Principles of Sterilisation

Dr. M. Siyawamwaya

1 Sterilization
 INTRODUCTION
⚫ Microorganisms are the agents of
contamination, infection, and decay.

⚫ Hence it becomes necessary to remove

them from materials and areas.

⚫ Early civilization practiced salting, smoking,

pickling and exposure to sunlight.
Bacteriostatic Agent:
An agent that inhibits the growth of
bacteria, but does not necessarily kill them.
Effect is reversible.

Bactericide:
An agent that kills bacteria. Most do not kill
endospores.

Sporicide:
An agent that kills spores.
In mid 1800s Lister developed Aseptic
techniques to prevent contamination of
surgical wounds. Prior to this development:
• Nosocomial infections caused death in
10% of surgeries
• Up to 25% mothers delivering in hospitals
died due to infection
Sepsis: Comes from Greek for decay or putrid.
Indicates bacterial contamination

Asepsis: Absence of significant contamination

Asepsis is usually achieved by first cleaning

followed by sterilization or disinfection.

Asepsis is accomplished by:

I. Sterilization
II. Disinfection
Aseptic techniques are used:
⚫ to prevent contamination of surgical
instruments, medical personnel and the
patient during surgery
⚫ to prevent bacterial contamination in food
industry
⚫ preparation of pharmaceutical products
using sterile ingredients and ensuring that
the final product is free from micro-
organisms
 I. STERILIZATION
• It is one of the basic steps in surface
asepsis.
• Definition: Sterilization refers to the use of
different procedures to destroy all forms of
micro-organisms including bacterial
spores.

7 Sterilization
 What to sterilize?
• It is mandatory to sterilize :
➢ all instruments that penetrate soft tissues
and bone.
➢ instruments that are not intended to
penetrate the tissues, but that may come
into contact with oral tissues.
• If the sterilization procedure may damage
the instruments, then, sterilization can be
replaced by Disinfection procedure
8 Sterilization
Instrument packing for sterilization
• Often instruments are packed for sterilization to
be stored and handled without being
contaminated.
• Packing depends on the intended shelf life after
sterilization.
• The available packing options are:
– Textile has shelf life of 1 month
– Paper has shelf life of 1 – 6 months
– Nylon, glass, and metal have shelf life of 1 year
if tightly closed
9 Sterilization
 Methods of Sterilization

Microbial
Control
Methods

Mechanical
Chemical
Removal Physical Agents
Agents
Methods
MECHANICAL METHOD OF
STERILIZATION
Filtration

• Aqueous liquids may be sterilized by forced

passage through a filter of porosity small
enough to retain any micro-organisms present
in them
• Used to sterilize serum, carbohydrates
solution, filtrates of toxins & bacteriophages,
in water bacteriology, in examination of
Schistosoma eggs
 FILTRATION
• Types of filters
– Earthenware candles
• Unglazed ceramic & diatomaceous earth filters
• Eg. Chamberland filters, Doulton filters
– Asbestos filter – Seitz, Carlson, Sterimat
– Sintered glass filter
– Membrane filters – cellulose nitrate, cellulose acetate,
polycarbonate, polyester filters
– Pore size: 0.015 – 12 μm
• HEPA filters – for large volumes of air

READ
Advantages of membrane filters
Sterilization control – bubble pressure test, bacteriological
tests
EARTHENWARE CANDLES

Widely used for purification

of water

Two types:
(a) Unglazed ceramic
filter – Chamberland filter
(b) Diatomaceous earth
filters – Berkefeld filter
 ASBESTOS FILTER

SEITZ FILTER
ASBESTOS DISCS
 ASBESTOS FILTER
⚫ Disposable single use discs

⚫ High adsorbing tendency

⚫Carcinogenic
Eg: Seitz filter
 SINTERED GLASS FILTER
⚫ Prepared by heat
fusing powdered
glass particles of
graded size

⚫ Slow filtration,
difficulty in
cleaning, brittle,
expensive.
MEMBRANE FILTERS
MEMBRANE FILTERS

Made of cellulose esters or

other polymers

Uses
Water purification &
analysis
Sterilization & sterility
testing
Preparation of solutions for
parenteral use
 May be done under either negative or
positive pressure.
 Membrane made from cellulose acetate
and recommended pore size is 0.22
micrometers.
 Generally removes most bacteria but
viruses and some small bacteria e.g.
Chlamydias & Mycoplasmas may pass
through. Thus filtration does not
technically sterilize items but it is
adequate for circumstances under
which it is used.
 Main use: for heat labile substances
e.g. sera, antibiotics.
 2. PHYSICAL METHOD OF
STERILIZATION

