# Defensive Ferments of the Animal Organism/Methods in Use/Performance of the Experiment

Performance of the Experiment.
In carrying out a dialysation test the following fundamental rules must be obeyed, of which not one is unimportant:—
(1) Extreme cleanliness is the first condition for ensuring success in the experiment. This applies to the surroundings, and to all the utensils employed. Pipettes, test-tubes, Erlenmeyer flasks, &c., must be thoroughly cleansed and absolutely dry.
(2) The water used must be thoroughly sterilized distilled water. So-called distilled water often proves to contain numerous germs of every description. If one uses water like this as the outer fluid in dialysis, then the way is laid open for all kinds of mistakes.
(3) The work is performed, as far as possible, aseptically and antiseptically.
(4) In the room in which the experiments are in progress neither bacteriological nor chemical work should be allowed. Above all, an incubator must be ​specially reserved for these experiments, nor is it possible to allow the incubator to be used at the same time for bacteriological purposes.
(5) Before starting it must be ascertained that all utensils are to hand and in perfect condition.
(6) Experiments can only be carried on with good light. It is impossible to carry on more than five or six experiments at the same time with the necessary care.
(7) Before successful tests can be expected, one must not only be certain of a perfect knowledge of all the details of the method, but a thorough knowledge of their fundamental principles is most essential. It is not sufficient to know the method thoroughly, one must have a perfect command of it, and, as it were, live in it. No one is able to stain tissues perfectly for the first time, even though he be guided by the strictest directions. Even simple chemical methods require practice, and the most elementary analyses sometimes fail. Even the Kjeldahl method, which is so easily handled, requires to be thoroughly learnt. Should a failure result, no one would think of communicating it while blaming the method; he would never rest until the cause of the error was found. The statement, "We have been working in the strictest manner according to the given directions," I treat with scepticism on the basis of a rich experience. Such great offences are often ​committed against the fundamental rules of the whole method, that errors are bound to occur. Therefore, we must not ignore a method because it requires careful working. It is quite possible that with time we shall arrive at more simplicity in our manipulations, and technique may place some further means at our disposal. But it is as yet too early to try to introduce modifications in the manner of working of the two methods, after a whole number of observers have obtained good results by their means in their present form. The principal requirement of any method is, that we should not rest until the cause of error is found in each case that occurs. This is the only way of avoiding them.
First of all blood is taken. If there be any doubt regarding the suitability of the substrate it is advisable first to test the organ, so as to avoid withdrawing the blood unnecessarily. This test should be repeated immediately before performing the experiment. The blood is allowed to coagulate spontaneously at the temperature of the room.
Immediately before beginning each experiment the organ is tested, and this important rule must never be neglected. It may so happen that all the parts of an organ have been freed from all extractive substances reacting with ninhydrin, except a piece here or there. It should be the duty of the observer to note down each time in his record: "Organ tested."
​So much of the tissues as are necessary for the experiments to be performed are taken, and to them is added at most five times their quantity of water. If any difficulty arises in the boiling, which may be traced to the insufficient quantity of tissue used, then more tissue is added, the excess of the organ being immediately put back into the bottle that contains the rest, in case it may be wanted later on. If the organ is left lying about for any time it becomes infected. An organ should never be boiled without being previously tested. It should not show any places that contain blood.
Further, the tissue must be shredded into small particles before it is boiled. It would be a great mistake to boil the tissues in large pieces and to use them later in the form of little pieces, for it might often happen that inside the big pieces products were enclosed which diffuse and react with ninhydrin, and they would not be noticed because they have not reached the outside. If, for instance, a lentil is boiled as a whole, the water does not readily show any ninhydrin reaction, but as soon as the lentil is broken up and boiled an intense reaction is observed. In the process of boiling the outer part coagulates, and thus tightly encloses the inner contents. Exactly the same thing may happen with other tissues. Therefore, before the experiment, the organ must be boiled in the same way as it is intended to be used, i.e., in a shredded form.
​The best way is to carry out the boiling in a test-tube for live minutes. It must be boiled energetically, and then filtered through a small hardened filter; after which, at least 1 c.c. of the 1 per cent. ninhydrin solution is added to 5 c.c. of the filtrate. Should one have less than 5 c.c. of the filtrate there is no harm in boiling with 1 c.c. of ninhydrin, because the stricter the conditions of these tests the better.
Boiling is performed (as described on p. 161) for one minute with the aid of a boiling rod. Only in cases, where the solution gives no traces whatsoever of a violet coloration, can the organ be used, and one must wait half an hour before one can establish its presence or absence. Should the organ not be required for immediate use, it must at once be covered with a layer of toluol. Should this test still give a coloration, then the substrate must be boiled over again with five times as much distilled water, until the test shows negative results.
