Disinfection, Antiseptic &

Sterilization
By:
Tsegaye A.
Assis. Prof. of Diagnostic & Public Health
Microbiology

Tsegaye A.(Assis, Prof) 1

 Learning Objectives
Up on completion of this session, You will be able to:
 Define disinfection, antiseptic and sterilization.

 Describe chemical means of sterilization and disinfection.

 Classify chemical means of sterilization and disinfection.

 Discuss the principles of chemical means of sterilization and

disinfection.
 List the physical means of sterilization.

 Discuss hot air oven and autoclaving sterilization.

 Identify the sterilization methods using low temperatures.

2
 Describe the methods of controlling sterilization
Tsegaye A.(Assis, Prof)
 What is

•Disinfection?
•Antiseptic?
•Sterilization?

Tsegaye A.(Assis, Prof) 3

 Definition

Disinfection: Destruction of microbes that cause disease;

may not be effective in killing spores.

Antisepsis: destruction or inhibition of micro-organisms

in living tissue there by limiting or preventing harmful
effect of infection.

Sterilization: is the destruction or complete removal of

all forms of micro-organisms including their spores.
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 Cont’d…

• The agents, which are used for sterilization &

disinfection, can be divided into two broad groups:

I. Chemical mean's

II. Physical mean's

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 Cont’d…

I. Chemical mean's of sterilization & disinfection

• This agent destroy any type of microbes without
showing any form of selectivity unlike antibiotics.

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 Cont’d…
• The efficacy of these agents depends on the following
factors
1.Concentration of the agent - there is a relationship b/n
the concentration of the agent & the time required to kill
a given fraction of the microbial population.
2. Time of exposure - microbes are killed with a
reasonable length of time with chemical agents.
3. pH of the medium - pH may determines degree of
ionization of the chemical & bacterial surface charge.
• E.g. The non-ionized form passes through the bacterial
cell membrane more readily than the ionized form.
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 Cont’d…
4. Temperature
• Bactericidal potency of the chemical agent increases with an
increase in temperature.
• An increase in 10 0C doubles the bacterial death rate.
5. Nature of the organism
• Species of the bacteria
• Growth phase of bacteria in culture
• Presence of capsule, spore and other special structures
• Number of bacteria in test system
6. Presence of extraneous materials
• Organic materials like serum, blood or pus makes chemicals inert
that are highly active in their absence.
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 Classification of Chemical Mean’s of
Sterilization & Disinfection
1. Chemical agents that damage the cell membrane
 Surface active agents - cationic & ionic agents
 Phenols
 Organic solvents
2. Chemical agents that denature proteins
 Acids & Alkalis
3. Chemical agents that modify functional groups of proteins
& nucleic acids
 Heavy metals
 Oxidizing agents
 Dyes
 Alkylating agents Tsegaye A.(Assis, Prof) 9
 1. Chemical Agents that Damage
the cell Membrane
1.1 Surface Active Agents
A. Cationic agents
• Quaternary ammonium compounds (Quates)
• It causes loss of cell membrane semi-permeability
leading to loss of nutrients & essential metabolites
• It can also denature protein.
• More active by inorganic material.
• Inactivated by organic materials.

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 Cont’d…
B. Anionic agents
•Soaps & fatty acids
•It causes gross disruption of cell membrane lipoprotein
framework.
•More active in GPB than in GNB.
•Active at acidic pH

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 Cont’d…
1.2. Phenolic Compounds
•Phenol is highly effective in GPB.
NB: Phenolic compounds are not clinically advised to be used because
of their neurotoxic effects.
1.3. Organic Solvents
Alcohol e.g. Ethyl alcohol, Isopropyl alcohol
•Disorganize cell membrane lipid structure.
•Denatures protein.
•Active against GPB, GNB and AFB.
Uses:
•Potent skin disinfectants
•Disinfects equipment's like clinical thermometer
N.B: Ethanol is potent at concentration of 70%
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 2. Chemical Agents that
Denature Proteins

E.g. Acids, alkalis, Quates, and Alcohol

• Causes conformational alteration of proteins.

