Sterilization means the freeing of an article from all living organisms, including viruses, bacteria and their spores, and fungi and their spores, both pathogenic and non- pathogenic. It is an absolute state.
Uses:
1. Culture medias
2. Suspending fluids
3. Reagents
4. Containers
5. Equipment used in microbiology.
6.Medical & Surgical instruments
7.Materials used in surgical operations, intravenous infusions, hypodermic injections.
Methods:
Four main methods are used for sterilization:
1. Heat
2. Filtration
3. Radiation
4. Chemical.
PHYSICAL METHOD:
1. Heat
A. Dry heat
I. Red heat
II. Flaming
III. Hot air oven
IV. Infra red radiation
B. Moist heat
At a temperature below 100 °C
1.Pasteurization
II. Vaccine bath
III. Water bath
At a temperature of 100 °C
I. Boiling
II. Steaming
1.Single exposure
II. Tantalization
At a temperature above 100 °C
1 Autoclave
2.Filtration
3. Radiation
A. High energy radiation
B. Ultraviolet rays
4. Sonic waves
5. Cold
CHEMICAL METHODS:
A Solid
B. Liquid
C .Gas
Sterilization by moist heat
Moist heat is more effective than dry heat.. Killing of the most resistant spores, requires exposure to moist heat at 121 °C for 10 to 30 minutes. Moist heat kills micro-organisms by coagulation and denaturation of enzymes and structural proteins.....a process in which water participates
• At temperatures below 100 °C
•At a temperature of 100 °C
•At a temperature above 100 °C i.e.in saturated steam under increasing pressure in an autoclave..
The first two procedures may be used for disinfection but only the third ensures sterilization and killing of highly resistant spores.
1. Moist heat at a temperature. below 100 °C:
• Pasteurization:
1. The holder method. 63 °C to 66 °C for 30 min.
2. The flash method. 72 °C for 20 sec.
These processes destroy all the non- spore forming pathogens
like MTB, M. bevies, B. aborts, Salmonella, Shhigella and
V. cholera.
• Vaccine Bath Vaccines may be inactivated in a special waterbath (vaccine
bath) at a low temp. 1 hour at 60 °C is sufficient.
• Washing Machine
Eating utensils, clothing, bed- pan and nursing equipment, hollow appratus. This is attained by washing in water and detergent for several minutes at 70 °C to 80 °C.
2. Moist heat at a temperature of 100 °C:
• Boiling at 100 °C:
Boiling at 100 °C for 5-10 minutes is sufficient to kill all non- sporing and few spore forming
organisms. Used for dental equipment in an OPD setting.
• Steaming at 100 °C:
I. Single exposure to pure and dry steam at 100 °C for 60 minutes. Kills all organisms.
II. Intermittent exposure at 100 °C or 'Tantalization' e.g. for 20 to 45 minutes on each of three
successive days. This method is used for culture medias containing sugars and gelatin media. Due to
initial heat aerobic spores germinate at room temperature and are killed on the next steaming
3. Moist heat at a temperature above 100 °C:
Saturated steam is a more efficient sterilizing agent than hot air because it has
•A greater lethal action.
•• Rapid in heating exposed subjects.
•Penetrates porous material quickly.
•Can be used to sterilize liquids and culture media.
Autoclave:
•Principle:
Water boils when its vapor pressure equals the pressure of the surrounding atmosphere. This
occurs at 100 °C at normal atmospheric pressure (i.e. 760 mm Hg, 14.7lb in absolute pressure)
When water is boiled within a closed vessel at increased pressure, the temperature at which it bois
and that of the steam it forms, will rise above 100 °C.
•This principle is employed in a pressure cooker and in an autoclave.
•Autoclaving is widely used to sterilize surgical supplies and bacteriological culture media.
•All parts of load must be permeated by steam. The steam should be not only saturated but also dry.
•All the air must be removed from the autoclave to expose its load to pure steam.
Sterilization by dry heat
"“Dry heat requires a temperature of about 160 °C for 60 minutes to kill highly resistant spores.. Dry heat kills the microorganisms by a destructive oxidation of essential cell constituents.
1. Red heat:
Inoculating wires, points of forceps are sterilized in the flame of Bunsen burner until they are red hot
2. Flaming:
Scalpels and needles are sterilized on direct exposure for few seconds in a flame.
3. Hot air oven:
It is the primary means of sterilization through dry heat. A temp. of 160 °C is maintained for 1 hour. Used for
sterilizing dry glassware, forceps, scalpels, scissors, throat swabs and syringes. It is also used for dry
Contents in sealed containers, powders, greases, oils and fats.
4. Infra-red radiation:
The infra red rays are directed onto the objects to be sterilized. Temperature of 180 °C is attained.
Heating at or above 200 °C is used for sterilizing surgical instruments. Cooling is hastened and oxidation prevented during the cooling period by admitting filtered nitrogen inside the chamber.
2. Sterilization by radiation:
I. Ultraviolet radiation
•Ability of sunlight to kill bacteria is due to UV rays.
•UV ray's are bactericidal at 200-290 nm. They induce thyamine dimmers in DNA thus interfering with replication.
•Effectiveness of UV rays as a sterilizing agent increases with decrease in wavelength.
II. lionizing radiation:
•High speed electrons, X-rays and gamma- rays are produced from isotope Cobalt 60.
•Lethal to all cells, bacteria, by production hydroxyl radicals which break down DNA inducing thiamine dimmers.
•For sterilizing disposable items in Bio medical industry e.g. plastic syringes and catheter.
