the elimination of all transmissible agents
Sterilization (such as bacteria, prions and viruses) from a
surface, a piece of equipment, food or
biological culture medium.
Prof. S.T. Yang Disinfection:
the elimination of only the pathogenic
Dept. Chemical & Biomolecular Eng. microorganism
The Ohio State University
Why Sterilization? Source of Contaminants
“To reduce the amount of contaminants
present in the culture medium” Raw materials
Equipment and Instruments
Containers and closure systems
Cause loss of productivity and product Using pure inoculum to start the
Contaminate and degrade final products Sterilizing medium, fermentor, air, etc
Could outgrow the production organisms Maintaining aseptic conditions
Could results in lysis of the culture (phage Use natural selection to resist the growth
contaminants) of contaminants:
pH, EtOH, high Temperature, etc.
Control of Microbial Growth Control of Microbial Growth
Early civilizations practiced salting, In mid 1800s Semmelweiss and Lister
smoking, pickling, drying, and exposure of helped developed aseptic techniques to
food and clothing to sunlight to control prevent contamination of surgical wounds.
microbial growth. Before then:
• Nosocomial infections caused death in 10% of
Use of spices in cooking was to mask surgeries.
taste of spoiled food. Some spices • Up to 25% mothers delivering in hospitals
prevented spoilage. died due to infection
Sterilization: Killing or removing all forms of Disinfection: Reducing the number of pathogenic
microbial life (including endospores) in a microorganisms to the point where they no longer
material or an object. cause diseases. Usually involves the removal of
Heating is the most commonly used method of vegetative or non-endospore forming
Commercial Sterilization: Heat treatment that May use physical or chemical methods.
kills endospores of Clostridium botulinum the Disinfectant: Applied to inanimate objects.
causative agent of botulism, in canned food. Antiseptic: Applied to living tissue (antisepsis).
Does not kill endospores of thermophiles, which Degerming: Mechanical removal of most microbes in a
are not pathogens and may grow at temperatures limited area. Example: Alcohol swab on skin.
above 45oC. Sanitization: Use of chemical agent on food-handling
equipment to meet public health standards and minimize
chances of disease transmission. E.g: Hot soap & water.
Biological Terms Biological Terms (cont’d)
Sepsis: Comes from Greek for decay or putrid. Bacteriostatic Agent: An agent that inhibits the
Indicates bacterial contamination. growth of bacteria, but does not necessarily kill
them. Suffix stasis: To stop or steady.
Asepsis: Absence of significant contamination.
Germicide: An agent that kills certain
Aseptic techniques are used to prevent microorganisms.
contamination of surgical instruments, medical Bactericide: An agent that kills bacteria. Most do not
personnel, and the patient during surgery. kill endospores.
Aseptic techniques are also used to prevent Viricide: An agent that inactivates viruses.
bacterial contamination in the food industry. Fungicide: An agent that kills fungi.
Sporicide: An agent that kills bacterial endospores or
Factors Affecting Sterilization Types of Sterilization
Number of Microbes: The more microbes present, the Heat
more time it takes to eliminate population.
Type of Microbes: Endospores are very difficult to Chemicals
destroy. Vegetative pathogens vary widely in
susceptibility to different methods of microbial control. Filtration
Environmental influences: Presence of organic Irradiation
material (blood, feces, saliva) tends to inhibit
antimicrobials, pH etc. Others – pressure, desiccation, freezing,
Time of Exposure: Chemical antimicrobials and etc.
radiation treatments are more effective at longer times.
In heat treatments, longer exposure compensates for
Heat Sterilization Heat Sterilization
Heat: Kills microorganisms by denaturing their enzymes
When bacterial populations are heated or
and other proteins. Heat resistance varies widely among treated with antimicrobial chemicals, they usually
microbes. die at a constant rate.
Thermal Death Point (TDP): Lowest temperature at which
all of the microbes in a liquid suspension will be killed in ten
Thermal Death Time (TDT): Minimal length of time in which
all bacteria will be killed at a given temperature.
Decimal Reduction Time (DRT): Time in minutes at which
90% of bacteria at a given temperature will be killed. Used in
Heat Sterilization – Moist Heat Heat Sterilization – Moist Heat
Moist Heat: Kills microorganisms by coagulating Moist Heat (Continued):
their proteins. Reliable sterilization with moist heat requires
In general, moist heat is much more effective than temperatures above that of boiling water.
dry heat. Autoclave: Chamber which is filled with hot steam under
Boiling: Heat to 100oC or more at sea level. Kills pressure. Preferred method of sterilization, unless material
vegetative forms of bacterial pathogens, almost all viruses, is damaged by heat, moisture, or high pressure.
and fungi and their spores within 10 minutes or less. Temperature of steam reaches 121oC at 15 psig (2× atmospheric pressure).
Endospores and some viruses are not destroyed this Most effective when organisms contact steam directly or are contained in a
quickly. However brief boiling will kill most pathogens. small volume of liquid.
