Defensive Ferments of the Animal Organism/Methods in Use/The Dialysation Process
Methods in Use.
I.—The Dialysation Process.
The principle of the method: Albumen being a colloid does not diffuse through animal membranes, while on the other hand peptones—the first products of its decomposition—are diffusible. If we put albumen in a dialysing tube and place the latter in water, no albumen appears in the surrounding fluid even after a considerable time. If, however, substances such as pepsin and hydrochloric acid are added to the albumen in the tube, we can soon trace, in the water surrounding the tube, substances which are produced from the decomposition of the albumen. These substances are the so-called peptones and some other simpler products of decomposition. If we desire to test any liquid to ascertain whether it contains any proteolytic—i.e., albumen-decomposing—ferments, we place it in a dialysing tube together with albumen, and note whether peptones appear in the liquid surrounding the dialysing tube. If none are present, we may be sure that the tested liquid contains none of the active ferments capable of decomposing albumen. Should we detect the presence of peptones, we may be certain that some decomposition of the albumen has taken place. In our special case the fluid to be tested is blood serum. It is obvious that the method is exactly the same, when we test, for their capacity of decomposing albumen, such substances as cerebro-spinal fluid, lymph, or extracts from various organs—e.g., juices obtained by means of high pressure.
Dialysing Tubes.—The result of tests for albumen-decomposing ferments by the dialysation process depends in the first place upon the quality of the membrane used. The latter must above all answer two requirements. First of all it must be absolutely impermeable to albumen, and further, evenly permeable to decomposites of albumen. If the tube allows albumen to pass through it, the latter may be mistaken for peptones, unless we apply special tests for albumen. Should dialysing tubes be used which allow peptones to diffuse through at a variable rate, then we should be at a loss in our judgment upon the results of a test, because, as will presently be shown, a control test of the fluid to be tested must always be made, without the presence of albumen, and the results of this test be compared with those of the tests in which albumen has been mixed in the dialysing tube with the fluid under research. Should one tube be very dense and allow little or no passage at all of the peptones, we should naturally have in this a considerable source of error.
Numerous dialysing membranes are known, of which very few have any real value for our purpose. The dialysation process requires dialysing tubes which can be used over and over again. The best are those supplied by Schleicher and Schüll, of Düren in Rhineland. The tubes of this firm should in no case be used without a thorough preliminary examination, because tubes are nearly always met with which allow albumen to pass through, while others are found through which peptones diffuse with difficulty, so that careful testing of the tubes is indispensable.[1] Further, the tubes must be short ones. No. 579A is a tube specially prepared for our purpose. If tubes be used, which project too much over the surface of the surrounding fluid towards which the dialysing process acts, this gives rise to a very uneven evaporation of the dialysate. The latter soaks into the tube, is carried upwards, and evaporates. Indeed, as we shall see later, everything depends upon the fact that, in comparative experiments, the concentration of the dialvsates shall not be affected by unequal evaporation; and every precaution must be taken to avoid this source of error.
The first duty to be undertaken in making use of the dialysation process, is the testing of the tubes, the so-called standardization of the dialysing tubes. This standardization, as we have already emphasized, implies the impermeability of the tubes towards albumen, and a perfectly equal permeability for the products of its decomposition.
(a) Test for Impermeability by Albumen.—A solution of albumen is prepared. The simplest way is to take the white of a new-laid egg. 5 c.c. of perfectly fresh white of egg are diluted with distilled water in a graduated tube to 100 c.c., and thoroughly mixed by shaking. Of the white of egg, which must be absolutely fresh, only the more fluid portion is used, while all flaky matter or bits of skin—in short, all solid parts—are rejected, as otherwise it is impossible to get a good mixture. Instead of the white of an egg, blood serum may be employed.
