72 MEDICAL MYCOLOGY 



Because of the particular advantages celloiclin has over paraffin in mak- 

 ing sections of agar cultures for cji:ologic purposes, I shall list steps that I 

 follow in my work. The general procedure is that improved by Jeffrey (1928) 

 and recently reviewed by Wetmore (1932), but with some slight changes as 

 are better adapted to this type of growth. When the organism is sufficiently 

 developed on a suitable medium, the fixing agent, Hermann's fluid, is poured 

 slowly down the side of the tube. After fixation for from 6-12 hours, depend- 

 ing on the type of organism and growth (a surface growth requiring less time 

 than a deep growth), the fixed culture is washed overnight in slowly running 

 tap water. Care should be taken not to let the water run strongly or the sur- 

 face mycelium and yeast cells will be washed away. The agar slant is now 

 taken from the test tube, which is broken, and cut up into pieces or blocks 

 approximately 1 sq. cm. or if large tubes are used, approximately 1 cm. by 

 the diameter of the tube. These blocks are next dehydrated in the following 

 alcohols, 2 hours in each: 15%, 25%, 35%, 50%, 70%, 85%, 95%, 100%. 



Celloidin may be used warm or cold. For best results it should be used 

 warm. An oven is kept regulated at 45° C. The material is next transferred 

 to a bottle with a collar, containing a solution of ether and absolute alcohol in 

 equal proportions. The bottle is corked and secured by passing a wire around 

 the collar and over the cork as shown by Wetmore. This is placed in the 

 oven and allowed to lie on its side for 24 hours. The preparation is now ready 

 to be run up in celloidin, which has been washed, thoroughly dried, and made 

 up in 2, 4, 6, 8, and 10 per cent solutions in ether-alcohol. Higher percentages 

 may be made, but are not necessary. The material is transferred to each suc- 

 cessive dilution every 24 hours, tightly corked, and placed in the 45° C. oven. 

 After the 10% solution, blocks are poured as with paraffin, care being taken not 

 to form bubbles. The blocks are arranged carefully and then set in chloroform to 

 harden for about 12 hours or overnight. When found to be sufficiently hard, 

 the material is placed in 70% alcohol for a few hours to allow for some 

 softening and then it may be stored in glycerol alcohol (95% alcohol in- 

 definitely). 



The blocks may be cut with a razor blade to get rid of excess celloidin 

 and to make a uniform block. In order to make sections, the preparations 

 are mounted on small wooden blocks as described by Wetmore. Sections are 

 then cut, as thin as possible, placed in 95% alcohol and then run down to dis- 

 tilled water: 95%, 85%, 70% alcohol, distilled water, 5 minutes in each 

 change, or else run down to the percentage alcohol of the stain used. The 

 sections are now ready to be stained. There are various stains used, but I 

 have found it best to use a 4% mordanting solution of iron-alum (ferric am- 

 monium sulphate) for 10 minutes, washing 4 times with water so that the 

 excess of mordant may be removed. Then 2 drops of Haidenhain's or Ehr- 

 lich's hematoxylin are added to a watch glass of sections in water and allowed 

 to stand overnight. The sections are then examined to see whether the mate- 

 rial is sufficiently stained. If heavily overstained, they can be decolored with 

 dilute iron alum. They should be slightly overstained, however, because the 