20 Sterilization
 HEAT STERILIZATION
• Advantages: It is the simplest, most
effective and inexpensive method.
• There are 2 procedures depending on
the tolerance of the material used:
a) Steam sterilization (Autoclaving)
b) Dry heat sterilization

21 Sterilization
 2a) Heat – Steam Sterilization
• Use saturated steam above 100º C on packed items.
• Objects occupy 4/5 of autoclave volume to facilitate
circulation.
• Process is divided into 3 periods as follows:
– Pre-vacuum period: air is
withdrawn from device
– Sterilizing period: steam is
introduced under pressure to
specific temperatures &
times.
– Post-vacuum: steam is
withdrawn to dry autoclave
Table shows samples of time-temperature relationships for
22
steam sterilization Sterilization
 Steam Sterilization
Advantages & Disadvantages
• Advantages:
– good penetration
– maintains integrity of liquids (e.g. Lubricants)
due to the 100% humidity within the chamber.
• Disadvantages:
– Non stainless steel metal items corrode
– may damage plastic and rubber items
– sharp instruments get dulled.

23 Sterilization
 Moist Heat
Terminal sterilization of aqueous injections,
ophthalmic preparations, irrigation &
haemodialysis solutions, equipment used in
aseptic processing
Not suitable for non-aqueous/dry
preparations
Preferred method of sterilization

24 Sterilization
Efficient Autoclaving
⚫ All instruments must be double
wrapped in linen or special paper or
placed in a special metal box
equipped with a filter before
sterilization.
⚫ The white stripes on the tape change
to black when the appropriate
conditions (temperature) have been
met.
⚫ Expiration dates should be printed on
all equipment packs.
⚫ There should be a uniform
development of bars throughout the
length of the strips.
⚫ Ready made plastic bags with strips
printed with a sensitive ink.
 Control of autoclave efficiency

• Bowie-Dick test: Used for high-vacuum autoclaves, Done

every day:
• In the middle of a test pack of towel, a paper on to which a strip of
a specific tape is put, for testing.
• Uniform development of dark color indicates that the steam has
passed freely and rapidly to the center of the load.
• Biological sterilization indicators: Spores of a non-
pathogenic organism: Done weekly
• They are killed at 121°C after 15 minutes.
• Attempts to culture them is subsequently made
Some causes of failure to produce
a sterile load are:

Faults in the autoclave and the way it is

operated
It maybe:
– Poor quality steam
– Failure to remove air and
condensate
– Faulty gauges and timings
– Leaking door seals
Errors in loading
Examples:
– Large packs
– Excessive layers of wrapping materials
– Over packing
Recontamination after sterilization due to:
o An inadequate air filter and leakage
into the chamber
o Wet or torn packs
o Incorrect storage
 Preparation of Item Before
Sterilization

1. Decontamination
2. Disassembly
3. Washing
4. Drying
5. Packing
6. Loading in sterilizer
 Five Phases of the Steam
Sterilization Process

PHASE I
The loading phase - objects or items are packaged
and loaded in the sterilizer.

PHASE II
The heating phase – steam is brought to the set
temperature and allowed to circulate and
penetrate through the objects in the chamber.
PHASE III
The destroying phase (time-temperature cycle) - all
microbial life is exposed to the killing effect of the
steam.

PHASE IV
The drying and cooling phase – objects are dried and
cooled, filtered air is introduced into the chamber, the
door is opened and the objects are removed and
stored.

PHASE V
Testing phase - efficiency of the sterilization process is
checked. All mechanical parts of sterilizers, including
gauges, steam lines and drains, should be
periodically checked by a competent engineer.
 Storage of Sterile Packages

⚫ Sterile packages/items should be left

untouched and allowed to be cooled
before storage to avoid condensation
inside the packs.

