Sterilization and Disinfection. Moist Heat: Moist heat may be used in three forms to achieve...
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- Slide 1
- Sterilization and Disinfection
- Slide 2
- 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).
- Slide 3
- 1. Autoclave (At temperature above 100oC): Moist heat
sterilization involves the use of steam in the range of 121- 134 0
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).
- Slide 4
- 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.
- Slide 5
- Moist heat kills microorganisms by: 1. Degrading nucleic acids
(DNA/RNA). 2. Denaturation of enzymes and other essential proteins.
3. Disrupting cell membranes.
- Slide 6
- 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).
- Slide 7
- Slide 8
- Slide 9
- 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 10-20
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.
- Slide 10
- 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.
- Slide 11
- 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.
- Slide 12
- 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.
- Slide 13
- 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).
- Slide 14
- 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.
- Slide 15
- 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.
- Slide 16
- 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.
- Slide 17
- 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.
- Slide 18
- 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.
- Slide 19
- 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.
- Slide 20
- 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.
- Slide 21
- 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.
- Slide 22
- 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
Lwenstein-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.