• Sterilization, disinfection and antisepsis
• Types of sterilization
• An ideal disinfectant
• Sterilization methods
refers to any process that effectively kills or
eliminates transmissible agents (microorganisms)
Protozoa and helminths,
from a surface, equipment, foods, medications, or
biological culture medium.
refers to any process that effectively kills or
eliminates microorganisms from in-animate
(non-living) objects -
• Disinfectants are antimicrobial agents that are
applied to non-living objects to destroy
microorganisms, the process of which is known
removal of microorganisms from living
• Antiseptics are antimicrobial agents that
are applied to living tissue to destroy
microorganisms, the process of which is
known as antisepsis.
Give examples of
General methods of sterilization/disinfection
Sterilizers and disinfectants
Qualities to look for in an ideal disinfectant
There are two types of 2. Chemical sterilization
sterilization: physical includes:
1. Physical sterilization Aldehydes
heat Quaternary ammonium
filtration ethylene oxide gas
An Ideal Disinfectant
A perfect disinfectant would offer
1) Complete sterilization,
2) Broad spectrum activity
3) Without harming other forms of life
4) Be inexpensive, and
A. PHYSICAL STERILIZATION
1. Heat Sterilization
Radiation I. Dry heat
Filtration II. Moist heat
l. Dry Heat
A. Flaming It is used to sanitize medical and
is done to loops and straight- other biohazardous waste before
wires in microbiology labs. it is discarded with non-
Leaving the loop in the flame of a
Bunsen burner or alcohol lamp C. Oven
until it glows red ensures that any Dry heat has the advantage that
infectious agent gets inactivated. it can be used on powders,
laboratory glassware and steel,
This is commonly used for small and other heat-stable items that
metal or glass objects, but not for are adversely affected by steam
The standard setting for a hot air
B. Incineration oven is at least two hours at 160
Incineration is burning of °C (320 °F).
contaminated waste to destroy
and kill micro-organisms.
will burn any organism to ash.
ll. Moist Heat
A. Autoclaving Biological - spores of the heat
resistant microbe Bacillus
Proper autoclave treatment will stearothermophilus
inactivate all fungi, bacteria,
viruses and also bacterial spores.
It will not necessarily eliminate all
To achieve sterility, a holding
time of at least 15 minutes at 121
Indicators of sterility
Physical - meters and charts that
record or display pertinent
information such as temperature
and pressure as a function of
Chemical - tape is often placed
on packages of products prior to
Autoclaves in medicine
2. RADIATION STERILIZATION
I. Gamma rays are very • It also damages many plastics,
penetrating and are commonly such as polystyrene foam.
used for sterilization of
disposable medical equipment, III. Electron beam processing is also
such as syringes, needles, commonly used for medical
cannulas and IV sets. device sterilization.
II. Ultraviolet light irradiation (UV,
from a germicidal lamp) is
useful only for sterilization of
surfaces and some transparent
• UV irradiation is routinely used
to sterilize the interiors of
safety cabinets GUE
• Ultraviolet light fixtures are
often present in microbiology
labs, and are activated only
when there are no occupants in
a room (e.g., at night).
3. FILTRATION STERILIZATION
This method is commonly used for To ensure sterility, the filtration
sensitive pharmaceuticals and system must be tested to ensure
protein solutions in biological that the membranes have not been
research. punctured prior to or during use.
A filter with pore size 0.2 µm will To ensure the best results,
effectively remove bacteria. pharmaceutical sterile filtration is
If viruses must also be removed, a performed in a room with highly
much smaller pore size around 20 filtered air (HEPA filtration) or in a
nm is needed. laminar flow cabinet or "flowbox", a
Prions are not removed by device which produces a laminar
filtration. stream of HEPA filtered air.
The filtration equipment and the HEPA filters are critical in the
filters themselves may be prevention of the spread of
purchased as presterilized airborne bacterial and viral
disposable units in sealed organisms and, therefore,
packaging, infection. Typically, medical-use
HEPA filtration systems also
or must be sterilized by the user, incorporate high-energy ultra-violet
generally by autoclaving at a light units to kill off the live bacteria
temperature that does not damage and viruses trapped by the filter
the fragile filter membranes. media.
