Physical Control of Microorganisms: Boiling Water

Introduction and Advantages:

Immersion in boiling water is the first of several moist-heat methods that we shall consider. Moist heat penetrates materials much more rapidly than dry heat because water molecules conduct heat better than air. Lower temperatures and a shorter exposure time are therefore required than for dry heat.

Mode of Action:

Moist heat kills microorganism by denaturing their proteins. Denaturation is a change in the chemical or physical property of a protein. It includes structural alterations due to destruction of the chemical bonds holding proteins in a three-dimensional form. As proteins revert to a two-dimensional structure, they coagulate (denature) and become nonfunctional. Egg protein undergoes a similar transformation when it is boiled. Details in chap 2. The coagulation of proteins requires less energy than oxidation, and, therefore, less heat need be applied.

The effectivenesss of different method according to temperature range and time of sterilization

Conditions for perfect Sterilization:

If it imperative that boiling water be used to destroy microorganisms, materials must be thoroughly cleaned to remove traces of organic matter, such as blood or feces. The minimum exposure period should be 30 minutes, except at high altitudes, where it should be increased to compensate for the lower boiling point of water. All materials should be well covered. Washing soda must be added at a 2 percent concentration to increase the efficiency of the process.

Disadvantages and Microbes Heat Resistance:

Boiling water is not considered a sterilizing agent because the destruction of bacterial spores and the inactivation of viruses cannot always be assured. Under ordinary circumstances, with microorganisms at concentrations of less than 1 million per milliliter, most species of microorganisms can be killed within 10 minutes. Indeed, the process may require only a few seconds. However, fungal spores, protozoan cysts, and large concentrations of hepatitis A viruses require up to 30 minutes exposure while endospores can resist boiling for more than 20 hours. Bacterial spores often require 2 hours or more. The effectiveness of boiling can be increased by adding 2 % sodium bicarbonate to the water. Because inadequate information exists on the heat tolerance of many species of microorganisms, boiling water is not reliable for sterilization purposes. Free-flowing (unpressurized) steam is equivalent in temperature to boiling water.

Uses of Boiling:

The use of boiling to sanitize baby bottles is a familiar example.

Physical Control of Microorganims: Dry Heat Methods

The Direct Flame:

One of the simplest methods of dry heat sterilization is direct flaming.

Mode of Action :

Dry heat probably does most of its damage by oxidizing molecules. Moist heat destroys microorganism mainly by denaturing proteins; the presence of water molecules helps disrupt the hydrogen bonds and other weak interactions that hold proteins in their three-dimensional shapes. A simple analogy is the slow charring of paper in a heated oven, even when the temperature remains below the ignition point of paper.

Procedure:

To effectively sterilize the inoculating loop, one must heat the wire to a red glow. The flame of the Bunsen burner is employed for a few seconds to sterilize the bacteriology loop before removing a sample for a culture tube and after preparing a smear as shown in (figure A) below.

Figure A: Indicating Dry Heat Sterilization Using Bunsen Burner

Applications:

Perhaps the most rapid sterilization method, to sterilize and dispose of contaminated paper cups, bags, and dressings, is the use of a direct flame in the process of incineration. It is still common practice to incinerate the carcasses of cattle that have died of anthrax and to put the contaminated field to the torch because anthrax spores cannot adequately be destroyed by other means. Indeed, British law stipulates that anthrax-contaminated animals may not be autopsied before burning.

The Hot-Air Oven:

Mode of Action:

The effect of dry heat on microorganisms is equivalent to that of baking. The heat changes microbial proteins by oxidation reactions and creates an arid internal environment.

Procedure:

Items to be sterilized by this procedure are placed in an oven. Generally, a temperature of about 170^oC maintained for nearly 2 hours ensures sterilization. The longer period and higher temperature (relative to moist heat) are required because the heat in water is more readily transferred to a cool body than is the heat in air. For example, imagine the different effects of immersing your hand in boiling water at 100^oC (212oF) and of holding it in a hot air oven at the same temperature for the same amount of time.

Disadvantages:

The hot-air oven utilizes radiating dry heat for sterilization. This type of energy does not penetrate materials easily, and therefore, long period of exposure to high temperatures are necessary. For example, at a temperature of 160^oC, a period of 2 hours is required for the destruction of bacterial sores. Higher temperatures are not recommended because the wrapping paper used for equipment tends to char at 180^oC.

Uses of Hot Air Ovens:

The hot-air method is useful for sterilizing dry powders and water-free oily substances, as well as for many types of glassware, such as pipettes, flasks, and syringes. Dry heat does not corrode sharp instruments as steam often does, nor does it erode the ground glass surfaces of nondisposable syringes.

Important Factors to Determine the Exposure Time:

Thereby burning microorganisms removed from the materials, because organic matter insulates against dry heat. Moreover, the time required for heat to reach sterilizing temperatures varies according to the material. This factor must be considered in determining the total exposure time.

