3. In the third series (Table 3), where the inoculation period, and the size of the tumor, were noted, the average result was similar to that in the previous experiment (1); but we see that the size of the tumor had no effect, on the numerical values.

4. In the fourth series of experiments serum was always taken from pairs of animals, to insure reliable results. (Table 4.) We see, from the foregoing data, that nitrogen was very much higher, and phosphorus, sulfur, chlorin and molecular depression slightly higher, in normal serum. In the Rous sera, amino-nitrogen and sugar were slightly higher. Two tumor sera (Nos. 14 and 18) gave contradictory values. This may be due to the resistance of the body to tumor growth. In these two animals the tumor was older than in others but attained only a very small size, with a capsule separating it entirely from the rest of the muscle tissue.

TABLE 5

Data pertaining to serum analyzed directly. (Serum of rats with Jensen's sarcoma: values per 100 gm. of serum.)

Serum

N

P

S Sugar A

Remarks

Sarcoma..... 1.050 0.075 0.356 0.070 0.61 Tumors 17 days old, hemolytic sera. Normal.. 0.955 0.078 0.072 0.051 0.63

Hemolytic.

5. In the fifth experiment six rats with small tumors were bled by cutting the throats; the blood volumes were combined. Five normal rats were treated in the same way. (Table 5.)

We see, also, chemical changes in the same direction for rats. This result will be checked by further study.

CONVENIENT METHODS FOR DEMONSTRATING THE BIOCHEMICAL ACTIVITY OF MICRO

ORGANISMS, WITH SPECIAL REFER

ENCE TO THE PRODUCTION

AND ACTIVITY OF
ENZYMES

C. H. CRABILL AND H. S. REED1

(WITH PLATE I)

INTRODUCTION. A large amount of work has been done on the biochemistry of microorganisms and many reliable tests for demonstrating these activities have been evolved. Some of the tests are not well adapted for ocular demonstration to classes, however, because of their transitoriness or for other reasons. We have thought it worth while to describe in the following paragraphs a few methods, for making semi-permanent demonstrations of these activities, which seem adapted to the needs of class room or laboratory instruction. Some of the methods given here are adapted from those of other investigators; other methods are original with the present writers.

The methods are designed to show the presence and action of products of cellular activity upon appropriate substances incorporated in thin layers of agar in Petri dishes. Agar seems to serve very well for this purpose, since it is not difficult to prepare a clear solution, and most solutes diffuse readily through the gel which it forms. When solid zymolytes are used they are suspended in the agar. In order to procure uniform distribution of these solids through the medium, the plates are poured directly from the flask in which the medium is cooked and sterilized. By frequent shaking between pourings, settling of the solids is prevented. With this procedure, tubing of the medium is entirely unnecessary, if not detrimental to the best results. Stain reduction cannot be well

shown by this Petri dish method, because the process of chemical reduction is usually counteracted by rapid oxidation of the products in contact with atmospheric oxygen.

For a number of the experiments a stock agar was prepared according to the following formula, filtered, and sterilized in the autoclave: Distilled water, 1000 c.c.; magnesium sulfate, 0.5 gm.; di-potassium hydrogen phosphate, 1.0 gm.; potassium chlorid, 0.5 gm.; ferrous sulfate, 0.01 gm.; agar, 20.0 gm. This stock medium is slightly acid in reaction. It presents no carbon-containing nutrient and consequently does not support microorganic growth. To this various zymolytes in the form of carbon containing compounds are added and inoculated with the organisms whose activities are to be tested.

AMYLASE. The action of this enzyme may be conveniently demonstrated by cultivating organisms on starch-agar, made by adding to 500 c.c. of the melted stock agar, 10 gm. of corn starch

suspended in a little cold water. The medium is then sterilized in the autoclave and poured with frequent shaking directly from the flask into sterile Petri dishes. This gives a clear white substratum in which the starch is suspended. As soon as the agar has solidified, the centers of the dishes are inoculated with the organisms to be tested. The dishes are inverted and incubated for two to five days under bell jars to prevent loss of moisture. Typical results are shown in Table I and Plate 1, Fig. 1.

Some of the organisms produce extracellular amylase which diffuses from the colony, dissolves the starch suspended in the agar, and renders the space about the colony clear. Such an appearance is designated as a "halo." The presence of a halo, then, around the edge of a bacterial or fungous colony on starch-agar indicates the production, by that organism, of a readily diffusible extracellular amylase.

Some organisms, especially fungi, do not produce a halo on starch-agar, but grow well and dissolve the starch from the agar immediately in contact with them. In such a case the secretion of extracellular amylase is apparently weak or the amylase is not so diffusible as that produced by other organisms.

Among the organisms so far tested, Streptothrix, Oospora scabies and Aspergillus niger were found to be the most active producers of extracellular amylase. The first of these is able to produce abundant amylase and to dissolve large quantities of starch even in the presence of an abundance of sugar.

INULASE. Inulin is hydrolyzed by the enzyme inulase to reducing sugars, and as such may be utilized by microorganisms.

Inulin-agar. To 1000 c.c. of the stock agar, 0.5 percent of pure inulin was added, and Petri dish cultures made as in the case of the starch-agar. Inulin is soluble in hot water and consequently dissolves during sterilization. Evidence of the ability of the organisms to dissolve inulin in this medium is afforded only by the amount of growth and spore production.

Only a few fungi have been tested, but some of them grew well upon this medium. See Table 2.

EMULSINS: glucoside-splitting enzymes. The glucosides are complex organic compounds capable of hydrolysis. Various end products, one of which is always a sugar, are the result. Since the glucosides are readily soluble, the agar containing them is transparent and the action of enzymes can be determined only indirectly. If the organisms can use the glucosides as sources of carbon, it is assumed that cleavage of the glucosides occurs in such consumption. The glucosides in a pure state are added to the stock agar to the amount of I percent, and plates poured and inoculated.

Esculin-agar. A bluish color pervades the agar made with esculin. In the event of the successful growth of the organism this blue color is reduced, sometimes throughout the plate. The successful growth of the organism is, however, a better indication of its ability to produce emulsin than is the color reduction.

Arbutin-agar. Arbutin, by hydrolytic cleavage, yields sugar and hydroquinone, which gives a brown color. The successful growth of the organism, and production of a brown stain on agar containing this chemical as the only source of carbon, are regarded as evidences of its ability to produce emulsin.

Amygdalin-agar. Success or failure to grow on this medium is our only indication of the ability or inability of an organism to produce emulsin. Typical data are given in Table 3.

LIPASE. Demonstration of the presence of lipase depends upon its power to split fats into glycerol and free fatty acids. The presence of the acids may then be shown by a convenient indicator.

Litmus-cream-agar. Fifty c.c. of 48 percent separator cream are diluted to 600 c.c. with distilled water and fractionally sterilized in an Arnold sterilizer. Twenty gm. of agar-agar are melted in 400 c.c. of water; the liquid is filtered, sterilized and added to the diluted cream while hot; and enough sterile litmus solution is poured into the fluid to impart a deep blue color. Plates are poured, and