All organisms and endospores are killed within 15 minutes.
Hepatitis virus: Can survive up to 30 minutes of boiling.
Require more time to reach center of solid or large volumes of liquid.
Endospores: Can survive up to 20 hours or more of boiling.
Autoclave Moist Heat - Pasteurization
Pasteurization: Developed by Louis Pasteur to prevent the
spoilage of beverages. Used to reduce microbes
responsible for spoilage of beer, milk, wine, juices, etc.
Classic Method of Pasteurization: Milk was exposed to 65oC for
High Temperature Short Time Pasteurization (HTST): Used
today. Milk is exposed to 72oC for 15 seconds.
Ultra High Temperature Pasteurization (UHT): Milk is treated at
140oC for 3 seconds and then cooled very quickly in a vacuum
Advantage: Milk can be stored at room temperature for several
Heat Sterilization – Dry Heat Filtration Sterilization
Removal of microbes by passage of a liquid or gas
Dry Heat: Kills by oxidation effects. through a screen-like material with small pores.
Direct Flaming: Used to sterilize inoculating loops and Used to sterilize heat sensitive materials like
needles. Heat metal until it has a red glow. vaccines, enzymes, antibiotics, and some culture
Incineration: Effective way to sterilize disposable items media.
(paper cups, dressings) and biological waste. High Efficiency Particulate Air Filters (HEPA): Used in
Hot Air Sterilization: Place objects in an oven. operating rooms and burn units to remove bacteria from air.
Require 2 hours at 170oC for sterilization. Dry heat Membrane Filters: Uniform pore size. Used in industry
transfers heat less effectively to a cool body, than moist and research. Different sizes:
heat. 0.22 and 0.45 μm Pores: Used to filter most bacteria. Don’t retain
spirochetes, mycoplasmas and viruses.
0.01 μm Pores: Retain all viruses and some large proteins.
Freezing Sterilization Desiccation Sterilization
Low Temperature: Effect depends on microbe Dessication: In the absence of water, microbes
and treatment applied.
cannot grow or reproduce, but some may remain
Refrigeration: Temperatures from 0 to 7oC.
viable for years. After water becomes available,
Bacteriostatic effect. Reduces metabolic rate of
they start growing again.
most microbes so they cannot reproduce or
Susceptibility to dessication varies widely:
Freezing: Temperatures below 0oC. Neisseria gonnorrhea: Only survives about one hour.
Flash Freezing: Does not kill most microbes. Mycobacterium tuberculosis: May survive several months.
Slow Freezing: More harmful because ice crystals Viruses are fairly resistant to dessication.
disrupt cell structure.
Over a third of vegetative bacteria may survive 1 year. Clostridium spp. and Bacillus spp.: May survive decades.
Most parasites are killed by a few days of freezing.
Pressure-driven Sterilization Radiation Sterilization
Osmotic Pressure: The use of high Three types of radiation kill microbes:
concentrations of salts and sugars in foods is - Ionizing Radiation
used to increase the osmotic pressure and
create a hypertonic environment. - Ultraviolet light (Nonionizing Radiation)
Plasmolysis: As water leaves the cell, plasma - Microwave Radiation
membrane shrinks away from cell wall. Cell may
not die, but usually stops growing.
Yeasts and molds: More resistant to high osmotic
Staphylococci spp. that live on skin are fairly resistant
to high osmotic pressure.
Ionizing Radiation Ultraviolet Radiation
Gamma rays, X rays, electron beams, or higher Nonionizing radiation
energy rays. Have short wavelengths (less than
1 nanometer). Wavelength is longer than 1 nanometer.
Dislodge electrons from atoms and form ions. Damages DNA by producing thymine dimers,
Cause mutations in DNA and produce which cause mutations.
peroxides. Used to disinfect operating rooms, nurseries,
Used to sterilize pharmaceuticals and cafeterias.
disposable medical supplies. Food industry is
interested in using ionizing radiation. Disadvantages: Damages skin, eyes. Doesn’t
Disadvantages: Penetrates human tissues. May penetrate paper, glass, and cloth.
cause genetic mutations in humans.
Microwave Radiation Chemical Sterilization
Wavelength ranges from 1 millimeter to 1 meter. Phenol and Phenolics
Heat is absorbed by water molecules. Halogens
May kill vegetative cells in moist foods. Alcohols
Bacterial endospores, which do not contain Heavy Metals
water, are not damaged by microwave radiation.
Quaternary Ammonium Compounds
Solid foods are unevenly penetrated by
Trichinosis outbreaks have been associated with Gaseous
pork cooked in microwaves. Peroxygens
Chemical - Phenolic Chemical - Halogens
Phenol (carbolic acid) was first used by Lister as a
Rarely used today because it is a skin irritant and has strong Tincture of iodine (alcohol solution) was one of first
odor. antiseptics used.