Now the tubes to be tested are prepared. They are soaked in cold water for about half an hour. The tubes are then placed in small Erlenmeyer flasks (fig. 7) and 2.5 c.c. of the thoroughly mixed solution of white of egg in water are poured into them. The solution is measured by means of a pipette. While filling the tubes the pipette is placed far down in them, and the greatest precautions must be taken not to spill any of the egg solution upon the exterior of the dialysing tube. Should this occur, the dialysate would incorrectly show a positive reaction for peptones, when, for instance, the biuret test is applied, since both albumens and peptones give this reaction. To avoid any chance of such an error, the dialysing tube, after having been filled, is closed at the top between the thumb and forefinger and well rinsed in running water. Then the tube is closed in the same manner half way down, and water is allowed
Fig. 7.
to enter the upper part of the tube, so as to wash that part of the dialysing tube which, during the dialysing process, projects out of the dialysate and above the layer of toluol. By moving the thumb and forefinger towards the upper end of the tube we expel the water remaining after washing. All these manipulations have the following object:—
When filling the tube with albumen its interior, near the free edge, may easily come into contact with the pipette. Some of the albumen may adhere to the edge of the tube and dry up in time. At the conclusion of the test some parts of this albumen may fall into the dialysate and pollute it. During the operation of cleansing the inside of the tubes, care must be taken to prevent water from entering the tubes. Before touching the tubes the hands should be thoroughly cleansed. The use of forceps is much recommended, and these must have wide, parallel, smooth arms.
The rinsed tubes are again put into Erlenmeyer flasks which contain 20 c.c. of sterile distilled water. The filling of the dialysation tubes must never be done in the same flasks in which it is intended to carry out the dialysation; something out of the pipette may too easily get into the flask. In order to prevent contamination the surrounding fluid, as well as the contents of the tubes, is covered with a layer of toluol about ½ cm. thick (fig. 7, p. 151). It is best to cover the flasks with watch glasses, unless one is prepared to use stoppered vessels. The dialysation is carried on at the temperature of the room, or, better still, in a closed space at a constant temperature—i.e., in an incubator.
After about sixteen hours—time is of no importance in this test, since the tubes are in this case merely tested for their permeability towards colloids—the dialysation is interrupted. The Erlenmeyer flasks, which should bear corresponding numbers, are placed in a row. By means of a pipette, which is closed at its upper extremity by the finger and rapidly passed through the layer of toluol, 10 c.c. of the dialysate are taken out, and placed in a test-tube bearing the same number as the corresponding Erlenmever flask. This is the best way to avoid mistakes. Of course, for each dialysate a separate and absolutely clean pipette must be used. We do not recommend transferring the dialysates to the test-tubes by means of the same pipette, rapidly cleansed each time after use, because by this means some impurity or other may easily be introduced into the dialysate. Some saliva may very easily enter that part of the pipette which, during the so-called cleansing, remains untouched by the water, alcohol and ether. On the contrary, new saliva is drawn in at each operation if the suction is made by the mouth. Now, when the dialysate is taken up, it is almost certain to be drawn above the level marked upon the pipette, and may then become mixed with the saliva. If test-tubes, graduated to 10 c.c., are to hand, then these tubes may be employed in the following manner: After removing the dialysing tubes, the toluol is drawn off and the dialysate is poured directly into the test-tube. It is of no great importance, in the biuret reaction, to consider quantities to the minutest exactness, nor does a little toluol do any harm.
Now, to each test-tube is added about 2.5 c.c. of a 33 per cent. caustic soda solution. The whole is shaken sideways to and fro. The mouth of the tube should not be closed with the linger, as in this manner some impurities may easily enter the mixture. Very often the dialysates become turbid upon the addition of the caustic soda solution, but this does not interfere with the reaction. In order to test for diffused albumen we have different methods at our disposal, of which the biuret reaction has been found to be the best. One could also make use of the precipitin formation that appears when prepared serum is employed, but such serum is not always at hand. Further, we may use ninhydrin, but it is not so sensitive to albumen.
