SPECIFIC EFFECTS OF CERTAIN TISSUE EXTRACTS ON T H E CRYSTALLIZATION PATTERN OF CUPRIC CHLORIDE DANIEL LUZON MORRIS AND CAROL TILDEN MORRIS Putney School, Putney, Vermont Received November 18, 1038
Numerous investigators have noticed that the pattern in which salts crystallize from solution is influenced by the presence of impurities. Ord (13) noted this effect particularly in connection with the growth of concretions in the body. Marriage (11) showed that adulterants in commercial jams and jellies could be detected by the size and arrangement of lead iodide crystals grown in them. Alexander ( 5 ) speaks of the effect of traces of foreign matter on the tensile strength of metals, and repeatedly mentions (1,2,3,4,6,7) the effects of colloids on crystallization. du Noiiy (9) found that a trace of adsorbable matter influenced the crystallization of sodium chloride, and that normal serum gave different results from immune sera (10). Michaud (12) has described some amusing results in the production of crystalline arborescences with urea in the presence of various colloids. Except for the work of Marriage, Michaud, and possibly that of du Noiiy, there is little in these references to indicate that a given colloid may produce a characteristic effect. PfeifIer (14, 15, 16) found that cupric chloride crystals formed characteristic patterns if they crystallized from solutions that contained extracts of living tissues. This work has been criticized because it starts with a full-fledged hypothesis which is by no means confirmed by his experimental work. Trumpp and Rascher (17) have applied Pfeiffer’s methods in an attempt to determine the presence of specific hormones in urine as a possible method for the diagnosis of pregnancy. Their success was about the same with this as with other methods based on the detection of hormones. The present paper is an attempt to clarify some of Pfeiffer’s work and to extend it. The methods are, in general, modifications of Pfeiffer’s. METHODS
The crystallization dishes are circular glass plates, each provided with a rim of Pyralin 10 cm. in diameter. The rim is applied by wrapping a strip of Pyralin 123 in. x Q in. x 0.015 in. around a petri dish, gluing it to the carefully cleaned glass plate with a 10 per cent solution of Pyralin in acetone, and removing the petri dish. 623
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DANIEL LUZON MORRIS AND CAROL TILDEN MORRIS
A stock solution of cupric chloride is prepared by dissolving 250 g. of the salt in water and diluting to 1 liter. This is boiled with activated charcoal and filtered; if a faint turbidity due to cupric hydroxide appears, a very little hydrochloric acid is added. For crystallization 2 cc. of the stock solution is placed in a teat tube, the solution to be tested is added, and the volume is brought to 8 cc. with distilled water. The solution is mixed thoroughly by pouring it back and forth from one test tube to another; it is then poured into one of the crystallizing dishes on a level table and allowed to evaporate spontaneously. It is important that the temperature be constant (27-30°C.) during evaporation, that the air be entirely free from draughts, and that the relative humidity be below 60 per cent. These conditions have been obtained by the use of an entirely enclosed and insulated cabinet 4 ft. x 4 ft. x 5 ft. or larger, with the temperature thermostatically controlled and with trays of crude calcium chloride present to absorb water. Satisfactory results have never been achieved in a space smaller than that mentioned, or in a room with windows in it. Evaporation may take from 8 to 14 hr. Under these conditions, when the cupric chloride solution alone is used a “blank” plate results, viz., irregular heaps of crystals scattered over the plate, often leaving patches of clear glass (see figure 10). RESULTS
The solutions tested may be grouped into three classifications: (a)Those which have no influence, or practically none, on the crystallization of the cupric chloride. These include inorganic salts, simple sugars, glycine, and other substances of low molecular weight. (b) Those which produce a pattern characteristic of the substance used. Outstanding members of this class so far studied have been solutions of starch and glycogen, and extracts of the cereal grains. (c) Those which give a pattern with cupric chloride, but one which is non-specific. I n this class have been most of the proteins studied, and some seed extracts; also cereal extracts that have autolyzed. Most of the work so far has been done on the cereal grains. The extractg are made by grinding the grain either in a meat chopper or a mortar, then extracting with cold water, and filtering. The amount of water for extraction and the activity of the extract vary with the kind and grade of grain. PfeiEer allows the grains to germinate for a few days before extraction. This does not affect the results appreciably. Figure 1 shows the results with 0.2 cc. of a n extract from oats, which was prepared with 5 parts of water and filtered or centrifuged. Figure 3 shows the results with 0.2 cc. of an extract from wheat, prepared with
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DANlEL LUZON MORHlS AND CAHOl, TILDEN MORKIS
Figlire 7 shows the results when a soiution containing 40 mg. of glycogen Figure 8 shows the results when a solution containing a solu-
was used.
PES. F A Fit;. 5. Effect of extrsct frum maize FIG. 6 . Effect of Oxtract from m s i x ( X 28) Prc.7. Etfeet of glycogen FIG.8. Etfeet of atareh Fro. 9. Etfect of g l u ~ o ~ e
tion of wheat starch was used. There ii no apparelit difference in the patterns produced hy starches from the different grains. Figure 9 shows the results with solutions containing 10 mg. and 1 mg. of glucose.
CllYSTAliLlZATlON OF CUPRIC CHLOKSDE:
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Figure 10 sliows typical blanks. Figure 11 shows the effect upon aii extract fmni niaize of tlic rooiri in which the solutions evaporated. Figure 1 l a was obtained by evaporating tire solution in an entirely cneiosed room (a respiration chamber), arid IIh wafi ohta.inrd a,t,t,he same time with the same solution iii an opm room. Figure 12 shows a typical non-specific pattern, ohtainctf with hemolyzed blood. TI~.TSON O F AN
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PI~ACTION
l'h wod ";tciive" nil1 bc uscd subscqueritly to dr.;nrilw the ahility of
