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Decontamination and Sterilization
Methods
Laboratory Biological Safety

Definitions
Sterilization: any process, physical or chemical, which results
in the absence of all life on or in an object. This term applies
especially to the destruction of microorganisms, including
bacteria, fungi, and their spores, and the inactivation of viruses


Decontamination: to destroy, remove, or neutralize living
organisms, toxic agents or chemical carcinogens on a surface or
object (this does not imply either total destruction or total
removal); to make an object safe for unprotected individuals

Disinfection: to use a chemical agent to kill or inactivate most
vegetative bacteria, fungi, and viruses but not necessarily
spores. This term applies to a chemical used on inanimate
surfaces not to living tissues

Germicide: a substance used to destroy a specific
microorganism
Algicide an agent that kills algae
Bacteriocide - an agent that kills vegetative bacteria and
possibly some less resistant spores (commercial term)
Fungicide an agent that kills fungi
Sporicide an agent that kills spores
Virucide an agent that inactivates, destroys or kills viruses
Tuberculocide an agent that kills mycobacteria
Definitions

General Precautions
All areas where infectious materials have been handled need to
be decontaminated a minimum of daily. Ideally an area will be
decontaminated before and after work is performed

All infectious materials and/or contaminated equipment or
apparatus should be decontaminated before being washed,
removed, stored, or discarded

All biohazardous materials should be placed in an appropriately
marked refrigerator, waste or incubator, sterilized or otherwise
confined at the end of each work day

Do not store materials in the autoclave!


General Precautions
Dry hypochlorites, or any other
strong oxidizing material, MUST
NOT be autoclaved with organic
materials such as paper, cloth, or
oils

If organic material in the autoclave
catches fire, the oxidizer will
stimulate the fire and may cause an
explosion because of the high
pressure in the autoclave

General Precautions
All floors, laboratory benches, and other surfaces should be
decontaminated after completion of operations involving
biohazardous materials

Floor drains should be flooded with water at least once each
week in order to fill traps and stop the backflow of sewer gases

Floors should be swept with push brooms (swiffers) only. The
use of a floor sweeping compound is recommended because of
its effectiveness in limiting the generation of airborne organisms

Stock solutions of suitable decontaminants should be maintained
in each laboratory for decontamination

Lab Benches
GOOD
BAD

Examples of Biological Waste
Waste Type Examples
Microbiology laboratory
(Biohazardous)
laboratory cultures, stocks of specimens of micro-
organisms, live or attenuated vaccines, human or
animal cell cultures used in research, and laboratory
material that has come into contact with any of the
above
Animal

animal tissues, organs, body parts, carcasses,
bedding, animal blood and blood products
Human anatomical human tissue, organs, body parts
Human blood and blood
products

human fluid blood/blood products, items saturated or
dripping with blood, body fluids contaminated with
human blood, human body fluids removed for
diagnosis or during surgery, treatment or autopsy
Clinical & laboratory
waste sharps
needles, syringes, blades or laboratory glass capable
of causing punctures/cuts
defined as per BC Hazardous Waste Regulation

Biological Waste Minimization Measures
SEGREGATE uncontaminated solid waste from biomedical and
biohazardous waste (all risk groups)

Use products with less environmental impact:
Petri dishes with 35% less plastic
Glassware that can be decontaminated and reused
Refillable pipette racks
Other reusable or recyclable products

Methods of Decontamination
Heat or autoclaving
Autoclaving is the most dependable procedure for ensuring the
complete destruction of microorganisms. It generally involves
heating in a chamber employing saturated steam under a relative
pressure of 103 kPa (15 psi) to achieve a chamber temperature of
at least 121
O
C for a minimum of 60 minutes

Chemical decontaminants
Chemical decontaminants are toxic chemicals that can kill certain
types of microorganisms

Radiation
This method is not used in academic laboratories


Sterilization by Heat
Moist Heat Dry Heat
Temperature Time Temperature Time
100
o
C 20 hours 120
o
C 8 hours
110
o
C 2.5 hours 140
o
C 2.5 hours
115
o
C 50 minutes 160
o
C 1 hour
121
o
C 15 minutes 170
o
C 40 minutes
125
o
C 6.5 minutes 180
o
C 20 minutes


The following table demonstrates the heat equivalency between
Moist and Dry Heat

Autoclave
When autoclaving, the materials being
sterilized must come into contact with steam
and heat for actual sterilization to result. It is
for this reason that the use of some form of
efficacy indicator must be done with each cycle

Sterilization Indicators:
Routine Testing
Autoclave Tape
Autoclave Charts
Chemical
Biological


This protocol describes the autoclave
treatment conditions and requirements for
laboratory solid waste contaminated with Risk
Group 1 and 2 Biohazardous Materials
(Category B Infectious Substances), in
Laboratories in UBC Point Grey Campus.
This treatment will allow the safe
management of the resulting waste as non-
hazardous solid waste in accordance with the
waste delisting approved by the Ministry of
Environment.
The new procedure can be found here along
with the training videos.
New Disposal Procedure: Solid Waste
Contaminated with Microbiological Risk Group 1
and Risk Group 2 Agents

Resistance to Chemical Decontaminants
Bacterial Spores
Bacillus subtilis, Clostridium sporogenes
Mycobacterium
Mycobacterium tuberculosis
Nonlipid or Small Viruses
Poliovirus, Coxsackievirus, Rhinovirus
Fungi
Trichophyton sp., Cryptococcus sp., Candida sp.
Microorganisms can be more or less resistant to decontamination
and the general order is (most to least resistant):

Resistance to Chemical Decontaminants
Fungi
Trichophyton sp., Cryptococcus sp., Candida sp.


