Download 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