Microbiology
STERILIZATION AND DISINFECTION
Dr. Tan | September 18, 2020 | Topic 4
Overview
I. Definition of Terms
II. Methods of Sterilization
III. Physical Methods of Sterilization
IV. Chemical Methods of Sterilization
V. Evaluation of Disinfectants
DEFINITION OF TERMS
• Sterilization: process of completely
destroying all microbial forms, including
bacteria spores
• Disinfection: process by which most
bacterial forms in non-living objects are
destroyed, without necessarily destroying
saprophytes and bacterial spores
• Biocide: germicide; kills organisms
• Sanitization: lowering microbial counts on
eating and drinking utensils to safe levels
• Aseptic technique: minimizes
contamination
• Asepsis: absence of significant
contamination
• Decimal reduction time (DRT): time in
minutes, in which 90% of a population of
bacteria at a given temperature will be killed
METHODS OF STERILIZATION
1) Physical
2) Chemical
PHYSICAL METHODS OF STERILIZATION
1) Heat
- Most reliable and universally
applicable method of sterilization
- A gradual process; kinetics of death
is exponential
- Thermal death point (TDP): lowest
temperature at which all the
microorganisms in a particular liquid
suspension will be kill in 10 minutes
- Thermal Death Time: the minimum
time required to kill a suspension of
organism at a predetermined
temperature in a specified
environment
Irelia: The Blade Dancer
MECHANISMS OF THERMAL INJURY (From
Beshywap)
1) Production of single strand breaks in
the DNA
§ primary lethal event
§ Enzymatic in nature and the
result of activation of nuclease
2) Loss of functional integrity of the cell
membrane
§ damage to the cell membrane
causes small molecules to leak
out of the membrane
§ Ribonucleases are activated by
heat → cause degradation of the
ribosomes
3) Denaturation and coagulation of
proteins
4) Oxidative damage
§ There is ATP being produced by
certain structures in the
microorganism, so when we say
oxidative damage- these ATP
that is being produced is
decreased or lowered, making it
easier to kill the microorganism
by physical method of
sterilization
5) Toxic effects of elevated levels of
electrolytes
§ There is no effective exchange of
electrolytes (e.g. Na, K) causing
it to elevate and later create
1. METHODS OF HEAT STERILIZATION
A. Moist heat
• Preferred over dry heat because of
its more rapid killing 
action
• Destroy vegetative forms of
microorganism at a 
temperature of
80oC for 5 to 10 min. 

• Spores are more resistant and
would require 4 minutes exposure at
120oC or 5.5 hours at 100oC 

FORMS OF MOIST HEAT
1) Boiling: kills all vegetative forms of
microorganism at 80-100oC BUT NOT
bacterial spores
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 Microbiology
STERILIZATION AND DISINFECTION
Dr. Tan | September 18, 2020 | Topic 4
2) Fractional Sterilization
(Tyndallization): used to sterilize 2. DESSICATION (DRYING)
materials that would be damaged by
autoclaving

• Mechanism of action: removal of
- The material to be sterilized is exposed
moisture → bacteria cannot grow in
to live steam for 30 mins. for 3
an environment without moisture →
consecutive days
bacterial multiplication is inhibited
• Bacterial spores are resistant to
3) Pasteurization: used in the
processing of milk, foods and other drying or desiccation

beverages.
 • Used only in the preservation of
- It does not sterilize the milk but it only food
inactivates disease producing
microorganism 
 3. FREEZING

- Rapid heating at temperature of 60o to

• Many microorganisms can survive
65oC followed by rapid cooling 
 low temperature for very long
periods of time → freezing cannot
4) Steam Under Pressure be used as a means of sterilization
(Autoclaving): most efficient method of • Used to preserve microorganism
sterilization
 and bacterial culture

- Steam is confined in a closed vessel → • Not reliable because spores are not
pressure reaches 15 psi → temperature destroyed
of the steam reaches 121oC → would • Lyophillization: a technique of
only take 15-20 mins. to sterilize the preserving microorganisms wherein
material. (The times required for the organism is frozen rapidly and
sterilization varies with bulk of the dehydrated in high vacuum and then
material.) 
 stored under vacuum in sealed
- Used In sterilization of surgical ampules in cold storage
bandages and instruments, culture
media, and other contaminated 4. RADIATION
materials 

