CHAPTER VIII

DISINFECTION ,
DECONTAMINATION
AND STERILIZATION

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 Disinfection , decontamination and
sterilization
Learning objectives

Up on completion of this chapter, the student will be able to:

1. Define disinfection, antiseptic and sterilization.

2. Describe chemical means of sterilization and disinfection.

3. Classify chemical means of sterilization and disinfection.

4. Discuss the principles of chemical means of sterilization

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and disinfection.
5. List the physical means of sterilization.

6. Discuss hot air oven and autoclaving sterilization.

7. Identify the sterilization methods using low

temperatures.

8. Describe the methods of controlling sterilization

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Disinfection: Destruction of microbes that cause
disease; may not be effective in killing spores.
Antisepsis: destruction or inhibition of micro-
organisms growth on 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. 4
 The agents, which are used for sterilization and
disinfection,
can be divided into two broad groups:
I. Chemical means’s of sterilization & disinfection
II. Physical means’s of sterilization & disinfection
I. Chemical Means’s of sterilization and disinfection
 These chemical agents destroy any type of microbes with
out showing any form of selectivity unlike antibiotics.
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The efficacy of these agents depends on the following
factors.
1. Concentration of agent
There is a relationship between the concentration of the
agent and 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. 6
3.pH of the medium
 The hydrogen ion concentration determines degree of
ionization of the chemical and bacterial surface charge.
 The non-ionized form passes though the bacterial cell
membrane more readily than the ionized form.

4. Temperature
 Bactericidal potency of the chemical agent increases with an
increase in temperature.

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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
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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 and Disinfection
1. Chemical agents that damage the cell membrane
 Surface Active Agents
 Phenols
 Organic solvents .
2. Chemical agents that denature proteins
 Acids and Alkalis
3. Chemical agents that modify functional groups of proteins
and nucleic acids
 Heavy metals
 Oxidizing agents
 Dyes 10
 Alkylating agents
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 and essential metabolites
 It can also denature protein.

 More active by inorganic material.

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 Inactivated by organic materials.
1.3 Organic Solvents

Alcohol e.g. Ethyl alcohol, Isopropyl alcohol

 Disorganize cell membrane lipid structure.
 Denatures protein.
 Active against Gram-positive bacteria, Gram-negative
bacteria and acid fast bacilli.
Uses:
 Potent skin disinfectants
 Disinfects clinical thermometer
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N.B: Ethanol is potent at concentration of 70%
2. Chemical Agents that Denature Proteins

E.g. Acids and alkalies, Quates, Alcohol

 Causes conformational alteration of proteins.

 Acids like benzoic acid, citric acid and 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 and nucleic acids
3.1 Heavy metals
 Various metals and 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.
e.g. Merthiolate
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 Silver compounds

e.g. Silver nitrate,

 Used as ophthalmic and wound (e.g. in burn patients)
antiseptic.

3.2 Oxidizing Agents

Converts functional-SH group into non-functional-S-S group.

e.g. -Halogens like Chlorine and iodine

- Hydrogen peroxide 15
Uses:
 Hypochlorite: sanitizing dairy and food processing
industries, households and hospitals.
 Organic or inorganic chloramines: Is effective water
disinfectant acting by liberating chlorine.
 Iodine: effective skin disinfectant.
 Hydrogen peroxide (3%): Used for cleansing of wound,
disinfecting medical-surgical devices and plastic contact
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lenses.
3.3 Dyes
Example:
 Malachite green
 Brilliant green
 Crystal violet/gentian violet

Uses:
 Highly selective for Gram-positive bacteria.
 For treatment of dermatological lesions. 17

 For formulation of selective culture media.

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

and toxins.
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B. Glutaraldehyde
 Is ten times more effective than formaldehyde
 Used for cold sterilizing medical -surgical instruments
C. Ethylene oxide
 is gaseous sterilizing chemical.
Use:
 Used to sterilize medical-surgical devices that would be
damaged by heat. 19
II. Physical means of sterilization and disinfection
 Physical means of sterilization and disinfection include
Heat, Filtration and Radiation
1. Heat: is the most reliable and universally applicable
method of sterilization.
Mechanism of Action:
 Dry heat-denatures protein.
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 Moist heat-denatures and coagulates protein.
1.1. Dry heat

It is less efficient and requires high temperature and long period

heating than moist heat.

