cal normal equilibrium of the tissue and tissue elements and when the restraint one tissue has upon another is removed. According to him the tendency for cell proliferation lies entirely within the cell and it is prohibited from abnormal growth by its environment. This theory held prominence for a long time.

Ribbert acknowledges many factors in addition to cell pressure as being responsible for this restraint, such as the relationship of the cell to vessels and nerves and its differentiation for functional purposes. He regards new growth on the part of the cell as being initiated by one and all the disturbances in function or cell relationship which disturb the equilibrium between the cells forming tissues. Like Weigert, he is of the opinion that cells proliferate from a tendency inherent within the cell and that stimuli from without do not initiate growth.

Morgan also states that the power of regeneration is closely related to the power of growth inherent in the protoplasm.

Adami, on the other hand, claims that stimuli from without are responsible to a great extent for cell proliferation, as, for example, when an amoeba ingests food particles it does so in consequence of an external stimulus, this stimulus preceding assimilation.

There can be no doubt that various stimuli do influence growth. Increase of temperature accelerates growth. Bizzozero and Penzo have shown that if one ear of a rabbit be kept at a temperature of 12° to 15° C. and the other at 37° to 39° C. the latter will outgrow the former in a comparatively short time.

Certain chemicals and toxins stimulate the growth of cells. It is known to pathologists that an endothelial swelling and proliferation of lymph glands occurs in certain fevers and after burns. Loeb has demonstrated the effects of elements such as calcium, magnesium, potassium ions, etc., upon the development of certain sea animals. He has shown also the relation and effects of heliotropism, light, geotropism, and oxygen to regeneration and growth.

The relation of water and osmotic pressure has a very important bearing upon regeneration and proliferation. Bezold,105 Davenport, 108 Schaper, 107 Loeb,108 Bialaszewicz,100 and Morgulis 110 have contributed to our knowledge of these factors to growth. The observations of these men, among others, demonstrate beyond doubt that external stimuli are important factors in the causation of cell proliferation in many forms of animal life and doubtless play a very important rôle in all cell growths.

The capacities for regeneration among animal-life forms may be summarized as follows:

(1) The lower and more simple the animal forms are the greater and more complete the capacity for regeneration. This can be accepted only in a general sense, as numerous exceptions to this rule occur.

(2) Regeneration is more complete in younger animals. Embryos have far greater powers of regeneration than do adults.

(3) In higher organisms: (a) Cells of different orders exhibit different degrees of specialization and differentiation. It is the less differentiated cells which regenerate most easily. (b) The higher and more specialized the tissue the less its capacity for regeneration. (c) If an organ is completely removed it can not regenerate.

A review of the literature on the various types of regeneration and tissue growths, both within and without the body, having a direct bearing on our problem has been made as fully as was thought essential. A brief summary of our knowledge regarding metaplasia was included also, as it is believed that this may play an important rôle in tissue proliferation in culture media.

A discussion of the results obtained from investigations conducted in this laboratory, with the technique used, may now be considered.

PART II.

TECHNIQUE.

1. CULTURE PREPARATIONS.

In beginning these experiments the technique of Carrel and Burrows was used, namely, the collection of blood in paraffined test tubes, which were surrounded by ice, followed by centrifugalization and removal of supernatant plasma into other paraffined tubes, which were placed in refrigerator until time for using. This method works very well, so far as we have tried it, with the lower forms of vertebrates; also, in certain mammals, such as the cat and dog, but it was found that the blood of the guinea pig has a tendency to such rapid coagulation that one could not be positive just when appropriate plasma was obtainable. Coagulation was far more frequent than was the desired noncoagulation, occurring about nine times out of ten in spite of every precaution.

Attempts were made to reduce this tendency by various methods. In the first place, an all-green diet was given the animals, as it has long been known that a diet composed chiefly of cereals, such as oats, has a tendency to increase the coagulation time. Noticeable desired results followed, but still it was difficult to secure noncoagulated plasma in most cases. Intravenous injections of peptone (Howell) in the proportion of 0.3 gram per kilogram of the animal's weight were tried. This method retarded coagulation, but required a great deal of time, as the animal must twice be prepared with all the care necessary for a strictly aseptic surgical operation, and even then one could not be certain of obtaining the desired liquid plasma. Also, the animal often died from the effects of the peptone.

The use of sodium citrate to counteract the rapid coagulation was suggested by Dr. Anderson, director of the Hygienic Laboratory, and proved satisfactory. The minimum citrate necessary for the desired effect was determined, as well as the minimum amount of calcium chloride to coagulate the citrate plasma when cultures were ready for it. Molecular sodium citrate solution (709.2 grams of sodium citrate in 1 liter of physiological salt solution) and a molecular solution of calcium chloride (111.01 grams in 1 liter of physiological salt solution) were made. By a series of experiments it was found that, as a minimum, 1 c. c. of the molecular citrate solution prevented 9 c. c. of guinea pig's blood from coagulating for an indefinite period and

that 0.5 c. c. of the molecular calcium chloride solution was sufficient to coagulate the plasma after the blood cells had been removed by centrifugalization.

In the above proportion the amount of citrate used was 0.7092 gram for 9 c. c. of blood, while for the coagulation of this plasma 0.0555 gram were sufficient for 9 c. c. of blood. The use of citrate and calcium in these proportions did not retard the growth of tissues to any appreciable extent, nor did it necessitate the use of ice-cold paraffined tubes-ordinary sterile test tubes sufficed.

Because of the inhibitory action which these substances have upon growths they can not be used in greater proportions than those given above. The use of calcium may be entirely obviated by using only 0.5 c. c. of the above citrate solution. In this case ice-cold paraffined tubes are used, as described later.

The usual process for collecting blood was as follows: Large guinea pigs weighing from 500 to 800 grams were used. The animal was etherized, the hair of the chest removed by clipping and shaving, and the skin rendered aseptic. A needle of a sterilized syringe was projected into the heart and 9 c. c. of blood was drawn into the syringe, which contained 1 c. c. of the citrate solution already described when calcium was to be used for coagulating the plasma. The syringe. was sufficiently agitated to permit of general diffusion of the citrate throughout the blood. The contents were then forced equally into two sterilized test tubes and were immediately centrifugalized. After this the supernatant plasma was removed by means of a graduated pipette and was placed in other sterile test tubes-1 cubic centimeter of plasma in each tube. The plasma remained in this condition until time for use.

When only 0.5 c. c. of the normal citrate solution was used, as a preventive against coagulation the blood was forced immediately from the syringe into paraffined tubes surrounded by ice. These tubes were packed with ice in the cups of a large centrifuge. After centrifuging, the supernatant plasma was transferred to other cold paraffined tubes and was kept in an ice chest until time for using. It coagulated, as a rule, in about 10 minutes after dropping on tissues.

By either of these methods an animal may be bled a number of times at intervals of one week, for the projection of the needle into the heart does not affect it. After removing the blood 10 c. c. of warm sterile salt solution was injected intraperitoneally and the animal soon regained its normal condition. By too frequent bleeding, however, the coagulative properties of the blood are increased to such an extent that even in a second bleeding an increase of citrate is necessary. About twice the original amount of citrate must be used in the third bleeding if it is done in less than a week after the