Growth and multiplication of microorganisms can be controlled by removing, killing or inhibiting them using various physical or chemical agents.
Physical Methods of Sterilization
The various physical methods of
sterilization are given in flowchart 4.1
Heat is the most rapid and best method of sterilization. It is the method of choice that the material to be sterilized is stable enough to withstand the required temperature necessary to kill the microbes. The time needed for sterilization depends on the initial number of organisms present, type of materials to be sterilized (hence washed and cleaned items are easier to sterilize than dirty ones) and also on the temperature used. Spores need higher temperatures while vegetative bacteria can be destroyed at lower temperatures.
Heat resistance varies among different microorganisms. These differences can be expressed in terms of thermal death point. Thermal Death Point (TDP) is the lowest temperature at which all the microorganisms in a particular liquid suspension will be killed in 10 minutes.
Another factor to be considered in sterilization is the duration of time required. This is expressed as Thermal Death Time (TDT). TDT is the minimal time required for all microorganism in a particular liquid culture to be killed at a given temperature. Both TDP and TDT are useful guidelines that indicate the degree of treatment required to kill a given population of bacteria.
Decimal Reduction Time (DRT) is related to bacterial heat resistance. DRT is the time, in minutes, in which 90% of a population of microorganism at a given temperature will be killed.
Heat is employed either as dry heat or moist heat.
Dry heat is frequently used for the sterilization of glassware and laboratory equipments. In dry heat sterilization, microbial cells are apparently killed by oxidation of their constituents and protein denaturation. Dry heat is applied in the following ways:
Inoculating wires, points of forceps and searing spatulas are sterilized by holding them in the flame of a bunsen burner until they are seen to be red hot.
This method is used for sterilizing scalpels, needles, mouths of culture tubes, slides and cover slips. It involves passing the article through the bunsen flame without allowing it to become red hot.
This is an excellent method for destroying materials such as contaminated clothes, cotton wool stoppers, animal carcasses and pathological materials. It involves burning of materials in incinerators.
This is the most widely used method of sterilization using dry heat. The oven is usually heated by electricity and it has a thermostat that maintains the chamber air constantly at the chosen temperature.
It has a fan or turbo-blower to assist the circulation of air and to ensure rapid, uniform heating of the load. In Hot Air Oven, the air is heated at a temperature of 160oC for one hour. Figure 4.1 shows laboratory hot air oven.
This is the best method of sterilizing dry glass ware such as test tubes, petri dishes, flasks, pipettes and instruments such as forceps, scalpels and scissors. It is also used to sterilize some pharmaceutical products such as liquid paraffin, dusting powder, fats and grease.
The spores of a nontoxigenic strain of Clostridium tetani are used to test the efficiency of dry heat sterlization.
Moist heat kills microorganisms primarily by the coagulation of proteins (denaturation), which is caused by breakage of the hydrogen bonds that hold the proteins in three dimensional structure.
There are three methods employed in moist heat sterilization.
· Temperature below 100°C.
· Temperature at 100°C.
· Temperature above 100°C.
The process of heating a liquid food or beverage either at 62.8°C for 30 minutes or 72°C for 15 seconds to enhance their shelf life and destroy harmful microorganisms. It should be noted that pasteurization process kills only vegetative cells but not the spores. Pasteurization named in honour of its developer Louis Pasteur. Table 4.1 gives comparison between Sterilization and Pasteurization.
Pasteurization can be done in the following methods,
In this method milk, beer and fruit juices are maintained at 62.8°C for 30 minutes.
Products are held at 72°C for 15 seconds.
Milk can be treated at 141°C for 2 seconds (This method employ temperature above 100°C).
Boiling is one of the moist heat sterilization methods. It kills vegetative forms of bacterial pathogens, almost all viruses and fungi (including their spores) within 10 minutes, usually much faster.
· Most vegetative bacteria will die in 5-10 minutes when immersed in boiling water, but some spores will survive at this temperature for several hours.
· Articles sterilized by this method cannot be stored for a long time.
It is a process discovered by John Tyndall in 19th century for sterilizing substances to kill the spores of bacteria. The process of exposure of materials to steam at 100°C for 20 min for three consecutive days is known as tyndallization. First exposure kills all the vegetative forms and in the intervals between heating, the remaining spores germinate into vegetative forms which are killed on subsequent heating. Tyndallization is also called fractional sterilization or intermittent boiling.
Moist heat sterilization can be carried out at temperature above 100°C in order to destroy bacterial endospores. This requires the use of saturated steam under pressure. This is achieved using autoclave.
