Transcript Introduction to Biotechnology

BIOL 3340
Chapter 8
Chapter 8
Control of Microorganisms
by Physical and Chemical
Agents
Figure 8.1-Microbial Control Methods
Definition of Frequently Used
Terms in Microbial control methods
Sterilization

destruction or removal of all viable organisms from an
object or habitat.
Disinfection


killing, inhibition, or removal of pathogenic organisms
that may cause disease:substantial reduction of total
population.
Disinfectants

agents, usually chemical, used for disinfection; not necessary
kills viable spores
More Definitions…
Sanitization

reduction of microbial population to levels deemed
safe (based on public health standards)
Antisepsis


prevention of infection or sepsis. of living tissue by
microorganisms using antiseptics.
antiseptics

chemical agents that kill or inhibit growth of
microorganisms when applied to tissue-should not be
toxic as disinfectants to kill host tissues.
Antimicrobial Agents
agents that kill microorganisms or inhibit
their growth e;g Chemotherapy- chemical
agent to kill or inhibit growth of
microorganisms within host tissues.
 -cidal agents to kill
 -static agents to inhibit growth

-cidal Agents
-cide
 suffix
indicating that agent kills
 germicide
 kills
pathogens and many nonpathogens but
not necessarily endospores
 include
bactericides, fungicides, algicides,
and viricides
-static Agents
-static
 suffix
indicating that agent inhibits growth
 include bacteriostatic and fungistatic
The Pattern of Microbial Death
A microbial population is not killed instantly
 population death usually occurs exponentially as
growth rate (Fig 8.2)
 microorganisms were previously considered to be
dead when they did not reproduce in conditions that
normally supported their reproduction


however we now know that organisms can be in a viable
but nonculturable (VBNC) condition

once they recover they may regain the ability to reproduce and
cause infection
…Microbial death
Decimal Reduction Time (D):
D is the time required to kill 90% of the
microorganisms or spores in a sample
under specified conditions.
Microorganisms is defined as dead when
they don’t grow or reproduce when
inoculated in culture medim.
Fig 8.2-Pattern of Microbial death-an exponential plot of survivors
against mins of exposure to heating at 121 0C. Note D121is decimal
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. Bacterial
spores are much more resistant to Microbial
agents e.g Mycobacterium tuberculosis.
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 to antimicrobial agents
more organisms killed
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. Organic matter in
biofilms protects biofilm microorganisms.
The Use of Physical Methods in
Control
Heat
 Low temperatures
 Filtration
 Radiation

Moist Heat Sterilization
must be carried out above 100oC which
requires saturated steam under
pressure
 Moist heat destroys viruses, bacteria
and fungi
 carried out using an autoclave (Fig8.3)
also known as Steam Sterilizer
 effective against all types of
microorganisms including spores
 degrades nucleic acids, denatures
proteins, and disrupts membranes

The Autoclave or Steam Sterilizer
Figure 8.3-Autoclave Steam Sterilizer
Table 8.2
Measuring Heat-Killing Efficiency
Thermal death time (TDT)

shortest time needed to kill all microorganisms in a
suspension at a specific temperature and under
defined conditions
Decimal reduction time (D or D value)

time required to kill 90% of microorganisms or spores
in a sample at a specific temperature (def previously)
Pasteurization
controlled heating at temperatures well
below boiling- Louis Pasteur
 used for milk, beer and other beverages
 process does not sterilize but does kill
pathogens present and slow spoilage by
reducing the total load of organisms
present

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
Filtration
reduces microbial population or sterilizes
solutions of heat-sensitive materials by
removing microorganisms
 also used to reduce microbial populations
in air

Filtering Liquids
Depth filters



thick fibrous or granular materials bonded in thick
layers that remove microorganisms by physical
screening(size), entrapment, and/or adsorption to the
surface of the filter materials.
Solution contain microorganisms are sucked under
vacuum
membrane filters

porous membranes with defined pore sizes that
remove microorganisms primarily by physical
screening. This has replaced Depth Filters.
Figure 8.5-Membrane Filter Sterilisation
Figure 8.6-Membrane Filter
Filtering Air
surgical masks used
in hospitals and
Labs
 cotton plugs on
culture vessels
 high-efficiency
particulate air
(HEPA) filters used
in laminar flow
biological safety
cabinets (remove
99.97% of particles)

Figure 8.7 (a)-Laminar
Floor
Ultraviolet (UV) Radiation

UV (260nm) quite lethal is
limited to surface
sterilization because it does
not penetrate glass, dirt
films, water, and other
substances.

