Desiccation

Mode of Action:

In the absence of water, a condition known as desiccation, microorganisms cannot grow or reproduce but can remain viable for years. Then, when water is made available to them, they can resume their growth and division. This ability is used in the laboratory when microbes are preserved by lyophilization, or freeze-drying. Certain foods are also freeze-dried (for example, coffee and some fruit additives for dry cereals).

Resistance and Desiccation:

The resistance of vegetative cells to desiccation varies with the species and the organism's environment. For example, the gonorrhea bacterium can withstand dryness for only about an hour, but tuberculosis bacterium can remain viable for months. Viruses are generally resistant to desiccation, but they are not as resistant as bacterial endospores, some of which have survived for centuries. This ability of certain dried microbes and endospores to remain viable is important in a hospital setting. Dust, clothing, bedding, and dressing might contain infectious microbes in dried mucus, urine, pus, and feces.

Osmotic Pressure

Mode of Action:

The use of high concentrations of salts and sugars to preserve food is based on the effects of osmotic pressure. High concentrations of these substances create a hypertonic environment that causes water to leave the microbial cell, this effect is also called as plasmolysis. Loss of water severely interferes with cell function and eventually leads to cell death. This process resembles preservation by desiccation, in that both methods deny the cell the moisture it needs for growth.

Application of Osmotic Pressure:

The use of sugar jellies, jams, and syrups or salts solution in curing meat and making pickles plasmolysis most organisms present and prevents growth of new organisms. A few halophilic organisms, however, thrive in these conditions and cause spoilage, especially of pickles, and some fungi can live on the surface of jams.

Limitations:

As a general rule, molds and yeasts are much more capable than bacteria of growing in materials with low moisture or high osmotic pressures. This property of molds, sometimes combine with their ability to grow under acidic conditions, is the reason fruits and grains are spoiled by molds rather than by bacteria. It is also part of the reason molds are able to form mildew on a damp wall or a shower curtain.

High Pressure

Mode of Action:

High pressure applied to liquid suspensions in transferred instantly and evenly throughout the same. If the pressure is high enough, the molecular structures of proteins and carbohydrates are altered, resulting in rapid inactivation of vegetative bacterial cells.

Limitation and Solutions:

Endospores are relatively resistant to high pressure. They can however, be killed by other techniques, such as combining high pressure with elevated temperatures or by alternating pressure cycles that cause spore germination, followed by pressure-caused death of resulting vegetative cells. Fruit juices preserved by high pressure treatments have been marketed in Japan the United States. An advantage is that these treatments preserve the flavors, colors, nutrient values of the products.

Freeze Drying

Freeze-drying, or lyophilization, is the drying of a material from the frozen state. This process is used in the manufacture of some brands of instant coffee; freeze-dried instant coffee has a more natural flavor than other kinds. Microbiologists use lyophilization for long-term preservation rather than destruction of cultures of microorganisms. Organisms are rapidly frozen in alcohol and dry ice or in liquid nitrogen and are then subjected to a high vacuum to remove all the water while in the frozen state. Rapid freezing allows only very tiny ice crystal to form in cells, so the organisms survive this process. Organism so treated can be kept alive for years, store under vacuum in the freeze-dried state.

Drying

Drying can be used to preserve foods because the absence of water inhibits the action of enzymes. Many foods, including peas, beans, raisins, and other fruits, are often preserved by drying. Yeast used in baking also can be preserved by drying. Endospores presents on such foods cab survive drying, but they do not produce toxins. Dried pepperoni sausage and smoked fish retain enough moisture for microorganism to grow. Because smoked fish is not cooked, eating it poises a risk of infection. Sealing such fish in plastic bags creates conditions that allow anaerobes such as Clostridium botulinum to grow.

Drying also naturally minimizes the spread o infectious agents. Some bacteria, such as Treponema pallidum, which causes syphilis, are extremely sensitive to drying and die almost immediately on a dry surface; thus they can be prevented from spreading by keeping toilet seats and other bathroom fixtures dry. Drying of laundry in dryers or in the sunshine also destroys pathogens.

