Hot Oil

Method:

Some dentists and physicians use hot oil at 160^oC for the sterilization of instruments. A time period of 1 hour is usually recommended.

Advantages:

Hot oil does not rust metals, and minimal corrosion takes place. However, once sterilization is complete, the instruments must be cleaned and dried for storage, and this steam may reintroduce contamination.

Another Method:

Silicone is sometimes used as an alternative to oil.

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Pasteurization

Introduction:

Louis Pasteur found a practical method of preventing the spoilage of beer and wine. Pasteur used mild heating, which was sufficient to kill the organisms that caused the particular spoilage problem without seriously damaging the taste of the product. The same principle was later applied to milk to produce what we now call pasteurized milk.

Purpose of Pasteurization:

Pasteurization is not the same as sterilization. Its purpose is to reduce the bacterial population of a liquid such as milk and to destroy organisms that may cause spoilage and human disease. Spores are not affected by pasteurization. The intent of pasteurization of milk is to eliminate pathogenic microbes. It also lowers microbial numbers, which prolongs milk's good quality under refrigeration. Many relatively heat-resistant (thermoduric) bacteria survive pasteurization, but these are unlikely to cause disease or cause refrigerated milk to spoil.

Pasteurization Methods:

In the classic pasteurization treatment of milk, the milk was exposed to a temperature of about 63^oC for 30 minutes, called the holding method. Most milk pasteurization today uses higher temperatures, at least 72^oC, but for only 15 seconds. This treatment, known as high-temperature short-time (HTST) pasteurization, is applied as the milk flows continuously past a heat exchanger. In addition to killing pathogens, HTST pasteurization lowers total bacterial counts, so the milk keeps well under refrigeration.

Milk can also be sterilized – something quite different from pasteurization --- ,Ultrahigh temperature (UHT) processing raises the temperature from 74 ° C to 140 ° C and then drops it back to 74 ° C in less than 5 seconds. In the United States, sterilization is sometimes used on the small containers of coffee creamers found in restaurants. To avoid giving the milk a cooked taste, a UHT system is used in which the liquid milk never touches a surface hotter than the milk itself while being heated by steam. The milk falls in a thin film through a chamber of superheated steam and reaches 140^oC and drops back to 74^oC .

Aims of pasteurization:

For decades, pasteurization has been aimed at destroying mycobacterium tuberculosis, long considered the most heat resistant bacterium. More recently, however, attention has shifted to destruction of Coxiella burnetii, the agent of Q fever, because these organisms have a higher resistance to heat. Since both organisms are eliminated by pasteurization, dairy microbiologists assume that other pathogenic bacteria are also destroyed.

Factors of Pasteurization:

Products other than milk, such as ice cream, yogurt, and beer, all have their own pasteurization times and temperatures, which often differ considerably. There are several reasons for these variations. For example, heating is less efficient in foods that are more viscous, and fats in food can have a protective effect on microorganisms.

Perfect Pasteurization Indicators:

The dairy industry routinely uses to test to determine whether products have been pasteurized: the phosphatase test (phosphatase is an enzyme naturally present in milk). If the product has been pasteurized, phosphatase will have been inactivated.

Concept of equivalent Treatment:

The heat treatments we have just discussed illustrate the concept of equivalent treatments: as the temperature is increased, much less time is needed to kill the same number of microbes. For example, the destruction of highly resistant endospores might take 70 minutes at 115^oC, whereas only 7 minutes might be needed at 125^oC. Both treatments yield the same result. The concept of equivalent treatments also explains why classic pasteurization at 63^oC for 30 minutes, HTST treatment at 72^oC for 15 seconds, and UHT treatment at 140^oC for less than a second can have similar effects.

Significance of Pasteurization:

Some years ago certain strains of bacteria of the genus Listeria were found in pasteurized milk and cheeses. This pathogen causes diarrhea and encephalitis and can lead to death in pregnant women. A few such infections have prompted questions about the need to revise standard procedures for pasteurization. However, finding these pathogens in pasteurized milk has not become a persistent problem, and no action has been taken.

Although most milk for sale in the United States is pasteurized fresh milk, sterile milk also is available. All evaporated or condensed canned milk is sterile, and some milk packaged in cardboard containers also is sterile. The canned milk is subjected to steam under pressure and has a "cooked" flavor. Sterilized milk in cardboard containers is widely available in Europe and can be found in some stores in the United States. It is subjected to a process that is similar to pasteurization but uses higher temperatures. It too has a "cooked" flavor but can be kept unrefrigerated as long as the container remains sealed. Such milk is often flavored with vanilla, strawberry, or chocolate.