UV prevent replication and
transcription of Microbial
DNA.
has been used for water
treatment

Figure 7.9
Ionizing Radiation
 Excellent
sterilization agent e.g
Gamma radiation
 penetrates deep into objects
 destroys bacterial endospores; not
always effective against viruses
 used for sterilization and
pasteurization of antibiotics,
hormones, sutures, plastic disposable
supplies, and food
Figure 8.8-Sterilization with Ionization; Radiation machine which uses
Cobalt 60 as a Gamma radiation to sterilize fruits, veg, fish, meat, etc..
Chemical Control Agents Disinfectants and Antiseptics
Phenolics
 commonly used as laboratory and hospital
disinfectants
 act by denaturing proteins and disrupting cell
membranes
 tuberculocidal, effective in presence of
organic material, and long lasting
 disagreeable odor and can cause skin
irritation
Alcohols
bactericidal, fungicidal, but not sporicidal
 inactivate some viruses
 denature proteins and possibly dissolve
membrane lipids

Halogens
any of five elements: fluorine,
chlorine, bromine, iodine, and
astatine
 iodine and chlorine are important
antimicrobial agents

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
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, but not
spores
can react with organic matter to form carcinogenic
compounds
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

Ammonium Compounds
These are detergents that have antimicrobial activity
and are effective disinfectants
organic molecules (cleansing agents) with hydrophilic
and hydrophobic ends for food utensils, small
instruments and skin antiseptics.
 act as wetting agents and emulsifiers
 Because of its positively charged nitrogen, cationic
detergents/ammonium compound, are effective
disinfectants.
 Hhey disrupt microbial membrane , may denature
protein.
 kill most bacteria, but not Mycobacterium tuberculosis or
endospores
 safe and easy to use, but inactivated by hard water and
soap
 E.g Benzalkonium chloride and Cetylpyridinium

Aldehydes
highly reactive molecules
 sporicidal and can be used as chemical
sterilants
 combine with and inactivate nucleic acids and
proteins
 E.g Formal dehyde and glutaraldehyde

Sterilizing Gases
used to sterilize heat-sensitive materials
such as disposable petri dishes,
syringes, heart lung machine
components, sutures, catheters
 microbicidal and sporicidal
 combine with and inactivate proteins
 E.g Ethylene oxide gas (EtO)

Figure 8.11-Ethylene Oxide Sterilizer
Chemotherapeutic Agents
chemicals that can be used internally to kill or
inhibit the growth of microbes within host
cells (covered later in book)
 their selective toxicity allows them to target
the microbe without harming the host
 most are antibiotics, chemicals synthesized by
microbes that are effective in controlling the
growth of bacteria

Evaluation of Antimicrobial
Agent Effectiveness
Testing of antimicrobial agents is a complex
process regulated by US federal agencies
such as:
 Environmental
Protection Agency (EPA)_
regulates disinfectants
 Food and Drug Administration (FDA)- agents
used against humans and animals
Biological Control of
Microorganisms
Biological Control:
 control of pathogenic microorganisms by
predation on one-another,
 Physical barrier
 Viral, fungal, bacterial mediated lysis,
 Toxin-mediated
Bibliography
Lecture PowerPoints Prescott’s Principles of
Microbiology-Mc Graw Hill Co.
 http://en.wikipedia.org/wiki/Scientific_metho
d
 https://files.kennesaw.edu/faculty/jhendrix/bi
o3340/home.html
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