Sterilization and Disinfection

Moist Heat:  Moist heat may be used in three forms to achieve microbial inactivation: 1. Autoclave (At temperature above100oC). 2. Boiling water/ steam at atmospheric pressure ( At temperature 100oC). 3. Hot water below boiling point (At temperature below 100oC).

1. Autoclave (At temperature above 100oC): Moist heat sterilization involves the use of steam in the range of C for 15 minutes. Steam under pressure of 1.15 BAR (The bar is a widely used metric unit of measurement for pressure) is used to generate high temperature needed for sterilization to destroy microorganisms. Uses of moist heat: 1. Decontamination of laboratory waste. 2. Sterilization of thermostable products (laboratory glassware, clothes and media used for microbial culture).

How to use the autoclave: 1) Fill the autoclave with distilled water to a point just below the basket bottom. 2) Place the material to be sterilized, close the lid and screw down the clamps firmly. 3) Open the air outlet valve. 4) Turn on the heating. 5) Close outlet valve when a constant jet of steam starts to escape. 6) Let the pressure rise to the required level and maintain at the level for the required period of time (121 ˚C for 15 min. under 1.15 BAR). 7) When the time required for sterilization has passed, turn off the heat. 8) When the temperature falls below 100˚ C, open the outlet valve. 9) Open the lid after the whistling sound stops. 10) Leave the sterilized material to cool before its removal from the autoclave.

Moist heat kills microorganisms by: 1. Degrading nucleic acids (DNA/RNA). 2. Denaturation of enzymes and other essential proteins. 3. Disrupting cell membranes.

Advantages : 1. It has more penetrative power than dry air, it kills the spores. 2. It is the most frequently used method of sterilization because bacterial spores are resistant to boiling. Disadvantages: It is not acceptable for heat sensitive substances (rubber, plastics, and equipment that would be damaged by high temperatures).

2- Boiling water ( At temperature 100oC): Advantages: 1. Kills most vegetative bacteria and viruses immediately. 2. Glass wares can be disinfected by placing them in boiling water for minutes (The lid of the boiler must not be opened during the period). Disadvantages: 1. Certain bacterial toxins such as Staphylococcal enterotoxin are heat resistant. 2. Some bacterial spores are resistant to boiling and can survive.

3. Hot water below boiling point (At temperature below 100oC). This type of sterilization such as (Pasteurization). Heating to below 100 o c followed by immediate cooling to below 10 o c.

B- Chemical Methods: Disinfectants are those chemicals that destroy pathogenic bacteria from inanimate surfaces. Those chemicals that can be safely applied over skin and mucus membranes are called antiseptics.  Chemical substances are mainly used as disinfectants for: 1) Solid objects: floors and benches. 2) Living tissues: wounds and skin lesions.  Chemicals that are commonly used: Ethyl alcohol, Hydrogen peroxide, Phenol and Iodine.

Chemical agents act primarily by one of the three mechanisms: (1) Disruption of the lipid-containing cell membrane. (2) Modification of proteins (3) Modification of DNA. but some of the chemicals act by more than one mechanism.

I- Disruption of Cell Membranes: 1- ALCOHOLS: Ethanol is widely used to clean the skin before immunization or venipuncture. Mode of action: It acts mainly by disorganizing the lipid structure in membranes, but it denatures proteins as well. Examples: Ethyl alcohol, isopropyl alcohol and methyl alcohol. Application: A 70% aqueous solution is more effective at killing microbes than absolute alcohols as with water exhibit optimal activity (facilitate diffusion through the cell membrane).

2- PHENOL: It was the first disinfectant used in the operating room, but it is rarely used as a disinfectant today because it is too caustic. Mode of action: Act by disruption of membranes, precipitation of proteins and inactivation of enzymes. Examples: 5% phenol, hexachlorophene, chloroxylenol (Dettol). Applications: 1) They act as disinfectants at high concentration and as antiseptics at low concentrations. 2) They are bactericidal, fungicidal, mycobactericidal but are inactive against spores and most viruses.

