The methods of sterilization.

There are 3 groups of methods of sterilization: physical, chemical and physico-chemical methods.

Physical methods include high temperature (heat), UV irradiation, ionizing irradiation, ultrasound, filtration through sterile filters.

Heat:

Heating is still one of the most popular ways to destroy microorganisms. Fire and boiling water have been used since the time of Greeks for sterilization and disinfection. For sterilization always consider type of heat, time of application and temperature to ensure destruction of all microorganisms. Endospores of bacteria are considered the most thermoduric of all cells so their destruction guarantees sterility. Either moist heat or dry heat may be applied.

The type of heat sterilization:

1.Flaming is the process of exposing metallic device like the needle, scalpels, scissors to flame for few minutes. The fire burns the microbes and other dust on the instrument directly.

2. Incineration is done especially for bacteriological loops used in microbe cultivation. The metallic end of the loop is heated to red hot on the flame. This exposure kills all the germs.

3. Boiling: + 100°C for 30 minutes. Kills everything except some endospores (actually, for the purposes of purifying drinking water +100°C for five minutes is probably adequate though there have been some reports that Giardia cysts can survive this process). Exposure to boiling water for 10 min is sufficient to kill or destroy vegetative cells and eukaryotic spores., but not enough to kill or destroy bacterial endospores. To kill endospores, and therefore sterilize the solution, very long or intermittent boiling is required.

In order to destroy bacterial endospores, moist heat sterilization must be carried out at temperatures above 100°C and this requires the use of saturated steam under pressure. This can be carried out with an autoclave (Chamberland, 1884).

4. Autoclaving (steam under pressure or pressure cooker): +121°C for 15
-20 minutes (15 pounds pressure). Water is boiled to produce steam, which is released through the jacket and into the autoclave's chamber. Hot, saturated steam enters the chamber and the desired temperature and pressure, usually 121°C and 15 pounds is reached. At this temperature saturated steam destroys all vegetative cells and endospores. Moist heat is thought to kill so effectively by degrading nucleic acids and by denaturing enzymes and other essential proteins. It also may disrupt cell membranes.

Good for sterilizing almost anything, but heat-labile substances will be denatured or destroyed.

Device of autoclave. The autoclave consists of 2 metal cylinders, inserted into each other with a hermetically sealed cover, having screws. It is equipped with a pressure gauge, steam valve, safety valve, water-glass. External cylinder - water-vapor chamber, internal - sterilization chamber. In the upper part of the sterilization chamber there is an opening through which steam flows from the water-steam chamber into sterilization chamber. The pressure gauge serves to determine the pressure in the sterilization chamber. There is a definite relationship between pressure and temperature: 121°C - 15 pounds ( 0.5 atm - 112 ° C, 1-01.1 atm - 119-121 ° C, 2 atm - 134 ° C). Safety valve - to protect against excessive pressure: when the pressure rises above the preset, it opens and releases excess steam.

Operating procedure. The water is poured into the autoclave, the level of which is controlled by the water glass. The material is placed in the sterilization chamber and the cover hermetically is closed. The steam valve is open. Turn on the heat. After the boiling of water, the steam valve is closed only when all air is expelled (the steam flows continuously with a strong, dry spray). If the valve is closed earlier, the pressure gauge reading will not match the desired temperature. After the valve is closed, the pressure in the boiler gradually increases. The beginning of sterilization is the moment when the needle of the pressure gauge shows the preset pressure.

After the sterilization, the heating is stopped and the autoclave is cooled before returning the pressure gauge needle to 0. If steam is released earlier, the liquid may boil up due to a rapid change in pressure and push out the plugs (o bjects will lose their sterility). When the cursor of the pressure gauge returns to 0, open the steam valve. Steam should flow out completely and then remove the sterilized objects. If steam is not released after returning the cursor to 0, water can condense and moisten plugs and sterilizable material (o bjects will lose their sterility).

