Microbiome Techniques

ORBG 8022

Dr. Nezar Al-hebshi, DDS, PhD

POST BACCALAUREATE PROGRAM

2020

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 Introduction to the Course
and Microbial Cultivation I
Lecture 1; ORBG 8022, Microbiome Laboratory

Dr. Sumant Puri, MSc, PhD

May 27th, 2020

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 Course outline
Date Lecture Topic
Introduction to the course and Microbial Basic
May 27 1
cultivation I (SP)
microbial growth
June 1 2 Microbial cultivation II (SP)
techniques;
June 3 3 Microscopy (SP) observation and
June 8 4 Immunological techniques (SP) protein-based
June 15 Exam on lectures 1- 4 ONLY
detection

Date Lecture Topic

Introduction Molecular Biology
June 17 5
Techniques(NA) DNA/RNA-
June 22 6 Nucleic acid hybridization techniques (NA) based techniques
June 24 7 Nucleic acid amplification techniques (NA)
June 29 8 Microbiome Sequencing (NA)

July 1 Exam on lectures 5 - 8 ONLY

 Course outline
Date Lecture Topic

July 6 9 Critical analysis of a research paper (SP)

Research paper presentation - groups A and
July 8
B (NA)
Research paper presentation - groups C and
July 13
D (NA)
Research paper presentation - groups E and
July 15
F (NA)
July 20 Research paper presentation - groups G and H (NA)
Group scores!
Date Lecture Topic
Research paper presentation - groups I and J
July 22
(SP)
Research paper presentation - groups K and
July 27
L (SP)
Research paper presentation - groups M and
July 29
N (NA)
August Research paper presentation – groups O and
3&5 P (SP) & groups Q and R (SP)

August 10 Research paper, open book test

 Overview of the Clinical
Microbiology Laboratory
• clinical microbiologist
– major function is to isolate and identify
microbes from clinical specimens rapidly
• clinical specimen
– portion or quantity of human material that
is tested, examined, or studied to determine
the presence or absence of specific microbes

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 Specimen Collection
• numerous methods used; choice of
method depends on specimen
– specimen should represent the diseased area
– quantity should be adequate
– collection to avoid contamination
– proper container, promptly sent to
laboratory
– obtain specimen before antimicrobial
treatment
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 Specimen Collection
• numerous methods used; choice of
method depends on specimen
– specimen should represent the diseased area
– quantity should be adequate
– collection to avoid contamination
– proper container, promptly sent to
laboratory
– obtain specimen before antimicrobial
treatment
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 https://www.youtube.com/watch?v=PF8GVs5gXqU

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 Standard Microbiological
Practices
• are minimum guidelines that should
be supplemented with other
precautions based on exposure risks
and lab biosafety level regulations
• goal is to protect workers from
contact with agents by taking
precautions and working in a safe
laboratory environment
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 Standard Microbiological
Practices…
• e.g., workers can limit their contact with
microbes by not eating or smoking in lab and
by preventing injuries caused by sharp objects
• e.g., coverings such as lab coats, gloves
etc..should be used PPE
• e.g., workers should know how to use
emergency eye wash and shower stations
• e.g., work space should be disinfected
• e.g., hands should be washed thoroughly after
any exposure and before leaving lab
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 Biosafety Levels
• recommended guidelines for additional
precautions reflect the laboratory’s
biosafety level (BSL)
– BSL 1 – not known to cause disease in
healthy adults
– BSL 2 – associated with human disease
– BSL 3 – disease may have serious or lethal
consequences
– BSL 4 – agent poses high risk of life-
threatening disease
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 Identification of
Microorganisms from
Specimens
• preliminary or definitive identification of
microbe based on numerous types of
diagnostic procedures
– microscopy
– growth and biochemical characteristics
– immunologic tests
– bacteriophage typing
– molecular methods
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 Preparing sterile medium,
tools, and environment for
growing microorganisms

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 Definition of Frequently Used
Terms
• sterilization
– destruction or removal of ALL viable
organisms
• disinfection
– killing, inhibition, or removal of disease
causing (pathogenic) organisms
– disinfectants
• agents, usually chemical, used for disinfection
• usually used on inanimate objects

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 More Definitions…
• sanitization
– reduction of microbial population to
levels deemed safe (based on public
health standards)
• antisepsis
– prevention of infection of living tissue
by microorganisms
– antiseptics
• chemical agents that kill or inhibit growth
of microorganisms when applied to tissue
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 • Antiseptics
• Disinfectants

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 Antimicrobial Agents
• chemotherapy
– use of chemicals to kill or inhibit
growth of microorganisms within host
tissue
• agents that kill microorganisms or
inhibit their growth
– cidal agents kill
– static agents inhibit growth
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 -cidal Agents
-cide
– suffix indicating that agent kills
– germicide
• kills pathogens and many nonpathogens
but not necessarily endospores
– include bactericides, fungicides,
algicides, and viricides

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 -static Agents

-static
– suffix indicating that agent inhibits
growth
– include bacteriostatic and fungistatic

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The Pattern of Microbial Death
• microorganisms are not killed instantly
• population death usually occurs
exponentially
• measure of agent’s killing efficiency
– decimal reduction time – time to kill 90%
– must be sure persister cells (viable but
nonculturable (VBNC) condition) are dead
• once they recover they may regain the ability to
reproduce and cause infection

