Sterilization is the removal of objectionable / specified microorganisms by control of microbial population, reduction in count, or complete elimination. Bacterial spores, mold spores, vegetative cells, viruses, and prions are the major target to be sterilized.
What is the Order of resistance of target to be sterilized?
Microorganisms are arranged in the following order for sterilization:
- Bacterial spores
- Non lipid or small virus
- Mold spores
- Vegetative bacteria
- Lipid or medium sized virus 3
What are the basic terminologies of sterilization?
The predominant process of sterilization includes:
- Sterilant: A sterilizing agent
- Commercial sterilization: Sufficient heat treatment to destroy endospores of Clostridium botulinum in canned food.
- Disinfection: Killing of vegetative pathogens on non- living surface.
- Antisepsis: Killing of vegetative pathogens on living tissue.
- Biocide/ germicide: To kill Bacteriostatic decay/ putrid, to stop or steady
- Sepsis: Indicates bacterial contamination
What are the items to be sterilized?
While fixating on killing the most resistant microbe in the process, it may result in over treatment of many items especially product, media & elastomeric materials. This creates numerous problems including degradation of product, loss of flexibility, increased particles, increased process time, change in essential properties, lost capacity, changes in color, increased extractables & leachables, increased costs, and loss of integrity.
Proper processes of sterilization must be capable of eliminating undesirable microbes while maintaining the essential quality attributes. No more, no less just achieving a perfect balance. Consider the items below to sterilize:
- Fluids: Liquids (formulated products, solutions, suspensions, media, waste streams) and gases
- Glass: Containers and vessels
- Solid & other metals: Tanks, valves, fill parts, and utensils
- Elastomeric materials: Containers, closures, filters, tubing, gaskets, valve components, and medical devices
What is the Probability of non-sterile Unit?
A minimum probability of non- sterile Unit (PNSU) of 10-6 is required. That means that in daily use of the sterilizer, the chance for a surviving microorganism must be less than 1 in 1,000,000, or 1 non-sterile unit in 1,000,000 units.
What Influences the antimicrobial treatment in sterilization?
Microbial control agents act by alteration of membrane permeability, damage to protein, or destruction to nucleic acids. Antimicrobial process in sterilization depends on number of microbes, environmental influences, time of exposure, and microbial characteristics.
What is D-Value?
The D-value is the time required to reduce a population of microorganisms by one log or a 90% reduction in count. It is only meaningful if referenced to specified lethal conditions. For example, it should always be referenced to a temperature, without that reference they have no meaning, i.e., moist heat D121.1°C or dry heat D170°C. For D-values in gases / liquids the agent concentration, RH and temperature must be indicated, i.e., D900 PPM, 75% RH, and 30°C.
What are the methods of Microbial Control in sterilization?
Physical and chemical methods are involved in the removal of microbes. Let’s discuss physical methods of sterilization first in which microbes are removed by using mechanical methods is included in physical methods.
It includes destruction of microbes by denaturing the enzymes. TDP (Thermal Death Point) is the lowest temp at which all the microorganisms in a particular liquid suspension will be killed in 10 minutes while TDT (Thermal Death Time) is the minimal length of time for all bacteria in a particular liquid to be killed at given temperature. DRT (decimal reduction time) or D value is the time in minutes, in which 90% of bacteria at a given temperature will be killed.
In moist heat, proteins of microorganisms by coagulation by boiling or autoclaving at 121°C, 15 psi in about 15 minutes. This method is effective only when organisms are either contacted by steam directly or contained in small volume of aqueous liquid. It is used to sterilize culture media, instruments, dressings, intravenous equipment, solutions, and transfusion equipment. Solid does not develop convection currents so aluminum foil is used which is impervious to heat and avoid trapped air in the container. Petroleum jelly and mineral oil cannot be sterilized by moist heat.
