R. M. RAUTER AND L. ZUFA 



DESCRIPTION OF TECHNIQUE 



Using a smooth, even stroke with a sharp scalpel, cut thin longitudi- 

 nal sections through to the xylem in the suspect area of the stem of the 

 tree. If a canker is present, take the sample from living tissue in an 

 area adjacent to the canker, not from the canker itself. 



Place the freshly collected material in small vials containing 50% 

 ethyl alcohol. Staining fresh material gives the best results. When 

 staining, pour the alcohol from the vial and replace it with the modified 

 Gram-Jorgensen stain. The formula for this stain is as follows: 



Fast green 0.5 gm 

 Safranin 1 . 5 gm 

 60% EtOH 200 ml 

 No filtering 



Place the cap on the vial and shake periodically. After 5 minutes, pour 

 off the stain and replace with 100% ethyl alcohol. Use three 30-second 

 changes of absolute alcohol or continue the changes until no red is 

 removed from the sections. Use Euparol to mount the material directly. 

 With Permount take the material through two changes of xylene. 



DISCUSSION 



The method of embedding or freezing parts of the stem of the host 

 tree and sectioning the material on a microtome may be satisfactory for 

 cytological studies when large amounts of material cannot be prepared and 

 analyzed immediately. Embedding tissue and using a microtome requires 

 time. This method is not satisfactory if positive confirmation of 

 infection is needed quickly, or if the host tree is a young seedling 

 which cannot be destroyed. In such cases, it is quicker and more 

 advantageous to collect thin slices and to stain, mount and preserve 

 them directly. Permanent slides can be prepared in about 10 minutes 

 using this technique and the modified Gram-Jorgensen staining procedure. 



Gram-Jorgensen ' s staining procedure was modified as follows: 



1. The hydrochloric acid was omitted from the original stain. The 

 nuclei were more distinct without the acid even though the hyphal walls 

 were harder to define. 



2. Staining was extended from 4 to 5 minutes, as the hyphal walls 

 and particularly the nuclei appeared more visible and clearer. 



The lignified cells of the host and all the nuclei stain red. 

 Unlignified cells of the host and the hyphal cells stain a green or 

 greenish-blue. The mycelium can be easily and readily detected by its 

 bright red nuclei, which appear as dots, in comparison to the larger and 

 less distinct nuclei in the cells of the host (Figs. 1 and 2). 



The blister rust mycelium can be distinguished from the other fungi 

 as it is intercellular and has very wide hyphae . Hirt (1964) states that 

 the vegetative hyphae are 2.8 to 4.2 y in diameter and the reproductive 

 hyphae even wider. 



