Decontamination

2019 V2

GLOBAL BIORISK MANAGEMENT CURRICULUM

 This course is part of the
Global Biorisk Management Curriculum Library (GBRMC)

The GBRMC Library is funded by the

US DOD/DTRA Biological Threat Reduction Program

with additional support from the

US DOS Biosecurity Engagement Program

The library is managed by

Sandia National Laboratories
Global Chemical and Biological Security

For more information on the GBRMC Library:

web: https://gcbs.sandia.gov/human_capacity_development/hcd-gbrmc.html
email: GBRMC@sandia.gov

2
 Introductions
• Instructors
• Students
– Your name?
– Where are you from?

3
 Action Plan
By the end of this lesson, I would like to:

BE
KNOW FEEL ABLE
TO DO

Your learning doesn’t stop with this lesson. Use this space to think about what else you need to do or learn to
put the information from this lesson into practice.
What more do I need to How will I acquire the How will I know that I’ve How will I use this new
know or do? knowledge or skills? succeeded? learning in my job?

4 Use space on back, if needed

 Key Messages
• Biological decontamination is a process to
reduce or remove unwanted or hazardous
pathogens. There are many methods of
decontamination.
• No one decontamination method is ideal,
each method has strengths and limitations.
Understanding the strengths and
limitations is key to their use.
• Disinfection and sterilization are types of
decontamination methods. Disinfection is
less rigorous than sterilization. Sterilization
is the complete removal of all organisms.
5
 Key Messages – Continued
• Many factors determine the efficacy of a
particular disinfectant. These must be
understood when selecting the appropriate
disinfectant.
• Some pathogens have resistance to various
disinfectants and decontamination methods.
• Autoclaves can be used to sterilize things
through wet heat and the application of
appropriate time, pressure, and temperature.
Wet heat is more effective than dry heat.
• Decontamination validation is a vital process
to confirm that pathogens are successfully
reduced to ensure that risk is mitigated.
6
 Biorisk Management:

The AMP Model

7
 Key Components of Biorisk
Management
• Biorisk Assessment
– Process of identifying the hazards and
evaluating the risks associated with
biological agents and toxins, taking into
account the adequacy of any existing
controls, and deciding whether or not the
risks are acceptable

8
 Key Components of Biorisk
Management
• Biorisk Mitigation
– Actions and control measures that are put
into place to reduce or eliminate the risks
associated with biological agents and
toxins

9
 Key Components of Biorisk

Management
• Biorisk Performance
– Improving biorisk management by
recording, measuring, and evaluating
organizational actions and outcomes to
reduce biorisk.

10
 Decontamination
Group Exercise:

• What is decontamination?

In your groups, spend 5 minutes developing a

definition for decontamination. Write your
definition on your flip chart. Be prepared to
report to the class.

11
 Def inition
Decontamination
A procedure that
eliminates or reduces
biological agents and
toxins to a safe level with
respect to the
transmission of infection
or other adverse effects 1
1 CWA 15793 – Laboratory biorisk management (CEN, 2011 from ISO 15190:2003)
12
 Def initions
Individual Exercise:
• Sterilization
• Disinfection
• Antiseptic
• Decontamination

Once receiving your term/definition

card, spend 5 minutes finding the
student that has the matching
definition/term card.
13
 Decontamination
Group Exercise:

• What types of decontamination

methods are used in your laboratory?

In your groups, spend 10 minutes

discussing common types of
decontamination methods used in your
laboratory. Write your examples on sticky
notes and place them on your flip chart.
Be prepared to report to the class.
14
 Decontamination Methods
1. Sterilization (Thermal)
• Wet/Steam and Dry
2. Disinfection
• Chemical and Antiseptic
3. Radiation
• Ionizing and Non-ionizing
4. Cleaning
• Soap/Water and Enzymatic Cleanser
5. Filtration
• Millipore Filters and HEPA
15 Radiation image: http://clipart-library.com/clipart/413132.htm
 Decontamination Methods
1. Sterilization (Thermal)
• Wet/Steam and Dry
2. Disinfection
• Chemical and Antiseptic
3. Radiation
• Ionizing and Non-ionizing
4. Cleaning
• Soap/Water and Enzymatic Cleanser
5. Filtration
• Millipore Filters and HEPA
16
 Thermal Sterilization
• A physical
process/procedure
• Destroys or eliminates
all forms of life,
especially pathogens
• The definition is
categorical and absolute
– an item is either sterile
or not!