Ninhydrin reacts, amongst others, with compounds which carry an amino group in a position to the carboxyl group; when it produces a bluish-violet colour, if the concentration of the reacting compounds is sufficiently strong. The albumen molecule contains a few free amino and carboxyl groups, and as soon as it is decomposed, these groups are set free. The ninhydrin reaction becomes stronger the more the albumen is decomposed, provided the various stages of decomposition are not withdrawn. At each stage an amino and carboxyl group are set free. The biuret reaction manifests itself quite differently. The greater the fractional decomposition of the albumen, the weaker is the biuret reaction. As soon as we pass a certain limit of decomposition the reaction ceases.
The biuret reaction is unfortunately rather difficult to detect when it is a case of demonstrating slight traces of the reddish-violet coloration. This is due to the fact that the eye is but slightly sensitive to these tints. Again there are great individual differences. If the observer is unable to detect a light biuret reaction then he has to rely on standardized tubes; or else he must make use of the ninhydrin reaction and try, by means of lengthy dialysation, to. increase the quantity of albumen in the dialysate, so far as the tubes are permeable to albumen. Seeing that white of egg, as well as serum, always contains substances which diffuse and react with ninhydrin, we are bound to find out, by means of a standardized tube, what quantity of a given albumen solution we may use without running the risk of the dialysate showing a ninhydrin reaction. How to perform the ninhydrin test we shall describe later, when we give the test for equal permeability to decomposites of albumen.
The biuret reaction is performed as follows: To the mixture of the dialysate with caustic soda about 1 c.c. of a very much diluted copper sulphate solution—e.g., 1 in 500 c.c.—is added. This solution is run down by means of a pipette along the inside of the test-tube, so as to obtain a surface layer. Then we observe by transmitted light the dividing line between the blue layer, which often, however, appears turbid owing to the deposition of copper hydroxide, and the quite colourless liquid below. The slightest trace of a pinkish-violet colour is a proof that the tube from which the dialysate was procured is unsuitable. Often the presence of albumen is shown by the fact that the precipitated copper oxide dissolves after a time—in about half an hour—and a clear violet layer appears which gradually diffuses into the other liquid. With this test it is better to be over-cautious, and the tubes should be rejected each time the biuret reaction gives doubtful results.
(b) Testing of the Dialysing Tubes for equal Permeability to Decomposites of Albumen.—Tubes, which do not allow the passage of albumen, must first of all be thoroughly cleansed. Their contents are poured out, and they are then placed on a sieve and rinsed for about half an hour in clean running water.
For the sake of security they are put in boiling water for not more than half a minute. We may also point out that experience has shown that boiling the tubes is not very good for them, for they easily become too dense. After this, 2.5 c.c. of a 1 per cent. solution of silk-peptone are poured into them; the tubes are again carefully rinsed in cold water, one by one, and are then placed in Erlenmeyer flasks filled with 20 c.c. of sterilized distilled water (compare pp. 150-152). The latter is covered with toluol. In this case also the dialysis is carried on in an incubator, in order to expose all the tubes to approximately equal conditions.
After some sixteen hours the ninhydrin reaction is applied. As this reaction depends so much upon the degree of concentration, it is advisable to carefully guard against the following sources of error. First of all, the dialysate must not be allowed to evaporate unevenly. To avoid this, an excess of toluol is added, and the Erlenmeyer tube is preferably covered with a watch glass. It is clear that, should the different dialysates evaporate unevenly, the ninhydrin reactions would be of varying intensity. The second source of error lies in the boiling of the separate test-tubes, which is applied in order to produce the formation of the colouring substances. We shall return to this presently.
