1. Control of Microorganisms
Bio3124 Lecture #6

2. 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

3. 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

4. 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
Survivorsx109 (CFU/ml)
Survivors( log of CFU/ml)
Time

5. 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

6. 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)

7. 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

8. The Autoclave or Steam Sterilizer
121°C, 15 psi (2 atm) for 20 minutes
Kills all bacteria
Kills endospores
Clostridium botulinum
Botulism
Bacillus anthracis
Anthrax

9. 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 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)

10. Dry Heat Sterilization
less effective
Clostridium botulinium spores killed in 2-3 hours
Ovens: higher temperatures & longer exposure time
( oC for 2 to 3 hours)
oxidizes cell constituents and
denatures proteins
Bench-top incinerators
inoculating loops
Institutional incineration

11. 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

12. Kinetics of thermal reduction
Time
106
105
104
103
100oC
D100
1 log
D is the time required for one log reduction (90% kill)
Can be calculated using:
DT= Δt
log N1-logN2
Δt: total exposure time
N1: initial population
N2: population size after treatment
# Bacteria
T= applied Temperature

13. 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.
DT= Δt
log N1-logN2
Δt: 15 minutes
N1: 109 cfu/ml
N2: 106 cfu/ml
T= 85˚ C
D85= 15
log 109-log106
D85= 15
9- 6
D85= 5 minutes

14. 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?
DT= Δt
log N1-logN2
Δt: ? minutes
N1: 108 cfu/ml
N2: 1 cfu/ml
T= 90˚ C 2= Δt
log 108-log100 2= Δt
8- 0
Δt = 16 minutes

15. The D value: an index for sensitivity to thermal killing
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.coli
DB.subtilis
# Bacteria
Time

16. 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
# Bacteria
120oC
110oC
100oC
D110
D100
D120

17. Kinetics of thermal reduction: the Z value
increase in temperature required to reduce D by 1/10 (one log reduction)
100
Z = ΔT
log D1-logD2 10
D value (min)
ΔT: Temperature change
D1: initial D value
D2: secondary D value
1 log 1
Z =10˚C
100
105
110
115
120
Temperature (T)

18. 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

19. 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.

20. 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

21. 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

22. 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

23. 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

24. Filtering air surgical masks cotton plugs on culture vessels
high-efficiency particulate air (HEPA) filters
used in laminar flow biological safety cabinets
laminar flow biological safety cabinet

25. 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?

26. 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

27. Chemical Control Agents -Disinfectants and Antiseptics

28. 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

29. 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

30. 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

31. 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

32. 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

33. Aldehydes highly reactive molecules that cross link proteins
sporicidal and can be used as chemical sterilants
combine with and inactivate nucleic acids and proteins

34. 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

35. Evaluation of antimicrobial efficiency: Phenol coefficient
5 Minutes TEST Minutes
5 Minutes Phenol Minutes

36. 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

37. Solution for take home problem:
using formula the D75 would be,
54x60 =
54x60
9 - 6 =
54x60 3
D75=
= 1080 sec
log109-log106
With the same token to achieve same killing rate over 10.8 seconds the
Dx should be equal to:
Dx =
10.8 3
= 3.6 sec
Using D1, Dx and T1 in Z expression, we can solve for T2:
Z =
T2 – T1
log D1-logD2
2(log 300) = T2 – 75
2x2.48 = T2 – 75
T2=
T2 = or ~ 80 C
2 =
T2 – 75
log 1080-log 3.6
NOTE: indeed this is what is done in so called ultra high temp short time
pasteurization where a thin layer of milk is passed through hot plates over
a few seconds
2(log 1080/3.6) = T2 – 75