Pettenkofer's Test.-This is undoubtedly the best test yet proposed for the detection of the biliary substances. It consists in mixing with a watery solution of the bile, or of the biliary substances, a little cane sugar, and then adding sulphuric acid to the mixture until a red, lake or purple color is produced. A solution may be made of cane sugar, in the proportion of one part of sugar to four parts of water, and kept for use. One drop of this solution is mixed with the suspected fluid, and the sulphuric acid then immediately added. On first dropping in the sulphuric acid, a whitish precipitate falls, which is abundant in the case of ox-bile, less so in that of the dog. This precipitate redissolves in a slight excess of sulphuric acid, which should then continue to be added until the mixture assumes a somewhat syrupy consistency and an opalescent look, owing to the development of minute bubbles of air. A red color then begins to show itself at the bottom of the test-tube, and afterward spreads through the mixture, until the whole fluid is of a clear, bright, cherry red. This color gradually changes to a lake, and finally to a deep, rich, opaque purple. If three or four volumes of water be then added to the mixture, a copious precipitate falls down, and the color is destroyed.

Various circumstances modify, to some extent, the rapidity and distinctness with which the above changes are produced. If the biliary substances be present in large quantity, and nearly pure, the red color shows itself at once, after adding an equal volume of sulphuric acid, and almost immediately passed into a strong purple. If they be scanty, on the other hand, the red color may not show itself for seven or eight minutes, nor the purple under twenty or twenty-five minutes. If foreign matters, again, not of a biliary nature, be also present, they are apt to be acted on by the sulphuric acid, and, by becoming discolored, interfere with the clearness and brilliancy of the tinges produced. On this account it is indispensable, in delicate examinations, to evaporate the suspected fluid to dryness, extract the dry residue with absolute alcohol, precipitate the alcoholic solution with ether, and dissolve the ether precipitate in water before applying the test. In this manner, all foreign substances which might do harm will be eliminated, and the test will succeed without difficulty.

It must not be forgotten, furthermore, that the sugar itself is liable to be acted on and discolored by sulphuric acid when added in excess, and may therefore by itself, give rise to confusion. A little care and practice, however, will enable the experimenter to avoid

all chance of deception from this source. When sulphuric acid is mixed with a watery solution containing cane sugar, after it has been added in considerable excess, a yellowish color begins to show itself, owing to the commencing decomposition of the sugar. This color gradually deepens until it has become a dark, dingy, muddy brown; but there is never at any time any clear red or purple color unless biliary matters be present. If the bile be present in but small quantity, the colors produced by it may be modified and obscured by the dingy yellow and brown of the sugar; but even this difficulty may be avoided by paying attention to the following precautions. In the first place, only very little sugar should be added to the suspected fluid. In the second place, the sulphuric acid should be added very gradually, and the mixture closely watched to detect the first changes of color. If bile be present, the red color peculiar to it is always produced before the yellowish tinge which indicates the decomposition of the sugar. When the biliary matters, therefore, are present in small quantity, the addition of sulphuric acid should be stopped at that point, and the colors, though faint, will then remain clear, and give unmistakable evidence of the presence of bile.

The red color alone is not sufficient as an indication of bile It is in fact only the commencement of the change which indicates the biliary matters. If these matters be present, the color passes, as we have already mentioned, first into a lake, then into a purple; and it is this lake and purple color alone which can be regarded as really characteristic of the biliary reaction.

It is important to observe that Pettenkofer's reaction is produced by the presence of either or both of the biliary substances proper; and is not at all dependent on the coloring matter of the bile. For if the two biliary substances, crystalline and resinous, be extracted by the process above described, and, after being dissolved in water, decolorized with animal charcoal, the watery solution will still give Pettenkofer's reaction perfectly, though no coloring matter be present, and though no green tinge can be produced by the addition of nitric acid or tincture of iodine. If the two biliary substances be then separated from each other, and tested in distinct solutions, each solution will give the same reaction promptly and completely.

Various objections have been urged against this test. It has been stated to be uncertain and variable in its action. Robin and Verdeil' say that its reactions "do not belong exclusively to the

Op cit., vol. ii. p. 468.

bile, and may therefore give rise to mistakes." Some fatty substances and volatile oils (olein, oleic acid, oil of turpentine, oil of caraway) have been stated to produce similar red and violet colors, when treated with sugar and sulphuric acid. These objections, however, have not much, if any, practical weight. The test no doubt requires some care and practice in its application, as we have already pointed out; but this is the case also, to a greater or less extent, with nearly all chemical tests, and particularly with those for substances of organic origin. No other substance is, in point of fact, liable to be met with in the intestinal fluids or the blood, which would simulate the reactions of the biliary matters. We have found that the fatty matters of the chyle, taken from the thoracic duct, do not give any coloration which would be mistaken for that of the bile. When the volatile oils (caraway and turpentine) are acted on by sulphuric acid, a red color is produced which afterward becomes brown and blackish, and a peculiar, tarry, empyreumatic odor is developed at the same time; but we do not get the lake and purple colors spoken of above. Finally, if the precaution be observed-first of extracting the suspected matters with absolute alcohol, then precipitating with ether and dissolving the precipitate in water, no ambiguity could result from the presence of any of the above substances.