In the application of the ninhydrin reaction we must never forget the fact, that ninhydrin is a most delicate reacting agent for albuminous substances, peptones, polypeptides, and amino-acids. Perspiration reacts very readily with ninhydrin, as do also the epidermic scales, &c. It is most important to avoid any contact of the dialysing tube with the hand; only sterilized forceps should be employed for holding them, and all the apparatus in use should be absolutely clean and dry. One must never rely upon any rapid drying methods. In the first place, it will not do to transfer the dialysates into the test-tubes by means of one pipette. It is essential to have at one's disposal for the actual tests as many different pipettes, graduated to 10 c.c., as there are dialysates to be handled. The test-tubes must also be absolutely clean and dry, and they must be of exactly the same width. Pouring the dialysates into the test-tubes is not admissible, because the toluol may easily spoil the reaction, chiefly by preventing satisfactory boiling.
In detail one proceeds as follows: As before, the pipette, closed at the top with the finger, is passed through the toluol layer, and 10 c.c. of the dialysate are withdrawn. The pipette is kept closed when passing through the toluol layer, in order to prevent any toluol from entering it. After transferring 10 c.c. of all the dialysates into the test-tubes, using separate pipettes for each, we add to each test exactly 0.2 c.c. of an accurately prepared 1 per cent. solution of ninhydrin.
For accurate measurements a capillary pipette of 1 c.c. is used. The ninhydrin solution is prepared as follows: ninhydrin is usually sold in 0.1-gr. packets, and this quantity is shaken out of the tube into a measuring flask marked to 10 c.c. The tube is best emptied by tapping it against the inside of the mouth of the measure, though it is not possible by this means to transfer the whole of the 0.1 gr. of ninhvdrin into the measure. The rest of the ninhydrin must be dissolved with distilled and sterilized water; this solution is poured into the measure, and the tube is again rinsed several times, after which the measure is filled up nearly to the mark. Ninhydrin dissolves sparingly in water, and in order to dissolve it quickly it must be heated a little. For this purpose it is best to stand the measure in the incubator. As soon as the solution is effected, the contents are cooled, and filled up to the mark on the flask.
The ninhydrin solution is not absolutely stable. It is liable to infection, and is also sensitive to the action of light. It may be kept in a brown flask, but this is not necessary so long as one prepares only 10 c.c. at a time, a quantity which is quickly used up.
After all the test-tubes containing 10 c.c. of the dialysate have been filled with 0.2 c.c. of the ninhydrin solution, a boiling-stick is placed in each. The latter is absolutelv essential, because only a very even ebullition will produce a properly comparable colour reaction. The boiling-sticks used in the trade are divided into segments about 10 cm. long; these must be boiled in distilled water, dried at 60° to 70°C., and kept in a tightly closed glass vessel. Boiling-sticks must not be stored in a damp condition; for on the one hand their use in this state may give rise to error, owing to the uneven amount of water present, while on the other hand mould may easily appear. Again, the boiling-sticks should never be dried at too high a temperature, otherwise they may turn brown, and in that case they give off a brown colouring matter during boiling, and thus render an exact reading impossible. They must never be touched with the hands, but should always be placed in the test-tubes by means of forceps.
The process of boiling is now started, and the manner in which this is carried out is of the greatest importance. Boiling must be intensive; at the same time every precaution must be taken to avoid the slightest spilling, as also to prevent uneven evaporation. When all the liquids to be tested have been boiled, we must assure ourselves that they are at the same level in all the test-tubes. It is best to use large test-tubes upon which the volume of 10 c.c. is conspicuously marked. It is then easy to ascertain whether the very important point of even boiling has been accurately carried out.
The test-tube is first held by means of a holder in the centre of a Bunsen burner, the flame of which must be a full one. One then watches carefully for the moment when the first bubbles of gas appear on the sides of the test-tube, which only takes a few seconds, and calculating from this moment one boils for exactly one minute. After ten to fifteen seconds a vivid ebullition is observed, and as soon as this point is reached the test-tube is brought to the edge of the flame, and the boiling is continued at the middle height of the flame (see fig. 8). In this way it is possible to carry out the boiling continuously and energetically, so that the liquid travels over more than half of the test-tube, without any danger of over-boiling. Not for a single moment should the attention be allowed to wander from this process, for
Fig. 8.
