Sterilization is defined as the process where all the living microorganisms, including bacterial spores are killed.
Filtration Filtration does not kill microbes, it separates them out. Membrane filters with pore sizes between 0.2-0.45 μm are
commonly used to remove particles from solutions that can't be autoclaved. It is used to remove microbes from heat
labile liquids such as serum, antibiotic solutions, sugar solutions, urea solution. Various applications of filtration
include removing bacteria from ingredients of culture media, preparing suspensions of viruses and phages free of
bacteria, measuring sizes of viruses, separating toxins from culture filtrates, counting bacteria, clarifying fluids and
purifying hydatid fluid. Filtration is aided by using either positive or negative pressure using vacuum pumps. The
older filters made of earthenware or asbestos are called depth filters.
Autoclaving Autoclaving is a method of sterilizing with water vapor under pressure. Nearly all microbes are killed by
xposure to the super-heated steam of an autoclave for 10-15 minutes. All objects should be
sterilized at 121°C and 15 psi for 15-20 min. Cotton plugs, gauze, labware, plastic caps, glassware,
filters, pipettes, water, and nutrient media can all be sterilized by autoclaving, but autoclaving is not
advisable for metal instruments due to the rust it may cause.
Sterilizing by boiling Boiling does not kill bacterial spores.Thereefore it is not best method for sterilizing.In this method instruments
should be heated in boiling water bath for 30 min.
Hot-dry sterilization Metal instruments, glassware, aluminum foil, etc., can be sterilized by exposure to hot dry air (130°-170°C)
for 2-4 hr in a hot-air oven. All items should be sealed before sterilization but not in paper, as it decomposes
at 170°C.
Flame sterilization Instruments can be dipped in ethyl alcohol and flamed to burn off all bacteria and fungi. Safety is a major
concern when using ethyl alcohol. Alcohol is flammable and if spilled near a flame will cause an instant flash fire.
This problem is compounded in laminar flow hoods due to the strong air currents blown towards the worker.
Fires commonly start when a flamed instrument is thrown back into the alcohol beaker. In case of fire do not panic.
Limiting the supply of oxygen can easily put out fires.This can use for metal instruments.
Red heat Articles such as bacteriological loops, straight wires, tips of forceps and searing spatulas are
sterilized by holding them in Bunsen flame till they become red hot. This is a simple method for effective
sterilization of such articles, but is limited to those articles that can be heated to redness in flame.
Sterilization Using Chemicals ALCOHOLS-
Alcohols dehydrate cells, disrupt membranes and cause coagulation of protein.(Examples: Ethyl alcohol,
isopropyl alcohol and methyl alcohol).A 70% aqueous solution is more effective at killing microbes than
absolute alcohols. 70% ethyl alcohol (spirit) is used as antiseptic on skin. Isopropyl alcohol is preferred to ethanol.
It can also be used to disinfect surfaces. It is used to disinfect clinical thermometers. Methyl alcohol kills fungal
spores, hence is useful in disinfecting inoculation hoods.
ALDEHYDES-
Acts through alkylation of amino-, carboxyl- or hydroxyl group, and probably damages nucleic
acids. It kills all microorganisms, including spores.(Examples: Formaldehyde, Gluteraldehyde)
40% Formaldehyde (formalin) is used for surface disinfection and fumigation of rooms, chambers,
operation theatres, biological safety cabinets, wards, sick rooms etc.
PHENOL-
Act by disruption of membranes, precipitation of proteins and inactivation of enzymes.
(Examples: 5% phenol, 1-5% Cresol, 5% Lysol (a saponified cresol), hexachlorophene, chlorhexidine, chloroxylenol
(Dettol) )Joseph Lister used it to prevent infection of surgical wounds. Phenols are coal-tar derivatives. They
act as disinfectants at high concentration and as antiseptics at low concentrations. They are bactericidal, fungicidal,
mycobactericidal but are inactive against spores and most viruses.
HYDROGEN PEROXIDE-
It acts on the microorganisms through its release of nascent oxygen. Hydrogen peroxide produces
hydroxyl-free radical that damages proteins and DNA.It is used at 6% concentration to decontaminate the
instruments, equipments such as ventilators.
3% Hydrogen Peroxide Solution is used for skin disinfection and deodorising wounds and ulcers.
Strong solutions are sporicidal.
Sterilization using Radiation Two types of radiation are used, ionizing and non-ionizing. Non-ionizing rays are low energy rays with poor
penetrative power while ionizing rays are high-energy rays with good penetrative power. Since radiation does not
generate heat, it is termed "cold sterilization". In some parts of Europe, fruits and vegetables are irradiated to
increase their shelf life up to 500 percent. Non-ionizing rays:- Rays of wavelength longer than the visible light are non-ionizing. Microbicidal
wavelength of UV rays lie in the range of 200-280 nm, with 260 nm being most effective. UV rays are
generated using a high-pressure mercury vapor lamp. It is at this wavelength that the absorption by the
microorganisms is at its maximum, which results in the germicidal effect. UV rays induce formation of
thymine-thymine dimers, which ultimately inhibits DNA replication. UV readily induces mutations in cells
irradiated with a non-lethal dose. Microorganisms such as bacteria, viruses, yeast, etc. that are exposed to
the effective UV radiation are inactivated within seconds. Since UV rays don’t kill spores, they are
considered to be of use in surface disinfection. UV rays are employed to disinfect hospital wards, operation
theatres, virus laboratories, corridors, etc. Disadvantages of using uv rays include low penetrative power,
limited life of the uv bulb, some bacteria have DNA repair enzymes that can overcome damage caused by
uv rays, organic matter and dust prevents its reach, rays are harmful to skin and eyes. It doesn't penetrate
glass, paper or plastic.
Ionizing rays:- Ionizing rays are of two types, particulate and electromagnetic rays.
Electron beams are particulate in nature while gamma rays are electromagnetic in nature. Highspeed
electrons are produced by a linear accelerator from a heated cathode. Electron beams are
employed to sterilize articles like syringes, gloves, dressing packs, foods and pharmaceuticals.
Sterilization is accomplished in few seconds. Unlike electromagnetic rays, the instruments can be
switched off. Disadvantage includes poor penetrative power and requirement of sophisticated
equipment.
Electromagnetic rays such as gamma rays emanate from nuclear disintegration of certain
radioactive isotopes (Co60, Cs137). They have more penetrative power than electron beam but
require longer time of exposure. These high-energy radiations damage the nucleic acid of the
microorganism. A dosage of 2.5 megarads kills all bacteria, fungi, viruses and spores. It is used
commercially to sterilize disposable petri dishes, plastic syringes, antibiotics, vitamins, hormones,
glasswares and fabrics. Disadvantages include; unlike electron beams, they can’t be switched off,
glasswares tend to become brownish, loss of tensile strength in fabric. Gamma irradiation impairs
the flavour of certain foods. Bacillus pumilus E601 is used to evaluate sterilization process.
