Bio3124Lecture #6

Control of MicroorganismsControl of Microorganisms

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

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

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