Sterilization using an autoclave is most effective when the organisms are either contacted by the steam directly or contained in a small volume of aqueous liquid (primarily water). The temperature used in autoclave is 121°C at 15 lbs (pounds) pressure for 15 minutes (Figure 4. 2a & b).
Autoclaving is used in sterilizing culture media, instruments, dressings, applicators, solutions, syringes, transfusion equipment, pharmaceutical products, aqueous solutions and numerous other items that can withstand high temperatures and pressures. The same principle of autoclaving applies for the common household pressure cooker used for cooking food.
Sterilization by heat depends upon various factors such as time, temperature employed, number of microorganisms, spores and nature of material to be sterilized.
To check the efficiency of moist heat sterlization, the indicator commonly used is the paper strips containing spores of Bacillus sterothermophilus.
Radiation is commonly employed for sterilizing heat sensitive materials such as disposable plastic products and materials that cannot withstand moisture.
The most effective type of radiation to sterilize or reduce the microbial burden in the substance is through the use of electromagnetic radiations. Figure 4.3 shows different types of electromagnetic radiations. Radiation has various effects on cells, depending on its wavelength, intensity and duration of explosure (Flowchart 4.2). Radiation that kills microorganism is of two types namely ionizing and nonionizing.
Infra-red rays and ultra-violet rays are non ionizing radiation.
These are electromagnetic rays with wavelengths longer than those of visible light. These are low energy type. It kills microorganisms by oxidation of molecules as a result of heat generated. Infrared radiation is used for rapid mass sterilization of pre-packed items such as syringes and catheters.
The ultraviolet (UV) portion of the electromagnetic spectrum includes all radiations from 150-3900A°, UV radiation around 2600A° is most lethal to microorganisms. UV has a very little ability to penetrate matter. Thus, only the microorganisms on the surface of an object, exposed directly to the ultraviolet light are susceptible to destruction. UV radiations are used to sterilize operation theaters, laboratories and entry ways.
Ionizing radiations (X-rays, Gamma rays and Cosmic rays) are an excellent sterilizing agents and they penetrate deep into the objects. These radiations do not produce heat on the surface of materials. Hence, sterilization using ionizing radiations is referred as cold sterilization. It will destroy bacterial endospores and vegetative cells, both Prokaryotic and Eukaryotic; however ionizing radiation is not always effective against viruses. Gamma radiation from Cobalt 60 source is used in the cold sterilization of antibiotics, hormones, sutures and plastic disposables supplied such as syringes and in pasteurization of meat.
Filtration is an effective and reasonably economical method of sterilization. It is used to sterilize heat-sensitive fluids, and air. It is particularly useful for solutions containing toxins, enzymes, drug, serum and sugars. Sugar solutions used for the cultivation of microorganisms tend to caramelise during autoclaving and so they are best sterilized by filtration. Filtration is also used extensively in beer and wine industries. Filters with known pore sizes which are sufficiently small to hold back bacteria are employed. Recently filters that can remove viruses are also available.
Filtration is an excellent way to remove the microbial population from solution containing heat sensitive material.
There are two types of filters namely (Figure 4.4):
1. Membrane filter (surface filtration) and
2. Depth filter
Membrane filtration is used for preparing heat-labile culture media components. It
Membrane filters are made up of either cellulose acetate, cellulose nitrate, polycarbonate, polyvinylidene fluoride or other synthetic porous materials. These filters remove microorganisms by screening them out, such as a sieve separates large sand particles from small ones. Membranes with pore size of 0.2μm in diameter are used to remove most vegetative cells but not viruses. These filters are used to sterilize pharmaceutical products, ophthalmic solutions, culture media, oils, antibiotics, and other heat sensitive solutions (Figure 4.5a, b & c).
Depth filters are the oldest type of filters and they consist of overlapping layers of fibrous sheets of paper, asbestos or glass fibers. The overlapping fibers create
Depth filter are made up of diatomaceous earth (Berkefeld filters) which are used as water purifiers. Examples of types of depth filters (Figure 4.6) contains unglazed porcelain (Chamberl and filters) and asbestos (Seitz Filter).
Air also can be sterilized by filtration.
The air is freed from infection by passing it through High Efficiency Particle Arrester (HEPA) filter. Laminar air flow biological safety cabinets are one of the most important air filtration systems (Figure 4.7). It employes HEPA filters which remove 99.97% of 0.33μm particles size. Some operation theaters and rooms occupied by burn patients receive filtered air to lower the numbers of airborne microbes. HEPA filters remove almost all microorganisms above 0.3μm in diameter.
Various physical methods of sterlization is summarized in Table 4.2