A. Ultraviolet Light
B. Dry heat • The most effective bactericidal
• Requires higher temperature and wavelength of UVL is 240- 
280 nm
longer exposure to heat
with an optimum of 260 nm that
• Its effectiveness depends on the
corresponds to 
the maximum
penetration of heat 
through the
absorption of the bacterial DNA 

material to be sterilized
• Mechanism of action: disrupt H
• Most widely used type is the hot air
bonds in microbial cell resulting to
oven
the formation of thymine dimers →
• Sterilization takes place at 180oC produce 
lethal frame shift
for 2 hours 
 mutations 

• Used in the sterilization of powders, • Has limited application because of
oils, jellies and glasswares its poor penetrating 
ability and its
• Other useful forms: incineration and
absorption by glass and water 

open flame 

• Primarily used in the reduction of
airborne infections in 
enclosed
areas like operating rooms and
hospital wards 


B. Ionizing radiation
• Has greater penetrating ability than

Irelia: The Blade Dancer 2

 Microbiology
STERILIZATION AND DISINFECTION
Dr. Tan | September 18, 2020 | Topic 4
UVL but are 
potentially hazardous
to human cells 
 CHEMICAL METHODS OF STERILIZATION
• Mechanism of action: cause the
formation of free radicals 
that
chemically interact with proteins and
nucleic acids causing cell death 
 1) Concentration of the chemical agent
5. FILTRATION
• A form of mechanical sieving or
physical separation of
microorganisms from the fluid
• Makes use of high-efficiency
particulate air filters or cellulose
ester membranes but the filter pore
size of 0.22 μm allows very small
organism to pass through (e.g.
viruses, Mycoplasma, Chlamydia
and Rickettsia)
6. OSMOTIC PRESSURE
• When the concentration of the
solution surrounding the bacterial
cell is altered, it may cause the
bacterial cell to collapse or become
turgid
• Used in food preservation
7. ULTRASONIC AND SONIC VIBRATIONS
• Principle: sound vibrations at high
frequency in the upper audible and
ultrasonic range are very useful in
disrupting the cell
 6) Nature of the organism
• Sound wave generators employed
operate at a range of 9-100 kc
• Mechanism: passage of sound
through the liquid → produce
alternating pressures → cause
cavities to form in the liquid →
cavities continue to grow in size →
burst violently → cause the cells to
disintegrate
• Cavitation also produces chemical
and physical changes in the
suspending medium which may be
deleterious to certain enzymes
Irelia: The Blade Dancer
FACTORS AFFECTING DISINFECTANT
POTENCY
• dependent on the material being
disinfected and the organism to be
destroyed
• Higher concentration will be
bactericidal and a weaker
concentration will be bacteriostatic
2) Time
3) Temperature
• The killing of bacteria by chemical
agents increases with an increase in
temperature 

• Increase in temperature speeds up
the rate of chemical reaction
4) pH
• The hydrogen ion concentration
influence both the organism and the
chemical agent 

• Determines the degree of ionization
of the chemical agent
5) Nature of the Medium
• The presence of extraneous
materials decreases the efficiency of
chemical agent by:
a) Surface absorption of the
disinfectant by protein colloids
b) Formation of chemically inert or less
active compounds 

c) Binding of disinfectant by active
groups of the foreign proteins 

EVALUATION OF DISINFECTANTS
PHENOL COEFFICIENT TEST
Ø Phenol is used as the standard in the
evaluation of new chemical agents
Ø Dilution test (tube dilution) is
designed to determine the ratio of the
highest dilution that will kill the organism
within a specified time to the greatest
dilution of phenol showing the same
result
MECHANISMS OF ACTION OF CHEMICAL
AGENTS
1) Damage to the cell membrane
2) Denaturation of proteins
3) Modification of the functional groups and
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 Microbiology
STERILIZATION AND DISINFECTION
Dr. Tan | September 18, 2020 | Topic 4
nucleic acids
DAMAGE TO THE CELL MEMBRANE
Chemical agents interfere with the normal
membrane function → release of small
metabolites and interference with active
transport and energy metabolism
 region
1) Surface active disinfectants
• Agents that alter the energy
relationships at interfaces producing
a reduction of surface tension
a) Cationic agents: quaternary
ammonium compounds
Mechanism
§ Distortion of cell membrane and

