20 
AMERICAN JOURNAL OF BOTANY 
[Vol. 9 
(17-34 microns), the escaping protoplasm immediately diffused in the 
water, rendering it very difficult to estimate the extent of the protoplasmic 
loss. In a few cases, when the hole was very small (17-34 microns), a 
delicate film formed over the globule of protruding protoplasm. This 
globule continued to increase in size, apparently by the absorption of water. 
The only movement of chloroplastids loosened by the puncturing 
occurred within a distance of 34 to 68 microns from the margin of the wound. 
Even when the hole was very small, rotation of the protoplasm immediately 
ceased and was not resumed. The original turgidity was also lost and was 
not regained. This was clearly shown by the invagination of the cross 
walls from the pressure of the adjacent cells. 
Nitella. The internodes of Nitella proved to be unusually favorable 
material for the study of punctured cells. By puncturing at intervals of 
five minutes, it is possible to puncture an internode several times without 
disastrous results. Cells less than one centimeter in length I have punctured 
six or seven times. Each puncture was followed by a momentary pause 
in the protoplasmic movement. Even when the puncture was large and 
deep, with a corresponding loss of protoplasm, the regular rotation was 
resumed in less than two minutes. After the sixth and seventh punctures 
the pause was slightly longer than at first. Ultimately the successive 
drains on the protoplasm result in the cell losing its turgidity; and when 
that occurs the wall bends in front of the needle, rendering it difficult or 
impossible to puncture successfully. Although the cell may not be suffi- 
ciently turgid to puncture, this condition does not seem to cause any retarda- 
tion in the healing of the previous wounds. The large size of the internodes 
and the regular arrangement of the plastids, as well as the moving proto- 
plasm, aid in estimating the effect of the wounds. 
Each puncture results in a rapid outflow of the cell contents. The 
protoplasm does not diffuse in the water, but forms globular or ovoid masses 
enclosed by a delicate film just outside the opening (PI. Ill, fig. i). The flow 
is slightly spasmodic — periods of rapid flow interrupted by brief pauses re- 
sulting in the formation of a number of separate globules. The plastids 
and other particles carried through the opening continue to move outward 
until they are lodged against the enclosing film. At first this film is ex- 
tremely delicate and can be detected only by careful adjustment of the 
light or by staining. Gradually the entire mass of globules assumes a 
brownish tint which brings the films into sharp contrast with the surrounding 
water. After a few days this protruding mass disintegrates and disappears. 
When the cell is punctured, the chloroplastids immediately surrounding 
the opening are torn from the plasma membrane (fig. i), and either pass 
through the opening with the outflowing protoplasm, or are carried along 
in the rotating protoplasmic stream. The amount of internal disturbance 
caused by the wound is impossible to determine definitely, but some slight 
impression of it may be gained by observing the movement of granules. 