Now, as many standardized dialysing tubes as are required are placed into empty, dry Erlenmeyer flasks, and about ½ grm. of the organ is poured into the tubes. This quantity is previously placed upon a piece of blotting paper, and dried by squeezing it strongly. Were the organ placed in a wet state directly into the tubes, a reaction which would give a weakly positive result might turn out negative, ​owing to the dilution of serum so caused. The tissue should never be handled with the fingers.
To the tubes containing the tissue 1 to 1.5 c.c. of serum are now added. A rule should always be made of arranging this experiment first. Afterwards from 1 to 1.5 c.c. of serum are placed in an empty tube (control test). Then the tubes are thoroughly rinsed with distilled water (as described on p. 151), and placed in Erlenmeyer flasks which have previously been filled with about 20 c.c. of sterilized water. Then a large amount of toluol is poured into the tubes and over the liquid outside, care being taken that the part of the tube which projects from the liquid should be soaked with toluol. At this stage of the experiment the following sources of error may arise. First of all, water may get into the tubes while they are being rinsed. If the work is not carried on in a scrupulously accurate manner considerable dilutions may occur. The tube must be completely closed during this operation. I have lately been in a position to observe a second source of error which may arise. Contrary to instructions the flask was filled with 20 c.c. of water and a large quantity of toluol, and only then was the tube and its contents immersed. In this case the liquid in the flask was raised to such a level that it passed from the outside to the inside of the tubes. Besides, the tube came into contact with the neck of the Erlenmever flask ​in many places, and here part of the liquid became enclosed by capillary action, thus forming a kind of communication between the contents of the tube and the liquid outside. From these observations it follows, that the toluol should never be introduced before the dialysing tube has previously been immersed in the 20 c.c. of water, in which case the quantity of toluol added can be accurately controlled, and care can be taken that both the inner and outer surfaces of the tubes shall project at least 0.5 c.c. over the toluol layer. Moreover, only wide-mouthed Erlenmeyer flasks should be used.
Then the flasks are placed in an incubator at a temperature of 37°C. At a higher temperature the ferments would be destroyed, and at a lower temperature the decomposition would be too slow.
After about sixteen hours the experiment is stopped. A thick layer of toluol must still be found upon the contents of the tubes, as well as on the surrounding liquid, at the end of the experiment. The Erlenmeyer flasks, carefully numbered, are best arranged in no special order. Then the tubes are taken out of the flasks, and placed, right up to the end of the experiment, into empty Erlenmeyer flasks. In withdrawing the tubes one at the same time effects a uniform mixing of the dialysate. Particular care must be taken to avoid a source of error that often arises at this point, which is, that if the flask has been supplied ​with too much toluol, or if the tube at the beginning of the test has not been immersed sufficiently deeply, then it may easily happen that, during the introduction of the pipette, some of the liquid passes from the outside into the dialysing tube. While, if one sucks strongly with the pipette at that moment, then the reverse may occur, and the contents of the tube may enter the pipette.
Ten cubic centimetres of the dialysate are taken, by means of a closed pipette passed through the toluol layer, and poured into a dry, wide, and absolutely clean test-tube. For each dialysate, as a matter of course, a separate, absolutely clean and dry pipette is used. One's work should never be arranged in such a manner that the pipette has to be hurriedly cleansed with alcohol, water, or ether after use, for the cleaning in this case may very easily be insufficient.
The danger of soiling the pipette with saliva is particularly great. (Compare p. 153.)
Then 0.2 c.c. of the 1 per cent. aqueous solution of ninhydrin are added to each test, together with a dry boiling rod (see p. 159), and one test after another is boiled absolutely evenly for a whole minute (see p. 160). After half an hour we ascertain which tests show a coloration and which do not, and only then do we compare our results with the original dialysates. If there be any tests which have evaporated more than the others, they are rejected, if they show a positive ​reaction. It may sometimes happen that, for instance, the dialysate of the serum gives a negative reaction, while serum + organ shows a slight violet coloration. If both samples have been boiled equally according to the directions, then both will have evaporated equally, and in this case the slightest coloration may be considered as unconditionally positive. If, on the contrary, the sample, organ + serum, has evaporated more, then we are confronted with the possibility that the stronger concentration is the cause of the coloration. In spite of the presence of absolutely equal quantities of substances, capable of reacting with ninhydrin, in the dialysate of the serum and that of the sample serum + organ, a higher concentration has been obtained owing to stronger evaporation. If it is impossible to effect even boiling by any other means, then it is necessary to resort to a water bath. The samples to be compared are placed in a stand, and immersed in a water bath. The boiling must be continued longer than when heating in an open flame, but two to three minutes are ​sufficient. As this method has not yet been applied to large quantities, the most suitable time has yet to be actually found.