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 Conditions Influencing the
Effectiveness of Antimicrobial
Agent Activity
• population size
– larger populations take longer to kill
than smaller populations
• population composition
– microorganisms differ markedly in
their sensitivity to antimicrobial agents
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 More Conditions…
• concentration or intensity of an
antimicrobial agent
– usually higher concentrations or
intensities kill more rapidly
– relationship is not linear
• duration of exposure
longer exposure  more organisms
killed
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 More Conditions…
• temperature
– higher temperatures usually increase
amount of killing
• local environment
– many factors (e.g., pH, viscosity, and
concentration of organic matter) can
profoundly impact effectiveness
– organisms in biofilms are
physiologically altered and less
susceptible to many antimicrobial
agents
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 Physical Control Methods

Heat
Radiation

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 Moist Heat
• destroys viruses, fungi, and bacteria
• boiling will not destroy spores and
does not sterilize
• degrades nucleic acids, denatures
proteins, and disrupts membranes

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 Steam Sterilization
• must be carried out above 100oC
which requires saturated steam
under pressure
• carried out using an autoclave
• effective against all types of
microorganisms including spores
• quality control - includes strips with
Geobacillus stearothermophilus
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 Dry Heat Sterilization
• less effective than moist heat
sterilization, requiring higher
temperatures and longer exposure
times
– items subjected to 160–170oC for 2 to 3
hours
• oxidizes cell constituents and
denatures proteins
Mostly used for instruments and containers
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 Dry Heat Incineration

• bench top
incinerators are
used to sterilize
inoculating loops
used in
microbiology
laboratories

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 Ultraviolet (UV) Radiation
• wavelength of 260 is most bactericidal
(DNA absorbs)
• causes thymine dimers preventing
replication and transcription
• UV limited to surface sterilization
because it does not penetrate glass, dirt
films, water, and other substances
• Used in microbiology biosafety bench
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cabinets
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 Ionizing Radiation
• gamma radiation penetrates deep
into objects (X rays and Gamma rays have wavelength
shorter than the wavelength of ultraviolet light)

• destroys bacterial endospores; not

always effective against viruses (dislodge
electrons from atoms, creating ions, damages DNA and produces
peroxides, which act as powerful oxidizing agents in cells )
• used for sterilization and
pasteurization of antibiotics,
hormones, sutures, plastic disposable
34supplies, and food
 Mechanical Methods-
Filtration
• reduces microbial population or
sterilizes solutions of heat-sensitive
materials by removing
microorganisms
• also used to reduce microbial
populations in air

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 Filtering Liquids
• membrane filters
– porous membranes with defined pore
sizes that remove microorganisms
primarily by physical screening

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 Filtering Air
• surgical masks
• cotton plugs on
culture vessels
• high-efficiency
particulate air
(HEPA) filters
– used in laminar
flow biological
safety cabinets
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 Filtering Air
• surgical masks
• cotton plugs on
culture vessels
• high-efficiency
particulate air
(HEPA) filters
– used in laminar
flow biological
safety cabinets
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 Chemical Control Agents

Disinfection
Antisepsis
Sterilization

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 Chemical Agents
• disinfectant must be effective against
wide variety of infectious agents at low
concentrations
• must be effective in the presence of
organic matter; should be stable in
storage
• overuse of antiseptics such as triclosan
has selected for triclosan resistant
bacteria
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 Phenolics
Both disinfectant and antispetic

• commonly used as laboratory and

hospital disinfectants (kill bacteria, fungi, some viruses
but not spores)

• act by denaturing proteins and

disrupting cell membranes
• effective in presence of organic material,
and long lasting
• disagreeable odor and can cause skin
irritation
41 • triclosan is used in hand sanitizers
 Alcohols
• among the most widely used disinfectants
and antiseptics
• two most common are ethanol and
isopropanol
• bactericidal, fungicidal, but not
sporicidal
• inactivate some viruses
• denature proteins and possibly dissolve
membrane lipids
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 Halogens
• any of five elements: fluorine,
chlorine, bromine, iodine, and
astatine
• important antimicrobial agents

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 Halogens - Iodine
• skin antiseptic
• oxidizes cell constituents and iodinates
proteins
• at high concentrations may kill spores
• skin damage, staining, and allergies can
be a problem
• iodophore
– iodine complexed with organic carrier
– released slowly to minimize skin burns
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 Halogens - Chlorine
• oxidizes cell constituents
• important in disinfection of water
supplies and swimming pools, used in
dairy and food industries, effective
household disinfectant
• destroys vegetative bacteria and fungi,
• chlorine gas is sporicidal
• can react with organic matter to form
carcinogenic compounds
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 Heavy Metals
• e.g., ions of mercury, silver, arsenic,
zinc, and copper
• effective but usually toxic
• combine with and inactivate
proteins; may also precipitate
proteins

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 Quaternary Ammonium
Compounds
• detergents that have antimicrobial activity and
are effective disinfectants (irreversibly bind to the
phospholipids and proteins of the membrane, thereby impairing permeability )
– amphipathic organic cleansing agents
– act as wetting agents and emulsifiers
• cationic detergents are effective disinfectants
– kill most bacteria, but not Mycobacterium
tuberculosis or endospores
– safe and easy to use, but inactivated by hard water
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and soap
 Aldehydes
• commonly used agents are formaldehyde
and glutaraldehyde
• highly reactive molecules
• sporicidal and can be used as chemical
sterilants
• combine with and inactivate nucleic acids
and proteins

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 Sterilizing Gases
• used to sterilize heat-sensitive materials
• microbicidal and sporicidal
• ethylene oxide sterilization is carried out
in equipment resembling an autoclave
• betapropiolactone and vaporized
hydrogen peroxide
• combine with and inactivate DNA and
proteins
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 Evaluation of Antimicrobial
Agent Effectiveness
• complex process regulated by U.S.
federal agencies
– Environmental Protection Agency
– Food and Drug Administration

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 Figure 8.1

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