• Acids like benzoic acid, citric acid & acetic acid are
helpful as food preservatives: extending storage life of
food products.

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 3. Chemical agents that modify
functional groups of proteins &
nucleic acids
3.1 Heavy metals
• Various metals & metal salts are commonly
employed to prevent microbial growth or kill
microbes
• Mercury compounds
e.g. Mercuric chloride - limited use because of its
toxicity.
• It can be used as antiseptics.
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 Cont’d…
• Silver compounds
e.g. Silver nitrate
• Used as ophthalmic and wound (e.g. in burn patients)
antiseptic.
3.2 Oxidizing Agents
• Converts functional-S-H group into non-functional-S-S
group.
e.g.
• Halogens like chlorine & iodine
• Hydrogen peroxide
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 Uses
• Hypochlorite: sanitizing agent in dairy & food
processing industries, households & hospitals.
• Organic/inorganic chloramines: is effective water
disinfectant acting by liberating chlorine.
• Iodine: effective skin disinfectant.

• Hydrogen peroxide (3-6%): used for cleansing of

wound, disinfecting medical-surgical devices &
plastic contact lenses.
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 Cont’d…
3.3 Dyes
Eg:
• Malachite green
• Brilliant green
• Crystal violet/gentian violet
Uses:
• Highly selective for Gram-positive bacteria.
• For treatment of dermatological lesions.
• For formulation of selective culture media.
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 Cont’d…
3.4 Alkylating Agents
A. Formaldehyde
 Formaldehyde 37% aqueous solution is named as
Formalin.
Uses:
 Preservation of fresh tissues.
 Preparation of vaccines from bacterial surfaces, viruses
& toxins.

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 Cont’d…
B. Glutaraldehyde
•Is ten times more effective than formaldehyde
•Used for sterilizing medical – surgical instruments
C. Ethylene oxide
•is gaseous sterilizing chemical.
Use:
•Used to sterilize medical - surgical devices that
would be damaged by heat.

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 II. Physical means of sterilization
& disinfection
It includes Heat, Filtration & Radiation
1. Heat: is the most reliable & universally applicable
method of sterilization.
Mechanism of Action:
•Dry heat - denatures protein.
•Moist heat - denatures & coagulates protein.

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 1.1. Dry heat

• It is less efficient
• Requires high temperature
• Long period heating than moist heat.
A. Incineration
• It is an efficient method of sterilization & disposal of
contaminated needles, syringes & cover slips at high
temperature.

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 B. Flaming

• Inoculating wires loops

• Points of forceps are sterilized by holding
them in the flame of a Bunsen burner until
they are red hot.
• Scalpels, neck of flasks, bottles and tubes are
exposed for a few seconds, but it is of
uncertain efficacy.

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 C. Hot air sterilizer (hot air
oven/ dry oven)
 It is essential that hot air should circulate between the
objects being sterilized.
 These must be loosely packed & adequate air space to
ensure optimum heat transfer.
 It is done by applying 140 -160 0C for 45-60 min or
180 0C for 30 minutes .
Use:
• Usually used to sterilize glass wares & metallic
objects.
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 1.2. Moist heat
 It is preferred to dry heat due to more rapid killing action.
 Moist heat can be used by the following methods.
Temperature below 100 0c
• This includes the method of pasteurization where objects are
subjected to a temperature of 72 oC for 15 seconds.
• This method does not destroy spores.
• Pasteurization: It is the process of application of heat at
temperature of 62 0c for 30 minutes or 72 0c for 15 seconds
followed by rapid cooling to discourage bacterial growth.
Uses:
• Pasteurization of milk (disinfecting milk)
• Preparation of bacterial vaccines.
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 Cont’d…
Temperatures around 100oC
A.Boiling - (Hot water boilers) - are still a common methods in
many hospitals.
•The maximum temperature is 100oC and will therefore, not kill all
the spores, but for 20 minute exposure all vegetative forms of
bacteria and viruses can be destroyed provided instruments are
cleaned before putting them in boilers.
B. Tyndallization: Intermittent steaming
•Steaming of the material is done at 100 0c for 30 minutes for three
consecutive days.
•The principle is that spores which survived the heating process would
germinate before the next thermal exposure and then would be killed.
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 Temperature above 100oC
Autoclave: Steam under pressure
• It is based on the principle that when microorganism is
boiled at increased pressure (in closed container), hot
saturated steam will be formed which penetrates and
gives up its latent heat when it condenses on objects.
• Hot saturated stem in autoclaving acts as an excellent
agent for sterilization because of:
 High temperature
 Wealth of latent heat (stem under pressure)
– It destroys bacterial endospores & vegetative cells

