Chitin in Mollusks — Winkler 
305 
The difference in preparation was a result of 
convenience. Commerically prepared chitin was 
used for controls in all cases. 
The chitosan color test of Campbell, as 
adapted by Richards (1951), was used as fol- 
lows: 
The chitin to be tested was placed in a test 
tube and a saturate solution of KOH was 
added. The tube was closed with a Bunsen 
valve (a piece of rubber tubing sealed at one 
end with a clamp and having a longitudinal slit 
0.5-0 cm. long made on its lateral surface) . The 
test tube was then placed in a glycerine bath 
and heated to 160° C. over a period of 15-20 
min. It was held at this temperature for about 
15 min. and allowed to cool to room tempera- 
ture. The persistent material assumed to be 
chitosan 2 was then rinsed and divided for the 
following tests: 
( 1 ) Lugol’s iodine was added, resulting in a 
brown color when positive. The excess iodine 
was removed and replaced with 1 per cent sul- 
furic acid. When chitin was present it changed 
color to what appeared black. However, upon 
mashing the specimen under a microscope, the 
true color — a reddish purple — was apparent 
(the chitosan color test). Adding 75 per cent 
sulfuric acid resulted in an immediate change 
to brown, followed by an eventual disappear- 
ance of the color as the material passed slowly 
into solution. (2) Three per cent acetic acid 
was added to another portion of the original 
KOH persistent material. When positive the 
tested substance passed slowly into solution. 
Addition of a drop of 1 per cent sulfuric acid 
then gave a white precipitate. 
For the enzymochromatographic determina- 
tion the chitin was mashed in a mortar or minced 
and placed in a miniature test tube. To this 
1 ml. of snail enzyme preparation was added, 
after which it was incubated overnight at room 
temperature. After incubation small samples 
were applied in spots about 1 V 2 in. apart on a 
line V 2 in. from the base of 8 X 8-in. squares 
of Whatman No. 1 chromatographic paper. The 
paper was then formed into a cylinder and 
fastened with staples so that the base line 
described the circumference at one end. This 
2 Few organic substances other than chitin can resist 
the KOH treatment. 
end was then placed in a Petri dish containing 
water-saturated phenol (Mallinckrodt liquid) 
which was not deep enough to reach the sample. 
This in turn was placed in a 10-gal. aquarium 
and covered with a glass plate. When develop- 
ment was completed the paper was dried over- 
night and then washed in ether to remove all 
the phenol possible. After further drying the 
chromatogram was dipped in benzidine-tri- 
chloroacetic acid reagent and heated at 100° C. 
for about 15 min., or until the brown spots 
indicating reducing substances had thoroughly 
formed. The spots disappear after a few days 
so it is necessary to mark the spots and soak 
the papers in water to have a permanent record. 
The snail enzyme reagent was prepared by 
excising the digestive gland of the common 
garden snail, Helix aspersa, and macerating it 
in a mortar with sand and acetate buffer at pH 
5.2. It was then centrifuged and the liquid was 
used in the digestion as described. 
The benzidine-tricholoracetic acid reagent was 
made as follows: 0.5 gm. of benzidine was dis- 
solved in 5 ml. of glacial acetic acid and added 
to 4 gm. of trichloroacetic acid dissolved in 5 
ml. of water. To this was added 90 ml. of ace- 
tone. If refrigerated it keeps about 2 weeks. 
EXPERIMENTAL RESULTS 
Both the nuclear area of the shell and the 
stomach teeth of Aplysia calif ornica were re- 
sistant to the action of the hot alkali and gave 
very strong chitosan color tests. The confirmatory 
tests mentioned were also positive. The organic 
periostracum of Bulla contained small amounts 
of chitin although the major portion was not 
persistent. The stomach teeth of Bulla were 
composed principally of chitin. The decalcified 
organic shell of Oncomelania , however, did not 
withstand the action of the hot alkali. Some 
white powder released was tested but gave no 
color. This latter shell material was completely 
resistant to the snail enzymes. 
Chromatographic spots at Rf .34 for all spec- 
imens and controls involving the snail enzyme 
preparation were the result of free sugars in 
the enzyme extract. The additional spots at an 
Rf value of approximately .69 appeared only in 
the aplysiid material tested and the chitin con- 