17
 Thermal Sterilization
Two types of thermal sterilization:

1. Wet Heat

2. Dry Heat

18
 Autoclaves

19
 Heat Kills!
― 160 C Spores killed 2 hrs dry heat
o

― 134-138 C Prions inactivated

o

― 121 C Spores killed in 20 min (autoclave)

o

― 100 C Only spores survive after 10 minutes

o

― 82 C Bacteria killed 3 secs (pasteurization)

o

― 72 C Bacteria killed 17 secs

o

― 63 C Bacteria killed in 30 mins

o

― 56 C HIV inactivated 30 mins

o

― 41 C Protein denaturing starts

o

― 37 C Body temperature
o

― 20 C Room temperature
o

20
 Principles of Autoclave Sterilization
• Direct exposure to steam at the required
temperature and pressure for a specific time
– 121 C – 123 C
– 15 psi; 1.05 kg/cm2

• Time required depends on the nature of the

material to be sterilized. (Generally 1 hour for
waste)

21
 Steam Penetration
• Steam must directly contact all areas
of the load (bags should be loosely
gathered)
• If the steam cannot penetrate a dry
container, you have dry heat, which
takes much longer to achieve kill
• Add ~ 50 - 250 ml of water to bags
prior to autoclaving to facilitate steam
saturation
22
 Autoclave Operation
Group Exercise:
In your groups, spend 10 minutes do
develop a list of the advantages and
disadvantages for using an autoclave to
decontaminate laboratory materials.
Record answers in your Student Guide.

• Based on your answers:

− When would using an autoclave be
advantageous?
23
 Autoclave Validation
• Biological indicators
– Spore strips or vials (place http://www.sterilizationproducts.com/wp-

within the load) content/uploads/2015/08/Crosstex-Ethylene-Oxide-Catalog-High-Res.pdf

– Indicates the autoclave’s

performance https:
//www.indiamart.com/progentechnologiesindia/ster
ile-assurance-products.html

• Chemical indicator
– Autoclave tape https://www.medline.com/product/Chemical-Indicator-Tapes-by-SPS-
Medical/Record-Cards/Z05-PF28030

• Thermocouple
– Measures temperature https://www.omega.ca/en/sensors-and-sensing-
24 equipment/temperature/sensors/thermocouple-probes/p/TJ36-ACL
 Autoclave Safety
• Follow manufacturers’ guidelines
• Inspect seals prior to using
• Do not open pressurized chamber
• Avoid standing directly in front when opening
• Clean autoclaves and inspect seals after use
(especially if spills occur)
• Establish a preventive maintenance schedule
and annual inspection by certified technician
• Wear appropriate PPE
• Be careful – liquids are hot
• Open door slowly, allow steam to vent before
opening fully
25
 Autoclave Safety
• Do not place sealed containers into
autoclave
• Do not autoclave items containing
solvents, volatiles, radioactive or corrosive
chemicals
• Use shallow metal pans for best results
and heat transfer
• Check drain and seals

26
27
 Decontamination Methods
1. Sterilization (Thermal)
• Wet/Steam and Dry
2. Disinfection
• Chemical and Antiseptic
3. Radiation
• Ionizing and Non-ionizing
4. Cleaning
• Soap/Water and Enzymatic Cleanser
5. Filtration
• Millipore Filters and HEPA
28
 Disinfection
A process to reduce the number of pathogens,
but not usually of bacterial spores, without
necessarily killing or removing all organisms

Three types:
1. Chemical Liquid
2. Chemical Vapor/Gas
3. Antiseptic

29
 Classes of Chemical

Disinfectants
• Halogens - Chlorine
• Aldehydes - Glutaraldehyde/Formaldehyde
• Phenolics - Phenol, Chloroxylenol
• Alcohols - Ethanol, Isopropanol
• Acids - Peracetic acid, Alkalis
• Oxidizing Agents - Hydrogen peroxide, Potassium
peroxymonosulfate
• Quaternary Ammonium compounds - Alkyl
dimethyl ammonium chlorides, Alkyl dimethyl benzyl
ammonium chloride
• Biguanidines - Chlorhexidine
30
 Ideal Chemical Disinfectant
Group Exercise (Part One):
You are developing the ideal or perfect
chemical disinfectant to eliminate all
pathogens.

In your groups, please spend 5 minutes to list

properties of an ideal chemical disinfectant.
Write one property per sticky note and post
them on your flip chart.

31
 Factors Affecting Disinfection
Group Exercise (Part Two):
Now consider the conditions and factors that
might affect how well a chemical disinfectant
will work.

In your groups, please spend 5 minutes listing

all of the factors. Write one factor per sticky
note and post them on your flip chart.

