Defensive Ferments of the Animal Organism/Methods in Use/Preparation of Peptones for use in the Optical Method
Preparation of Peptones for use in the Optical Method.
Organs are first deprived of their blood, in exactly the same way as has been described on p. 164. They can then be subjected directly to hydrolysis, after the pieces of tissue have been dried, as much as possible, between filter papers. If it is desired to collect larger quantities of the same tissue, then the tissue, freed from blood, is boiled for ten minutes in water, and is subsequently preserved in sterilized water with chloroform and toluol. It is, of course, not necessary, in this case, to boil the organ to such an extent, as to deprive it of all substances reacting with ninhydrin. Boiling is merely resorted to here, in order to destroy any cell ferments that may be present; otherwise autolysis may manifest itself. As soon as enough of the organ has been collected, then it is similarly freed from water, as far as possible, before being placed in sulphuric acid, which is kept cool by means of ice. Nervous tissue, after it has been deprived of all blood and boiled, must first be extracted with carbon tetachloride, as otherwise its lipoidal sheath makes decomposition very difficult. Tubercle bacilli must also be freed from lipoids.
For hydrolysis, we use 70 per cent. (by weight) of sulphuric acid, which must be cold. We take three times as much of this, as of the tissue to be decomposed. The vessel is energetically shaken, and then carefully stoppered. From time to time it is shaken again. The tissue is soon dissolved, the solution becoming more or less brown. After standing for exactly three days, at the temperature of the room (20° C. at most), the vessel containing the hydrolysate is placed into iced water, and diluted with ten times its quantity of distilled water. The addition must be made very gradually. The temperature of the solution is controlled by means of a thermometer, and must never be allowed to rise above 20° C. If the vessel is too small, then the solution is transferred into a larger one, and the water with which we are diluting is used to rinse the first vessel.
We now begin the neutralization of the sulphuric acid with barium hydroxide. Pure crystalline hydroxide is employed for this purpose, and this is gradually added, until the solution gives no precipitate, either with barium hydroxide solution or with sulphuric acid. In the test with barium hydroxide it may happen, that a precipitate appears, even though no more sulphuric acid is present. These are barium salts of peptones, which separate out. They can be dissolved in nitric acid, while barium sulphate is insoluble in this.
Neutralization is carried out in such a way, as to calculate the quantity of barium hydroxide necessary, by the amount of sulphuric acid used. The barium hydroxide is best added in the solid form, and is well stirred until the action is complete. The neutralization of the sulphuric acid is first tested by means of litmus paper. Finally, small samples are filtered through a small funnel,[1] and then one sample is tested with barium hydroxide,[2] and another with sulphuric acid. If, in the first case, the solution becomes turbid, or precipitates are formed, then nitric acid is added, and the solution is slightly warmed. If the sediment remains, it is a sign that more barium hydroxide is to be added to the original solution. It is advisable, always to work with very dilute solutions of sulphuric acid and barium hydroxide, otherwise one may easily overshoot the mark.
When the solution is free from sulphuric acid and baryta, we proceed to filter it through a doubled sheet of folded filter paper, or, by means of a filter pump, through a hardened filter impregnated with animal charcoal. This process can be hastened by the use of a centrifuge. The precipitate of barium sulphate is stirred up with distilled water, well kneaded in a mortar with water, and then filtered again. It is advantageous, in order to ensure a good output of peptone, to repeat this washing out with cold water many times. The ninhydrin test can be applied at this stage, as a test of the satisfactory washing out of the precipitate. To a portion of the filtrate about 1 c.c. of ninhydrin is added, and the mixture is boiled for one minute. If the coloration is faint, or even negative, then the process of washing-out is discontinued.
In the meantime, the process of concentration has been begun. As solutions of peptones produce a great deal of scum, the apparatus represented in fig. 9 is used. The latter allows the peptone solution to evaporate to dryness, at about 40° C., under highly reduced pressure. The drop funnel serves the purpose of conducting the peptone solution, in drops, into the flask. These drops evaporate immediately, and no scum is formed.
Fig. 9.
The peptone solution must never be strongly evaporated, until we have repeatedly satisfied ourselves, that it is actually free from sulphuric acid and barium. With very dilute solutions traces of these compounds may escape detection. During the concentration of the solution, that of the sulphuric acid and barium hydroxide naturally increases, so that we may eventually get an hydrolysis of the peptone mixture.
Finally, we are left with a light yellow syrupy residuum. The latter is mixed with about 100 times its amount of methyl-alcohol, and the mixture is boiled. The boiling hot solution is filtered through a filter paper into about five times its amount of cold ethyl alcohol. It is well to put the latter into iced water. Precipitation is aided by the addition of ether. The whole is filtered, directly the precipitate begins to be formed. During the filtration, the filter should not be allowed to become empty. It is best to use a filter pump. Only at the end is the liquid allowed to pass entirely through the filter, after which the latter is immediately placed in a vacuum exsiccator. After a day or two the peptone is absolutely dry, and may then be weighed. First, a 10 per cent. solution, in 0.9 per cent. solution of common salt, is prepared, and the deviation of rotation of the solution is determined. If this is more than 1°, the solution is diluted, until it shows a rotation of about 0.75°. The higher degree of rotation would not be injurious. Dilution is only effected in order to make the best use of the costly material.
