Post on 24-Dec-2015
Definitions
sterilizationdestruction /removal of all viable organisms
disinfectionkilling, inhibition, removal of pathogensdisinfectants
usually chemical used on inanimate objects sanitization
reduction of microbial population to levels deemed safe
antisepsisprevention 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
killed instantly
death curves are
exponential
Plot: log of survivors vs
antimicrobial exposure
time
The slope: average death
rate
Bacterial Death Curve
Su
rviv
ors
x109
(CF
U/m
l)
Su
rviv
ors
( lo
g o
f C
FU
/ml)
Time
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
Autoclave121°C, 15 psi (2 atm) for
20 minutes Kills all bacteriaKills endospores
Clostridium botulinum Botulism
Bacillus anthracis– Anthrax
The Autoclave or Steam Sterilizer
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 #
Bac
teri
a
DT=Δt
log N1-logN2
D is the time required for one log reduction (90% kill)Can be calculated using:
Δt: total exposure timeN1: initial populationN2: population size after treatment
Time
106
105
104
103
100oC
D100
1 log
T= applied Temperature
Example 1:
DT=Δt
log N1-logN2
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 densitywas reduced to 106 cfu/ml.
Δt: 15 minutesN1: 109 cfu/ml N2: 106 cfu/ml T= 85˚ C
D85=15
log 109-log106
D85=15
9- 6
D85= 5 minutes
Example 2:
DT=Δt
log N1-logN2
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: ? minutesN1: 108 cfu/ml N2: 1 cfu/ml T= 90˚ C
2=Δt
log 108-log100
2=Δt
8- 0
Δt = 16 minutes
The D value: an index for sensitivity to thermal killing
Time
# B
acte
ria
• Which one is more sensitive to heat killing at 100˚C? Bacillus subtilis or E.coli?
• At 100C the time required to reduce Bacillus population is longer than that required for E.coli
106
105
104
103 100oC
DE.coliDB.subtilis
The D value is temperature dependent
D value decreases as the temperature increases ie. there is less time required to reduce the population by one log at higher temperatures
Time
106
105
104
103
# B
acte
ria
120oC 110oC 100oC
D110 D100D120
Kinetics of thermal reduction: the Z value D
val
ue
(min
)
Z value
increase in temperature required to reduce D by 1/10 (one log reduction)
Temperature (T)
100
10
1
1 log
100 105 110 115 120
Z =10˚C
Z =ΔT
log D1-logD2
ΔT: Temperature changeD1: initial D valueD2: secondary D value
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, D111=Δt
log1012-log100 2 =
Δt
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=80Answer=8000CC
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 heat-sensitive materials by removing microorganisms
also used to reduce microbial populations in air
Filtration
Enterococcus faecalisTrapped on a polycarbonate Membrane with 0.4 um pore size
Bacillus megaterium Trapped on a nylon Membrane with 0.2 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
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
5 Minutes PhenolPhenol 10 Minutes
5 Minutes TESTTEST 10 Minutes
Evaluation of antimicrobial efficiency: Phenol coefficient
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
Evaluation of antimicrobial efficiency