Low Temperature

Mode of Action:

The effect of low temperatures on microorganisms depends on the particular microbe and the intensity of application. For example, at temperatures of ordinary refrigerators (0 ˚ C), the metabolic rate of some microbes is so reduced that they cannot reproduce or synthesize toxins. In other words, ordinary refrigeration has a bacteriostatic effect, but does not kill many microbes. Heat is much more effective than cold at killing microorganism.

Disadvantage:

Yet psychrotrophs do grow slowly at refrigerator temperatures and will alter the appearance and taste of foods after a time. For example, a single microbe reproducing only three times a day would reach a population of more than 2 million within a week.

Advantage by Medical Point of View:

Pathogenic bacteria generally will not grow at refrigerator temperature.

Uses of Cold temperature:

Refrigeration is used to prevent food spoilage. Freezing, drying, and freeze-drying are used to preserve both foods and microorganism, but these methods do not achieve sterilization.

Optimum Conditions:

Surprisingly, some bacteria can grow at temperatures several degrees below freezing. Most foods remain unfrozen until -2^oC or lower. Rapidly attained subfreezing temperatures tend to render microbes dormant but do not necessarily kill them. Slow freezing is more harmful to bacteria; the ice crystals that form and grow disrupt the cellular and molecular structure of the bacteria. Thawing, being inherently slower is actually the more damaging part of a freeze-thaw cycle. Once frozen, one third of the population of some vegetative bacteria might survive a year, whereas other species might have very few survivors after this time.

Results of Low Temperature Treatment:

Many eukaryotic parasites, such as the roundworms that cause trichinosis, are killed by several days of freezing temperatures.

Conditions:

Many fresh foods can be prevented from spoiling by keeping them at 5 ° C (ordinary refrigerator temperature).

Limitations:

However, storage should be limited to a few days because some bacteria and molds continue to grow at this temperature. To convince yourself of this, recall some of the strange things you have found growing on left over of the back of your refrigerator. In rare instances strains of Clostridium botulinum have been found growing and producing lethal toxins in a refrigerator when the organism were deep within a container of food, where anaerobic conditions exist.

Freezing:

Uses of Freezing:

Freezing at -20 ° C is used to preserve foods in homes and in the food industry. Although freezing does not sterilize foods, it does significantly slow the rate of chemical reactions so that microorganism does not cause food to spoil. Frozen foods should not be thawed and refrozen. Repeated freezing and thawing of foods causes large ice crystals to form in the foods during slow freezing. Cell membranes in the foods are ruptured, and nutrients leak out. The texture of foods is thus altered, and they become less palatable. It also allows bacteria to multiply while food is thawed, making the food more susceptible to bacterial degradation.

Freezing can be used to preserve microorganisms, but this requires a much lower temperature than that used for food preservation. Microorganism are usually suspended in glycerol or protein to prevent the formation of large ice crystal (which could puncture cells), cooled with solid carbon dioxide (dry ice) to a temperature of -78 ° C, and then held there. Alternatively, they can be placed in liquid nitrogen and cooled to – 180 ° C.

Ultrasonic Vibrations

Ultrasonic vibrations are high-frequency sound waves beyond the range of human hearing.

Optimum Conditions and Mode of Action for Ultrasonics:

Ultrasonic waves or wave's frequencies above 15000 cycles per second, can cause bacteria to caviatate. Cavitation is the formation of a partial vacuum in a liquid—in this case, the fluid cytoplasm in the bacterial cell. Bacteria so treated disintegrate, and their proteins are denatured.

Cold Boiling:

When propagated in fluids, ultrasonic vibrations cause the formation of microscopic bubbles, or cavities, and the water appears to boils. Some observers call this "cold boiling." The cavities rapidly collapse and send out shock waves. Microorganisms in the fluid are quickly disintegrated by the external pressures. The formation and implosion of the cavities are known as cavitation. (Figure) illustrates this process.

 

Applications:

Ultrasonic vibrations are valuable in research for breaking open tissue cells and obtaining their parts for study. A device called the cavitron is used by dentists to clean teeth, and ultrasonic machines are available for cleaning dental plates, jewelry, and coins. A major appliance company has also experimented with an ultrasonic washing machine. Many research laboratories us e ultrasonic probes for cell disruption and hospitals use ultrasonic devices to clean their instruments. When use with an effective germicide, an ultrasonic device may achieve sterilization, but the current trend is to use ultrasonic vibrations as a cleaning agent and follow the process by sterilization in an autoclave.