⚫ Sterile packages must be handled as

little as possible to reduce the risk of
contamination.
Sterile packages should be stored on
open shelves.
– The lowest shelf should be 8 inches
from the floor
– The highest shelf should be 18
inches from the ceiling
– All shelves should be at least 2
inches from the walls
⚫ The storage room must be subjected to adequate pest
control to prevent contamination from rodents, ants and
cockroaches.
⚫ Traffic is restricted to CSSD personnel and trainees only.
Physical Methods of Disinfection

DRY HEAT
• Kills by Destructive oxidation of cell constituents
• Surest incineration
• Hot-air oven:
➢ Inefficient, poor conductor of heat, penetrates
feebly.
➢ 160°C for 1 hour: damages fabrics, melts
rubber
➢ Used for: Glassware, oily fluids, powders
37 Sterilization
 3. CHEMICAL METHOD OF
STERILIZATION

40 Sterilization
 Types of Chemical Sterilization

• The chemical compounds used can be:

a) Gas Sterilization
b) Liquid Sterilization
• Generally, chemical sterilization
procedures have the disadvantages of
presenting health hazards to users (e.g.
poisonous, flammable,)

41 Sterilization
 3a) Gas Sterilization
• The gas used in this procedure is ethylene
oxide.
• Procedure:
– Keep objects in constant atmospheric
humidity.
– Heat to temperatures between 30º and 60º C.
– maintain for a period of 10 hrs.
– Residual ethylene oxide must be ventilated
• Formalin gas autoclaves have been developed
for sterilizing endoscopes and anaesthetic
apparatus.
42 Sterilization
 Gaseous Disinfection
Ethylene Oxide Sterilization: EO Gas

⚫ Colorless gas, available as cartridges

⚫ Toxic and flammable, Odor similar to ether
⚫ Has an extremely well penetration, even through
plastics
⚫ Microorganism destruction is caused by a
chemical reaction
⚫ Effective sterilization is dependent on
concentration of gas, exposure time,
temperature, and relative humidity
⚫ Powerful sterilizer: Kills all known viruses,
43 Sterilization
bacteria (including spores), and fungi
 EO Gas Sterilizer
⚫ Is used in large hospitals, as it is expensive,
dangerous, needs more expertise.
⚫ Used for heat sensitive instruments: fabrics,
plastics, suture material, lenses, endoscopes,
electrical equipment and finely sharpened
instruments.
⚫ At 20°C-25°C: sterilization takes 18hours
⚫ At 50°C-60°C: sterilization takes 4 hours
⚫ The average of the cycle is 8-14 hours

44 Sterilization
 Ethylene Oxide Gas
• Either pure or in mixtures with other inert gases
• Requires presence of moisture
• Complex process
• Typical cycles:
– 1-24hours
– 25-1200 mg/L gas
– 25-65°C
– 30-85% relative humidity

45 Sterilization
Gas Sterilization – Disadvantages

• Ethylene oxide (& formalin) autoclaves

have the following disadvantages:
– Difficult to operate
– Unsuitable for hospitals but used in
industry (e.g. for sterilizing disposable
materials that can not tolerate high
temperatures).

46 Sterilization
 3b) Liquid Sterilization

• Can be performed with buffered glutaric

aldehyde.
• Procedure:
– Immerse object in liquid for several
hours.
– Rinse with sterile water after end of
procedure.
47 Sterilization
3. RADIATION STERILIZATION

48 Sterilization
 Radiation Sterilization
• Provides effective way of sterilization
when used in high doses.
• Gamma radiation (from 60Co).
• Procedure:
– Objects on belt conveyer
– Expose objects to 25 to 30 kGy for
about 24 hrs.

49 Sterilization
 Radiation Sterilization
Advantages and Disadvantages
• Advantages:
– Clean process
– Dry process
– Ensures full exposure of object from all
directions
• Disadvantages:
– Posses threat to humans (radiation)
– Lengthy process
– Requires very qualified personnel
50 Sterilization
 In summary
• Selection of sterilization process must be
appropriate for product
– terminal sterilization is the method of choice
– moist heat (autoclaving) is the most common process
used for terminal sterilization
– product must not be affected by heat
– container/closure integrity must be established
– items being sterilized must contain water (if sealed) or
material must allow for removal of air and penetration
of steam for steam (moist heat) sterilization

51 Sterilization
 II. DISINFECTION

52 Sterilization
 II. Disinfection
• It a is less lethal process than sterilization.
• Definition: It is a procedure intended to
reduce microorganisms as far as possible
(but not bacterial spores).
• Thus, disinfection can never replace
sterilization

