a. The Fermentation Test.—To 30 c.c. of the suspected urine a piece of ordinary yeast the size of a pea (or better a small quantity of a pure culture of saccharomyces apiculatus) is added, and the whole well shaken. Then the urine is poured into a so-called "fer mentation tube" and set aside in a warm place (20° to 30° C.-68° to 86° F.). If much sugar be present (0.5 per cent. or more), a large quantity of gas (carbonic acid) will have collected by the end of two hours at the upper extremity of the tube; if but little sugar be pres ent, we shall have to wait from five to eighteen hours. The forma tion of gas is very evident when not more than 0.15 per cent. of sugar is present in the urine.
It is advisable, for comparison, to place another glass containing normal urine and yeast alongside of the first one, for the yeast itself often contains traces of sugar. In that case there will be a small bubble of gas at the top of the tube containing normal urine, but that in the other tube, containing saccharine urine, will be much greater in amount.
The gas set free is carbonic acid, as the yeast fungus splits up the grape sugar into alcohol and CO,. If caustic potash be added to the fermented urine the gas will disappear as the potash seizes upon the carbonic acid.
b. E. Fischer's Phenylkyclicain c.c. of urine and 2 drops of a saturated solution of neutral acetate of lead (sugar of lead) are mixed together and then filtered. The filtrate is acidulated with one drop of glacial acetic acid, and to it are added a piece the size of a pea of phenylhydrazin and one the size of a beau of acetate of sodium. The test-tube is then placed in a water-bath for an hour and heated. At the end of this time, if sugar be present, there will be a yellow precipitate formed in which there can be seen raider the microscope a cluster of beautiful, long, yellow, needle-shaped crystals. These needles consist of phenylglycosazon. This test gives positive results in the presence of only 0.05 per cent. of sugar. It must be remem bered, however, that it is only these yellow, needle-shaped crystals that are indicative of sugar.
c. Raiier's Test.-10 c.c. of urine are mixed with an equal amount of a concentrated solution of neutral acetate of lead and then filtered. To the filtrate ammonia is added drop by drop until a thick cheesy precipitate is formed. It is then cautiously heated up to not more than S0° C. (176° F.). When grape sugar is present, the previously white precipitate is colored a beautiful rose-red. Further heating
changes the color to a coffee-brown. A positive reaction is obtained when sugar is present in the proportion of at least 0.25 per cent.
d. The Cireumpolarization Test.—Solutions of grape sugar have the property of turning the plane of polarized light to the right. In order to examine the urine for this property we may use either the saccharimeter of Soleil-Ventike or the so-called half-shadow appara tus of Laurent, Lippich, and others. Marked dextrorotation of po larized light always points to the presence of grape sugar; a slight dextrorotation may be caused by milk sugar (in lying-in women), by compounds of glycuronic acid often present in the urine, or by many medicinal substances. On the other hand saccharine urine may lose the property of dextrorotation when at the same time levorotatory substances are present in the urine (oxybutyric acid, levulose, and many drugs, such as benzosol).
c. Differentiation of Milk Sugar from Grape Sugar.
Grape sugar. Milk sugar.
Trommer's test Positive. Positive.
Nylander's " Positive. Positive.
Moore's " Positive. Positive.
Fermentation test Positive. Negative.
Phenylbydrazin test Yellow needles, with a melt- Globular collections of yellow ing point of 204° to 205° C. needles, with a melting point of 200° C.
Rubver's test Cherry-red color. Orange-red or brown color.
Quantitative Estimation of Grape Sugar.
a. By Jeans of Polarization. —The different saccharimeters either indicate directly on a scale the percentage of sugar in the urine or mark only the degree of rotation of the polarized light. In the latter case an accompanying table shows to what percentage of sugar the degree of rotation corresponds. A description of the apparatus is given in every text-book on the technique of physiological chemistry.
b. By 1l.1ea»s of Fehlinifs Solution.—Reagents. (1) A solution of 34.639 gm. pure crystallized sulphate of copper in 500 c.c. water; (2) 173 gin. sodium tartrate are dissolved in 350 c.c. pure caustic soda of a specific gravity of 1.14, and water is added to bring the entire vol ume up to 500 c.c. (3) Immediately before being used, a mixture is made of 10 c.c. of each of the two solutions. We then have 20 c.c. of a dark blue fluid which is called "Fehling's solution." Principle : Upon boiling a thin solution of grape sugar with Feh ling's solution, the copper sulphate is reduced to red copper oxide, exactly 0.1 gm. of grape sugar being necessary to reduce completely 20 c.c. of Fehling's solution.