Limitations of Method:

As a sterilizing agent, ultrasonic vibrations have receive minimal attention because liquid is required and other method are more efficient.

Ionizing Radiation: (Radiation Sterilization)

Mode of Action:

Both, X rays and Gamma rays have wavelength shorter than the wavelength of ultraviolet light. X rays, which have wavelength of 0.1 to 40 nm, and gamma rays, which have even shorter wavelength, are forms of ionizing radiation, so named because it can dislodge electrons from atoms, creating ions. (Longer wavelengths comprise nonionizing radiation.) These forms of radiation also kill microorganisms and viruses and ionizing radiation damages DNA and produces peroxides, which act as powerful oxidizing agents in cells. This radiation can also kill or cause mutations in human cells if it reaches them.

Production of Gamma rays:

Gamma rays are emitted by certain radioactive elements such as cobalt, and electron beams are produced by accelerating electrons to high energies in special machines.

Production of X rays:

X rays, which are produced by machines in a manner similar to the production of electron beams, are similar in nature to gamma rays.

Advantage of Gamma rays:

Gamma rays penetrate deeply but may requite hours to sterilize large masses.

High energy Electron Beams:

Effectiveness of Electron Beam:

High energy electron beams have much lower penetration power but usually require only a few seconds of exposure.

Mode of Action of Ionizing Radiations:

The principal effect of ionizing radiation is the ionization of water, which forms highly reactive hydroxyl radicals. These radicals react with organic cellular components, especially DNA.

The so-called target theory of damage by radiation supposes that ionizing particles, or packets of energy, pass through or close to vital portions of the cell; these constitute "hits." One, or a few, hits may only cause nonlethal mutations, some of them conceivably useful. More hits are likely to cause sufficient mutations to kill the microbe.

Application of Method:

The food industry has recently renewed it interest in the use of radiation for food preservation. It can be used to prevent spoilage in seafood by doses of 100 to 250 kilorads, in meats and poultry by doses of 50 to 100 kilorads, and in fruits by doses of 200 to 300 kilorads. (one kilorad equals 1000 rads) many consumers in the United States reject irradiation foods for fear o receiving radiation, but such foods are quite safe --- free of both pathogens and radiation. In Europe, mil and other foods are often irradiated to achieve sterility. Especially high energy electron beams, is used for the sterilization pharmaceuticals and disposable dental and medical supplies, such as plastic syringes, surgical gloves, suturing materials, and catheters. As a protection against Bioterrorism, the postal service often uses electron beam radiation to sterilize certain classes of mail. These radiations can be used to differentiate between Gram positive and negative bacteria. Gram-positive bacteria are more sensitive to ionizing radiations than gram-negative bacteria. Ionizing radiations are currently used to sterilize such heat sensitive pharmaceuticals as vitamins, hormones, and antibiotics, as well as certain plastics and suture materials.

Worldwide Importance and Controversy:

Ionizing radiations have also been approved for controlling microorganisms, and for preserving foods, as noted in MIcrofocus 21.4. The approval has generated much controversy, fueled by activists concerned about the safety of factory workers and consumers. First used in 1921 to inactivate Trichinella spiralis, the agent of trichinosis, irradiation is now used as a preservative in more than 40 countries for over 100 food items, including potatoes, onions, cereals, flour, fresh fruit, and poultry. The US Food and Drug Administration (FDA) approved cobalt-60 irradiation to preserve poultry in the early 1990s, and in 1997, it extended the approval to preserve red meat such as beef, lamb and pork.

Strong Visible Light: (Radiation Sterilization)

Sunlight has been known for years to have a bactericidal effect; nut the effect is due primarily to ultraviolet rays in the sunlight.

Introduction :

Strong visible light, which contains light of wavelength from 400 to 700 nm (violet to red light).

Mode of Action:

Visible light can have direct bactericidal effects by oxidizing light-sensitive molecules such as riboflavin and porphyrins (components of oxidative enzymes) in bacteria. For that reason, bacterial cultures should be exposed to strong light during laboratory manipulations.

