As shown in the Figure 1, the temperature in the nests gets below the freezing temperature of painted turtles (represented by the dotted line) during the winter when hatchling turtles remain in the nest. Until at least the mid-1990s, biologists believed that painted turtles survived these freezing temperatures by freezing.

Figure 1: Temperature inside a painted turtle's nest from December to March. Dashed line represents equilibrium freezing point of neonatal animals. From Packard and Packard 2001. Reproduced with permission of American Institute of Biological Sciences. http://giorgio.ingentaselect.com/vl=7811771/cl=35/nw=1/fm=docpdf/rpsv/cw/aibs/00063568/v51n3/s5/p199

According to one study, painted turtles' body fluids freeze when the temperature is lowered to -3.06*C. As the temperature is lowered further to -3.89*C, the ice formation moves inward from the skin to the core of the turtles. When freezing is complete, about 53% of their body water is frozen, there is no heartbeat, and only minimal brain activity can be monitored. The ice formation is restricted to extracellular fluids, for ice formation in the cells would destroy the organization and structure of the cells, thereby killing them and the turtles. The turtles all can recover fully after 24 hours at this temperature (Storey and Storey 1992). According to further Storey and Storey studies, the turtles could withstand this temperature for 11 days.

Another study looked further into the adaptations that allowed the freezing by using proton MRI. Most freeze tolerant animals have high concentrations of sugar compounds, called colligative cytoprotectants, that reduce the amount of ice that can form. This reduction in ice formation is necessary because when the water freezes outside the cell, the osmotic pressure increases, so water flows out of the cell and solute moves in to equalize the pressure. This leads to reduced cell volume which can harm the animal. With the cytoprotectants, and the consequential decrease in the amount of ice, the reduction of cell volume is not as severe. However, painted turtles do not show a significant increase in the concentration of the normal cytoprotectants glucose and lactate. Instead, water remains around glycogen particles in frozen turtles. Turtles used this noncolligative method instead of the colligative cytoprotectants. This leads to some other differences in the freezing, like the thawing does not occur "from the inside out," but uniformly, meaning the core organs do not thaw earlier (Rubinsky et. al. 1994).