The vacuolar surface of Nitella is covered with a non-aqueous film too thin to be visible as a separate membrane. The motion of the protoplasm may subject this film to a good deal of mechanical disturbance.

Apparently this does not rupture the film for no dye escapes into the protoplasm as the result of such disturbance when the vacuolar sap is deeply stained with neutral red or brilliant cresyl blue.

When the deeply stained central vacuole breaks up into several smaller vacuoles, leaving the outer protoplasmic surface in its normal position, there is no evidence of the escape of dye into the protoplasm through the film surrounding the vacuole.

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