(a) Test-tube holder; (b) boiling-stick.
everything depends upon the accuracy of the operation. If the ebullition is too weak, then under certain circumstances the reaction may fail altogether, while, if the rate of ebullition differs in the different tests, we get a difference in the intensities of the colorations. The results, in short, are inaccurate.
After the lapse of half an hour a comparison of the intensity of the blue coloration is made in each case. It is soon found that a particular intensity prevails. All the tests which show a greater or smaller intensity than this are carefully noted, and the tubes from which the dialysates in question were obtained are rejected. In this case, too, it is necessary to be very accurate, otherwise the actual experiments may easily lead to deceptive results. Thus it may happen that serum alone, and serum added to a given organ, contain diffusible compounds which react with ninhydrin with precisely equal difficulty; yet on testing the dialysate in the experiment serum + organ, we may get a positive reaction, because the tube was more permeable to decomposites of albumen than the tube used as a control.
The tubes that are equally permeable in this respect are now carefully rinsed, then plunged into boiling water for thirty seconds, and finally brought into a sterilized flask. Sterilized water is added, together with an equal quantity of toluol, the fluid being calculated to fill the flask exactly. The tubes are now ready for use. They are taken out of the flask with sterilized forceps and must, if possible, not come into any contact with the fingers during all the manipulations.
Preparation of the Substrates (Organs).—As material for these tests we use either an albuminous body or else a mixture of these bodies—e.g., an organ. The manner in which a substrate is prepared is of the greatest importance for the whole success of the dialysation process, and, unless one adheres to the directions in every particular, one is bound to meet with unsuccessful results. These, however, can be successfully avoided if the preparation of the substrate be carried out with proper attention. The principle of the matter is, to obtain substrates which contain coagulated albumen and are absolutely free from diffusible substances which react with ninhydrin. We shall demonstrate the method of obtaining the substrates by means of the preparation of coagulated placenta. Other organs are treated in exactly the same way; only, those which are rich in fats and lipoids have to be previously extracted with carbon tetrachloride in a Soxhlet apparatus. The same applies also to tubercle bacilli. Placenta can always be procured in a fresh state, whereas in other cases we have to deal with organs from dead bodies. In the latter case the dissection should be made at the earliest opportunity. The best corpses are those of accidents. If prolonged agony has been undergone previous to death, the organs are almost useless. It is very important to test the organs for pathological changes; and it is absolutely essential to state in what condition the organ used was found, for different results could easily be obtained if one observed used normal organs while another made use of abnormal ones. The question whether the organs of animals may be used will be discussed later.[2]
The organ must be absolutely freed from blood, a condition that can be attained in the case of different organs with varying facility. Placenta and lungs, for instance, can be easily washed so as to free them from blood, or the blood may be rinsed out through the large blood-vessels; whilst the liver, kidneys, and particularly the uvea, are freed from blood with great difficulty. With the latter there is scarcely any other means of proving its suitability, than by experimenting comparatively with serum from individuals with healthy and diseased uvea respectively. The pigment prevents us from discovering the last traces of blood.
The fresh and still warm placenta is first freed from blood clots by mechanical means, the membranes and the umbilical cord being removed at the same time. Then the placenta is cut into small pieces, about one inch square or less, and these are crushed in a current of water, for which purpose they are best placed on a sieve. Water is allowed to run continuously upon the pieces of placenta, each piece being pressed between the fingers. From time to time the pieces are placed in a cloth and squeezed in it. The washing of the placenta must never be interrupted. Pieces to which coagulated blood adheres, which cannot easily be removed, are rejected. Finally, they are placed in a mortar and broken up with a pestle, by which process the last traces of blood are eliminated; and then the connective tissue can be removed. We now have a snow-white tissue, which is immediately boiled. The whole process takes from one to at most three hours, according to the kind of tissue employed.
