Sterilization & Disinfection

DR. WAJID HUSSAIN

MBBS, DCP, M.Phil
 LEARNING OBJECTIVES
• Terminology related to control of microbial Growth

• Most and least resistant organism

• Sterilization methods

• Sites of action of chemical antimicrobial agents

• Physical methods of microbial control

 Terminology relating to the control of microbial
Growth
Sterilization

Disinfection

Biocide

Preservation

Sanitization
 Terminology relating to the control of microbial Growth (cont:)

Bacteriostatic

Bactericidal

Fungicidal & Fungistatic

Virucidal

Antibiotics
Most and least resistant organism
 Sterilization methods
• Chemical
• Physical methods

• Chemical methods are used b/c many matters, substance

and object cannot be sterilize with Physical methods.
Sites of action of chemical antimicrobial agents
 Disruption of the cell wall
Phenols (carbolic acid) & phenolics
• (1827- 1912) Joseph.Lester
• Disinfectant, antiseptic & preservative

• used in household
• industrial products like mouth wash.
• ==Bactericidal, fungicidal and virucidal
• == not sporicidal
M.A ## denaturation of protein
## lysis of the cell membrane
• Commonly used phenol derivatives are
o Chlorhexidine - e.g Savlon
o Chloroxylenol - e.g Dettol
o Hexachlorophane - has been used in powders, creams and lotions.
 Alcohols
• Ethyl (60 – 90% solutions) and
isopropyl alcohol
• water is essential for activity
• Poorly effective against viruses
and fungi and no activity
against spores.
• Also used in hand sanitizers
Mode of action:
• Denature proteins, dissolve
lipids
 Surface Acting Ingredients / Surfactants
❑ Soaps and Detergents
Regular -
Antibacterial -
• Detergents
Detergents (cont:)
➢ Acidic-Anionic → Sanitizing
Anion reacts with plasma membrane.
Nontoxic, non-corrosive, and fast acting.

➢ Cationic →
➢ Strongly bactericidal
denature proteins
disrupt plasma membrane.
 Modification of proteins:

Chlorine
▪ Oxidizing agent
▪ Widely used as disinfectant
▪ Broad spectrum and sporicidal (pools, drinking
water tank)
▪ 25% of medical devices
Iodine
More reactive, rapidly bactericidal, fungicidal, tuberculocidal,
virucidal & sporicidal.
Tincture of iodine (solution with alcohol) → wound antiseptic
Iodophors combined with an organic molecule
→ iodine detergent complex (e.g. Betadine®).
Mode of action:
• O2 requirement of aerobic m.o
Act on nucleotides and fatty acids
 Heavy Metals:
Used for disinfection and preservation.
• Mercury: for skin lesions; Thimerosal
• Silver (AgNO3): Antiseptic for eyes of newborns and burn
wound →disruption of enzyme functions
• Zinc (ZnCl2) in mouthwashes, ZnO in antifungal in paint
• Copper is more potent against antibiotic resistant bacteria
(MRSA) and chlorophyll containing organisms → Algicides
.
• Brass is an alloy of copper
and zinc.

• storage of river water in brass

containers is a best way to
prevent disease.

• The river water may have up to

1 million fecal bacteria per ml.
That count could be reduced to
undetectable by 2 days of
storage in a brass container!
Hydrogen Peroxide:
• == Oxidizing agent
• == bleaching agent
• == high-level disinfectant (25%)
• == antiseptic (3%)
• == cleansing of wounds & ulcers

catalase in human cells

• H2O2 H2O + O
• ==sporocidal
• ==contact lenses preservative
Mode of action:
• oxidises & denaturates proteins
& lipids of m.o.
 Ethylene oxide:
• It is a liquid at temperatures below 10.8o C, above it
inflammable gaseous.
• It is generally used in a chamber (90% CO2, 10%
ethylene oxide),

• Uses:
• Disposable healthcare product e.g. fabric surgical
implements, cardiac catheters and plastics equipment.
• At 20 -25°C: sterilization takes 18hours
• At 50°C-60°C: sterilization takes 4 hours to kill
bacteria, spores and viruses.
M/ A
• Disrupts the DNA of m.o which prevent them for
reproducing.
Aldehydes
Glutaraldehyde:
• Brand name is Cidex (disinfectant and preservative).

• Used as a cold sterilant to disinfect and clean heat-sensitive

equipment such as dialysis instruments, suction bottles,
bronchoscopes, endoscopes, and ENT instruments.

Formaldehyde: Virucidal, sporicidal &bactericidal.

• Formalin (37%)

• acts on

## protein by denaturation

## nucleic acid by alkylation

 Disadvantages :
• Cause skin irritation.
• Glutaraldehydes are expensive.
• Vapors from formaldehyde (classified as a potential
carcinogen).
• Formaldehyde cannot be mixed with chlorine or
chlorinated water because a dangerous gas (bis-
chloromethyl-ether) is produced
 Physical Methods of Microbial Control

▪ Heat is very effective (fast and cheap).

▪ Thermal death point (TDP): Lowest temperature at which all cells

in a culture are killed in 10 min.