Application of Strong Light:

The fluorescent dyes cosin and methylene blue can denature proteins in the presence of strong light because they absorb energy and cause oxidation of proteins and nucleic acids. The combination of a dye and strong light can used to rid materials of both with bacteria and viruses.

Radiation Spectra is available here.

Microwave: (Radiation Sterilization)

Introduction :

Microwave radiation, in contrast with gamma, X ray, and ultraviolet radiation, falls at the longer wavelength end of the electro-magnetic spectrum. It has wavelengths of approximately 1 mm to 1 m, a range that includes television and police radar wavelengths.

Sterilization by Microwave Oven:

A specialized microwave oven has recently become available that can be used to sterilize media in just 10 minutes. It has 12 pressure vessels, each of which holds 100 ml of medium. Microwave energy increases the pressure of the medium inside the vessels until sterilizing temperatures are reached.

Applications of Microwaves:

Microwave oven frequencies are tuned to match energy levels in water molecules. In the liquid state, water molecules quickly absorbs the microwave energy and than release it to surrounding materials as heat. The molecules are set into high-speed motion, and the heat of friction is transferred of foods, which become hot rapidly. Thus, materials that do not contain water, such as plates made of paper, china, or plastic, remain cool while the moist food on them becomes heated.

Limitations of Microwave Ovens:

Other than the heat generated, there is no specific activity against microorganisms.

For this reason the home microwave cannot be used to sterilize items such as bandages and glassware. Conduction of energy is metals leads to problems such as sparking, which makes most metallic items also unsuitable for microwave sterilization. Moreover, bacterial endospores, which contain almost no water, are not destroyed by microwaves.

Laser:

A final form of radiation we shall consider is light energy. When concentrated by sophisticated devices, light energy forms a laser beam. The word laser is an acronym of light amplification by stimulated emission of radiation.

Advantage of Lasers

Recent experiments indicate that laser beams can be used to sterilize instruments and the air in operating rooms, as well as a wound surface. Microorganisms are destroyed in a fraction of a second, but the laser beam must reach all parts of the material to effect sterilization.

Caution:

Caution should be observed in cooking foods in the home microwave oven. Geometry and differences in density of the food being cooked can cause certain regions to become hotter than other, sometimes leaving very cold spots. Consequently, to cook foods thoroughly in a microwave oven, it is necessary to rotate the items either mechanically or by hand. For example, pork roasts must be turned frequently and cooked thoroughly to kill any cysts of the pork roundworm Trichinella. Failure to kill such cysts could lead to the disease trichinosis, in which cysts of the worm become embedded in human muscles and other tissues. All experimentally infected pork roasts, when microwaved without rotation, showed live worm remaining in some portion at the end of standard cooking time.

Radiation: A Sterilization Method

Nonionizing Radiation:

Nonionizing radiation has a wavelength longer than that or ionizing radiation, usually greater than about 1 nm. The best example of non ionizing radiation is ultraviolet (UV) light.

Mode of Action:

When microorganisms are subjected to UV light, cellular DNA absorbs the energy by purines and pyrimidine bases, and adjacent thymine molecules link together, as figure illustrates. Linked thymine molecules are unable to encode adenine on messenger RNA molecules during the process of protein synthesis. Moreover, replication of the chromosome in binary fission is impaired. The damaged organism can no longer produce critical proteins or reproduce, and it quickly dies. Ultraviolet light is especially effective in inactivating viruses. However, it kills far fewer bacteria than one might expect because of DNA repair mechanisms. Once DNA is repaired, new molecules of RNA and protein can be synthesized to replace the damaged molecules.

Conditions for Microbe control:

Ultraviolet (UV) light consists of light of wavelengths between 40 to 390 nm, but wavelength in the 200 nm range are most effective in killing microorganisms But according to some books wavelength between 260 – 265 nm is most effective.

Uses of Ultraviolet:

Ultraviolet light effectively reduces the microbial population where direct exposure takes place. It is used to limit airborne or surface contamination in a hospital room, morgue, pharmacy, toilet facility, or food service operation. In some communities, ultraviolet light is replacing chlorine in sewage treatment. When chlorine-treated sewage effluent is discharged into streams or other bodies of water, carcinogenic compounds form and may enter the food chain. The cost of removing chlorine before discharging treated effluent could add as much as $100 per year to the sewage bills of the average American family, and very few sewage plants do this. Running the sewage effluent under ultraviolet light before discharging it can destroy microorganism without altering the odor, pH, or chemical composition of the water and without forming carcinogenic compounds.