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 Uses:

• Sterilize solid and fluid culture media, gowns,

medical and surgical equipment.
• N.B: Most autoclave work by 1210c ,15 minutes
and 15 lb/inch2 (temperature, time and pressure
respectively).

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 Methods of Controlling
Sterilization in autoclave
Complete sterilization can be checked by:
1. Heat-sensitive autoclave tape fixed to the outside of each
pack.
• Color change of autoclave tape from blue to brown-black
indicates complete sterilization.
2. Biological indicator: Use of paper strips impregnated with
spores of Bacillus thermophiles.
• Put the paper strip in the culture medium.
• After autoclaving observe for germinating bacteria to check for
growth.
• In a complete sterilization there should not be bacterial growth.
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 Other methods using low
temperature
A. Freezing (At 0 oC or less temp) - is inactivation of
living bacteria by cold.
• It prevents active multiplication of bacteria by
decreasing the metabolic activity of bacteria.
• is more of preservative than disinfectant.
B. Lyophilization: (Freeze-drying) is a process which
involves rapid freezing with subsequent drying.
Use:
• Preservation of microbial cultures.
• Preservation of vaccines.
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 2. Filtration
• Liquids and gases can be sterilized by passing them
through filters.
• Filters that have pores smaller than the size of the
microbes retain micro-organisms.
• The filter acts as a strainer, a microbial sieve.
• Standard bacteriological membrane filters are
composed of nitrocellulose and have pore diameters of
0.45 µm, small enough to prevent passage of most
bacteria.

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 Uses

 Preparing heat-labile culture media components

 Preparing pharmaceuticals & biological solutions
 Microbial evaluation of water purity

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 3. Radiation

 Gamma rays, x-rays, beta rays, cosmic rays, ultraviolet light,

and even visible light are all forms of radiation.
 When these rays strike an organism, energy may be absorbed
by the cells, often causing cell damage or death.
 Are divided into Ionic and ultraviolet radiation

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 1. Ionizing Radiation
 Some rays possess so much energy that they cause
biologically active molecules to lose electrons.
 This results in production of ionised molecules that no
longer perform critical cellular functions.
 Such high energy ionising radiation is an effective
sterilizing agent. e.g. Gamma rays, Beta rays and X-rays
induce break down of single stranded or sometimes
double stranded DNA.Tsegaye A.(Assis, Prof) 33
 2. Ultraviolet Radiation

 It has less quantum energy with low penetrating than

ionic radiation.
 Cellular DNA absorbs the energy of radiation at
wavelengths between 250 and 260 nm & forms aberrant
chemical bonds between adjacent thymine nucleotide
bases.
 These thymine dimmers distort the DNA strands &
impair replication and transcription.
 This interferes with the expression of genes & DNA
replication is blocked.
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 Uses of radiation

• Sterilize surgical sutures

• Catheters
• Petri dishes
• Culture media while dispensing
• Pharmaceutical products like hormones, enzymes &
antibiotics

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