Introduction to Biotechnology

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Feb 20, 2013 (4 years and 4 months ago)

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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
0
C. Note D121is decimal
reduction time which is 1 min to kill 90%
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 100
o
C 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
-
170
o
C

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