Pettenkofer's test, then, if used with care, is extremely useful, and may lead to many valuable results. Indeed, no other test than this can be at all relied on to determine the presence or absence of the biliary substances proper.

VARIATIONS AND FUNCTIONS OF BILE.

With regard to the entire quantity of bile secreted daily, we have had no very positive knowledge, until the experiments of Bidder and Schmidt, published in 1852. These experiments were performed on cats, dogs, sheep, and rabbits, in the following manner. The abdomen was opened, and a ligature placed upon the ductus communis choledochus, so as to prevent the bile finding its way into the intestine. An opening was then made in the fundus of the gall-bladder, by which the bile was discharged externally. The bile, so discharged, was received into previously weighed vessels,

' Verdaungssaefte und Stoffwechsel. Leipzig, 1852.

and its quantity accurately determined. Each observation usually occupied about two hours, during which period the temporary fluctuations occasionally observable in the quantity of bile discharged were mutually corrected, so far as the entire result was concerned. The animal was then killed, weighed and carefully examined, in order to make sure that the biliary duct had been securely tied, and that no inflammatory alteration had taken place in the abdominal organs. The observations were made at very different periods after the last meal, so as to determine the influence exerted by the digestive process upon the rapidity of the secretion. The average quantity of bile for twenty-four hours was then calculated from a comparison of the above results; and the quantity of its solid ingredients was also ascertained in each instance by evaporating a portion of the bile in the water-bath, and weighing the dry residue.

Bidder and Schmidt found in this way that the daily quantity of bile varied considerably in different species of animals. It was very much greater in the herbivorous animals used for experiment than in the carnivora. The results obtained by these observers are as follows:

For every pound weight of the entire body there is secreted during 24 hours

Since, in the human subject, the digestive processes and the nutritive actions generally resemble those of the carnivora, rather than those of the herbivora, it is probable that the daily quantity of bile in man is very similar to that in the carnivorous animals. If we apply to the human subject the average results obtained by Bidder and Schmidt from the cat and dog, we find that, in an adult man, weighing 140 pounds, the daily quantity of the bile will be certainly not less than 16,940 grains, or very nearly 2 pounds avoirdupois.

It is a matter of great importance, in regard to the bile, as well as the other intestinal fluids, to ascertain whether it be a constant secretion, like the urine and perspiration, or whether it be intermittent, like the gastric juice, and discharged only during the digestive process. In order to determine this point, we have performed the

following series of experiments on dogs. The animals were kept confined, and killed at various periods after feeding, sometimes by the inoculation of woorara, sometimes by hydrocyanic acid, but most frequently by section of the medulla oblongata. The contents of the intestine were then collected and examined. In all instances, the bile was also taken from the gall-bladder, and treated in the same way, for purposes of comparison. The intestinal contents always presented some peculiarities of appearance when treated with alcohol and ether, owing probably to the presence of other substances than the bile; but they always gave evidence of the presence of biliary matters as well. The biliary substances could almost always be recognized by the microscope in the ether-precipitate of the alcoholic solution; the resinous substance, under the form of rounded, oily-looking drops (Fig. 52), and the other, under the form of crystalline groups, generally presenting the appearance of double bundles of slender, radiating, slightly curved or wavy, needle-shaped crystals. These substances, dissolved in water, gave a purple color with sugar and sulphuric acid. These experiments were tried after the animals had been kept for one, two, three, five, six, seven, eight, and twelve days without food. The result showed that, in all these instances, bile was present in the small intestine. It is, therefore, plainly not an intermittent secretion, nor one which is con

Fig. 52.

CRYSTALLINE AND RESINOUS BILIARY SUB

STANCES; from Small Intestine of Dog, after two days' fasting.

cerned exclusively in the digestive process; but its secretion is constant, and it continues to be discharged into the intestine for many days after the animal has been deprived of food.

The next point of importance to be examined relates to the time after feeding at which the bile passes into the intestine in the greatest abundance. Bidder and Schmidt have already investigated this point in the following manner. They operated, as above described,