sample/2021week-6-control-of-microorganismshaov-qw51qzc3.pdf
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Dr Hao Van
XXXXXXXXXX
Microbial Growth Control
&
Antimicrobial Resistance
Microbiology 2020
BIOL2158 / BIOL2173
Primary References
Brock 15th edition‐ Chapter 5 and 28
Brock 14th edition‐ Chapters 5 and 27
Brock 13th edition‐ Chapters 26 and 36
2
What do the following images
have in common?
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4
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Learning Objectives
• To understand the different microbial control
processes and how , when and why they are used
Physical Microbial Control,
Heat, filtration, radiation
Chemical Microbial Control in vitro
Sterilants, disinfectants, sanitizers, antiseptics,
preservatives
Antimicrobial agents used in vivo
Synthetic antimicrobial drugs
Naturally occu
ing antimicrobial drugs – Antibiotics
• To understand how microbial control processes
affect the cell
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Learning Objectives
• Overview of the tests used to measure Antimicrobial
activity
• Overview of Microbial Resistance
How resistance is acquired and spread.
Review Antibiotic resistance in some pathogens
( Students are advised to review the structure of the cell and
factors controlling microbial growth as these topics are linked)
• Humans are constantly exposed to bacteria
fungi and viruses from other people, air,
water, the environment, products and foods
• We employ many different methods to control
microbial growth with the aim of:
Eliminating or Reducing microbial load
Preventing or limiting growth
Limiting the effects of microbial growth
.
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Industrial – leather , plastics, wood ,paper,
petroleum, textiles, paints, metal working fluids
Environment – air, air conditioning units, water
potable and swimming pools, surfaces, home,
food, hospitals
Foods and food production facilities
Household cleaners and Personal care products
– shampoos, cosmetics, hand washes
Medical – in vitro and in vivo
o Sterilization of equipment, disinfection of surfaces
o Topical and in vivo antibacterial agents
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The mode of action of all physical and
chemical control agents can be divided into 2
asic categories ( the action of antibiotics will be
discussed later)
1. Alteration of cell walls or mem
anes
• Cell wall – cell integrity protects against
osmosis – damage by physical or chemical
agents – water moves in or out of the cell -
cell bursts or collapses
• Cell mem
ane controls passage of
chemicals in and out of the cell – damage to
proteins or phospholipids – leakage or failure
of uptake systems - can lead to cell death
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.
2. Damage to Proteins or Nucleic acids
Proteins
Regulate cellular metabolism
Function as enzymes
Structural components of mem
anes and cytoplasm
Function depends on 3D shape – hydrogen, disulphide
onds
Heat, chemicals – denature proteins – cellular death
Nucleic acids
Chemicals , heat, radiation alter or destroy nucleic
acids – lead to fatal mutations
Damage to enzymatic RNA molecule – interferes with
or stops protein synthesis
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.
Sterilization
The killing or removal of all viable organisms bacteria
and viruses.
Inhibition
Effectively limiting microbial growth – prevention or
slowing
Decontamination
The treatment of an object to make it safe to handle
eg used petri dishes, used surgical equipment
Disinfection
Directly targets the removal of all pathogens, not
necessarily all microorganisms, on non-living objects
Antisepsis
Inhibition or killing organisms (particularly
pathogens) on the skin
Sanitization (Sanitizing)
reduction of pathogens and non pathogenic
acteria from surfaces and utensils ( ‘sanitizers’ are
used in food industry)
Pasteurization
A heat process used to kill pathogens and control
some other microbes that can cause spoilage in
food and beverages
Temperature Sterilization
Pasteurization
Radiation Non ionizing - UV
Ionizing
Filtration HEPA filters
Mem
ane filters
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Heat sterilization is the most widely used method of
controlling microbial growth
Sterilization kills all living cells and spores
– High temperatures denatures macromolecules
– heat killing faster as temperature rises
– Moist heat works better than dry heat- has
etter penetrating power.
• Endospores can survive heat that would rapidly kill
vegetative cells.
• Autoclave at least 121ºC for 15 minutes to kill
endospores
• Amount of time required to reduce viability of the
organism tenfold is called the decimal reduction
time
• thermal death time: time to kill all cells at a given
temperature; affected by population size
Figure 26.1
Decimal
eduction
time (D)
Time (min)
Su
v
iv
al
f
ac
tio
n
(lo
g
sc
al
e)
70ºC
60ºC
50ºC
© 2012 Pearson Education, Inc.
1 log
edn
The autoclave (retort) is a sealed device that
uses steam under pressure
At 121ºC, the time to achieve sterilization of small
amounts of endospore-containing material is
about 15 minutes
– Not the pressure that kills, but the high
temperature
– Used to produce canned foods
– Sterilize microbiological media
– Sterilize dressings, surgical gowns, equipment
etc
Figure 26.3
Chambe
pressure
gauge
Steam
exhaust
valve
Doo
Thermomete
and valve
Steam supply
valve
Steam enters here
Steam exhaust
Jacket chambe
Air exits through vent
Total cycle time (min)
Te
m
pe
a
tu
e
(C
)
Autoclave time
Stop
steam
Begin
pressure
Flowing
steam
Sterilization time
Temperature
of object being
sterilized
Temperature
of autoclave
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Autoclave
Pasteurization
• is the process of using precisely controlled heat to
educe the microbial load in heat-sensitive liquids
– Does not kill all organisms
• Designed to kill relevant pathogens and reduce
potential spoilage organisms.
Is pasteurization a method of sterilization?