53 Sterilization
 Disinfection - Types
• As mentioned before, disinfection can not
kill all microorganisms but only reduce its
number.
• Disinfection can be accomplished with:
1. Heat disinfection
2. Chemical disinfection
• Object that can be disinfected are
bedpans, patient skin before operation
and surgeon hands before putting gloves.
54 Sterilization
 1. HEAT DISINFECTION
• It is accomplished by boiling water at
atmospheric pressure for at least 5 mins

55 Sterilization
 2. CHEMICAL DISINFECTION
• Number of different agents are used
according to tolerance of objects and
infectious agents.
– Using phenol with cleaning component
destroy the membrane of microorganisms.
– Using 70% alcohol for skin which denatures
proteins of microorganisms.
– Use soap containing hexachlorophene for
hands.
– In case of hepatitis use 5% solution of
56 Sterilization
chloramines or heat disinfection
 Chemical Disinfection
A SATISFACTORY AGENT SHOULD:
• Be active against a wide range of organisms and
spores. Only few are truly sterilizer
• Have a rapid action
• Should not be toxic or irritant to the skin
• Should be Persistent

• There is no one disinfectant which can be

used to kill all micro-organisms in all
situations.
57 Sterilization
Types of Chemical disinfectants
➢ Inorganic:
• Iodine
• Chlorine
➢ Organic:
• Alcohols
• Aldehydes
• Phenols
• Cationic surface-active agents

58 Sterilization
 Inorganic disinfectants
The halogens: Chlorine and Iodine
⚫ Have a rapid action against vegetative
organisms and spores = true sterilizers
⚫ Their action is annulled by foreign organic
material

59 Sterilization
 Iodine disinfectants
The broadest spectrum of all topical anti-infectives, with
action against bacteria, fungi, viruses, spores, protozoa,
and yeasts.
⚫ Tincture iodine:
– 2.5% iodine & 2.5% potassium iodide in 90% ethanol.
– Best skin disinfectant
– Irritating to raw surfaces: due to its alcoholic component
– Allergic dermatitis
⚫ Iodophors: Solutions of iodine in non-ionic detergents=
Povidone iodine= Betadine
– Less irritating and less staining
– Less disinfectant than tincture

60 Sterilization
 Chlorine disinfectants
⚫ Powerful, Disinfect water
⚫ Particularly active against viruses
⚫ Concentrated solutions too corrosive
⚫ Usually diluted with a compatible
detergent

61 Sterilization
 Organic Disinfectants
⚫ Alcohols: bactericidal: 50-70% ethanol
⚫ Aldehydes:
– Formalin: irritant, powerful=sterilizer
– Glutaraldehyde: less irritant, not volatile, more rapid action.
⚫ Phenols: continued activity in organic matter as human
excreta.
– Phenol: Toxic, expensive
– Cresols: Lysol
– Chloroxylenol: Dettol
– Chlorhexidine: Hibitane- Alkanol
– Hexachlorophane
⚫ Cationic surface-active agents:
– Cetrimide: Cetavlon

62 Sterilization
 Alcohol
⚫ Isopropyl Alcohol 70% (or Ethyl Alcohol
90%)
ADVANTAGES:
⚫ Causes protein denaturation, cell lysis,
and metabolic interruption.
⚫ Degreases the skin.
DISADVANTAGES:
⚫ Ineffective against bacterial spores and
poorly effective against viruses and
fungi.
63 Sterilization
 Glutaraldehyde (Cidex)
Cold Sterilization:
⚫ Instruments must be dry before immersion.
⚫ Glutaraldehyde is bactericidal, fungicidal,
viricidal, and sporicidal
⚫ Sterilization: a 10 hour immersion. This
prolonged chemical action can be more
detrimental to surgical instruments.
⚫ 3 hours exposure time is needed to
destroy spores.
⚫ If the instruments need to be "disinfected"
only, cold sterilization is okay as
disinfection will take place in only 10
minutes.
64 Sterilization
 Lysoformin

⚫ Formaldehyde & glutaral

⚫ Lysoformin: liquid concentrate with which any dilution
required can be made by simply adding water (20ml + 4-
8L)
⚫ The timing depends on the concentration used:
– flexible endoscopes
– deactivation of HBV & HIV
⚫ Used for heat labile instruments and cleaning
⚫ Does not harm metal instruments
65 Sterilization
 Chlorhexidine Gluconate
Broadest spectrum
Better residual activity than iodophors
Occasional skin sensitivity
ADVANTAGES:
Rapid action
Residual activity is enhanced by repeated use
Less susceptible to organic inactivation than povidone iodine
DISADVANTAGES:
Occasional skin sensitivity.
Inactive against bacterial spores
Activity against viruses and fungi is variable and inconsistent
May harm metal instruments
66 Sterilization
 Asepsis – Summary
Asepsis