The autoclave is sterilized:

a) glass, metal, porcelain, linen, rubber and cork stoppers, rubber, cellulose, wood products, dressings (cotton wool, gauze);

b) saline, solutions for injection, eye drops, distilled water, simple nutrient media (MPB, MPA);

c) mineral, plant oils in hermetically sealed flask.

5. Dry heat sterilization (hot air oven) can also be used on many objects in the absence of water. The items to be sterilized are placed in an oven at 160 to 170°C for 2 to 3 hours. Oxidation of cell constituents and denaturation of proteins results in the death of microbes. Most laboratories sterilize glass Petri dishes and pipettes with dry heat. This method though is not suitable for heat sensitive materials like many plastic and rubber items. Used also for objects that won’t melt or go bad: heat-resistant powdered medicinal products (talc, white clay, zinc oxide, etc.), mineral and plant oils, fats, lanolin, petroleum jelly, wax.

 

The device of hot air oven ( Pasteur oven)  and the order of work. The Pasteur oven is a double-skinned metal cabinet having outside with a material that does not conduct heat well (asbestos). Oven has an automatic temperature controller that maintains the set temperature. Heated air circulates in the space between the walls and exits through special openings. In the upper wall of the cabinet is a hole for the thermometer, which indicates the temperature inside the cabinet. When working, you must strictly monitor the correct temperature and sterilization time. If the temperature is higher, cotton plugs, paper in which the dishes are wrapped will be burned and at a lower temperature will require a longer sterilization time. At the end of the sterilization, the cabinet is opened only after it has cooled down, otherwise glassware may become cracked due to a rapid change in temperature.

Hot Air Oven

 

6. I ntermittent boiling. This method is used for sterilization of the media with gelatin, vitamins, carbohydrates, for some drugs, which are spoiled at temperatures above 100⁰C.

As after a single boiling (T100 ° C) there is not killing of endospores, intermittent boiling is used: 20-30 min daily for 3 days. In the intervals between boiling, the material is kept at room temperature so that the endospores grow into vegetative forms, which will be killed with subsequent boiling.

Methods of intermittent boiling include tindalization. Tindalization is carried out in a water bath at 56 ° C for 1 hour 5-6 days. It is used to sterilize objects which are subjected to denaturation at a temperature of 100 ° C: serum, ascitic fluid, vitamins.

Methods of microbial control with heating also include pasteurization. It is carried out at a relatively low temperature once for the objects that lose their quality at high temperatures. The pasteurization does not refer to sterilization as endospores remain viable, so these products need to be stored in the cold (in a refrigerator).

Pasteurization is a process where many substances such as milk, are treated with heating at temperatures well below boiling (in honour of its developer Louis Pasteur). Milk, beer and many other beverages are now pasteurized. Pasteur examined the spoiled wine and detected the presence of microorganisms like bacteria which were responsible for the production of lactic acid and acetic acid fermentations which resulted in the spoilage of wine. He then discovered that brief heating at 55 to 60°C would destroy these microbes and preserve wine for long periods. Hence, pasteurization does not sterilize a beverage or milk but kills any pathogens present and slows spoilage by reducing the level of non-pathogenic spoilage microbes.

Milk in older methods of pasteurization (batch
method) was held at 63°C for 30 min. Kills most vegetative bacterial cells
including pathogens such as streptococci, staphylococci and
Mycobacterium tuberculosis .

Now, mostly two methods are used, flash pasteurization or high temperature short-term (HTST) pasteurization, which consists of quick heating t about 72°C for 15 sec and then rapid cooling. The other method used in dairy industry is ultrahigh-temperature (UHT) sterilization, where milk and milk products are heated at 140 to 150°C for 1 to 3 sec. The products pasteurized by this method needs no refrigeration and can be stored at room temperature for about 2 months.