3. Sterilization by filtration:
Principle:
It is used to render heat labile fluids and bacterial cultures free from bacteria by passing them through special halters with a pore size of less than 0.75um. Filtration renders a liquid bacteria free but not mycoplasma- free or Virus free. e.g. serum treated by Seitz filtration is not safe for clinical use and is not sterile.
Uses:
Preparation of soluble products of bacterial growth and toxins. In sterilizing liquids damaged by heat e.g. serum and antibiotic solution. Cellulose membrane filters are much less adsorptive; used to separate viruses. Air falters e.g. HEPA used in safety cabinets and operation theatres.
DISINFECTION:
Disinfection means freeing an item from some or all of its load of live pathogenic microorganisms that could cause infection during its use. Kills all or most of the vegetative forms. It is a relative state.
Uses:
When sterilizing procedures are impracticable, disinfection is performed to decrease the load of the bacteria.
•Bed pans
•Baths
•Wash basins
•Furniture
•Eating utensils
•Bed clothes
•Skin
Methods:
1. Washing, cleansing, ventilation
2. Heat, boiling
3. Chemical disinfectants
Heat:
• Washing; or rinsing of eating utensils and clothing in water at 70-80 °C for several minutes will kill
non-spore forming pathogens.
•Boiling: A glass hypodermic syringe or surgical instrument is disinfected by boiling in water at 100 °C for at least 5 minutes.
Chemical disinfectants:
Mechanism of action:
•Denature or alter the cytoplasmic membrane proteins or lipids.
•Interfere with the energy yielding systems of the microorganism.
•Directly inhibit specific biosynthetic pathways.
Strong disinfectants:
1.These are potent microbicidal but relatively toxic substances.
2.They rapidly kill the vegetative forms, often ineffective against spores.
Mild disinfectants (antiseptic):
Non toxic and bland, used for superficial application to living tissues e.g. to intact mucous membrane, broken skin or the interior of wound.
Mechanism of action of chemical disinfectant:
1. Disruption of cell membrane
A. Phenol group (Cresol, Lysol, Sudol)
•Good general disinfectant, powerful, cheap.
• Disorganizes the cell membrane and denatures the membranous protein.
•Active against T.B.
• Not inactivated by organic matter if used in proper dilution.
• Uses: Decontamination of bed pans, infective discharges, baths, floors.
• At 0.5% conc. They are used to preserve sera/vaccine.
I. Chlorhexidine (Cresol, Lysol, Sudol)
•Uses: Decontamination of skin.
• Inactivated by soap and organic matter.
• No action against Pseudomonas.
II. Hexachlorophene
●Used in soaps and hand creams but it is neurotoxic. eg. Dettol soap.
B. Volatile solvents (Isopropyl Alcohol, Ether, Chloroform, Acetone)
•Uses: Disinfection of skin before hypodermic inj, venipuncture by 70% alcohol.
•Disorganizes the membrane and denatures the proteins.
•Good bactericidal used to disinfect oral thermometers in OPD settings.
•Kills HIV, HBV, TB and HCV.
C. Soaps/Detergents.
• Anionic and cationic
• Disrupts the bacterial cytoplasm membrane by interacting with the lipid through their
hydrophobic end and with the surrounding water with the hydrophilic polar end, and at
an emulsifier.
• Bacteriostatic, wetting and cleansing agents.
• Inactivated by organic matter.
• Pseudomonas are resistant.
2. Modification of protein
A. Halogen compounds are chlorine releasing agents, bactericidal, kill T.B., sporocidal, veridical &
fungicidal, Fast acting, cheap & stable.
I. Chlorine: (Milton, Eusol)
●Disinfection of swimming pool, dairy products at a dilution of 1 in 100.
●Active component of Hypochlorite solution (Household bleach).
●Powerful oxidizing agent but is corrosive.
II. Iodine (Providence, Butadiene)
●Rapid, powerful antiseptic
●2% tincture (iodine+ Potassium Iodide in 70% isopropyl alcohol) used to clean intact skin
●before surgery.
●Iodophore (iodine+ detergent) rapid, bactericidal, sporocidal, used to disinfect superficial
wounds.
B. Heavy metals (Mercuric chloride)
●Bacteriostatic
●Acts by binding to sulfhydryl group and blocking enzymatic activity.
●Uses: Preparation of vaccines, preservation of anti- toxin.
●1% Silver nitrate was used for treatment of eye infection and burns previously.
C. Hydrogen peroxide
Antiseptic to clean wounds/ contact lens. Degrades H₂O, by producing the enzyme catalase
D. Formaldehyde
●Rapid, lethal, cheap, irritant.
●Sporocidal, veridical, fungicidal.
●Available as a liquid, solid or gas.
●Used to disinfect room, furniture and articles liable to damage by heat.
E. Ethylene oxide 10%
Used commercially for sterilization of medical equipment
F. Acids & Alkalies
●Denature proteins and nucleic acids.
●MTB is resistent to 2% NaOH.
●Weak acids are use to preserve food.
G. Glutaraldehyde 2% (Cidex solution)
●Ten times more efficient than formalin.
●More effective, Less toxic.
●Disinfection of delicate, heat sensitive medical/ surgical equipment, cystoscopy, anesthetic.
equipment.
●Bactericidal, sporocidal & veridical.
3. Modification of nucleic acid
A. Dyes (Crystal Violet).
Stain and inhibit bacterial growth on skin and mucus membrane.
Acts by binding of the positively charged dye molecules to the negatively charged phosphate groups
of the nucleic acids.
B. Miscellaneous (Boric Acid, Sodium Azide)
●Used as a preservative for urine, vaccine and reagents.
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