Heat Inactivation Procedure

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1. Purpose
The following protocol is suggested for the heat inactivation of fetal bovine serum ( FBS ). FBS is heated to 56± 2°C for 30± 2 minutes in agitating water-bath.

2. Definition

Heat Inactivation (HI) – a process by which FBS is maintained at a temperature of 56± 2° for 30± 2 minutes.

3. Equipment

  • Water bath capable of maintaining temperatures of 56± 2°C. Preferably a agitating water bath.
  • Calibrated thermometers adept of measuring 56°C.

4.  Materials

  • Control bottles should be equivalent to the product bottle. Controls bottles are volume to the same level as the product being heat inactivated and fitted with a thermometer suitable for monitoring 56°C. The thermometer should not touch the sides or bottom of the bottle.
  • Circular weights to ensure bottles are stable.

5. Specifications

The temperature of the control must be 56± 2°C for 30 ± 2 minutes during the heat inactivation process.

6. Precautions

For laboratory use only.

7. Procedure

7.1 Thaw FBS.

7.1.1 If serum was thawed in a refrigerator allow serum to come to room
temperature prior to placing in water bath.

7.2 Fill the water-bath with sufficient water so that the product and control
bottles are immersed near the serum level.
7.3 Set water-bath temperature to maintain the product at 56± 2°C
7.4 Place a lead weight on the control bottle and place into the center of the water
7.5 Mix the contents of the product bottles using a gentle swirling motion until
the product is uniform.
7.6 Place circular weights over the tops of the bottles to keep them upright.
7.7 Place the bottles in the water-bath.
7.8 Swirl bottle thoroughly every 8-10 minutes or if applicable turn on the
oscillating shaker unit. Check the temperature of the control bottle frequently as the temperature approaches 56± 2°C.
7.9 When the temperature of the control reaches 56± 2°C start the timer for 30 minutes. If a shaking water bath is not available, ensure bottles are swirled every 8-10 minutes during the entire process.
7.10 After 30 minutes turn off the oscillating shaker and remove the bottles from the water bath. Cool to room temperature ( 1 hour ) and either aliquot or return to the freezer.