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Fractional Sterilization

Introduction:

In the years before the development of the autoclave, liquids and other objects were sterilized by exposure to free flowing steam at 100^oC for 30 minutes on each of 3 successive days, with incubation periods between the steaming. The method was called fractional sterilization because a fraction was accomplished on each day. It was also called tydallization after its developer, John Tyndall.

Procedure:

Sterilization by fractional method is achieved by an interesting series of events. During the first day's exposure, steam kills virtually all organisms except bacterial spores, and it stimulates spore to germinate to vegetative cells. During overnight incubation, the cells multiply and are killed on the second day. Again, the material is cooled and the few remaining spores germinate, only to be killed on the third day. Although the method usually results in sterilization, occasions arise when several spores fail to germinate. The method also requires the spores be in a suitable medium for germination, such as a broth.

Importance in modern Age:

Fractional sterilization has assumed renewed importance in modern microbiology with the development of high technology instrumentation and new chemical substances. Often, these materials cannot be sterilized at autoclave temperatures, or by long periods of boiling or baking, or with chemicals. An instrument that generates free flowing steam, such as the Arnold sterilizer, is used in these instances.

Autoclaving: Real Sterilization

Preparation of items for Autoclaving:

In preparing items for autoclaving, containers should be unsealed and articles should be wrapped in materials that allow steam penetration. Large packages of dressings and large flasks of media require extra time for heat to penetrate them. Likewise, packing many articles close together in an autoclave lengthens the processing time to as much as 60 minutes to ensure sterility. It is more efficient and safer to run two separate, uncrowded loads than one crowded one. Wrapping objects in aluminum foil is not recommended because it may interfere with steam penetration. Steam circulates through an autoclave from a steam outlet to an air evacuation port (figure ).

 

Importance:

Moist heat in the form of pressurized steam is regarded as the most dependable method for the destruction of all forms of life, including bacterial spores. This method is incorporated into a device called the autoclave. Over 100 years ago, French and German microbiologist developed the autoclave as an essential component of their laboratories.

Need of autoclaving:

Reliable sterilization with moist heat requires temperatures above that of boiling water. These high temperatures are most commonly achieved by steam under pressure in an autoclave. Autoclaving is the preferred method of sterilization, unless the material to be sterilized can be damaged by heat or moisture.

Effectiveness of Autoclave or Optimum Conditions:

Sterilization in an autoclave is most effective when the organisms are either contacted by the steam directly or are contained in a small volume of aqueous (primarily water) liquid. Under these conditions, steam at a pressure about 15 psi; attaining temperature (121^oC) will kill all organisms and their endospores in about 15 minutes.

Principle of Autoclaving:

A basic principle of chemistry is that when the pressure of a gas increases, the temperature of the gas increase proportionally. For example, when free flowing steam at a temperature of 100^oC is placed under a pressure of 1 atmosphere above sea level pressure – that is, about 15 pounds of pressure per square inch (Psi) --- the temperature rises to 121^oC. Increasing the pressure to 20 psi raises the temperature to 126^oC. The relationship between temperature and pressure is shown in table 2. In this way steam is a gas, increasing its pressure in a closed system increases its temperature. As the water molecules in steam become more energized, their penetration increases substantially. This principle is used to reduce cooking time in the home pressure cooker and to reduce sterilizing time in the autoclave. It is important to note that the sterilizing agent is the moist heat, not the pressure.

Table	The Relationship Between the Pressure and Temperature of Steam at Sea Level*
Pressure (psi in excess of atmospheric pressure)	Temperature (oC)
0 psi	100
5 psi	110
10 psi	116
15 psi	121
20 psi	126
30 psi	135

Rules implied for Autoclaving:

Sterilization by autoclaving is invariably successful if properly done and if two common-sense rules are followed:

First, articles should be placed in the autoclave so that steam can easily penetrate them.

Second, air should be evacuated so that the chamber fills with steam.

Working of Autoclave:

Most autoclaves contain a sterilizing chamber into which articles are place and a steam jacket where steam is maintained. As steam flows from the steam jacket into the sterilizing chamber, cool air is forced out and a special valve increases the pressure to 15 pounds/square inch above normal atmospheric pressure. The temperature rises to 121.5^oC, and the superheated water molecules rapidly conduct heat into microorganisms. The time for destruction of the most resistant bacterial spore is now reduced to about 15 minutes. For denser objects, up to 30 minutes of exposure may be required. The conditions must be carefully controlled or serious problems may occur.