RECOMMENDED USE OF HEAT TO CONTROL
MICROBIAL GROWTH

Treatment	Temperature	Effectiveness
Incineration	>500°C	Vaporizes organic material on nonflammable surfaces but may destroy many substances in the process
Boiling	1OO°C	30 minutes of boiling kills microbial pathogens and vegetative forms of bacteria but may not kill bacterial endospores
Intermittent boiling	100°C	Three 30-minute intervals of boiling, followed by periods of cooling kills bacterial endospores
Autoclave and pressure	121°C/15 minutes	Kills all forms of life including bacteria]
cooker (steam under pressure)	at 15# pressure	endospores. The substance being sterilized must be maintained at the effective T for the full time
Dry heat (hot air oven)	160°C/2 hours	For materials that must remain dry and which are not destroyed at T between 121°C and 170°C Good for glassware, metal, not plastic or rubber items
Dry heat (hot air oven)	170°C/1 hour	Same as above, Note increasing T by 10 degrees shortens the sterilizing time by 50 percent
Pasteurization (batch method)	63°C/30 minutes	Kills most vegetative bacterial cells including pathogens such as streptococci, staphylococci and Mycobacterium tuberculosis
Pasteurization (flash method)	72°C/15 seconds	Effect on bacterial cells similar to batch method; for milk, this method is more conducive to industry and has fewer undesirable effects on quality or taste

 

Filtration:

In order to sterilize solutions which is heat sensitive, filtration is an excellent way to reduce the microbial population. The filters simply remove the microbes instead of killing them. Depth filters consists of fibrous or granular materials that have been bonded into a thick layer filled with twisting channels of small diameter. The solution is passed through the filter which is sucked through this layer under vacuum and microbial cells are removed. The material used mostly is unglazed porcelain, asbestos or other similar materials. Membrane filters are also used and have replaced depth filters in recent times. These filters are made up of cellulose acetate, cellulose nitrate, polycarbonate, polyvinylidene fluoride, and other synthetic materials. These filters vary in size with pore sizes mostly of 0.2 to 0.5 µm in diameter and used to remove most vegetative cells, but not viruses, from solutions ranging in volume from 1ml to many litres. These filters are mostly used to sterilize pharmaceuticals, ophthalmic solutions, culture media, oils, antibiotics and other heat sensitive solutions.

The other way this method is used is in the laminar flow biological safety cabinets where the air is sterilized by filtration. These cabinets contain high-efficiency particulate air (HEPA) filters, which remove 99.97% of 0.3µm particles. The safety cabinets are most useful as the culturing of any organisms requires contamination free air to reduce the growth of other undesired organisms or for the preparation of media, examining tissue cultures etc (Fig. 10).

Radiation:

§ We have discussed about the effects of radiation on the growth of microorganisms earlier. The radiations like ultraviolet and ionizing can be used for sterilizing objects. Ultraviolet radiation around 260 nm is quite lethal but does not penetrate glass, dirt films, water and other substances very effectively. UV radiation is used as a sterilizing agent only in a few specific situations, like UV lamps are placed on the ceilings of rooms or in biological safety cabinets to sterilize air and other exposed surfaces. Commercial UV units are available for water treatment. Pathogens and microorganisms are destroyed when a thin layer of water is passed under the lamps (water purifiers). Ultraviolet radiation are safe to the operator of sterilization, they can be used even at the door entrances to prevent entry of live microbes through the air.

Ionizing radiation penetrates deep into objects and is an excellent sterilizing agent. It destroys bacterial endospores and vegetative cells of both prokaryotic and eukaryotic origin but not against viruses. Gamma radiation from a cobalt 60 source is used in the cold sterilization of antibiotics, hormones, sutures and plastic disposable supplies such as syringes, and Petri dishes, dressings, blood transfusion systems. Used for sterilization of objects that are not resistent for thermal and chemical treatment methods. It does not change the quality of the product, does not cause denaturation of the constituent parts of the product.

Also, ultra sound waves are being tested for sterilization. Though it is not as effective as other methods, it was found to be useful in tissue cultures. Here the aim is to sterilize or even prevent the growth of bacteria during culturing of tissue. For ultrasonic sterilization, special ultrasonic transducers are used. Sterilize food products (their nutritional value is kept as much as possible), vaccines and some objects of laboratory equipment that spoil under the influence of high temperature and chemical sterilization

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