Pasteurization
– To achieve pasteurization, the liquid is passed through a
tubular heat exchange
– Times and temperatures vary depending on the
organism and matrix
• milk - 71˚C for 15 seconds
– kills E, coli, Salmonella, Brucella, Mycobacterium (TB)
Coxiella burnetti (Q fever), Listeria monocytogenes etc
– Does not kill spores of Bacillus or Clostridium
– Some non sporing gram positive organisms will also survive
eg Lactobacillus
• UHT (Ultrahigh-temperature pasteurization) 135ºC 1min kills
spores- shelf stable 6-9 months
• Bulk pasterurization: 63-66 °C for 30 min: less satisfactory
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• Ultraviolet (UV) radiation (between 220 and
300 nm): cause mutations or other serious effects
on DNA XXXXXXXXXXdeath of the exposed organism.
• UV useful for disinfecting surfaces and air: laboratory
laminar flow hoods, air circulating in hospital
• Has very poor penetrating powe
© 2019 Pearson Education Ltd. Figure 5.32
Clean work station - Laminar flow cabinet HEPA Filter
UV light used to ‘decontaminate’ a clean surface after
use
© 2019 Pearson Education Ltd.
• Ionizing radiation
• electromagnetic radiation that has sufficient energy to
produce ions and other reactive molecules upon
collision
• Generate higher energy electrons, hydroxyl radicals
(OH·) and hydride radicals (H·)
• Can damage macromolecules and kill i
adiated
cells
Gamma rays
• Used for sterilizing disposable hospital equipment
syringes, needles, cannulas etc can affect material some
plastics change density and become opaque,
ittle
• Gamma ray continuously emitted from a radioactive
isotope Cobalt 60 – rods stored in a water filled pool
Electron beams
• On/ off operation
• Higher dose than gamma (less time) but less
penetrating
X rays
• Electricity based – on/off operation
• Used for large pallet loads – uniform dose
© 2012 Pearson Education, Inc.
© 2019 Pearson Education Ltd.
• amount of energy required to reduce viability tenfold
(D10) is analogous to D value for heat sterilization
Figure 5.33
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• Some microorganisms more resistant to radiation than
others (e.g., endospores vs. vegetative cells, viruses vs.
acteria)
Table 5.7
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• Radiation is used for sterilization in the medical field and
food industry.
• plastic labware, drugs, tissue grafts etc.
• fresh produce, meat products, spices etc.
• In Australia, the doses permitted range from a
maximum of 1 kilogray (kGy) for tropical fruits;
persimmons, tomatoes and capsicums, and up to
30 kGy for he
s and spices.
For sterilization of food products, ionizing
adiation is more effective than UV radiation
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• Filtration avoids the use of heat on sensitive
liquids and gases.
• pores of filter are too small for living organisms to pass
through but do not trap most viruses
• pores allow liquid or gas to pass through
Figure 5.35
Types of filters in routine use in microbiology
Depth filters
– Filtration material a
anged randomly
– Traps particulate matter, dust, pollens, allergens
– HEPA filters high energy particulate air filter - will
trap bacteria used in safety cabinets – control
airflow in and out, also clean rooms
Mem
ane filters
– Functions more like a sieve trapping particles and
acteria on the surface
– Most common in laboratory
– Made of high grade tensile polymers
– Size of pore can be adjusted
– Application - syringe, pump, or vacuum
• A type of mem
ane filter is the nucleopore filte
– Very uniform holes and an even surface
– Used for isolating specimens for electron microscopy
© 2019 Pearson Education Ltd. Figure 5.34
SEM showing the structure of (a) a depth filter, (b) a conventional mem
ane filter
and (c) a nucleopore filter (d) bacteria trapped on nucleopore mem
ane filte
(d)
Pore Size (micron) Smallest organism trapped
5 Multicellular algae, fungi (hyphae, some spores)
3 Yeasts and larger unicellular algae, some fungal
spores
1.2 Protozoa, small unicellular algae, most fungal spores
0.45 Largest bacteria
0.22 Largest viruses and most bacteria
0.025 Larger viruses and pliable bacteria (mycoplasmas,
chlamydia some spirochetes)
0.01 Smallest viruses
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.
Chemical Microbial Control
Chemical Antimicrobial Agents
External Use
In Products
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Chemical Microbial Control
An antimicrobial agent is a natural or
synthetic chemical that kills or inhibits the
growth of micro-organisms
Antimicrobial agents can be classified as
acteriostatic, bacteriocidal, and bacteriolytic
– Bacteriostatic - inhibit
– Bacteriocidal - kills
– Bacteriolytic - kills and destroys cells
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Chemical Microbial Control
Effect of Antimicrobial agents on growth
Bacteriostatic agents frequently inhibit protein
synthesis - eg bind to ribosomes
If the concentration drops the agent can be released
from the ribosome and growth of the organism
esumes
Bacterocidal agents bond tightly to cellular targets
Are not removed by dilution and eventually kill the
cell – however dead cells are not destroyed
Bacteriolytic agents kill by cell lysis and release of
cytoplasmic contents eg. detergents that rupture
cytoplasmic mem
ane.
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Figure 26.9
Total cell count
Viable cell count
Time
Lo
g
ce
ll
nu
m
e
Lo
g
ce
ll
nu
m
e
Lo
g
ce
ll
nu
m
e
Bacteriostatic Bacteriocidal
Bacteriolytic
Total cell count
Total cell count
Time
Time
Viable
cell count
Viable
cell count
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Viable cells
measured by
plate counts
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Total cell count
measured
microscopically
with a counting
chambe
Measuring Antimicrobial Activity
Minimum inhibitory concentration (MIC) is the