Advantages:

It does penetrate air, effectively reducing the number of airborne microorganism and killing them on surfaces on operating rooms and rooms that will contain caged animals. To help sanitize the air without irradiation humans, these lights can be turned on when there rooms are not in use. . Hanging laundry outdoors on bright, sunny days takes advantage of the ultraviolet light present in sunlight. Although the quantity of UV rays in sunlight is small, these rays may help kill bacteria on clothing, especially diapers.

Disadvantage of UV Light:

A major disadvantage of UV light as a disinfectant is that the radiation is not very penetrating, so the organism to be killed must be directly exposed to the rays. It is noteworthy microorganisms in the air and upper layers of the soil, but it may not the effective against all bacterial spores. Organisms protected by solids and such coverings as paper, glass, and textiles are not affected. Another potential problem is that UV light can damage human eyes, and prolonged exposure can cause burns and skin cancer in humans. And it may cause damage in human skin cells and permanent damage the eyes.

SUN--Free Source of UV:

Sunlight contains some UV radiation, but the shorter wavelengths – those most effective against bacteria – are screened out by the ozone layer of the atmosphere. The antimicrobial effect of sunlight is due almost entirely to the formation of singlet oxygen on the cytoplasm. Many pigments produced by bacteria provide protection from sunlight.

Filtration: Types of Filters

Several types of filters are available for use in the microbiology laboratory. Inorganic filters are typified by the Seitz filter, which consists of a pad of porcelain or ground glass mounted in a filter flask.

Organic filters:

Organic filters are advantageous because the organic molecules of the filter attract organic components in microorganisms. They are given below:

1) Berkefeld filter:

One example, The Berkefeld filter, utilizes as substance called diatomaceous earth. This material contains the remains of marine algae known as diatoms. Diatoms are unicellular algae that abound in oceans and provide important foundations for the world's food chains. Their remains accumulate on the shoreline and are gathered for use in swimming pool aquarium filters, as well as for microbiological filters used in laboratories.

2) Membrane Filter:

The membrane filter is at third type of filter that has received broad acceptance. It consists of a pad of organic compounds such as cellulose acetate (cellulose esters) or polycarbonate (plastic polymers), mounted in a holding device. These filters are only 0.1 mm thick. The pores of membrane filters include, for example, 0.22μm and 0.45μm sizes, which are intended for bacteria. This filter is particularly valuable because bacteria multiply and for colonies on the filter pad when the pad is place on a plate of culture medium. Microbiologists can then count the colonies to determine the number of bacteria originally present. For example, if a 100-ml sample of liquid were filtered and 59 colonies appeared on the pad after incubation, it could be assumed that 59 bacteria were in the sample. However, Some very flexible bacteria, such as spirochetes, or the wall less mycoplasma, will sometimes pass through such filters.

Membrane filters used to trap bacteria form air and water samples can be transferred directly to agar plates, and the quantity of bacteria in the sample can be determined. Alternatively, the filters can be transferred from one medium to another, so organisms with different nutrient requirements can be detected. Filtration is also used to remove microorganisms and other small particles from public water supplies and in sewage treatment facilities. This technique, however, cannot sterilize; it merely reduces contamination.

3) HEPA filters:

Air can also be filtered to remove microorganisms. The filter generally used is a high-efficiency particulate air (HEPA) filter. This apparatus can remove over 99 percent of all particles, including microorganisms with a diameter larger than 0.3 μm. The air entering surgical units and specialized treatment facilities, such as burn units, is filtered to exclude microorganisms. In some hospital wards, such as for respiratory diseases, and in certain pharmaceutical filling rooms, the air is recirculated through HEPA filters to ensure its purity. Used filter are soaked in formalin before they are disposed of.

Industrial Fluid Filtration:

Two examples of filters used in conjunction with fluids. (a) A filter of woven mesh Dacron (arrow) is used to trap clumps of unwanted blood cells that might otherwise enter the recipient's circulation during a transfusion. (b) A cartridge filter removes contaminants from fluids to be used for intravenous injections or for other medical purposes.