Physical Control of Microorganisms: Heat Sterilization

Introduction and Importance:

The killing effect of heat on microorganisms has long been known. Heat is fast, reliable, and relatively inexpensive, and it does not introduce chemicals to a substance, as disinfectants sometimes do. A visit to any supermarket will demonstrate that heat preserved canned goods represent one of the most common methods of food preservation. Laboratory media and glassware, and hospital instruments, are also usually sterilized by heat.

Mode of Action:

Above maximum growth temperatures, biochemical changes in the cell's organic molecules result in its death. These changes arise from alterations on enzyme molecules and the resultant changes to the three dimensional shape of proteins inactivate proteins or chemical breakdown of structural molecules, especially in cell membranes. Heat also droves off water, and since all organisms depend on water, this loss may be fatal. Heat appears to kill microorganisms by denaturing their enzymes.

Principles and Applications of Heat Killing:

Heat—Preferred Agent of Sterilization:

Heat is preferred agent of sterilization for all material not damaged by it. It rapidly penetrates thick materials not easily penetrated by chemical agents.

Measurements to Determine Killing Power:

The killing rate of heat may be expressed as a function of time and temperature. For example, tubercle bacilli are destroyed in 30 minutes at 58^oC, but in only 2 minutes at 65^oC, and in a few seconds at 72^oC. Several measurements have been defined to quantify and killing power of heat. The thermal death point is the temperature that kills all the bacteria in a 24-hour-old broth culture at neutral pH in 10 minutes. Factor to be considered in sterilization is the length of time required.The thermal death time is the time required to kill all the bacteria in a particular culture at a specified temperature. Both TDP and TDT are useful guidelines that indicate the severity of treatment required to kill a given population of bacteria.

Decimal reduction time (DRT or D value) is a third concept related to bacterial heat resistance. DRT is the time, in minutes, in which 90% of a population of bacteria at a given temperature will be killed (in table 1, DRT is 1 minute). (The temperature is indicated by a subscript: D80°C, for example.)

Table 1	Microbial Death Rate: An Example
Time (mm)	Death per Minute	Number of Survivors
0	0	1,000,000
1	900,000	100,000
2	90,000	10,000
3	9000	1000
4	900	100
5	90	10
6	9	1

Significance of Measurements:

These measurements have practical significance in industry as well as in the laboratory. For example, a food-processing technician wanting to sterilize a food as quickly as possible would determine the thermal death point of the most resistant organisms that might be present in the food and would employ that temperature. In another situation it might be preferable to make the food safe for human consumption by processing foods containing proteins that would be denatured, thereby altering their flavor or consistency. The processor would then need to know the thermal death time at the desired temperature for the most resistant organism likely to be in the food.

Factors Important for Determination of Time and Temperature:

When determining the time and temperature for microbial destruction with heat, certain factors bear consideration.

1) Type of Organism:

One factor is the type of organism to be killed. For example, if materials are to be sterilized, the physical method must be directed at bacterial spores. Milk, however, need not be sterile for consumption, and heat is therefore aimed at the most resistant vegetative cells.

2) Type of Material:

Second factor is the type of material to be treated. Powder is subjected to dry heat rather than moist heat, because moist heat will leave it soggy. Saline solutions, by contrast, can be sterilized with moist heat but are not easily treated with dry heat.

3) Presence of Organic, Acidic or Basic Material:

Third important factor is the presence of organic matter and the acidic or basic nature of the material. Organic matter may prevent heat from reaching microorganisms, while acidity or alkalinity may encourage the lethal action of heat.

Physical Methods of Controling Microorganisms: (A Historical Perspective)

Some Earliest Methods:

As early as the Stone Age, it is likely that humans were already using some physical methods of microbial control to preserve foods. Drying (desiccation) and salting (osmotic pressure) were probably among the earliest techniques. Ancient Egyptians dried perishable foods to preserve them. Scandinavians made holes in the centers of pieces of dry, flat, crisp bread in order to hang them in the air of their homes during the winter; likewise they kept seed grains in a dry place. Otherwise, both flour and grains would have molded during the long and very moist winters. Europeans used heat I the food-canning process 50 years before Pasteur's work explained why heating prevented food from spoiling.

Control in the Modern Age:

Today, physical agents that destroy microorganism are still used in food preservation and preparation. Such agents remain a crucial weapon in the prevention of infectious disease. Physical antimicrobial agents include various forms of heat, refrigeration, desiccation (drying), irradiation, and filtration.

Factors Effecting the Selection of Method:

When selecting methods of microbial control, consideration must be given to effects on things besides the microbes. For example, certain vitamins or antibiotics in solution might be inactivated by heat. Many laboratory or hospital materials, such as rubber and latex tubing, are damaged by repeated heating. There are also economic considerations; for example it may be less expensive to use presterilized, disposable plasticware than to repeatedly wash and re-sterilize glassware.

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