3- SURFACE ACTIVE AGENTS (Detergents): Detergents are "surface-active" agents composed of a long-chain, lipid-soluble, hydrophobic portion and a polar hydrophilic group. Mode of actions: 1. They disrupt membrane resulting in leakage of cell constituents. 2. These surfactants interact with the lipid in the cell membrane through their hydrophobic chain and with the surrounding water through their polar group and thus disrupt the membrane. Examples: Quaternary ammonium compounds, e.g., benzalkonium chloride, are cationic detergents widely used for skin antisepsis.

II- Modification of Proteins: 1- Chlorine Chlorine is used as a disinfectant to purify the water supply and to treat swimming pools. It is also the active component of hypochlorite (bleach, Clorox), which is used as a disinfectant in the home and in hospitals. Mode of action: Chlorine is a powerful oxidizing agent that kills by cross-linking essential sulfhydryl groups in enzymes to form the inactive disulfide.

2- Iodine Iodine is the most effective skin antiseptic used in medical practice and should be used prior to obtaining a blood culture and installing intravenous catheters because contamination with skin flora such as Staphylococcus epidermidis can be a problem. Tincture of iodine (2% iodine in 70% alcohol) is an antiseptic. Mode of action: Iodine, like chlorine, is an oxidant that inactivates sulfhydryl- containing enzymes.

3- HEAVY METALS: Mercury and silver have the greatest antibacterial activity of the heavy metals and are the most widely used in medicine. Mode of action: Act by precipitation of proteins and They act by binding to sulfhydryl groups, thereby blocking enzymatic activity. Examples: Mercuric chloride, silver nitrate, copper sulfate. Applications: 1. 1% silver nitrate solution is useful in preventing gonococcal ophthalmia neonatorum. 2. Silver sulfadiazine is used to prevent infection of burn wounds.

4- HYDROGEN PEROXIDE: Mode of action: 1. Hydrogen peroxide is an oxidizing agent that attacks sulfhydryl groups, thereby inhibiting enzymatic activity. 2. Its effectiveness is limited by the organism's ability to produce catalase, an enzyme that degrades H 2 O 2. (The bubbles produced when peroxide is used on wounds are formed by oxygen arising from the breakdown of H 2 O 2 by catalase.) Application: 1. It is used at 6% concentration to decontaminate the instruments, equipments such as ventilators. 2. 3% Hydrogen Peroxide Solution is used for skin disinfection,wounds and ulcers. 3. Strong solutions are sporicidal.

5- ALDEHYDES: Mode of action: Denatures proteins and nucleic acids. Examples: Formaldehyde, Gluteraldehyde. Application: 40% Formaldehyde (formalin) is used for surface disinfection and fumigation of rooms, chambers, operation theatres, biological safety cabinets, wards, sick rooms. 6- Acids & Alkalis Strong acids and alkalis kill by denaturing proteins.

7- Ethylene Oxide Ethylene oxide gas is used extensively in hospitals for the sterilization of heat-sensitive materials such as surgical instruments and plastics. Mode of action: It kills by alkylating both proteins and nucleic acids, i.e., the hydroxyethyl group attacks the reactive hydrogen atoms on essential amino and hydroxyl groups. Examples: Ethylene oxide, peracetic acid, formaldehyde and H 2 O 2 gases.

III- Modification of Nucleic Acids:  A variety of dyes not only stain microorganisms but also inhibit their growth. 1)One of these is crystal violet (gentian violet), which is used as a skin antiseptic. Its action is based on binding of the positively charged dye molecule to the negatively charged phosphate groups of the nucleic acids. 2)Malachite green, is a component of Löwenstein-Jensen's medium, which is used to grow M. tuberculosis. The dye inhibits the growth of unwanted organisms in the sputum during the 6-week incubation period.