FROZEN SECTIONS 



102 



FUNGI 



indicated. They are specified elsewhere 

 in this book under several headings 

 including : 



To make the sections take recently 

 excised still living tissue, or better fresh 

 tissue fixed for about 30 min. in 10% 

 formalin. First freeze a little water on 

 the block of a freezing microtome. 

 Then add the tissue and freeze it too 

 plus a drop more of water. Allow block 

 to thaw to optimum consistency, cut 

 sections 15-50m thick as desired, and 

 remove them with a camel's hair brush 

 from the microtome knife to formalin, 

 water or physiological saline. When 

 many are required, it may be necessary 

 to freeze several times as the tissue be- 

 comes too soft. If thinner sections are 

 wanted resort to Gelatin Imbedding 

 before sectioning. 



For quick staining Thibaudeau, A. A., 

 J. Lab. & Clin. Med., 1933, 19, 204-209 

 advises that sections of formalin fixed 

 tissue be stained in Harris' hematoxylin 

 5-15 sec, rinsed in aq. dest., blued in 

 aq. dest. + few drops NH4OH, passed 

 up through 70, 85 and 95% alcohol 

 counterstained in absolute alcohol and 

 eosin (5 sec), cleared in carbol xylol, 

 blotted with filter paper and mounted 

 in balsam. Proescher, F., Proc Soc. 

 Exp. Biol, and Med., 1933, 31, 79-81 

 recommends pinacyanol as giving ex- 

 cellent color contrasts. Perliaps the 

 simplest method advised by the Bens- 

 leys (p. 138) is to stain the sections in 

 Goodpasture's Acid Polychrome Meth- 

 ylene Blue (which see) 1 min. or longer, 

 wash and mount in aq. dest. This 

 colors nuclei dark purple and connective 

 tissue bright rose red. But methylene 

 blue is less permanent than hema- 

 toxylin. 



For reticular and collagenic fibers in 

 frozen sections proceed as follows 

 (Krajian, A. A., Arch. Path., 1933, 16, 

 376-378): After_ fixation in 10% for- 

 malin, cut sections 5-10 microns and 

 wash in aq. dest. Then 10% aq. 

 NH4OH at eO^C, 15 min. Wash in 3 

 changes aq. dest. and place in 0.3% 

 KMn04 for 5 min. Rinse in aq. dest., 

 decolorize in 1.5% oxalic acid until 

 brown color has entirely disappeared. 

 Wash 4-5 times in aq. dest. and soak 

 in 5% AgNOa at 60 °C. for 1 hr. Wash 



twice in aq. dest. Transfer to ammoni- 

 acal silver sol. (to make add 6 drops 10% 

 NaOH to 8 cc 10% AgNOa. Then add 

 freshly prepared 10% NH4OH drop by 

 drop until almost entirely clear. Dilute 

 to 28 cc. withaq. dest.) 16 min. at 60°C. 

 Wash 3 times quickly in aq. dest. 

 Change to 30 cc. formaldehyde + 70 cc. 

 aq. dest. 1-3 min. at 60 °C. Wash in 

 tap water. Mount on slide. Dehydrate 

 with a little absolute alcohol and blot 

 into position. Dehydrate more, blot, 

 3 changes equal parts anilin oil and xylol, 

 xylol, balsam. Reticular fibers jet 

 black, collagenic ones dark brown. 



For serial sections of brain (Marshall, 

 W. H., Stain Tech., 1940, 15, 133-138) 

 fix slices 24 hours or longer in 10 or 

 15% formalin and then treat them with 

 a 20-30% alcohol or in 15% formalin in 

 20% alcohol. The object of the alcohol 

 treatment is to avoid formation of hard 

 and brittle ice crystals which fracture 

 the sections as they are made. Cut 

 tissue into blocks about 1.0 cm. thick. 

 Place on a CO2 ice freezing disc which 

 has been covered by a piece of wet blot- 

 ting paper. (In our laboratory we use a 

 regular CO2 gas freezing disc which has 

 been adapted to a precision sliding 

 microtome.) Freeze the block of tissue 

 slowly throughout. The proper degree 

 of freezing depends on the thickness of 

 the sections to be cut. Marshall recom- 

 mends a paraffin knife, 20-30° angle with 

 block, knife set in a line perpendicular 

 to the direction of motion. Remove cut 

 sections by a camel's hair brush to 50% 

 alcohol and keep them in serial order. 

 Mount sections serially on slides coated 

 with Albumen-Glycerin. Smooth out 

 wrinkles and flatten sections by gentle 

 pressure with blotting paper moistened 

 with 50% alcohol. Remove slides to a 

 38 °C. oven for 4-6 hrs. when they are 

 ready for staining. (In some cases it 

 may be inadvisable to press the sections 

 flat upon the slide. Thin sections re- 

 quire less drying than thick ones. In 

 any case until one has gained confidence 

 in the use of the technique, the sections 

 should be observed at intervals in the 

 38°C. oven. At the least sign of exces- 

 sive drying (whitening of parts of the 

 section) the sections should at once be 

 removed to the stain.) The Cresyl 

 Violet method of Tress and Tress is 

 recommended. 



Fuchsin NB, see New Fuchsin. 



Fuchsin S, SN, SS, ST or S III, see Acid 

 Fuchsin. 



Fungi. Data contributed by Dr. Morris 



Moore of The Barnard Free Skin and 



Cancer Hospital. Revised bv him July 



17, 1946. 



1. Skin scrapings and hair. The 



