Haines' solution is prepared as follows: completely dissolve 30 gr. pure copper sulphate in ½ oz. distilled water, and add ½ oz. pure glycerin; mix thoroughly, and add 5 oz. liquor potassæ. The solution keeps well.
(2) Fehling's Test.—Two solutions are required—one containing 34.64 gm. pure crystalline copper sulphate in 500 c.c. distilled water; the other, 173 gm. Rochelle salt and 100 gm. potassium hydroxid in 500 c.c. distilled water. Mix equal parts of the two solutions in a test-tube, dilute with 3 or 4 volumes of water, and boil. Add the urine a little at a time, heating, but not boiling, between additions. In the presence of glucose a heavy red or yellow precipitate will appear. The quantity of urine should not exceed that of the reagent.
| FIG. 24.—Crystals of phenylglucosazone from diabetic urine—Kowarsky's test. X500. |
(3) Phenylhydrazin Test.—Kowarsky's Method.—In a test-tube take 5 drops pure phenylhydrazin, 10 drops glacial acetic acid, and 1 c.c. saturated solution of sodium chlorid. A curdy mass results. Add 2 or 3 cc. urine, boil for at least two minutes, and set aside to cool. Examine the sediment with the microscope, using a two-thirds objective. If glucose be present, characteristic crystals of phenylglucosazone will be seen. These are yellow, needle-like crystals arranged mostly in clusters or in sheaves (Fig. 24). When traces only of glucose are present, the crystals may not appear for one-half hour or more. Best crystals are obtained when the fluid is cooled very slowly. It must not be agitated during cooling.
This is an excellent test for clinical work. It requires slightly more time than Haines' test, but more than compensates for this by increased accuracy. It is fully as sensitive as Haines', and has practically no fallacies excepting levulose, which is a fallacy for all tests but the polariscope. Other carbohydrates which are capable of forming crystals with phenylhydrazin are extremely unlikely to do so when the test is applied directly to the urine by the method just detailed. Even if not used routinely, this test should always be resorted to when Haines' test gives a positive reaction in doubtful cases.
Quantitative Estimation.—In quantitative work Fehling's solution, for so many years the standard, has been largely displaced by Purdy's, which avoids the heavy precipitate that so greatly obscures the end-reaction in Fehling's method. The older method is still preferred by many, and both are, therefore, given. Should the urine contain much glucose, it must be diluted before making any quantitative test, allowance being made for the dilution in the subsequent calculation. Albumin, if present, must be removed by acidifying a considerable quantity of urine with acetic acid, boiling, and filtering. The precipitate should then be washed with water and the washings added to the urine to bring it to its original volume.
(1) Purdy's Method.—Take exactly 35 c.c. of Purdy's solution in a flask or beaker, add twice its volume of distilled water, heat to boiling, and, still keeping the solution hot, add the urine very slowly from a buret until the blue color entirely disappears. Read off the amount of urine added; considering the strength of Purdy's solution, it is readily seen that this amount of urine contains 0.02 gm. of glucose, from which the amount in the twenty-four-hour urine, or the percentage, can easily be calculated. Example: Suppose that 2.5 c.c. of urine discharged the blue color of 35 c.c. of Purdy's solution. This amount of urine, therefore, contains exactly 0.02 gm. glucose, and the percentage is obtained from the equation: 2.5:100 :: 0.02:x, and x equals 0.8 per cent. If, then, the twenty-four-hour quantity of urine were 3000 c.c., the twenty-four-hour elimination of glucose would be found as follows: 100:3000 :: 0.8:x, and x equals 24 gm.
It will be found that after the test is completed the blue color slowly returns. This is due to reoxidation, and should not be mistaken for incomplete reduction.
A somewhat simpler application of this method, which is accurate enough for clinical purposes, is as follows: Take 8¾ c.c. (roughly, 9 c.c.) of Purdy's solution in a large test-tube, dilute with an equal volume of water, heat to boiling, and, while keeping the solution hot but not boiling, add the urine drop by drop from a medicine-dropper until the blue color is entirely gone. Toward the end add the drops very slowly, not more than 4 or 5 a minute. Divide 10 by the number of drops required to discharge the blue color; the quotient will be the percentage of glucose. If 20 drops were required, the percentage would be 10÷20 = 0.5 per cent. It is imperative that the drops be of such size that 20 of them will make 1 c.c. Test the dropper with urine, not water. If the drops are too large, draw out the tip of the dropper; if too small, file off the tip.
Purdy's solution consists of pure crystalline copper sulphate, 4.752 gm.; potassium hydroxid, 23.5 gm.; ammonia (U.S.P.; sp. gr., 0.9), 350 c.c.; glycerin, 38 c.c.; distilled water, to make 1000 c.c. Dissolve the copper sulphate and glycerin in 200 c.c. of the water by aid of gentle heat. In another 200 c.c. of water dissolve the potassium hydroxid. Mix the two solutions, and when cool, add the ammonia. Lastly, bring the whole up to 1000 c.c. with distilled water. This solution is of such strength that the copper in 35 c.c. will be reduced by exactly 0.02 gm. of glucose.