32
 Factors Affecting the Ef ficacy of
Chemical Liquid Disinfection

Understanding how liquid disinfection works

against pathogens is dependent upon a
number of factors involved. Awareness of
these factors should lead to better use of
disinfection against the pathogen.

33
 Liquid Disinfectant
Individual Reflection:

List in your Student Guide:

All the biological agents you use in your
laboratory, AND the chemical disinfectants
you normally use in your laboratory (brand
name/chemical name).

Think about the chemical disinfectant you

use against the pathogen in your lab as we
go over the factors affecting disinfectants.
34
 Some Factors Affecting
Disinfection
Pathogen Factors:
• Type
• Number
• Location (examples: surface
type, size, and/or configuration
of material to be treated)
• Innate resistance to the
disinfectant
• Presence of organic material
(example: blood)
• Duration of exposure
• Room temperature of laboratory
35
 Some Factors Affecting
Disinfection Continued
Product Factors:
• Age of the product/solution
• Method of application
– spray vs. wipe
• Rate of application
• Storage conditions
– Opaque vs. clear containers
• Concentration and potency of the disinfectant
• Physical and chemical factors (e.g. pH,
temperature, humidity) of the disinfectant
36
 Some Factors Affecting
Disinfection Continued
Environmental Factors:
• Biofilms
– Bacterial aggregates embedded in
extracellular polymeric substances
(EPS)
• Dried spills
– Example: dried spills from media and
buffers may limit contact between
the disinfectant and the target
organism
– Pre-cleaning usually necessary for
spill
• Dirt, grease, and oils
– All can protect the organisms
– Grease and oils will repel water
based disinfectants
37
 Properties of Chemical
Disinfectants
Group Exercise
How do chemical disinfectants work against an
enveloped, non-enveloped, prions, or spore forming
pathogen?

In your groups, please spend 10 minutes discussing

the mode of actions for chemical disinfectants and
reflect on what disinfectants would work on your
assigned organism. Be prepared to report to the
class.

38
 Resistance to Disinfectants
Resistant
• Prions - BSE/Mad Cow
• Bacterial spores -Bacillus anthracis
• Coccidia - Toxoplasma
• Mycobacteria -Leprosy
• Nonenveloped/Lipid/naked or small viruses -
Polio, FMD
• Fungi -Aspergillus, Candida
• Bacteria -E. coli, S. aureus (MRSA)
• Enveloped/Lipid or medium-large virus - SARs,
Rabies
Susceptible
39
 Properties of Chemical
Disinfectants
Group Scenario:
Your group will be assigned a chemical disinfectant to
research the following information:
• Typical concentration used
• Uses in the laboratory
• Advantages
• Limitations/Disadvantages

In your groups, please spend 10 minutes reviewing the

resource material provided. Complete the table in your
Student Guide and be prepared to report to the class.
40
 Properties of Chemical Disinfectants

Criteria

Name of Chemical
Disinfectant

Typical Concentration Used

Uses in the Laboratory

Advantages

Limitations/Disadvantages
41
 Choosing a Chemical Disinfectant
Group Scenario:

In your groups, please read the scenario provided.

Take 10 minutes to discuss and select an

appropriate disinfectant for use in this scenario.
How will laboratory surfaces and reusable
materials be disinfected?

Write you answers on a sticky note and post onto

your flip chart.
42
 Scenario
• A researcher plans to grow various strains of Bacillus
cereus (a potential foodborne pathogen closely
related toB. anthracis ) on petri dishes.
• Individual colonies will then be used to inoculate
liquid broth cultures of up to 500 mLs. The cultures
are grown in glass reusable Erlenmeyer flasks in a
shaker incubator.
• Cultures will be transferred to plastic disposable
tubes to be spun down in a centrifuge. The pellet will
be washed, collected and analyzed for toxin
production. This will involve the use of micropipettes,
glass slides, and various stains and reagents.
• Sub cultures will be lyophilized for storage in small
(<1ml) cryovials and stored in the freezer.
• How will lab surfaces and reusable materials be
disinfected?
43
 Validation Methods
Group Activity:
In your groups, take 5 minutes to discuss
methods or ways in which you can ensure that
the following procedures actually result in
decontamination:
• Chemical disinfection – surfaces
• Chemical disinfection – liquids
• Autoclave sterilization
• Incinerator run

44
 Decontamination Methods
1. Sterilization (Thermal)
• Wet/Steam and Dry
2. Disinfection
• Chemical and Antiseptic
3. Radiation
• Ionizing and Non-ionizing
4. Cleaning
• Soap/Water and Enzymatic Cleanser
5. Filtration
• Millipore Filters and HEPA
45
 Radiation
A process by which energy is emitted as either
particles or wave
Two Types: 1) Ionizing, 2) Non-ionizing