The extraction of blood can also be effected by thoroughly washing out the organ through the blood-vessels; but in this case, the organ must be washed out again after being broken up. If the extraction presents any difficulties, one can often attain one's object by covering the tissue in the fresh state with a very thick layer of common salt. The mixture is allowed to stand for two to six hours in an ice-chest; the salt is then dissolved, and the washing carried on in the usual manner. One must never preserve an organ from which the blood has been incompletely abstracted, in any particular manner, with the intention of completing the process later on. All preservation media produce coagulation and alteration of the blood. The smallest blood-vessels always contain, in that case, small quantities of blood constituents. We must also give particular warning against the use of bleaching agents, as, for instance, hydrogen peroxide. The red colour of the blood indicates to us that it is still present. If we use hydrogen peroxide, then we lose any control over the blood contained in the tissue. If one is not quite certain of the fact that the organ is free from blood, one should squeeze out a few pieces of it in a little water, and examine the fluid with the spectroscope.
About a hundred times more distilled water than there is of the tissue is placed in an enamelled vessel and then brought to the boil. The tissue, having been absolutely freed from blood, is placed in the boiling water, for every litre of which it is advisable to add about five drops of glacial acetic acid. This is boiled for ten minutes, and the boiling water is passed through a sieve; the tissue is thoroughly rinsed for about five minutes with distilled water, and the same process of boiling is repeated, using fresh water without the addition of acetic acid. The boiling, the pouring off of the boiled water, the rinsing of the tissues, and the renewed boiling are repeated about six times without interrtiption. If it is necessary to cease boiling, then one must never forget to pour a fairly large quantity of toluol on the top of the boiled water containing the tissue. If this be omitted the tissue is liable to become infected, and then some hours of boiling may be necessary in order to free the organ again from extractive substances which react with ninhydrin.
If a centrifuge be at one’s disposal, the boiling water is centrifuged at a suitable speed. This is still more necessary when one is working with finely minced organs or bacteriological cultures and the like, otherwise too much of the material would be lost when pouring off the water.
After the sixth boiling only five times the amount of water at most is used. The smaller the amount of water employed, the more exact is the result of the test for the extractive substances that react with ninhydrin. In every case as much water must be present as will be needed to continue active boiling for five minutes without the risk of burning, the smallest possible vessels being used. Then a certain quantity of boiled water is filtered through a hardened filter paper. To 5 c.c. of the filtrate is added at least 1 c.c. of a 1 per cent. aqueous solution of ninhydrin, and the mixture is boiled (as described on p. 160 seq.) for one minute. If, after half an hour, not the slightest trace of a violet coloration manifests itself, the organ may be considered as suitable, provided it still remains snow—white. Only the tissues of the liver, the spleen, and the kidneys do not appear quite white. Should the tissue turn grey, or even brown, during boiling, this is a proof that it was not absolutely freed from blood, or that the boiling was not conducted properly. Should the particular test prove positive, the boiling must be continued—i.e., the water must be poured off, the organ be thoroughly rinsed in distilled water, and boiled over again for five minutes with not more than five times its own quantity of water. It is filtered again through a hardened filter; to 5 c.c. of the filtrate is added at least 1 c.c. of ninhydrin solution, and the mixture is boiled for one minute.
Before the organ is put by for keeping, it is spread upon a white glass plate or a sheet of white paper, and every separate piece is thoroughly examined. Should brown spots or other doubtful points, which cause one to suspect the presence of coagulated blood, be noticed, the pieces affected must be thrown away. Only by conscientiously and carefully adhering to these rules can results be expected which are free from all objection. An organ, which has given a whole series of correct results, may lead us astray if even one single piece containing blood happens to be used.