Vegetative Bacteria
Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella
choleraesuis
Lipid or Medium-Size Viruses
Herpes simplex virus, Cytomegalovirus, Respiratory syncytial virus,
Hepatitis B virus, Human Immunodeficiency virus

Chemical Decontaminants
Chemical decontaminants are usually supplied as either liquid
concentrates or solids which must be diluted or hydrated prior to
use. Once diluted, there is generally a limited time period in
which they maintain acceptable levels of antimicrobial activity.
This period is called the "use life" and it varies from hours to
weeks depending on the disinfectant

When to Use Chemical Decontaminants:
To decontaminate growth medium in CL1
To decontaminate pipettes before disposal or reuse
To decontaminate equipment that cannot be autoclaved
To decontaminate liquid waste

Microorganisms are affected by chemical decontaminants
through:
Cell lysis
Protein coagulation or denaturation
Enzyme denaturation or inactivation
Destruction of enzyme substrates

Chemical Decontaminants

Decontaminant Effectiveness
The effectiveness of a decontaminant is
influenced by a number of factors:

Organic Load
Surface Topography
Method of Application
Concentration
Contact Time
Temperature
Relative Humidity
pH and Stability


The ideal decontaminant would be a broad spectrum agent able to
act effectively against all biohazards. It would be fast acting, not
easily inactivated, non-toxic to the user, non-corrosive,
economical, easy to use, easy to dispose of and have a long "use
life". Unfortunately, there is no one chemical agent that can
fulfill all of these requirements

There are several categories of liquid decontaminants, each of
which has different characteristics and different ranges of
usefulness. Some agents are not compatible, so always follow the
manufacturers instructions regarding mixing agents, dilution,
storage and use of the decontaminant
Types of Chemical Decontaminants

Liquid Decontaminants
Alcohols
Ethyl or isopropyl alcohol in a concentration of 70 - 85%
by volume is often used
Pros: easy to use and non corrosive
Cons: extremely flammable, evaporates easily, inactivated
by organic matter, and no cleansing properties
Effective: lipid-containing viruses, enveloped viruses, and
vegetative bacteria. Longer contact times are needed for
fungi and some mycobacteria
Ineffective: non-enveloped viruses and bacterial spores

Chlorine Compounds
This halogen is a universal decontaminant active against
all microorganisms including bacterial spores. Chlorine
combines with protein and rapidly decreases in
concentration in its presence. It is a strong oxidizing
agent
Pros: inexpensive, readily available, and broad spectrum
Cons: corrosive, short use life, and inactivated by
organic material
Effective: everything
Ineffective:
14
C (produces radioactive CO
2
) should not
be used with any radioisotope where the reaction would
create a volatile compound
Examples: Bleach, Javex, Presept, Alcide
Liquid Decontaminants

Formaldehydes
Formaldehyde for use as a decontaminant is usually
marketed at about 37% concentration of the gas in water
solution (referred to as formalin), or as a polymerized
compound called paraformaldehyde. Formaldehyde, in a
concentration of 5% active ingredient (18.5 g/l
formaldehyde), is an effective liquid decontaminant
Pros: broad spectrum and less susceptible to inactivation
by organic material
Cons: long contact times (30+ minutes), loss of activity
at cooler temperatures, pungent odour, expensive, and
short use life
Effective: everything
Examples: Cidex 7, Sporocidin, and Sonacide
Liquid Decontaminants

Gas Decontaminants
Formaldehydes
The chemical of choice for space decontamination
Used at a concentration of 10,000 ppm (TLV 0.3 ppm with
a ceiling of 0.5 ppm)
It has limited penetrating abilities
It is a suspected carcinogen
It is a sensitizer

Radiation Decontaminants
Ionizing Radiation
Ionizing radiation will destroy all microorganisms
Gamma radiation - either Co-60 or Cs-137
None of these methods are presently used at UBC. The risk from
the radiation outweighs the decontamination benefits

Nonionizing Radiation
Ultraviolet radiation the short wavelengths of light beyond the
violet end of the visible spectrum
Produces thymine dimers in cells which leads to the death of the
microorganism
A narrow range of the UV spectrum has antimicrobial activity but
is limited by poor penetrating power
May not lead to total decontamination of the area
UV lights need to be changed every 3-6 months to be effective

Most chemicals are white
powders, so if you dont clean
up after yourself no one will
know what the white powder
left behind is
Clean-up

Clean-up
All spaces where biological work is
performed need to be
decontaminated at least once daily

Not only is this space messy, there
is also a very large possibility of
contamination and makes
reproducible data difficult to obtain

Decontamination and Sterilization are not equivalent.

There are several methods of decontamination, each with
advantages and dis-advantages:
Heat/autoclaving,
Chemical decontaminants
Radiation

It is the responsibility of the supervisor to ensure that workers
are appropriately trained on how to clean up a biological spill
for their specific projects.

The next module will discuss Lab Emergency Procedures
Summary