222 
Vol. XXVI, No. 5 
Journal of Agricultural Research 
“BERLIN BLUE” REACTION 
The reaction described is cited under tests for the localization of 
proteins by Dr. Sophia H. Eckerson 4 in “Notes on Microchemistry.” 
It was applied with varying success. One excellent result is shown in 
Plate 2, A. In this instance the hyphae of the molds assumed a bright, 
clear, blue color which caused them to stand out in striking contrast to 
the entirely uncolored background of the wood cells. The method used 
consisted in placing the sections in a dilute solution of potassium ferro- 
cyanide (i part potassium ferrocyanide to 2a parts water and 10 acetic 
acid, sp. gr. 1.063). After about an hour the sections were carefully 
washed with 60 per cent alcohol and a few drops of dilute ferric chlorid 
were added. The hyphae immediately turned a clear transparent blue. 
silver solutions 
A saturated solution of silver nitrate in distilled water was prepared 
as a stock solution and used in varying dilutions. This solution was 
effective in practically all cases. The mycelium in sections soaked in 
silver nitrate for periods varying from one to two hours to as many days 
assumed an orange, dark brown, or, in one case, violet brown color in 
contrast to the constantly lighter color of the wood tissue. 
Plate 2, D, shows what a striking differentiation may be obtained. In 
this specimen the organism was not a mold but a wood-destroying fun¬ 
gus. Plate 3, A, shows the mycelium of the mold Monilia sitophila in a 
piece of white oak. Here one of the worst difficulties encountered with 
this stain is apparent, namely, the precipitate which, although it does 
not interfere notably with the detection of the fungus, makes a dirty- 
looking preparation. No satisfactory means of removing the precipitate 
was devised. It occurred even with extremely dilute solutions. 
Whenever the mycelium was well stained, a precipitate might be 
found, although it was not necessarily present, as indicated by 
Plate 2, D. Dissolving the precipitate invariably also bleached the 
mycelium. Slight assistance was obtained by washing with ammonia, 
sometimes followed by very dilute acetic acid. Sets of sections were 
also suspended in the silver nitrate solution vertically on platinum hooks 
and kept in the dark and in the light, respectively. Although this 
tended to eliminate the precipitate, the resulting differentiation was not 
so marked as when the sections were laid flat in an ordinary staining 
dish. Treating the sections with glycerin tended to improve the quality 
of the differentiation secured. Long soaking (over-night) in dilute stain 
gave, on the average, good results. Permanent mounts of this material 
were made by passing the sections through the usual dehydrating alco¬ 
hols, clearing in xylol, and mounting in Canada balsam! Crystals 
present in the wood often appear dark with this treatment. 
Silver lactate was suggested for use instead of silver nitrate, and some 
was obtained through the Courtesy of Dr. Alfred Koehler, of the Univer¬ 
sity of Wisconsin. It was not found to be as effective as the nitrate, 
however. The precipitate was just as abundant and the mycelium was 
less well stained. It was particularly noticeable with this solution that 
in spruce the middle lamella and the “bars of Sanio” stained a marked 
orange, similar to the color acquired by the mold hyphae. 
1 Eckerson, Sophia H. notes on microchemistry. (Unpublished.) 