The Membrane Filter Technique:

a) The membrane filter consists of a pad of cellulose acetate, or similar material, mounted in a holding device. (b) The holding device is secured by a clamp, and a measured amount of fluid is filtered by pouring it into the cup. The solution runs through to a flask beneath, and bacteria are trapped in the filter material. (c) The filter pad is place onto a plate of nutritious medium, and the plate is incubated. (d) After incubation, colonies appear on the surface of the filter pad. The colony count reflects the original number of bacteria in the fluid sample.

Suitable Selection of Filters --- By pore size

In the manufacture of vaccines that require the presence of live viruses, it is important to select a filter pore size that will allow viruses to pass but prevent bacteria from doing so. By selecting a filter with a proper pore size, scientists can separate polioviruses from the fluid and debris in tissue cultures in which they were grown. This procedure simplifies the manufacture of polio vaccine. Cellulose acetate filters with extremely tiny pores are now available and are capable of removing many viruses (although not the very smallest) from liquids. However, these filters are expensive and clog easily.

Filtration

Importance of Filter in Microbiology history:

In the early days of microbiology, hollow candle shaped filters of unglazed porcelain were used to filter liquids. The long and indirect passageways through the walls of the filter adsorbed the bacteria. Filters came into prominent use in microbiology as interest in viruses grew during the 1890s. Previous to that time, filters had been utilized to trap airborne organisms and sterilize bacteriological media, but now they became essential for separating viruses from other microorganisms. Among the early pioneers of filter technology was Charles Chamberland, as associate of Pasteur. His porcelain filter was important to early virus research. Another pioneer was Julius Petri (inventor of Petri dish), who developed a sand filter to separate bacteria from the air.

Introduction and Action:

Filtration is the passage of a liquid or gas through a screen like material with pores small enough to retain microorganisms (often the same apparatus used for counting. A vacuum that is created in the receiving flask helps gravity pull the liquid through the filter. As fluid passes through the filter, organisms are trapped in the pores of the filtering material, as (figure) shows.

The solution that drips into the receiving container is decontaminated or, in some cases, sterilized.They are usually made of nitrocellulose and have the great advantage that they can be manufactured with specific pore sizes from 25 µm to less than 0.025μm. Particles filtered by various pore sizes are summarized in table.

Table 4	Pore sizes of membrane filters and particles that pass through them
Pore Size in (µm)	Particles that pass through them
10	Erythrocytes, yeast cells, bacteria, viruses, molecules
5	Yeast cells, bacteria, viruses, molecules
3	Some yeast cells, bacteria, viruses, molecules
1.2	Most bacteria, viruses, molecules
0.45	A few bacteria, viruses, molecules
0.22	Viruses, molecules
0.10	Medium-sized to Small Viruses, molecules
0.05	Small viruses, molecules
0.025	Only the very smallest viruses, molecules
Ultra-filter	Small molecules

Uses of Filtration:

Membrane-filters are used to sterilize heat sensitive materials include media, special nutrients that might be added to media, enzymes, vaccines, and pharmaceutical products such as drugs, sera, and vitamins. They are also used to sterilize the things such things as beverages, intravenous solutions and bacteriological media. Some operating theaters and rooms occupied by burn patients receive filtered air to lower the numbers of air borne microbes.

Some filters can be attached to syringes so that materials can be forced through them relatively quickly. Filtration can also be used instead of pasteurization in the manufacture of beer. When using filters to sterilize materials, it is important to select a filter pore size that will prevent any infectious agent from passing into the product.

Advantages:

Membrane filters have certain advantages and disadvantages. Except for those with the smallest pore sizes, membrane filters are relatively inexpensive, do not clog easily, and can filter large volumes of fluid reasonably rapidly. They can be autoclaved or purchased already sterilized.

Disadvantages:

A disadvantage of membrane filters is that many of them allow viruses and some mycoplasmas to pass through. Other disadvantages are that they may absorb relatively large amounts of the filtrate and may introduce metallic ions into the filtrate.

Different types of filters will be discussed in some future post.

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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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.