Standardization of the Peptone.—Let us assume that we have to deal with placenta peptone. This is mixed with the serum of individuals, who are certainly not pregnant, and then there should be no alteration of the original rotation. Should this not be the case, then the peptone is certainly not free from sulphuric acid or barium. With the serum of pregnant individuals a decomposition is bound to take place. At first, readings are taken every hour, and tests are made with many sera. A normal curve for the peptone is constructed, from the separate readings, by marking the angle of rotation on the abscissa and the time on the ordinate (cf. the curves given on pp. 62, 64, and 75-77). Once the normal alterations of rotation of the serum peptone mixture are known, then the readings for the diagnosis of normal cases need only be taken every four to six hours. If one has a special object in view, then the readings are taken more often.
The optical method supplements the dialysation process in many directions. In the first place, it is possible to determine quantitative differences in the speed of the decomposition. Further, qualitative differences may be observed. In the dialysation process, on the other hand, the dialysate may be used for experiments on animals and, for instance, be injected, in a state of concentration, for the purpose of deciding, whether certain products of decomposition, contained in it, have a toxic effect.
To determine the range of rotation, a perfect instrument is necessary. The polarizing apparatus of Schmid and Hänsch, of Berlin, is one that answers
Fig. 10.
(a) Ocular for taking readings; (b) polarization tube; (c) sodium flame; (d) for illumination; (e) battery.
all the requirements (fig. 10). It allows of readings to the hundredth part of a degree. Since everyone makes individual errors in taking readings, i.e., the range of rotation of the same solution is differently observed, it has to be determined how great the limits of error are, on the average. It has been found, that most observers are capable of reading with accuracy to 0.02 of a degree. In order to attain greater certainty, we shall consider even a difference of 0.04 of a degree as the limit of error. Only with a change of rotation of 0.05 of a degree can decomposition be assumed to have taken place. The limit can thus be fixed without any danger, because, when an hydrolysis of the peptone does take place, the alteration of rotation is certainly more than 0.04 of a degree.
This method, as such, presents hardly any sources of error. At most, errors may be occasionally produced through turbidity, precipitates, and the like. Fortunately, however, in such cases, which actually very seldom happen with proper working, the reading of the rotation is impossible, and so this source of error disappears of itself. Of course, we should get no result if we were to try to polarize a cloudy solution.
A very important source of error would arise, if the range of rotation of the cold solution were taken for the initial value. The readings must be taken from the moment the contents of the tubes reach a temperature of 37° C. It is best to take the reading at the end of one hour, and take another at the end of the second hour. Values obtained in such a manner should, in general, not be too distant one from the other, as decomposition begins, and manifests itself for a certainty, only after about six hours. Readings should not be followed up for more than thirty-six to thirty-eight hours.
Great progress would be made, if the reading of angles of rotation could be taken by means of some kind of automatic device. Objective values could be obtained, and we should be in a position to follow
Fig. 11.
up details which, during the long intervals between readings, at present escape observation. Experiments in this direction, with the collaboration of Dr. Wildermuth, are in progress.
To obviate the cooling of the polariscopic tubes, during polarization, tubes have been constructed with water jackets (fig. 11). Lately, an electric heating apparatus[3] has been added to the polariscope itself. The former holds six polarization tubes, which can be brought into the field of observation without opening the heated incubator. In this way, all variations of temperature are avoided during the test. (See Plate.)
The most important source of error lies in the observer himself. The eye soon becomes tired, and it is impossible to take many readings at a time. One must become so experienced, as to be able to record a reading in at least thirty seconds. So soon as the eyes become weary, the readings become dubious. It is not advisable to resort to the optical method, until one has attained to sufficient certainty in taking readings. 
- ↑ If there be a centrifuge at one's disposal, then we recommend centrifuging samples of the mixture. In this way a clear solution is obtained immediately without any loss of material.
- ↑ For testing purposes an aqueous solution of barium chloride gives better results than barium hydroxide, because the baryta water becomes turbid, owing to its affinity for carbonic acid, with consequent formation of barium carbonate. When using the above solution, the sample employed must never be returned to the original solution, but must be thrown away.
- ↑ Emil Abderhalden: "Ueber eine mit Polarisations-apparat kombinierte elektrisch heizbare Vorrichtung zur Ablesung und Beobachtung des Drehungsvermögens bei konstanter Temperatur. Zeitschr. f. physiol. Chem., 84, 300 (1913).