As soon as the organ has been tested in the above manner for the absence of any piece that may contain blood, and as being free from extractives which react with ninhydrin, it is immediately placed in a bottle, with a well-ground stopper; the bottle having been previously sterilized. Then a little sterilized distilled water and a good deal of chloroform and toluol are added, the bottle being filled in such a way that the stopper comes into contact with the liquid. A thoroughly well-prepared organ should preserve indefinitely, and it only becomes useless again by being contaminated. There are various contingencies that may spoil a perfect organ. In the first place, it must be taken out of the bottle only by means of sterilized forceps. None of the sample taken should be put back into the bottle if it has been exposed to the risk of infection, or been left lying about, and so on. The bottle must be kept filled with toluol, otherwise part of the tissue may adhere to the neck of the bottle. If such a piece protrudes from the level of the toluol it decays, and finally drops down on to the rest of the tissue. The bottle containing the organ should be kept in an ice cupboard.
Bacteria and other living organisms may be prepared exactly in the same way as tissues. Boiling is also resorted to in these cases; and the same rules hold good. It is obvious that organs can be separated into their tissues. The more special the problems to be dealt with, the more does one limit oneself to a very definite tissue.
All organs which are very dense in structure, and which become hard when boiled, require special treatment. Carcinomas, myomas, &c., may appear snow-white and still contain blood, so that in these cases the pieces have to be cut into very minute particles in order to prevent mistakes.
Every organ must be standardized. Placenta is only useful so long as it is not decomposed by the serum of carcinomatous subjects, or of individuals with salpingitis, tuberculosis, and the like. Carcinoma is correctly prepared if it is not attacked by the serum of pregnant individuals.
Above all, the organ should be tested by means of cases which contain ferments acting against the components of the red blood corpuscles. Cases of blood effusion are excellent testing agents for the absence of blood in the prepared organ. Or disharmonious blood—in this case human blood—is injected into an animal, and its serum is tested against coagulated red blood corpuscles and the organ to be employed.
In conducting these experiments we must be able, with absolute certainty, to prevent the decomposition of all proteins other than those belonging to the actual organ itself. It is clear that serum, which contains a defensive ferment against the components of the form-elements of the blood, will decompose every organ containing blood—that is, it will split up, not the proteins of the organ, but the components of the blood within the organ. The importance of a clear recognition of this circumstance may be gathered from the fact that serum of normal horses and cattle decomposed red blood corpuscles in about 40 per cent. of cases. Further, it was found that serum taken from animals that exhibited hæmatoma produced decomposition with every kind of organ containing blood, whilst organs freed from blood and subjected to parallel tests were left unattacked. This fundamental rule, of completely freeing the organ in question from its blood, is often transgressed. If the serum does not contain any defensive ferments against the form-elements of the blood, then, of course, even an organ containing blood may give correct results. As, however, mistaken results are liable to occur, such an organ should, as a rule, never be used.
It is advisable never to use one particular organ exclusively for testing a definite problem; and one should always work with controls. For instance, placenta is always tested with serum from obviously non-pregnant persons. Male serum should also be employed. Should cases of diabetes, for instance, be tested exclusively with faulty preparations of pancreatic gland, then in most cases a "decomposition" would be found. Such mistakes are avoided by using thoroughly prepared organs on the one hand, and by means of control experiments on the other.
It is of fundamental importance to establish the morphological state of the organ used, and its origin. It is possible that, in a given disease, a normal organ is not decomposed, although the same organ is readily attacked, if it has already undergone particular pathological changes. Thus it is quite possible that, for instance, a normal thyroid gland would not be decomposed by Basedow serum, while a gland originating from a morbus Basedowi would be subject to decomposition. Just as every case examined has to be thoroughly tested by clinical means, and its further course closely followed up, so also must the substrate to be employed be characterized exactly. A bare statistical compilation of cases, with percentage accounts of faulty diagnoses is unworthy of scientific publication. Each separate case must be clinically investigated. This is the reason why the fruits of these researches are bound to fall into the hands of clinical observers. The physiologist can only note one case after another without being able to characterize them individually, or even to observe them continuously, and, in consequence, we can expect little help from his side.
