CHAPTER V

DISINFECTION , DECONTAMINATION
AND STERILIZATION
 ACKNOWLEDGMENT
• ADDIS ABABA UNIVERSITY
• JIMMA UNIVERSITY
• HAWASSA UNIVERSITY
• HARAMAYA UNIVERSITY
• UNIVERSITY OF GONDAR
• AMERICAN SOCIETY OF CLINICAL PATHOLOGY
• CDC- Ethiopia
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
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
• 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.
• 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.
 The efficacy of these agents depends on the following factors.
1. Concentration of the 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.
3. pH of the medium- pH may determines degree of ionization of the
chemical and bacterial surface charge.
• E.g.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.
• An increase in 100C 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.
 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
– 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.
• Inactivated by organic materials.
B. Anionic agents
• Soaps and fatty acids
• It causes gross disruption of cell membrane lipoprotein
framework.
• More active in Gram-positive bacteria than in Gram-
negative bacteria.
• Active at acidic pH
1.2. Phenolic Compounds
• Phenol is highly effective in Gram positive bacteria..

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 equipments like clinical thermometer
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.
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.
• 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 and iodine
- Hydrogen peroxide
Uses:
• Hypochlorite: sanitizing agent in 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 (3-6%): Used for cleansing of wound,
disinfecting medical-surgical devices and plastic contact
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.
• 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.
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.
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.
• 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
• Inoculating wires, loops and 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.
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
and adequate air space to ensure optimum heat transfer.
 It is done by applying 140-160 0C for 45 to 60 min or 180
0
C for 30 minutes .
Use:
Usually used to sterilize glass wares and metallic objects.
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.
- Pasturization: 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 ( disinfecting milk)
• Preparation of bacterial vaccines.
Temperatures around 100oC
A. Boiling- (Hot water boilers) -are still a common methods in
many hospitals. the maximum temperature is 100 oC and will
there for, 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.
 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 and vegetative cells
Uses:
• Sterilize solid and fluid culture media, gowns, medical
and surgical equipment.
Table: 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

N.B: Most autoclave work by 1210c ,15 minutes and 15 lb/inch2 (temperature,
time and pressure respectively).
Methods of Controlling Sterilization: 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 thermophilus.
• 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.
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.

B. Lyophilization: (Freeze-drying) is a process which

involves rapid freezing with subsequent drying.
Use:
• Preservation of microbial cultures.
• Preservation of vaccines.
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.

Figure: Micropore filter apparatus

Uses :
 Preparing heat-labile culture media components
 Preparing pharmaceuticals and biological solutions
 Microbial evaluation of water purity
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 ultra violet radiation
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.
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.
Uses of radiation:
Sterilize surgical sutures, catheters, Petri dishes, and
culture media while dispensing and pharmaceutical
products like hormones, enzymes and antibiotics.
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