SECTION THREE 



3. Rinse in two changes of 70% alcohol for thirty seconds in 

 each. 



4. Repeat with two changes of 90% alcohol for the same time. 



5. Immerse in two changes of absolute alcohol for two or three 

 minutes in each. 



6. Stain in light green - clove oil for one minute. 



7. Rinse and wash in clove oil for about five minutes. 



8. Examine under the microscope and if it is found that the 

 safranin has been extracted by the alcohols, take the section down 

 through the alcohols and restain with safranin. 



9. Mount in balsam or in Cristalite mountant. 



Results: 



Cellulose tissues and cytoplasm are stained green. Lignified 

 tissues and nuclei, red. Chloroplasts, pink. 



SAFRANIN - PICRO ANILINE BLUE 



A rapid and simple method of demonstrating hyphae in 



wood sections 



Solutions required: 



A. Safranin 1% in distilled water. 



B. Aniline blue, water soluble . . 2-5 gm. 

 Distilled water . . . . . . 25 ml. 



Picric acid, saturated, aqueous . . 100 ml. 



Technique: 



1. Stain one to three minutes in Solution A ; then wash in water. 



2. Flood sections with Solution B and heat over a flame until it 

 begins to simmer ; then pour oif excess stain ; allow the slide to 

 cool before washing with water. 



3. Wash with 70%, followed by absolute, alcohol ; clear in clove 

 oil ; mount in balsam. 



Results: 



Lignified walls, red. Mycelia, clear blue. Areas where the wood 

 is badly decayed may appear bluish, but the hyphae are always well 

 defined. 



u 279 