Image: https://www.cdc.gov/nceh/radiation/nonionizing_radiation.html
46
 Radiation
Ionizing Radiation: low temperature or cold
sterilization that uses energy to remove
electrons from air, water and living tissues thus
causing ions to form. This method is not as
favorable due to cost
• gamma rays
• x-rays
• cathode rays

This Photo by Unknown Author is licensed under CC BY-

NC-SA
47
 Radiation
Non-ionizing Radiation: is any type of
electromagnetic radiation that excites atoms
but does not ionize and cannot penetrate
barriers
• Ultraviolet (UV)
• Microwave

S!
I O N
AT
IT
LI M
Image: This Photo by Unknown Author is licensed under CC BY-SA

48
 Decontamination Methods
1. Sterilization (Thermal)
• Wet/Steam and Dry
2. Disinfection
• Chemical and Antiseptic
3. Radiation
• Ionizing and Non-ionizing
4. Cleaning
• Soap/Water and Enzymatic Cleanser
5. Filtration
• Millipore Filters and HEPA
49
 Cleaning
Cleaning uses detergent or enzymatic
cleaners to remove dirt, blood,
pathogens, and debris from surfaces. It
is generally used to prepare items for
further decontamination. Examples:
• Soap/Water
• Enzymatic cleanser (can take several
hours to days to completely clean)

50
 Decontamination Methods
1. Sterilization (Thermal)
• Wet/Steam and Dry
2. Disinfection
• Chemical and Antiseptic
3. Radiation
• Ionizing and Non-ionizing
4. Cleaning
• Soap/Water and Enzymatic Cleanser
5. Filtration
• Millipore Filters and HEPA
51
 Filtration
A process that uses a filter to capture, trap, or
remove pathogens

Remember - Filtration does not kill pathogens .

The filter must be on for

it to work!

52
 Filtration
Examples:
• Use of 0.22 micron pore size membrane filter made
of materials like cellulose acetate/nitrate to
decontaminate liquid media or heat labile liquid
products
• Use of HEPA filters in a BSC to remove infectious
particles from the air
Note: Filters will be contaminated once used
and require their own decontamination!

Dispose micron filters properly and have

HEPA filters disposed by professionals.
53 This Photo by Unknown Author is licensed under CC BY-NC-SA
 Incineration
• A process that involves the
combustion of organic substances
contained in waste materials that
converts into ash, flue gas, and heat,
– Flue gases:
• The mixture of gases resulting from
combustion and other reactions in a furnace,
passing off through the smoke (flue),composed
largely of nitrogen, carbon dioxide, carbon
monoxide, water vapor, and often sulfur dioxide.
• Sometimes it serves as a source from which
carbon dioxide or other compounds are
recovered.
54
 Incineration
• Treatment of choice for animal
bedding, carcasses, and
pathological wastes; but not plastics!
• Incinerators are a way to
decontaminate waste for final
disposal and as complete
sterilization

55
 Review of Decontamination

For 10 minutes, let’s discuss what we

learned about decontamination in a
biological laboratory setting.

What did we What does it Where do we go

learn? mean? from here?

56
 Key Messages
• Biological decontamination is a process
to reduce or remove unwanted or
hazardous pathogens. There are many
methods of decontamination.
• No one decontamination method is ideal,
each method has strengths and
limitations. Understanding the strengths
and limitations is key to their use.
• Disinfection and sterilization are types of
decontamination methods. Disinfection
is less rigorous than sterilization.
Sterilization is the complete removal of all
organisms.
57
 Key Messages – Continued
• Many factors determine the efficacy of a
particular disinfectant. These must be
understood when selecting the appropriate
disinfectant.
• Some pathogens have resistance to various
disinfectants and decontamination methods.
• Autoclaves can be used to sterilize things
through wet heat and the application of
appropriate time, pressure, and temperature.
Wet heat is more effective than dry heat.
• Decontamination validation is a vital process
to confirm that pathogens are successfully
reduced to ensure that risk is mitigated.
58
 Action Plan
By the end of this lesson, I would like to:

BE
KNOW FEEL ABLE
TO DO

Your learning doesn’t stop with this lesson. Use this space to think about what else you need to do or learn to
put the information from this lesson into practice.
What more do I need to How will I acquire the How will I know that I’ve How will I use this new
know or do? knowledge or skills? succeeded? learning in my job?

59 Use space on back, if needed

 Thank You!
Don’t forget to complete your evaluation!

G LO B A L B I O R I S K M A N A G E M E N T
C U R R I C U LU M