Several factors can influence the total time that serum is exposed to high temperatures. Glass and plastic (PETG) differ in their heat capacities which directly affects heating rates. To reduce breakage and facilitate storage, plastic bottles have largely replaced borosilicate glass bottles. These plastic bottles increase the time required for the contents to reach 56°C by approximately 30 percent. This insulating effect also increases the amount of time required for the contents of plastic bottles to cool following heat inactivation. The cooling rate also depends on whether the serum is placed into a refrigerator, freezer, or ice bath following heat inactivation (Figure 5). Heat-inactivated serum required 30 minutes to cool to a temperature of 10°C in an ice bath versus 330 minutes when simply placed into a refrigerator. Dividing the serum into aliquots facilitates heating and cooling. The water level in the heat inactivation bath will also affect heating rates of the serum. It is often inconvenient to fill the water bath such that it equals the level of serum in the bottle since the bottles will have a tendency to float. Floating can be prevented by placing commercially available lead weights around the necks of the bottles. Figure 6 illustrates the heating rates in water baths in which levels were equal to either the 300 ml or the 500 ml graduation of a standard plastic bottle. At the 500 ml graduation, the serum required 40 minutes to reach 56°C. At the 300 ml graduation, the serum required 60 minutes to reach 56°C thus increasing the total exposure time to elevated temperatures.