This method is used in beer and wine preservation which includes mild heat for the elimination of pathogenic bacteria and lower microbial number. Pasteurization is categorized as:
- Classic: 63°C for 30 minutes
- HTST: 72°C for 15 seconds
- UHT: 140°C for 3 sec
Ice cream, yogurt, and beer have own pasteurization time.
By oxidation effect, dry heat is used as flaming, incineration, and hot air sterilization at 170°C for 2 hours. The process takes higher time than moist heat as heat in water is more readily transferred.
Screen like material is used in filtration for heat sensitive material. High efficiency particulate air filters are also included. Membrane filters are cellulose ester plastic polymer of pore size 0.22 micrometer or 0.45 micrometer as small as 0.01 um.
There is a reduction of metabolic rate of microorganisms like Pyschrotrophs which may survive at -20°C. Slow freezing is more harmful. At low temperature, only third population of microbes survives.
Applying high pressure to liquid suspensions containing microbes alter their carbohydrate and protein structures. Endospores are resistant to this technique.
Microorganisms are killed in the absence of water when they cannot grow or reproduce. Typically in this process, lypholization or freeze drying are used. The resistance varies with the species and organism’s environment as Gonorrhea bacterium can with stand dryness for only about an hour, but tuberculosis bacteria can remain viable for months. Viruses are resistant but less than the bacterial endospores.
Ionizing radiation uses gama rays, X-rays, high energy electron beams of wavelength less than 1nm. Gamma rays can penetrate deeply but may require hours to sterilize while high energy electron beams have lower penetrating power but usually require only few seconds of exposure. Principle of ionizing radiation is the ionization of water that forms reactive hydroxyl radicals. Non Ionizing radiations are greater than 1nm UV which Damage DNA of microbes by forming pyrimidine dimers. 260 nm most effective, as it is absorbed by DNA. These radiations are not very penetrating so direct exposure is required such as Sunlight and microwaves.
A few chemical methods of sterilization include:
Phenol and Phenolics
Phenol is used as a disinfectant in operating rooms. It controls odor in sewage. Local anesthetic in throat lozenges below 1% and derivatives of phenol (phenolics) are used to reduce irritating quality or increase antibacterial activity which exert effect by injuring lipids in plasma membrane and remain active in organic matter and persistent. Example includes Cresol (o-phenyl phenol): Lysol.
Two phenolic groups connected by bridge such as Triclosan that is antibacterial soaps and tooth paste. Bisphenol is incorporated into kitchen cutting boards, handles of knives and other plastic kitchen ware. It inhibits enzyme of microbes required for synthesis of lipids which affects the integrity of plasma membrane.
Halogens include iodine and chlorine which impairs protein synthesis and alters cell membrane of microbes by making complex with amino acids and unsaturated fatty acids. Examples are tincture, a solution in aqueous alcohol, and Iodophor which is a combination with organic molecule. Chlorine is used as a gas or hypochlorous acid which is a strong oxiding agent preventing enzymatic functions of microorganisms. Calcium hypochlorite and sodium hypochlorite used to disinfect dairy and restaurant utensils. Sodium hypochlorite (Bleach), sodium dichloroisocyanuate (used by US military forces), chlorine dioxide in anthrax, chloramines in sanitizer are the major examples.
They denature protein dissolve lipids to kill microbes. They evaporate which is not good for wounds, for example, ethanol and isopropanol of optimum concentration 70%.
Sulfur dioxide as a food preservative is used in wine making; sodium benzoate in soft drinks and cheese, sorbic acid, calcium propionate in bread, sodium nitrate and nitrite to inhibits iron containing enzymes, and sodium nitrate and sodium nitrite in meat products.
Formaldehyde and glutaraldehyde are the most effective method to kill microbes which inactivate proteins by forming cross links with several organic functional groups on proteins. Formalin is 37% aq sol of formaldehyde and Glutaraldehyde which is less irritating and more effective for the disinfection of hospital instruments.
They oxidize cellular contents such as ozone for water treatment, hydrogen peroxide, peracetic acid, ozone, benzoyl peroxide for acne, and peracetic acid for liquid sporicides.
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