Uses of Autoclave:

Autoclaving is used to sterilize culture media, instruments, dressings, intravenous equipment, applicators, solutions, syringes, transfusion equipment, and numerous other items that can withstand high temperatures and pressures. The laboratory technician uses it to sterilize bacteriological media and destroy pathogenic cultures. The autoclave is equally valuable for glassware and metalware, and is among the first instruments ordered when a microbiology laboratory is established. Autoclaves are also used on large industrial scale. Large industrial autoclaves are called retorts, but the same principle applies for common household pressure cooker used in the home canning of foods

Limitations and Disadvantages of Autoclave:

The autoclave also has certain limitations. For example, some plasticware melts in the high heat, and sharp instruments often become dull. Moreover, many chemicals breakdown during the sterilization process and oily substances cannot be treated because they do not mix with water.

Heat requires extra time to reach the center of solid materials, such as caned meats, because such materials do not develop the efficient heat-distributing convection currents that occur in liquids. Heating large containers also requires extra time. Table 3 shows the different time requirements for sterilizing liquids in various container sizes. Unlike sterilizing aqueous solutions, sterilizing the surface of a solid requires that steam actually contact it.

Table 3	The effect of Container Size on Autoclve Sterilization Times for Liquid Solutions*
Container Size	Liquid Volume	Sterilization Time (min)
Test Tube: 18×150 mm	10 ml	15
Erlenmeyer Flask: 125 ml	95 ml	15
Erlenmeyer Flask: 2000 ml	1500 ml	30
Fermentation Bottle: 9000 ml	6750 ml	70

Indicator of Sterilization Achievement:

Several commercially available methods can indicate whether sterilization has been achieved by heat treatment. Modern autoclaves have devices to maintain proper pressure and record internal temperature during operations. Regardless of the presence of such a device, the operator should check pressure periodically and maintain the appropriate pressure. Chemical reactions in which an indicator changes color when the proper times and temperatures have been reached. In some designs, the word "sterile" or "autoclaved" appears on wrappings or tapes. These tapes are not fully reliable because they do not indicate how long appropriate conditions were maintained. Tapes or other sterilization indicators should be placed inside and near the center of large packages of determine whether heat penetrated them. In another method, a pellet contained within a glass vial melts. A widely used test consists of preparations of specified species of bacterial endospores such as Bacillus stearothermophilus, impregnated into paper strips. The spore strip and an ampule of medium are enclosed in a soft plastic vial. The vial is placed in the center of the material to be sterilized and is autoclaved. After autoclaving, these can then be aseptically inoculated into culture media. Growth in the culture media indicates survival of the endospores and therefore inadequate processing. Other designs use endospore suspensions that can be released, after heating, into a surrounding culture medium within medium within the same vial.

Important Points to Remember For Autoclaving:

Steam under pressure fails to sterilize when the air is not completely exhausted. This can happen with the premature closing of autoclave's automatic ejector valve. The principles of heat sterilization have a direct bearing on home canning. To sterilize dry glassware, bandages, and the like, care must be taken to ensure that steam contacts all surfaces. For example, aluminum foil is impervious to steam and should not be used to wrap dry materials that are to be sterilized; paper should be used instead. Care should also be taken to avoid trapping air In the bottom of a dry container because trapped air will not be replaced by steam, which is lighter than air. The trapped air is the equivalent of a small hot-air oven, which, as we will see shortly, requires a higher temperature and longer time to sterilize materials. Containers that can trap air

should be placed in a tipped position so that the steam will force out the air. Products that do not permit penetration by moisture, such as mineral oil or petroleum jelly, are not sterilized by the same methods that would sterilize aqueous solutions. This precaution is necessary because when an object is exposed to heat, its surface becomes hot much more quickly than its center. (When a large piece of meat is roasted, for example, the surface can be well done while the center remains rare.)

Prevacuum Autoclave:

In large laboratories and hospitals, where great quantities of materials must be sterilized, special autoclaves, called prevacuum autoclaves, are often used. This machine draws air out of the sterilizing chamber at the beginning of the cycle. Saturated steam is then used at a temperature of 132^oC to 134^oC at a pressure of 28 to 30 lb/in2. The time for sterilization is now reduced to as little as 4 minutes. A vacuum pump operates at the end of the cycle to remove the steam and dry the load. The major advantages of the prevacuum autoclave are the minimal exposure time for sterilization, the reduced time to complete the cycle and the costs of sterilization are greatly decreased.

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