For continued proliferation, heparin plasma or serum must be repeatedly supplied with the digests. Without them, multiplication continues for only a few days, the coagulum in which the cells are embedded liquefies around the central fragment, and the cells disintegrate.

The enzymatic digests exert a marked effect on the morphological appearance of the cells and finally cause them to agglutinate. The extent of this effect is determined by the degree of hydrolysis of the digest, its concentration, and the length of time the cells are cultivated in it. The morphological appearance of the cells is also somewhat influenced by the nature of the plasma or serum used with the digest.

Digests having very little free amino nitrogen produce short, round, granular cells. Those more highly hydrolyzed produce large, long, slender forms.

An increase in the concentration of the digest in the medium causes a shortening and broadening of the cell and an increase in its granulations. Therefore, even a highly hydrolyzed digest may, if concentrated, give cells resembling those in a lower concentration of a less hydrolyzed one.

The digests have a cumulative effect on the cells, as the time of cultivation is extended. Therefore, cultivation for a long period in a dilute solution may give the same effect as a shorter time in a higher concentration.

A different effect is obtained if plasma is used with the digest than if serum is used, even when the plasma and serum are taken from the same animal. The monocytes cultivated in serum and digest are generally shorter, broader, and more granular than those cultivated in heparin plasma and digest. They also contain more fat and have a greater tendency to digest the clot.

Agglutination of the cells takes place more readily in highly hydrolyzed products than in those slightly hydrolyzed. It is hastened by increase in concentration of the digest in the medium. It occurs more readily at the periphery of the culture and sooner in serum and digest than in heparin plasma and digest.

Completely hydrolyzed proteins and mixtures of pure amino acids do not produce effects at all comparable to those of the enzymatic digests either in their effect on the rate of cell proliferation or their action on the morphology of the cell.

Arsenic pentoxide in dilutions from 1:20,000 to 1:80,000 is the only other agent known to bring about agglutinations of the monocytes when cultivated in vitro.

The early changes in the morphological appearance of the cell that are produced by these digests are reversible. When the digests are removed from the medium and the cells cultivated in plasma and Tyrode solution, they very gradually revert to their original form.