B. CHEMICAL STERILIZATION
I. usually ethanol or isopropanol, vi. Alcohols are more effective
are sometimes used as a combined with purified water—
disinfectant, but more often as 70% isopropyl alcohol or 70%
ethyl alcohol is more effective
than 90% alcohol, because the
II. They have wide microbicidal higher water content allows for
activity, greater diffusion through the cell
III. Are non corrosive, but can be a membrane.
fire hazard. vii. Alcohol is effective against
IV. They also have limited residual resistant fungal and bacterial
activity due to evaporation, spores.
which results in brief contact
V. Have a limited activity in the
presence of organic material.
1) Aldehydes, such as Glutaraldehyde, have a wide microbiocidal
activity and are sporocidal and fungicidal.
2) They are partly inactivated by organic matter and have slight
3) Glutaraldehyde and formaldehyde are volatile, and toxic by both
skin contact and inhalation.
4) Glutaraldehyde has a short shelf life (<2 weeks), and is
5) Formaldehyde is less expensive and has a much longer shelf life
if some methanol is added to inhibit polymerization to
paraformaldehyde, but is much more volatile
6) Formaldehyde is also used as a gaseous sterilizing agent; in this
case, it is prepared on-site by depolymerization of solid
i. Phenol is probably the oldest known disinfectant as it was first
used by Lister, when it was called carbolic acid.
ii. Phenolics are active ingredients in some household disinfectants. They
are also found in some mouthwashes and in disinfectant soap and
iii. Phenolics are tuberculocidal
iv. It is rather corrosive to the skin and sometimes toxic to sensitive
v. O-phenylphenol is often used instead of Phenol, since it is
somewhat less corrosive.
vi. Chloroxylenol is the principal ingredient in Dettol, a household
disinfectant and antiseptic.
vii. Thymol, derived from the herb thyme, is the active ingredient in
the only 100% botanical disinfectant with an EPA registration
(#74771-1), Benefect. Registered as "broad spectrum," or
hospital-grade, it is also the only disinfectant with a green
certification, Environmental Choice.
4. OXIDIZING AGENTS
act by oxidising the cell membrane of microorganisms, which results in a
loss of structure and leads to cell lysis and death.
I. Sodium hypochlorite is very commonly used. Common household
bleach is a sodium hypochlorite solution and is used in the home to
disinfect drains, toilets, and other surfaces.
II. Hydrogen peroxide is used in hospitals to disinfect surfaces. It is
sometimes mixed with colloidal silver.
It is often preferred because it causes far fewer allergic reactions than
Also used in the food packaging industry to disinfect foil containers. A
3% solution is also used as an antiseptic.
Its use as an antiseptic is no longer recommended
III. Iodine is usually dissolved in an organic solvent or as Lugol's iodine
solution. Although no longer recommended because it increases scar
tissue formation and increases healing time, tincture of iodine has also
been used as an antiseptic for skin cuts and scrapes.
IV. Paracetic acid is a disinfectant produced by reacting hydrogen peroxide
with acetic acid.
It is broadly effective against microorganisms and is not deactivated by
catalase and peroxidase, the enzymes which break down hydrogen
It also breaks down to food safe and environmentally friendly residues
(acetic acid and hydrogen peroxide), and therefore can be used in non-
It can be used over a wide temperature range (0-40°C), wide pH range
(3.0-7.5), in clean-in-place (CIP) processes, in hard water conditions,
and is not affected by protein residues.
V. Potassium peroxymonosulfate, the principal ingredient in Virkon, is a
wide-spectrum disinfectant used in labs.
Virkon kills bacteria, viruses, and fungi.
It is used as a 1% solution in water, and keeps for one week once it is
It is expensive, but very effective, its pink colour fades as it is used up so
it is possible to see at a glance if it is still fresh.
5. QUARTENARY AMMONIUM
Quaternary ammonium compounds (Quats), such as
benzalkonium chloride, are a large group of related compounds.
Some have been used as low level disinfectants.