A. Incineration

It is an efficient method of sterilization and disposal of

contaminated needles, syringes and cover slips at high

temperature.
B. Flaming
Scalpels , neck of flasks, bottles and tubes are exposed for a few
seconds, but it is of uncertain efficacy. 21
C. Red heat

Inoculating wire, loops and points of forceps are

sterilized by holding them in the flame of a Bunsen

burner until they are red hot.

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D. 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 and adequate air

space to ensure optimum heat transfer.

 It is done by applying 160 0C for 1 hour or 180 0C for 30
minutes .

Use:

Usually used to sterilize glass wares.

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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.
A. Boiling
 Hot water boilers are still a common methods in many hospitals.
 Maximum temperature is 100 0C and will there for, not kill all
the spores. However, when exposed for 20minutes, all vegetative
forms of bacteria and viruses can be destroyed provided that
instruments are cleaned before putting them in boilers.

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B. Tyndallization (Intermittent steaming)
 Steaming of the material is done at 100 0C for 30 minutes on
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.

It is used for sterilizing heat sensitive culture media containing

materials such as carbohydrate, egg or serum.

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C. Pasteurization:

It is the process of application of heat at temperature of

620c for 30 minutes or 720c for 15 seconds followed
by rapid cooling to discourage bacterial growth.

Uses:
Pasteurization of milk.
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Preparation of bacterial vaccines.
D. Autoclave (Steam under pressure)
 It is based on the principle that when micro-organism 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); 27

- It destroys bacterial spores and vegetative cells.

Table 1. Time-Temperature-Pressure Level Relationship in moist
heat sterilization (Autoclaving)

Temperature Time Pressure level

1210c 15 minutes 15 lb/inch2

1260c 10 minutes 20 lb/inch2

1340c 3 minutes 30 lb/inch2

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Uses:
Sterilize solid and fluid culture media, gowns, medical and
Surgical equipment

Other methods using low temperature

A. Freezing

(At 0oC 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.

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B. Lyophilization (freeze-drying)
Is a process which involves rapid freezing with
subsequent drying.
Uses:
 Preservation of microbial cultures.
 Preservation of vaccines

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C. 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
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bacteria.
Uses :
 Preparing heat-labile culture media components
 Preparing pharmaceuticals and biological solutions
 Microbial evaluation of water purity
 Bacterial viability count
 Determination of viral particle size
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C. 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 ultra violet 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 some times
double stranded DNA. 34
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 and forms aberrant
chemical bonds between adjacent thymine nucleotide
bases.
 These thymine dimmers distort the DNA strands and impair
replication and transcription. This interferes with the expression
of genes and DNA replication is blocked.
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Uses:

Sterilize surgical sutures, catheters, Petri dishes, and

culture media while dispensing and pharmaceutical

products like hormones, enzymes and antibiotics.

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4. Methods of controlling sterilization
 Recording of temperature, time and pressure of each sterilizing cycle

 By use of heat-sensitive autoclave tape fixed to the outside of each

pack . Colour change of autoclave tape from blue to brown-black
indicates complete sterilization.
 By the use of biological indicators: is made up of a carrier material,
on which bacterial spores with a defined resistance to
the sterilization process have been applied

- Use of paper strips impregnated with spores of Bacillus

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stereothermophilus.
• Put the paper strip in culture medium and after
autoclaving, observe for germinating bacteria to
check for growth.
• In a complete sterilization, there should not be a
bacterial growth.

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Sterilization & disinfection
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Spaulding Classification of Disinfection
•Chemical Germicide Activity Level:
1. High Level Disinfection
• Comprises high concentrations of chemical germicides (ex:
concentrated sodium hypochlorite).
• Kills vegetative microorganisms and inactivates viruses
• Does not kill high numbers of bacterial spores
• High level disinfectants are typically used for short time periods
(10-30 min) for disinfection purposes, but may achieve
sterilization if left in contact with the surface for long time
periods (6-10 hours).
• Not for use on environmental surfaces like floors or lab benches 40
2.. Intermediate Level Disinfection
• Kills vegetative microorganisms, including Mycobacterium
tuberculosis, all fungi, and inactivates most viruses
• EPA Approved Hospital Disinfectants which are also
tuberculocidal fall into this category
• May be used for housekeeping and disinfection of laboratory
benches
3. Low Level Disinfection
• Kills most vegetative bacteria, some fungi, and inactivates some
viruses.
• Does not kill M. tuberculosis
• Also known as “hospital disinfectants” or “sanitizer
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