Lecture #6 - Université d`Ottawa
Download
Report
Transcript Lecture #6 - Université d`Ottawa
Control of Microorganisms
Bio3124
Lecture #6
Definitions
sterilization
destruction /removal of all viable organisms
disinfection
killing, inhibition, removal of pathogens
disinfectants
usually chemical used on inanimate objects
sanitization
reduction of microbial population to levels deemed safe
antisepsis
prevention of infection of living tissue by microorganisms
antiseptics
chemical agents, kill or inhibit growth of microorganisms
when applied to tissues
Chemotherapy: internal use chemicals to kill or inhibit
microbes within host tissues
Definitions…
Antimicrobials:
-cidal agents: agent kills, commonly called
germicides
kills pathogens and many nonpathogens but not
necessarily endospores
include bactericides, fungicides, algicides, and virucides
-static agents: agent inhibits growth
include bacteriostatic and fungistatic
Microbial Death
microorganisms are not
Bacterial Death Curve
killed instantly
120
Plot: log of survivors vs
antimicrobial exposure
time
The slope: average
death rate
100
1E+09
1E+08
80
1E+07
1E+06
60
100000
10000
40
1000
100
20
10
0
1
1
2
3
4
5
6
7
Time
8
9
10 11 12
Survivors( log of CFU/ml)
exponential
1E+10
Survivorsx109 (CFU/ml)
death curves are
1E+11
Effectiveness of Antimicrobial Agent Activity
Depends on:
population size
population composition
vegetative vs dormant
concentration
duration of exposure
longer exposure more organisms killed
temperature
higher temperatures usually increase killing
local environment
e.g., pH, viscosity and concentration of organic matter
organisms in biofilms are physiologically altered and less
susceptible to many antimicrobial agents
Methods in controlling microorganisms
Two major methods are used,
Physical methods
Heat
Moist heat sterilization (autoclaves)
Pasteurization
Dry heat sterilization (ovens, incinerators)
Low temperature (refrigeration, freezing)
Filtration (for heat labile liquids)
Irradiation (UV and ionizing radiation)
Chemical methods
Disinfectants and antiseptics (phenolics,alcohols,
aldehydes, gases… etc)
Chemotherapeutic agents (internal use)
Moist Heat Sterilization
above 100oC , requires saturated steam
under pressure (autoclave)
effective against all types of microorganisms
and spores
degrades nucleic acids, denatures proteins,
and disrupts membranes
The Autoclave or Steam Sterilizer
Autoclave
121°C, 15 psi (2 atm) for
20 minutes
Kills all bacteria
Kills endospores
Clostridium botulinum
Botulism
Bacillus anthracis
– Anthrax
Pasteurization
Louis Pasteur and Claude Bernard (1862)
does not sterilize
logarithmic reduction of germs rather than killing them all
Most often ~5 log reduction; milk, beer, apple cider, fruit juice
and other beverages
Procedures
High temperature short time: holding milk at 72 C for 15-30
seconds
Ultra high temperature: exposure to ~130 C for a fraction of
second
Double pasteurization: 68C for 30 minutes followed by cooling
and again heating at 68C for additional 30 minutes (spores
germinate, killed upon entry to vegetative stage)
Dry Heat Sterilization
less effective
Clostridium botulinium spores killed in 2-3 hours
Ovens: higher temperatures & longer exposure
time
(160-170oC for 2 to 3 hours)
oxidizes cell constituents and
denatures proteins
Bench-top incinerators
inoculating loops
Institutional incineration
Measuring Heat-Killing Efficiency
To develop standards for killing efficiency:
specially important for industrial settings to
develop SOPs
decimal reduction time (D or D value)
time required to kill 90% of microorganisms or
spores in a sample at a specific temperature
One log reduction
Kinetics of thermal reduction
106
D is the time required for one log reduction (90% kill)
Can be calculated using:
Δt
# Bacteria
DT=
105
100oC
1 log
104
log N1-logN2
Δt: total exposure time
N1: initial population
N2: population size after treatment
T= applied Temperature
D100
103
Time
Example 1:
calculate the D value for a bacterial suspension of 109 cfu/ml
that was subjected to 85˚C for 15 minutes at which point its density
was reduced to 106 cfu/ml.
Δt
DT=
log N1-logN2
15
D85=
log 109-log106
15
D85=
9- 6
D85= 5 minutes
Δt: 15 minutes
N1: 109 cfu/ml
N2: 106 cfu/ml
T= 85˚ C
Example 2:
the D90 value for a bacterium is 2 minutes. If starting culture has
108 cfu/ml, how long should this suspension be kept at 90C
to kill the entire population?
Δt
DT=
log N1-logN2
Δt
2=
log 108-log100
Δt
2=
8- 0
Δt = 16 minutes
Δt: ? minutes
N1: 108 cfu/ml
N2: 1 cfu/ml
T= 90˚ C
The D value: an index for sensitivity to thermal killing
106
• Which one is more sensitive to heat killing at 100˚C?