They are effective against bacteria, but not against some species of
Pseudomonas bacteria or bacterial spores.
Quats are biocides which also kill algae and are used as an additive
in large-scale industrial water systems to minimize undesired
Quaternary ammonium compounds can also be effective
disinfectants against enveloped viruses.
6. ETHYLENE OXIDE GAS
1) is commonly used to sterilize objects sensitive to temperatures greater than
60 °C such as plastics, optics and electrics.
2) treatment is generally carried out between 30°C and 60°C with relative
humidity above 30% and a gas concentration between 200 and 800 mg/L
for at least three hours.
3) penetrates well, moving through paper, cloth, and some plastic films and is
4) sterilizers are used to process sensitive instruments which cannot be
adequately sterilized by other methods.
5) EtO can kill all known viruses, bacteria and fungi, including bacterial spores
and is satisfactory for most medical materials, even with repeated use.
6) However it is highly flammable, and requires a longer time to sterilize than
any heat treatment.
7) The process also requires a period of post-sterilization aeration to remove
8) is the most common sterilization method, used for over 70% of total
sterilizations, and for 50% of all disposable medical devices.
1. High-intensity shortwave ultraviolet light can be used for disinfecting
smooth surfaces such as dental tools.
2. Biguanide polymer polyaminopropyl biguanide is specifically bactericidal
at very low concentrations (10 mg/l).
It has a unique method of action: the polymer strands are incorporated
into the bacterial cell wall, which disrupts the membrane and reduces its
permeability, which has a lethal effect to bacteria.
It is also known to bind to bacterial DNA, alter its transcription, and cause
lethal DNA damage.
It has very low toxicity to higher organisms such as human cells, which
have more complex and protective membranes.
• General safety practices
– Work habits (personal, work space, material)
– Proper disposal of sharps and waste
– Disinfection of work areas
Maintain Clean & Orderly
• Keep work areas uncluttered
• Disinfect work surfaces daily
• Restrict or limit access when
• Keep supplies locked in a
safe and secure area
• Make provisions to handle
accidental eye splashes
(eye wash station or bottle of
Universal or Standard
Every specimen should
be treated as infectious
Apply Safety Practices Throughout
the Testing Process
• Before Testing (Pre-analytical)
– Specimen collection
– Specimen preparation
– Specimen transport
• Testing (Analytical)
• After Testing (Post-analytical)
– Disposal or
Develop Personal Safe Work
• Wash hands before
and after testing
• Wear fresh pair of
• Wear lab coat or apron
• Dispose of
Develop Personal Safe Work Habits
• Pipetting by mouth is
• Never eat, drink, apply
cosmetics or smoke in
• Keep food and drinks
out of the laboratory /
testing site refrigerator
Take Precautions to Avoid
Needle Stick Injury
What can cause needle stick injury?
• Lack of concentration
(e.g. worker burn-out)
• Lack of concern for others
• Improper disposal of sharps
Drop Used Sharps in
• If your site does not
have access to one of
containers, you can
use a pedal bucket,
thick plastic or metal
container having a
wide opening with lid
Do’s and Don’ts: Sharps and
• Do not re-cap
• Do Not break, bend,
or reuse lancets,
syringes or needles
• Do Not shake
sharps containers to
Do’s and Don’ts: Sharps and
What’s wrong with this picture?
Never Place Needles or Sharps
in Office Waste Containers
Sharp containers must be:
• Placed near
• Closed when not
Sharp containers must be:
• Sealed when ¾ full
Policy for Handling Sharps
Dispose of sharps yourself, immediately
Must dispose of sharps after each test
Must place sharps in sharps boxes
Do not drop sharps on the floor or in the
office waste bin
Place sharps container near your
Seal and remove when box is ¾ full
Incinerate all waste (or burn in a hole)
Incineration of Waste
• Incineration is burning of contaminated
waste to destroy and kill micro-organisms.
• Autoclave all hazardous waste before it
leaves the lab.
– Is effective against potential re-use
– Protects environment
– Must be supervised
An example of risk waste
• Needle Pit
Using the PPE
Collection and Handling of
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