▪ Thermal death time (TDT): Time to kill all cells in a culture

▪ Decimal Reduction Time (DRT):

Minutes to kill 90% of a population at a given temperature
 PHYSICAL AGENTS:

Act in the form of,

• Heat
• Radiation
• Filtration
 HEAT
1. Dry heat sterilization
• Principle : its killing effect is because of denaturation
of protein, oxidative damage and toxic effect of
elevated levels of electrolyte
• Method: There are different methods for dry heat
sterilization
• Red heat
• Incineration
• Flaming
• Hot air oven
RED HEAT: The article to be sterilized are put in the flame
directly until red hot.
Wire loop
Tip of needles & forceps.
• Incineration:

• Procedures in safety cabin.

• Destroying contaminated cloth

or pathological materials etc.
FLAMMING:
eg. Mouth of culture -tubes,
stoppers,
glass slides etc.
 HOT AIR OVEN :
• An electrically heated chamber that has a thermostat
which maintains the chamber’s air constantly at desired
temp.

• It is used to produce dry heat at 160OC for 2 h

• 170OC for 1 hour
• Used for sterilization of
Glass ware eg. Petri dishes, test tube.
Scissors
Forceps
Throat swabs
Syringes ,
Powders, oils etc
 HEAT(cont:)
2. Moist heat:
• TYPES:
• Temperature above 100 OC. (Boiling)
• Steam at atmospheric pressure (Tyndallization)
• Temperature below 100 OC (Pasteurization)
• Moist heat at temp above 100OC under Pressure.
(Autoclaving)
BOILING : At 100 OC For 5 to 10 minutes is sufficient to kill
all non-spore forming bacteria.

used for sterilization of ,

syringes ,
needles
 Tyndallization: (discontinuous heating).

• Heat labile media like those containing sugar, milk, gelatin

• Steaming at 100°C is done in steam sterilizer for 20 minutes

followed by incubation at 37°C overnight.

• This procedure is repeated for 3 successive days. Spores, if

any, germinate to vegetative bacteria during incubation and are
destroyed during steaming on second and third day.
 Pasteurization
• It is a process in which heat is applied up
to 63OC for 30 min followed by rapid
cooling.

– Used primarily for milk.

– TYPES
– Batch method – 30 minutes at 63ºC
– Flash pasteurization – 72ºC for 15
seconds
– Ultrahigh-temperature (UHT)
pasteurization – 134ºC for 1 second.
 AUTOCLAVING:
• It rely on moist heat.
• When pressure i-e 15 lb /15min
/inches squares is applied the temp
reaches 121 OC
• At this temp all microorganism with
their spores are completely destroyed.
• USES:
• For virus and highly resistant
endospores which are not destroyed
by boiling temp e.g. cl. Botulinum
• Sterilize culture media.
• Glass ware
• Surgical instruments like scalpel,
forceps and dressing kit.
Desiccation and Lyophilization

❑ Desiccation is drying (98% water is removed)

❑ Lyophilization (freeze drying)

• Product is frozen and sealed in a vacuum.
• Preservation of heat-sensitive pharmaceuticals such as live
virus vaccines and foods.
 Sterilization by Radiation:
Ionizing radiation is the use of short wave length, high-
intensity radiation to destroy microorganisms. This
radiation can come in the form of gamma or x-rays that
react with DNA resulting in cell damaged.
Sterilization of heat sensitive materials:
sutures, surgical gloves & plastic items, such as
syringes
.
▪ Nonionizing: UV light
uses longer wavelength and lower energy
as a result, non-ionizing radiation loses the
ability to penetrate substances, and can only
be used for sterilizing surfaces like hospitals
to kill airborne organisms.
• DISADVANTAGES:
• It requires a reliable source of electricity.
• It is not effective in areas of high relative humidity.
• UV bulbs require frequent cleaning to remain effective.
• Exposure to UV rays can burn the skin and eyes.
FILTERATION:
• Preferred method of sterilizing certain solution eg., those with
heat sensitive components.
Types:
• Seitz filter,
• HEPA filters
• Membrane filters
Function more like a sieve

• Most commonly used filter is composed of nitrocellulose

and has a pore size of 0.22 micro meter.
This size will retain all bacteria and spores.
Uses:
Anti-sera , Toxins, Blood products.
 Seitz filter
• It is disposable and single use disc
available in different grades.
• They have high adsorbing capacity
and tend to alkalinize the filtered
fluid.
• Used to sterilize heat sensitive
materials
e.g special nutrients that might be
added to media, enzymes,
vaccines,and pharmaceutical
products such as drugs,
sera, and vitamins.
• Their use is limited by the
carcinogenic potential of asbestos.
HEPA (high efficiency particulate air)
Filter
Clinical Use Commonly used disinfectant
Disinfect surgeon’s hands prior to surgery Chlorohexidine
Disinfect surgical site prior to surgery Iodophor
Disinfect skin prior to venipuncture 70% ethanol
Disinfect skin prior to blood culture Tincture of iodine followed by 70%
ethanol, chlorohexidine, iodophor
Cleanse wounds Chlorohexidine,Thimerosal,hydrogen
peroxide
Cleans burn wounds Silver sulfadiazine
Sterilize non heat sensitive materials(eg. Autoclave
Drapes, surgical gowns)
Sterilize intravenous solutions Filtration
Disinfect air in operating room (when not Ultraviolet light
in use)
Disinfect floor of operating use Benzalkonium chloride (Lysol)
Disinfect stethoscope 70% ethanol
Preservative in vaccines Thimerosal
Clean up of blood spill from a patient with Hypochlorite (bleach, clorox)
hepatitis B or C ( disinfect area)