A very important question is whether, instead of human organs, the corresponding organs of animals may be used in experiments with human serum.[3] It would naturally be a great advantage in all these researches if this were the case. Our earliest researches enabled us to state the fact that human placenta can be replaced by that of animals, and vice versa.[4] We have made further experiments with the brain and other organs, and have obtained good results. It seems that organs which have the same function to fulfil, in the animal kingdom have common properties in their structure. In spite of our favourable experiences we have not ventured generally to recommend the use of animal organs. It is still very difficult to find a proper balance amongst the contrary results of many observers, and were we to change the type of substrate without sufficient experience, we should arrive at still more divergent results. This is the reason why it is particularly necessary to use organs of the same species as that to which the serum under investigation belongs, as well as those of a different type. Only when it is established that harmonious results are obtained ought we to be satisfied with non-specific organs, and always under the condition that no substrate is used which shows definite pathological alterations.
Means of Obtaining Blood Serum.—Three conditions have to be complied with. The serum must be as poor as possible in diffusible substances which react with ninhydrin, and this is attained by taking the blood in a fasting condition. In all cases in which the albuminous metabolism is very rapid, in cases of disease which are accompanied by decay of the tissues, as in the case of carcinoma, in cases of absorption of exudates and transudates, in all purulent processes, and lastly, in effusions of blood, the blood always contains a larger quantity of such compounds. The blood serum must further be absolutely free from hæmoglobin, and in doubtful cases the spectroscope should be used.
The serum must be completely freed from its form-elements, a point which is often neglected. A serum may appear absolutely clear, and yet contain millions of red blood corpuscles. The serum must be treated with a good electric centrifuge until the tube shows no trace of blood corpuscles, either on its sides or at the bottom. The serum, after each treatment with the centrifuge, is drawn off with a pipette and transferred to another tube, and during this operation, in order to avoid any contact of the pipette with the red blood corpuscles, the tube is placed upon a mirror. One can then see exactly where the end of the pipette is at any moment. The blood is best taken with an absolutely dry needle and placed directly into a sterilized centrifuge tube, or, better still, into a small Erlenmeyer flask. The blood is allowed to clot spontaneously, and is watched until the serum separates out. Any mode of procedure which accelerates the separation of the serum increases the risk of hæmolysis. The blood should not be placed either in an ice-chest or in an incubator, but should be left simply at room temperature. In the first case, the risk of hæmolysis is very great; in the second, autolysis of the form-elements generally results. Serum is generally obtained in a considerable quantity after five or six hours, but if enough has not separated out one makes use of the centrifuge. In the first case the serum is poured into a centrifuge tube, and centrifuged for about five to ten minutes. It is then easy to ascertain that the serum, which was apparently free from solid elements, has now given off a whole layer of red blood corpuscles during the process of centrifuging a second time. Should this remain in the serum, then during the dialysis hæmolysis would take place in the dialysing tube, and the experiment would give faulty results.
It happens, usually, that the experiment is so arranged that, say, 1.5 c.c. of serum are taken from the centrifuge tube and employed as a control. Only after this do we remove more for the test, organ + serum. If at this point the directions are not followed exactly, it may easily happen that red corpuscles are found in the test, organ + serum. Hæmolysis appears during dialysis, and then we have exactly the same conditions as arise when organs are used which contain blood; only in this case the contents of the corpuscles are found, not in the tissue, but in the serum. It is from non-observance of the rules given that we get the observation that a serum, which is absolutely free from hæmoglobin, appears quite red at the end of the experiment. It is the diffusion of water from the outer fluid into the tube that has led to the hæmolysis of the red corpuscles which, though present, have been overlooked.
It is sufficient to use 15 to 20 c.c. of blood. For sending away, only serum should be used which has been centrifuged completely. The latter must in any case be centrifuged again. The serum should not be more than twelve hours old, even though it has been collected and preserved in a really sterile way. The taking of the blood, its collection, and its manipulation must be done aseptically.