Bacillus subtilis or E.coli?
# Bacteria
• At 100C the time required to reduce Bacillus population
is longer than that required for E.coli
105
104
DE.coli
103
DB.subtilis
100oC
Time
The D value is temperature dependent
# Bacteria
106
D value decreases as the temperature increases
ie. there is less time required to reduce
the population by one log at higher temperatures
105
104
D120
120oC
D110
D100
110oC
103
Time
100oC
Kinetics of thermal reduction: the Z value
Z value
100
increase in temperature required to reduce
D value (min)
D by 1/10 (one log reduction)
ΔT
Z=
10
ΔT: Temperature change
D1: initial D value
D2: secondary D value
1 log
1
Z =10˚C
100
105
log D1-logD2
110
115
120
Temperature (T)
Kinetics of thermal reduction: the Z value
by having D values for different temperatures
one can seek for altering the sterilization protocol to fit
to the industrial setting
One question would be: how much the temperature can
increase to reduce the D value to a given length
This would provide a pragmatic approach in setting up
SOPs in industrial settings
The use of Z value
Example:
A food processing company produces canned meat. Prevention of
Clostridium botulinum spores from growing is important. The D121
for botulinum spores is 0.2 minutes and the Z value is 10˚C. the
company wants to sterilize the canned food at 111˚C. what should
be the length of sterilization if they consider to kill 1012 spores per
can content.
since every 10˚C decrease in treatment causes 10-fold increase in
D value then:
D111= D121x10 ie. D111= 0.2x10 = 2 minutes
using,
Δt
D111=
log1012-log100
Δt
2=
12- 0
Δt= 2x12= 24 minutes
They should heat treat their product at 111˚C for 24 minutes.
Problem : try this on your own
The Z value for a microorganism is 2oC. it takes 54
minutes at 75oC to reduce the population from 109 to
106. At what temperature should the microorganism
be treated to achieve the same result in 10.8 sec.
Answer=800C
Low Temperatures
Freezing
stops microbial reproduction due to lack of liquid
water
some microorganisms killed by ice crystal
disruption of cell membranes
Refrigeration
slows microbial growth and reproduction
Does not prevent psychrophilic microorganisms
Filtration
Porous material with 0.1-0.45
um pore size
reduces microbial population or
sterilizes solutions of heatsensitive materials by
removing microorganisms
also used to reduce microbial
populations in air
Filtration
Bacillus megaterium
Trapped on a nylon
Membrane with 0.2 um pore size
Enterococcus faecalis
Trapped on a polycarbonate
Membrane with 0.4 um pore size
Filtering air
surgical masks
cotton plugs on culture
vessels
high-efficiency
particulate air (HEPA)
filters
used in laminar flow
biological safety
cabinets
Ultraviolet (UV) Radiation
limited to surface
sterilization because it
does not penetrate glass,
dirt films, water, and
other substances
has been used for water
treatment
Kills by inducing massive
number of mutations
How about escaping
mutants?
Ionizing Radiation
Gamma radiation from cobalt 60 is used
penetrates deep into objects
destroys bacterial endospores; not
always effective against viruses
used for sterilization of antibiotics,
hormones, sutures, plastic disposable
supplies, and food
Chemical Control Agents -Disinfectants and Antiseptics
Phenolics
commonly used as laboratory and hospital disinfectants
(2%)
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
Effective if diluted to 70% in water (95% is much less
active)
inactivate some enveloped viruses
denature proteins and possibly dissolve membrane lipids
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
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
Quaternary Ammonium Compounds
detergents that have antimicrobial activity and are effective
disinfectants
organic molecules with hydrophilic and hydrophobic ends
cationic detergents are effective disinfectants
kill most bacteria, but not Mycobacterium tuberculosis or
endospores
safe and easy to use, but inactivated by hard water and
soap
Aldehydes
highly reactive molecules
that cross link proteins
sporicidal and can be
used as chemical
sterilants
combine with and
inactivate nucleic acids
and proteins
Sterilizing Gases
used to sterilize heat-sensitive
materials
EtO penetrates plastic packages
Toxic, needs to be aerated
microbicidal and sporicidal
combine with and inactivate proteins
BPL used for sterilizing vaccines
Decomposes after use, but is
carcinogenic
Evaluation of antimicrobial efficiency: Phenol coefficient
5 Minutes
5 Minutes
TEST
Phenol
10 Minutes
10 Minutes
Evaluation of antimicrobial efficiency
calculation:
The reciprocal of the lowest concentration of the
test material that prevents the microorganism
from growing over 10 minutes exposure but not
at 5 minutes relative to that of phenol is
considered as phenol coefficient of the test
compound.
In this example:
PC= 320/160
PC= 2