Heating Methods

A variety of methods have been used for determining the heat resistance of a microorganism:

1. For liquids being heated at 100°C or below, a three-neck flask can be used. A thermometer is included in one neck of the flask, a stirrer in another, and the third is used to add microorganisms and to remove samples. This is a convenient, rapid method for determining the heat resistance of vegetative cells. Heating lag times are minimized because small volumes of inoculum are added to a large volume of preheated liquid, but splashing or flocculation may give erroneous results.

2. One of the most common methods uses thermal death time (TDT) tubes to which a given volume (usually 1-2 mL) of product containing the microorganism is added, and the tube is sealed and heated in an oil or water bath (depending on the heating temperatures). The procedure is useful for liquids and liquefied food homogenates. There are heating and cooling lag times (since heat must penetrate into the tube and heat the product) that must be considered, but a thermocouple in a replicate sample can be used to measure the temperature during heating and cooling to account for these lags in the TDT calculations. It is easy to heat many replicates at one time. Usually 10 tubes per time interval and five or six time intervals are heated at the same time at a single temperature, and the procedure is repeated at additional temperatures. In general, the TDT tubes will need to be opened and the contents subcultured in an appropriate growth medium or the food product. The procedure is suitable for vegetative cells and spores.

3. A similar method uses capillary tubes, which are much smaller versions of TDT tubes. Because of the small diameter and thickness of capillary tubes, heating and cooling lags are negligible. This procedure is limited to liquids, and the contents must be subcultured. The external surface of the tube must be sterilized prior to subculturing the contents, because the tube is generally crushed to release its contents. The procedure is suitable for vegetative cells and spores.

4. A specialized piece of equipment known as a thermoresistometer has been used to determine the heat resistance of microorganisms at very high temperatures (121-138°C in saturated steam and 121-260°C in superheated steam) for very short time intervals (seconds). The procedure is suitable for liquids or diluted food homogenates. Heating and cooling lag times are negligible, and the system allows for very precise timing, which is critical with high temperature-short time studies. There is a high initial cost for the equipment, and product must be subcultured for survivors. This procedure is suitable for spores.

5. Another specialized piece of equipment for TDT studies is the thermal death time retort. These retorts are used to heat either TDT tubes (containing liquid or food homogenates) or TDT cans (containing ground or homogenized foods) at temperatures of 100 to 121°C. Use of TDT cans closely simulates canning practices, and the cans can be incubated directly. Spoilage by gas producers is detected by swelling of the can. Heating and cooling lag times are substantial, and it is essential to account for the lethality that occurs by measuring the temperatures with a thermocouple inserted in replicate cans. The incubation time can be lengthy—several months to ensure all injured organisms are recovered. This procedure is suitable for spores.

Recovery of Survivors

The apparent heat resistance of an organism depends on the recovery medium, as well as the substrate in which it is heated. When testing the heat resistance of a microorganism in a particular food, recovery in the product is preferred, as this will reflect the environment the injured organisms would see after processing. This procedure measures whether or not there are organisms that can grow in the product after the heat treatment, not how many there are. Alternatively, the heated product can be subcultured to determine survivors. This may be either a qualitative procedure, where the heated product is transferred to a broth medium to determine if there are organisms that can grow, or a quantitative procedure, where the survivors in the heated product are enumerated. Use of a culture medium has several advantages. The media used generally allow better recovery of survivors, as the medium can be formulated to favor growth of the specific organism being tested, and components that enhance the recovery of injured organisms can be added. Also, organisms generally recover more quickly in the more favorable environment.

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