Exact comparisons are only possible, when the test-tubes are of the same dimensions and have exactly the same thickness. For this purpose we must always arrange to have a sufficient stock of test-tubes answering perfectly to this requirement. In order to realize the importance of this proceeding, we only have to pour a slightly bluish solution into a wide and a narrow test-tube respectively, when we see that the former will show a much deeper blue colour than the latter. A faulty diagnosis would thus result.
The following cases are possible. The usual result of the reaction is either, dialysate of serum and of serum + organ negative, in which case no decomposition has taken place; and, if placenta had been used, we should assert that there was no placenta in the living state that had any connection with the particular organism; or else serum alone gives negative, and organ - serum positive, results. The diagnosis would indicate pregnancy, or, better still, the existence of a placenta that still stands in effective relations with the organism of the mother.
It may happen that the serum alone gives off substances to the dialysate, in sufficient quantity for the positive reaction to appear under the conditions ​selected. If, in such a case, the sample of organ + serum shows a markedly stronger blue coloration, then the case has to be looked upon as positive in respect to the reaction. Should, however, the difference in the intensity of the coloration be very small, the experiment has to be performed again, using a less quantity, say 1 c.c., of serum. It would then be possible to ascertain whether decomposition had taken place or not, as the serum sample would be negative.
The appearance of the reaction should on no account ever be determined by artificial light. Again, it is not advisable to compare the test-tubes in their stands, but each one should be taken out separately, and examined against white paper by both transmitted and reflected light.
Breaches of this rule are very often committed. Many reactions are declared positive which, when thoroughly investigated, show not the slightest coloration. If a sample is marked as being just perceptibly positive, then a number of other samples should be changed about in the hand, and, only if the same sample can be unhesitatingly picked out as showing a coloration, should one's judgment concerning the reaction be relied on.
Difficulties are only experienced with reddish and yellowish-brown tones, but these have no relation whatever with the ninhydrin reaction. They can ​easily be recognized by diluting a truly violet solution with water until the intensity of the colour corresponds with that of the sample, when one can see at once that, though the solution has been very much weakened, the colour still appears violet. A reddish, or, rather, yellowish-brown tint means that either the work has not been properly carried out, or else that the blood contained acids or alkalies in excess. The experiment must be repeated, otherwise it may happen that the existing conditions conceal a positive reaction. We shall deal with this point in fuller detail, when we return to the question of the sources of faulty observations.
Under certain conditions a special control test may be needed. Such would be the case in dealing with micro-organisms cultivated on a medium which could not be readily separated by centrifuging. In this case the germ-free medium must be boiled by itself, until the filtered boiled water gives no traces of coloration with ninhydrin. Then the cultures are prepared in exactly the same way, and the following tests are performed: (i) Serum alone; (2) serum + medium; and (3) serum + culture. Should the experiment (2) produce decomposition, then a positive reaction in experiment (3) would certainly not prove that the micro-organisms had been decomposed.
A very important control test, for proving the suitability of the organ or the substrate used, is the ​following: About five to ten times more of the substrate than has been used for the test is taken together with 5 c.c. of water, and the whole is dialysed in an incubator for sixteen hours, against 20 c.c. of distilled water. Then the dialysate is evaporated on the water bath to 5 c.c., and the latter is boiled in the usual way with 1 c.c. of ninhydrin solution. The solution must remain absolutely colourless. According to my own experience this test always results negatively, when the substrates have been prepared in accordance with the directions. It is only necessary for the first testing of the organ, and is carried out if doubts arise as to the suitability of the latter. As the same organ is always used over and over again for experiments in which no decomposition is expected, we have a concurrent control over the suitability of the organ. Should errors occur in these experiments, then the dialysing tubes are immediately tested, as well as the organ, in the manner laid down. The statement that for the control experiment organ alone was used—0.5 gr. of the organ—and that 10 c.c. of the dialysate have given a negative result, always proves that the principles of the whole method have been misunderstood. An organ must have been very unsatisfactorily prepared, if the 20 c.c. of the dialysate contain such a quantity of substances reacting with ninhydrin that the reaction, after being conducted in the usual way, gives positive results.
​We have described the performance of the experiment as it is carried out at the present time. Previously it was usual to make use of the biuret reaction for proofs of decomposition of albumen. To 10 c.c. of the dialysate 2.5 c.c. of a 33 per cent. solution of caustic soda were added, and this was then covered with a layer of very dilute copper sulphate solution. (See here p. 155.) If a violet to red ring appeared, the reaction was recorded as positive.
The biuret test has been entirely given up for the ninhydrin test, because the majority of observers have a difficulty in distinguishing with certainty a feeble biuret reaction. Those, however, who are capable of distinguishing a biuret reaction, however slight, should adhere to this test as well under all conditions.
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