Sterilization Disinfection

Heat Chemical Radiation Heat Chemical

Steam Gas Gamma Boiling water Phenol

Dry heat Ethylene oxide 70% Alcohol

Formalin Hexachlorophene

Heat or
Liquid chloramines
solution

Glutaric
Aldehyde

67 Sterilization
 Which procedure to use?
Category Items in each category Procedure

Items that: (1) enter the tissue; (2)

Critical Enter vascular system; (3) through Sterilization
which blood flows
Items that: (1) touch mucous
membranes; (2) touch non-intact High level
Semi-critical skin (e.g. endoscopes, respiratory disinfection
therapy equipment, and diaphragms)

Items such as bedpans, blood Low level

Non-critical pressure cuffs, and bedside tables disinfection
68 Sterilization
 CONCLUSION
• In summary:
– Sterilization and disinfection are costly and
time consuming process
– However, it is essential in all health care
facilities to avoid spread of diseases.
– It depends on the ethics of the instrument
users.
– Users should keep in mind that contaminated
instrument present risk to patient as well as
the user himself.
69 Sterilization
Disinfection and Sterilization - New
methods

70 Sterilization
 Table 1. New methods in disinfection and sterilization

Process Agent Regulatory agency action

Disinfection Ortho-phthalaldehyde (Cidex FDA cleared, October 1999

OPA)
Antimicrobial coating Not FDA/EPA cleared
(Surfacine)
Superoxidized water Not FDA/EPA cleared
(Sterilox)

Sterilization Liquid sterilization process Not FDA cleared

(Endoclens)
Rapid readout ethylene oxide Not FDA cleared
biological indicator (Attest)
New plasma sterilizer FDA cleared, January 1999
(Sterrad 50)

71 Sterilization
 1. Ortho-Phthalaldehyde (OPA)
• Clear, pale-blue liquid (pH 7.5), which typically contains
0.55% OPA
• Chemical Sterilizer
• Advantages:
– not eye and nasal passages irritant
– has excellent stability over a wide range of pH (3 to 9)
– has a barely perceptible odor
– Like glutaric aldehyde, it has excellent material
compatibility
• Disadvantage:
– It stains proteins gray (including unprotected skin)
72 Sterilization
 OPA vs. Glutaric Aldehyde
Table 2. Activity of glutaraldehyde and ortho-phthalaldehyde
against Mycobacterium bovis

Disinfectant Time for 6-log10 reductiona

1.5% glutaraldehyde 28-36 minutes
2.5% glutaraldehyde 14-18 minutes
0.21% ortho-phthalaldehyde 4.8-6.3 minutes

aRange of values from two different laboratories (4).

73 Sterilization
 2. Surfacine
• Surface disinfectants such as phenols
• Advantages
– Effective in significantly reducing
microbial contamination
– Cost-effective
– Have long-term residual activity

74 Sterilization
 3. Superoxidized Water
• Based on the concept of electrolyzing
saline.
• Advantages:
– Cheap
– The end product (water) is not
damaging to the environment
• Available in the United Kingdom; Sterilox

75 Sterilization
 4. Endoclens
• A New rapid automated liquid Chemical Sterilization
System.
• The sterilant is produced, as needed, by automatic
mixing of solutions of hydrogen peroxide and formic acid.
• Advantages:
– automatic cleaning process
– capability to process two flexible scopes at same
time
– automated channel blockage and leak detection
– filter water rinsing and scope drying after sterilization
– hard-copy documentation of key process parameters
– user-friendly machine interface
– total cycle time less than 30 minutes
76 Sterilization
 5. Hydrogen Peroxide Plasma
• A New Low-Temperature Sterilization
Technique.
• Can be used for temperature-sensitive
equipment.

77 Sterilization
 6. Low-temperature plasma
• Technique for decontaminating thermolabile
products without the severe drawbacks of gas
sterilization methods.
• Advantages:
– Alternative to steam sterilization for sterilizing
easily corroding products or electronic
instruments
– Takes less time.
• However, Steam sterilization proved to be the
cheapest method of sterilizing

78 Sterilization
 Sterilization & Disinfection
End

79 Sterilization