116 STAINING OF BACTERIA 



Before use add 5 c.c. of glacial acetic acid. The fluid, without the 

 acetic acid, may be made up in bulk, as it keeps indefinitely, but the 

 acid can only be added shortly before use. Leave the tissues in this 

 fluid for from two to twenty-four hours, according to the size of the 

 piece and the hardness or softness of the tissue. Then wash for 

 twenty-four hours in running water. In spite of this washing an 

 insoluble sulphite of mercury will remain in the tissue which must be 

 removed before staining. How this is accomplished with the aid of 

 Gram's or Lugol's iodin solution is described under the steps of the 

 eosin-methylene-blue staining method following. 



After tissues have been fixed in a watery fluid, or in 95 per cent, 

 alcohol, they must always be completely dehydrated in absolute 

 alcohol or some other suitable medium. 1 Only after this has been 

 accomplished can the tissues be embedded. 



Embedding Methods. There are two principal embedding methods, 

 and the object of both is to get the tissue into such shape that it can 

 be cut into very thin sections with a razor, or, what is much better, 

 a machine with a special knife called a microtome. 



A. CELLOIDIN EMBEDDING METHOD. 1. After fixation, place the 

 tissue into absolute alcohol for twenty-four hours and change the 

 latter once. 



2. Place into equal parts of absolute alcohol and ether for one day. 



3. Place in thin celloidin at least for a day, better for several days. 



4. Place in thick celloidin at least for a day, better for several days. 



5. Paste the piece of tissue with thick celloidin on a block of wood, 

 or, better, on a block of vulcanized wood fiber (a certain wood fiber 

 material impregnated with gutta-percha). 



6. Allow the celloidin on the block and on the tissue to become 

 superficially hard; then place block and all in 80 per cent, alcohol. 

 After twenty-four hours the celloidin has hardened well and the 

 tissue can now be sectioned with the microtome. The thin and 

 thick celloidin used in this work are prepared from the solid imported 

 celloidin, which comes in a dark bottle put up with water. It is 

 prepared for use as follows: 



(a) Drain off the water and remove all traces of it by washing in 

 a little absolute alcohol. 



(6) Place the dry celloidin into a large glass-stoppered bottle, cutting 

 it up with scissors into small fragments if necessary. Add six to 

 eight ounces of equal parts of absolute alcohol and ether and shake 

 violently until all the celloidin is dissolved. This sometimes takes a 

 couple of hours. The thick syrupy fluid which results after complete 

 solution of the celloidin is the thick celloidin. Take some of this and 

 dilute it with ether to a thin consistency. This is the thin celloidin. 

 To get the best results it is necessary to place the tissue finally in 

 some thick celloidin kept in a Stender or Petri dish (see Chapter XII) 



i Complete dehydration means the complete abstraction of water. 