Used in some throat sprays and lozenges. Combines with amino acid tyrosine in proteins and
Acts as local anesthetic. denatures proteins.
Phenolics are chemical derivatives of phenol Stains skin and clothes, somewhat irritating.
Cresols: Derived from coal tar (Lysol).
Iodophors: Compounds with iodine that are slow
Biphenols (pHisoHex): Effective against gram-positive
staphylococci and streptococci. Used in nurseries. Excessive
releasing, take several minutes to act. Used as skin
use in infants may cause neurological damage. antiseptic in surgery. Not effective against bacterial
Destroy plasma membranes and denature proteins.
Advantages: Stable, persist for long times after applied, Isodine
and remain active in the presence of organic
Chemical – Halogens (cont’d) Chemical - Alcohols
Chlorine: Kill bacteria, fungi, but not endospores or naked viruses.
When mixed in water forms hypochlorous acid: Act by denaturing proteins and disrupting cell
Cl2 + H2O ------> H+ + Cl- + HOCl
Evaporate, leaving no residue.
Used to mechanically wipe microbes off skin before
Used to disinfect drinking water, pools, and sewage. injections or blood drawing.
Chlorine is easily inactivated by organic materials. Not good for open wounds, because cause proteins to
Sodium hypochlorite (NaOCl): Is active ingredient of coagulate.
bleach. Ethanol: Drinking alcohol. Optimum concentration is 70%.
Chloramines: Consist of chlorine and ammonia. Less Isopropanol: Rubbing alcohol. Better disinfectant than
effective as germicides. ethanol. Also cheaper and less volatile.
Chemical – Heavy Metals Chemical – Heavy Metals (cont’d)
Oligodynamic action: Very tiny amounts are effective.
Copper sulfate is used to kill algae in pools and fish
1% silver nitrate used to protect infants against
gonorrheal eye infections until recently. Selenium
Kills fungi and their spores. Used for fungal infections.
Mercury Also used in dandruff shampoos.
Organic mercury compounds like merthiolate and
mercurochrome are used to disinfect skin wounds. Zinc
Zinc chloride is used in mouthwashes.
Zinc oxide is used as antifungal agent in paints.
Chemical – Quats Chemical – Aldehydes
Widely used surface active agents. Inactivate proteins by forming covalent crosslinks with
Cationic (positively charge) detergents. several functional groups.
Include some of the most effective antimicrobials.
Effective against gram positive bacteria, less effective
against gram-negative bacteria.
Also destroy fungi, amoebas, and enveloped viruses.
Zephiran, Cepacol, also found in lab spray bottles.
Commonly used as formalin, a 37% aqueous solution.
Pseudomonas strains that are resistant and can grow Formalin was used extensively to preserve biological
in presence of Quats are a big concern in hospitals. specimens and inactivate viruses and bacteria in
Advantages: Strong antimicrobial action, colorless, vaccines.
odorless, tasteless, stable, and nontoxic. Irritates mucous membranes, strong odor.
Disadvantages: Form foam. Organic matter interferes Also used in mortuaries for embalming.
with effectiveness. Neutralized by soaps and anionic
Chemical – Aldehydes (cont’d) Chemical – Gaseous Sterilizers
Chemicals that sterilize in a chamber similar to an
Less irritating and more effective than formaldehyde. Denature proteins, by replacing functional groups with
One of the few chemical disinfectants that is a alkyl groups.
A 2% solution of glutaraldehyde (Cidex) is: Ethylene Oxide:
Bactericidal, tuberculocidal, and viricidal in 10 Kills all microbes and endospores, but requires
minutes. exposure of 4 to 18 hours.
Sporicidal in 3 to 10 hours. Toxic and explosive in pure form.
Commonly used to disinfect hospital instruments. Highly penetrating.
Also used in mortuaries for embalming. Most hospitals have ethylene oxide chambers to
sterilize mattresses and large equipment.
Chemical – Peroxygens Chemical – Peroxygens (cont’d)
Oxidizing agents Hydrogen Peroxide:
Disrupt membranes and proteins. Used as an antiseptic.
Oxidize cellular components of treated microbes. Not good for open wounds because quickly broken
down by catalase present in human cells.
Ozone: Effective in disinfection of inanimate objects.
Used along with chlorine to disinfect water. Sporicidal at higher temperatures.
Helps neutralize unpleasant tastes and odors. Used by the food industry and to disinfect contact
More effective killing agent than chlorine, but less lenses.
stable and more expensive.
Highly reactive form of oxygen. Benzoyl Peroxide:
Made by exposing oxygen to electricity or UV light. Used in acne medications.
Efficiency of Different Chemical
Chemical – Peroxygens (cont’d) Antimicrobial Agents
One of the most effective liquid sporicides available.
Kills bacteria and fungi in less than 5 minutes.
Kills endospores and viruses within 30 minutes.
Used widely in disinfection of food and medical
instruments because it does not leave toxic residues.
Sterilization in fermentation
Fermentors and other equipment