Disinfection & Sterilization (Chapter Seven)

Bacteriology I (MeLS-M3293) for MLS Students

By Temesgen W/medhin (MSc – Medical Microbiology)
Wolaita Sodo University CHSM
May, 2024
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 Learning objectives

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

– Define disinfection, antiseptic and sterilization.
– Describe means of sterilization and disinfection.
– Discuss the principles of sterilization and disinfection.

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 Sterilization & disinfection
Definition of terms:
Sterilization:
 Killing or removing all forms of microbial life in a material or an
object.
 involves the removal of both vegetative or endospores.
Disinfection:
 Reducing the number of pathogenic microorganisms capable of
giving rise to infection.
 Usually involves the removal of Vegetative form of pathogens.
 May not be effective in killing spores.

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 Definition of terms(cont…)

• Antisepsis: Are chemicals applied in living tissue which are

able to destruct or inhibit the growth of microorganisms
there by limiting or preventing the harmful effect of infection.
eg:- Savlon, tincture of iodine , 70% alchol
• Disinfectants: agents applied on an inanimate object that
destroys harmful organisms except spores.

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 Rate of Microbial Death
Several factors influence the effectiveness of sterilization & disinfection
1. Number of Microbes: The more microbes present, the more time it
takes to eliminate population.
2. Type of Microbes: Endospores are very difficult to destroy.
3. Environmental influences: pH, temperature
4. Presence of extraneous materials: Organic materials like serum,
blood or pus makes chemicals inert that are highly active in their
absence.
5. Time of Exposure: Chemical antimicrobials and radiation
treatments are more effective at longer time
6. The developmental stage of the microorganism

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Methods of sterilization & disinfection

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I. Physical means of sterilization and disinfection

– Physical means of sterilization and disinfection include

Heat, Filtration and Radiation
I. Heat: is the most reliable and universally applicable
method of sterilization.
Mechanism of Action:
• Dry heat-oxidative damage and denatures protein.
• Moist heat-coagulation and denaturation of proteins

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 Physical means…

1.1. Dry Heat

 Dry heat sterilization requires higher temperature and
often takes longer than moist heat sterilization.
 The longer time and higher temperature require in a dry
heat sterilization is because
 heat in water (moist) can ready transfer to cold object
than heat in air (dry).

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 Dry heat (cont’d)

A. Incineration:
 It is an efficient method of sterilization and disposal of
contaminated needles, syringes and cover slips at high
temperature.

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B. Flaming:
 It is a technique of passing an object over a flame with out
allowing it to become red hot.
 Used to disinfect glass slides, mouth of culture test tubes
etc.

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C. Red hot:
 Sterilization of an object by holding them in a flame
till they become red hot
 It is used for sterilizing needles and inoculating wires

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 Dry heat (cont’d)

D. Hot air sterilizer (hot air oven/ dry oven)

 It is essential that hot air should circulate b/n 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 0C for 30 min.
Use: Usually used to sterilize
glass wares and metallic objects.
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 Physical means…
1.2. Moist heat
 It is preferable than dry heat due to more rapid killing action.
 Moist heat can be used by the following methods.
I. Temperature below 100 0c
• 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
Then immediately cooling by <30c
Uses: 1. Pasteurization of milk
( disinfecting milk) and
2. Preparation of bacterial vaccines. 13
 Moist heat (cont’d)

II. 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.

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 Moist heat (cont’d)
Temperatures around 100oC (cont’d)
B. Tyndallization: Intermittent steaming
– Steaming of the material is done at 1000c 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.
– It is used for sterilizing heat sensitive culture media
containing materials such as carbohydrates, egg or
serum.. 15
 Moist heat (cont’d)

III. 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 steam 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 culture media, gowns, medical and surgical equipment.

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 Moist heat (cont’d)
Table: Time-Temperature-Pressure Level Relationship in moist heat
sterilization (Autoclaving)

Temperature Time Pressure level Routinely used

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).
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 Moist heat (cont’d)
• 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 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.

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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
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 2. 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 non-ionic (ultra violet radiation)

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 Radiation (cont’d)

A. 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 are not
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.

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Radiation (cont’d)
B. Ultraviolet Radiation
 It has less quantum energy with low penetrating than ionic radiation.
 They are powerful germicides that kill microorganisms near or on the surface of
clear solution and also on bench tops.
 Cellular DNA absorbs the energy of radiation at wavelengths between 250 & 260
nm & 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:
 Sterilize surgical sutures, catheters, Petri dishes, and culture media while
dispensing and pharmaceutical products like hormones, enzymes & antibiotics.
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 Radiation (cont’d)

Uses of radiation:
– Pharmaceutical products such as hormones,
antibodies and enzymes.
– Heat sensitive articles such as surgical suture,
disposable plastic syringes and catheter
– Used to sterilize large room like operation theatre

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 3. Filtration
• Liquids and gases can be sterilized by passing them through
filters.
• 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 24

 Filtration (cont’d)
Uses of filtration:
 Preparing heat sensitive ingredients of culture media like
blood, serum,
 Preparing pharmaceuticals and biological solutions such as
vaccines, enzymes, antibiotics, and injection fluid and IV fluids
 Microbial evaluation of water purity

