MICROSCOPY 71 



Fixing Agents. — It is at times desirable to kill and fix material to prepare 

 the fungus for staining and clearing. A number of fixatives are used, but 

 most authors recommend either Plemming's weak killing agent which con- 

 sists of two solutions which are mixed when ready to fix the material (A. 1% 

 chromic acid 25 c.c, 1% acetic acid 10 c.c, water 55 c.c. ; B. 1% osmic acid 

 10 c.c.) or Flemming's strong agent (A. 1% chromic acid 45 c.c, glacial acetic 

 3 C.C; B. 2% osmic acid 12 c.c). Ninety-five per cent alcohol is used, but it 

 is not so suitable for fine work since the material is frequently plasmolyzed. 

 There are various chromo-acetic acid formulae used, but the reader is referred 

 to the standard books on methods in histology for these. One of these for- 

 mulae, Benda's fluid, has been used favorably with yeastlike fungi. It is a 

 modification of Flemming's strong agent and works well for chromatin inves- 

 tigations. One per cent chromic acid 16 c.c, 2% osmic acid 4 c.c, and glacial 

 acetic acid 2 drops. One of the best, yet most expensive fixing agents I have 

 found for celloidin sections of pathogenic fungi is Hermann's fluid : 1% platinic 

 chloride 15 parts, glacial acetic acid 1 part, 2% osmic acid 2 parts. Fix for 

 6-12 hours; wash overnight. MerkeFs fluid gives good results in organs filled 

 with reserves of food materials. 



ParaJSn Method. — There are two general methods for embedding material 

 for cutting sections : the paraffin and the celloidin technic Recently, modifi- 

 cations have been reported, but they have not been sufficiently tested to report 

 here. The paraffin method is familiar to practically all technicians and is 

 in general use, although not very satisfactory for agar cultures. The diffi- 

 culty seems to lie in the fact that it may cause shrinkage of the material, and 

 it does not penetrate the agar sufficiently for good embedding. Masses of 

 mycelium scraped from the surface of the agar and embedded in paraffin may 

 give favorable results, but it is often desirable to see the characteristics de- 

 veloped in the substrate. 



Nitrocellulose Method. — The outstanding method of embedding agar cul- 

 tures is the second procedure. Although its particular disadvantage lies in 

 the fact that it is difficult to obtain very thin sections as with paraffin and 

 also more difficult to make serial sections, although possible, its advantages 

 surpass those of paraffin. Material if fixed properly will show no shrinkage. 

 The agar substrate is penetrated much better, so that celloidin blocks can be 

 made easily and sections, even if slightly thicker than paraffin sections, clear 

 sufficiently to permit study of the internal structure which is usually broken 

 up or distorted by paraffin. 



There are various products of nitrocellulose on the market, e.g., celloidin 

 and collodion. They are sold as shredded or granulated products and are in- 

 flammable, but not explosive. Schering's celloidin is in general use. Du- 

 pont's parlodion or the product of Mallinckrodt is a purified pyroxylin which 

 gives very good results in embedding and can be obtained as small strips sold 

 in 1-ounce jars. Celloidin may also be obtained in tablet form with directions 

 for making the dilutions accompanying the tablets. 



