute alcohol 9. Fix for not more 

 than twenty-four hours ; then place in absolute alcohol if the tissue is 

 to be embedded at once, in 50 per cent, spirit if it is to be kept for some 

 time. For small pieces of tissue fixation for twelve hours or even less is 

 sufficient. The method is a rapid and very satisfactory one. 



(d) Corrosive sublimate is an excellent fixing agent. It is best used 

 as a saturated solution in 75 per cent, sodium chloride solution. Dis- 

 solve the sublimate in the salt solution by heat ; the separation of 

 crystals on cooling shows that the solution is saturated. For small 

 pieces of tissue § inch in thickness, twelve hours' immersion is sufficient. 

 If the pieces are larger, twenty-four hours is necessary. They should 

 then be tied up in a piece of gauze, and placed in a stream of running 

 water for from twelve to twenty-four hours, according to the size of the 

 pieces, to wash out the excess of sublimate. They are then placed for 

 twenty-four hours in each of the following strengths of methylated 

 spirit (free from naphtha 1 ) : 30 per cent., 60 per cent., and 90 per cent. 

 Finally they are placed in absolute alcohol for twenty-four hours, and 

 are then ready to be prepared for cutting. 



If the tissue is very small, as in the case of minute pieces removed 

 for diagnosis, the stages may be all compressed into twenty-four hours. 



1 In Britain ordinary commercial methylated spirit has mineral naphtha 

 added to it to discourage its being used as a beverage. The naphtha being 

 insoluble in water a milky fluid results from the dilution of the spirit. By 

 law, chemists can only sell 8 ounces of pure spirit at a time. Most pathological 

 laboratories are, however, permitted by the Excise to buy "industrial spirit," 

 which contains only one-nineteenth of wood naphtha. 