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 II. Chemical Methods
Classification of disinfectants:
1. Based on consistency
– Liquid (E.g., Alcohols, Phenols)
– Gaseous (Ethylene oxide)
2. Based on mechanism of action
– Action on membrane (Eg., Alcohol, detergent, phenol)
– Denaturation of cellular proteins (E.g., Alcohol, Phenol)
– Oxidation of essential sulphydryl groups of enzymes (E.g., H2O2,
Halogens)
– Alkylation of amino-, carboxyl- and hydroxyl group (E.g., Ethylene
Oxide, Formaldehyde)
– Damage to nucleic acids (Ethylene Oxide, Formaldehyde)
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1. Phenolic compounds

– Mode of action: Act by disruption of membranes,

precipitation of proteins and inactivation of enzymes
– They act as disinfectants at high concentration and as
antiseptics at low concentrations
– They are bactericidal, fungicidal, mycobactericidal but
are inactive against spores and most viruses
– Phenolic compounds are not clinically advised to be
used because of their neurotoxic effects.

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 2. Surface active agents/Detergents

• Mode of actions: They have the property of concentrating at

interfaces between lipid containing membrane of bacterial cell
and surrounding aqueous medium.
– Disrupt plasma membrane and inactivate bacterial
enzymes
– Used as surgical scrub to disinfect floor and walls, and
mouth wash.
– On the basis of their polarity they are divided in to
anionic, cationic.

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 Detergents (cont’d)

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.
– are highly bactericidal for both the gram-positive and
negative bacteria in the absence of contaminating
organic matter.

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 Detergents (cont’d)

B. Anionic agents
– Soaps
– It causes gross disruption of cell membrane
lipoprotein framework.
– More active in Gram-positive bacteria than in Gram-
negative bacteria.
– Active at acidic pH

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3. Alcohols

• Mode of action: Alcohols dehydrate cells, disrupt

membranes and cause coagulation of protein.
• Eg: Ethyl alcohol, isopropyl alcohol and methyl alcohol
• Use: A 70% aqueous solution is more effective at killing
microbes than absolute alcohols.
– 70% ethyl alcohol (spirit) is used as antiseptic on skin.

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4. Hydrogen Peroxide

• Mode of action: Hydrogen peroxide produces hydroxyl-free

radical that damages proteins and DNA
• 3-6% hydrogen peroxide uses for cleansing of wound,
disinfecting medical-surgical devices and plastic contact
lenses
• Sporicidal at higher temperatures.

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 5. Aldehydes
• Mode of action: Acts through alkylation of amino-, carboxyl- or hydroxyl
groups, and probably damages nucleic acids.
• It kills all microorganisms, including spores.
A. Formaldehyde:
– Excellent disinfectant.
– Formaldehyde 37% aqueous solution is named as formalin.
Uses: Preservation of fresh tissues (to preserve tissues for routine histology
in many labs and funeral homes ) and preparation of vaccines from
bacterial surfaces, viruses and toxins.
B. Glutaraldehyde
– Is ten times more effective than formaldehyde
– Used for sterilizing medical -surgical instruments
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 6. Halogens

 Mode of action: They are oxidizing agents and cause damage

by oxidation of essential sulfydryl groups of enzymes.
A. Iodine: effective skin disinfectant. Tincture of iodine (2% iodine
in 70% alcohol) is an antiseptic

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B. Chlorine: a fast - acting oxidant, is a widely available and it has broad
spectrum of antimicrobial activity
 It is normally sold as bleach, an aqueous solution of 5% sodium hypo
chloride, which can be diluted with water (usually 1/10 immediately before
use) to provide various concentrations of available chlorine.
 They are effective against viruses as well as bacteria.
 It can eliminate both enveloped and non-enveloped viruses
 Dilution should not be kept for more than 24 hours so as to maintain its
activity.

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Sodium Hyporchlorite (chlorine bleach)
• Advantages
• Usually is the least expensive and most readily available
disinfectant.
• Easy to prepare and use.
• Quickly inactivates all viruses including HBV, HCV and HIV, as well as
killing tubercle bacillus.
• Very useful for decontaminating soiled surgical instruments, gloves
and other items and large surface areas

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• Limitation of Chlorine
• Inactivated by organic matter.
• Loses potency on standing if left in open container (replace
at least daily).
• May corrode metal instruments with prolonged exposure
(>20 minutes) to concentrations greater than 0.5%.

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7. Heavy metals
• Mode of action: Act by precipitation of proteins and oxidation
of sulfydryl groups
• Mercury compounds
e.g. mercuric chloride- limited use because of its toxicity.
- It can be used as antiseptics.
• Silver compounds
e.g. Silver nitrate,
– 1% silver nitrate solution can be applied on eyes as
treatment for ophthalmic neonatorum.

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8. Dyes

Mode of action: dyes are bactericidal because of their

interaction with bacterial nucleic acids
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
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 9. Ethylene Oxide
• Gaseous Sterilizers: Chemicals that sterilize in a chamber similar
to an autoclave & leaves no residue on items it contacts
• Used to sterilize medical-surgical devices that would be damaged
by heat.
• Mode of action: It is an alkylating agent. It acts by alkylating
sulfydryl-, amino-, carboxyl- and hydroxyl- groups.
– Kills all microbes and endospores, but requires exposure
of 4 to 18 hours.
– Toxic and explosive in pure form.
– Most hospitals have ethylene oxide chambers to sterilize
mattresses and large equipment.
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Thank You!

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