loss of membrane permeability
→ leakage of nitrogen and
phosphorous containing
compounds
§ Denaturation of proteins 
 normal temperature
• Bacteriostatic at low concentration
and bactericidal at high
concentration but NOT 
effective
against viruses & bacterial spores 
 greater than ethanol and less
• Most effective at alkaline pH
• Example: zephiran, benzalkonium
chloride, 
cetylpyridinium chloride 
 ethanol like narcosis due to the
b) Anionic agents
• Mechanism: disruption of the
lipoprotein framework of the cell
membrane 

• Most effective at acid pH 

• Example: soaps and detergents
2) Phenolic compounds
• Mechanism: cause disruption of
lipid-containing membranes causing
leakage of cell contents and
irreversible inactivation of
membrane bound oxidases and
dehydrogenases
• Phenols are now used only as
reference to 
testing new chemical
agents because they are toxic to
human cells 

• Cresols are alkyl phenols that are
less toxic 
and more active than
phenol
Irelia: The Blade Dancer
• Bis-phenols- linked phenol
compounds whose activity is
enhanced by halogenation 

3) Alcohols
• Disorganize lipid structure by
penetrating into the hydrocarbon
• Denatures cellular proteins
• Capable of destroying almost all
microbes except microbes
• Inactivated by organic matters
• Activity is greater in the presence of
water
a) Ethyl alcohol
§ Most effective at concentration
of 50-70%
§ Widely used as skin disinfectant
because it is bactericidal and
remove lipids from the skin
surfaces

§ Cannot destroy spores at
b) Isopropyl alcohol
§ Bactericidal activity is slightly
volatile

§ Has greater toxic effect than
absorption of vapors
AGENTS THAT DENATURE PROTEINS
• The backbone of cellular
structures is made up primarily
of proteins
• Enzymes that catalyzes
metabolic reactions are also
made up of proteins 

• Any change in these proteins
would greatly affect the
structure of the cell 

• Includes
§ Acids and alkalis
§ Alcohol and acetone
§ Phenol and its
derivatives
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 Microbiology
STERILIZATION AND DISINFECTION
Dr. Tan | September 18, 2020 | Topic 4
REFERENCE
AGENTS THAT MODIFY FUNCTIONAL • Jawetz’ Medical Microbiology
th
GROUPS OF PROTEINS AND NUCLEIC 28 Edition
ACIDS • Dr. Tan’s PowerPoint
presentation
• 2021 Transcript
1) Heavy Metals • Beshywap Transcript
• Mechanism: poison the enzyme
activity forming mercaptides with the
SH groups of cysteine residues
• Includes Mercurials and Silver
Compound

2) Oxidizing Agents
• Mechanism: inactivates enzymes by
converting functional SH groups to
oxidized S-S form
• Stronger agents also attack amino
groups, indole groups and phenolic
hydroxyl group of tyrosine

Table 1. List of oxidizing agents

Oxidizing Remarks
Agents
Halogens Bactericidal and effective against
sporulating organisms (Eg. Iodine
as best antiseptic)
Chlorine Water disinfectant
Dyes Treatment of dermatologic lesions
and also used in staining bacteria
Alkalating Preservation of specimens and
Agents preparation of vaccines

Factors that influence the degree of killing

1) Type of organism
2) Size inoculum
3) Concentration of disinfecting agent
4) Nature of surface to be disinfected
5) Contact time
6) Temperature: generally at room
0 0C)
temperature (20 – 22
7) pH
8) Biofilm formation: community of bacteria
or other microorganisms that have a
protective layer over them à protected
from outside environmental factors
9) Compatibility of disinfectants and
sterilants

Irelia: The Blade Dancer 5