presentation for students in medical school: general principles of the most important sterilisation methods applicable in microbiology labs and in healthcare facilities

1. Sterilization and Disinfection
(control of microorgansisms by
nonselective methods)

2. Contents
• Definition of terms
• Methods
• Equipments and agents
• Applicability in medicine and other fields
• How to choose the most appropriate
methods/agents

3. Definition of terms
• Sterilization = process that eliminates or kills
What? - all forms of life including microbial agents:
– bacteria (vegetative forms and spores),
– viruses,
– fungi
Where? - on surfaces, in fluids, in medication, in culture media,
How? - by use of heat, chemical agents, irradiation, vacuum & high
pressure, filtration or combinations thereof.

4. Definition of terms (II)
• Disinfection = cleansing techniques that:
– destroy or
– decrease the number or
– remove or
– prevent the growth
of infectious microorganisms on surfaces which cannot be
subjected to sterilization i.e. furniture, rooms, skin, water in
swimming pools

5. Sterilization Methods
I. Physical Methods & agents:
A. Heat ± vacuum ± high pressure
a. Dry (red heat, flame, heat in vacuum – Poupinel oven)
b. Moist (boiling, autoclavation, pasteurization, tyndallization)
B. Filtration
C. Irradiation
a. UV light,
b. ionizing radiation: gamma, electron, X-ray
I. Chemical Methods & agents:
A. Ethylene oxide
B. Formaldehide

6. Sterilization Methods (II)
I.A.a. Dry Heat
Flame sterilization: Bunsen burners - small gas burners with an
adjustable flame, manipulated at the base by controlling the amount
of gas and air admitted

7. Sterilization Methods (III)
I.A.a. Dry Heat (continued) - Sterilization ovens – dry heat + vacuum

8. Sterilization Methods (IV)
Sterilization ovens (Poupinel) (Continued)
- e.g. 180°C, 1 hour → destruction of all microorganisms incl. spores
- Components:
- Sterilization chamber
- Electrical heating system
- Thermal insulation (azbestos)
- Thermometer + thermostate
- Fan (mechanical air convection)

9. Sterilization Methods (IV)
Sterilization ovens (Poupinel) (Continued)
- Pre-sterilization procedures:
- Objects must be thoroughly cleaned, dried, packaged/wrapped
- Packaging: petri dishes, test tubes, small jars, stainless steel
trays or pans with lids + cotton / paper wrappers or aluminum foil
may be used, if the temperature does not exceed 204°C
- overloading must be avoided !

10. Sterilization Methods (IV)
- Sterilization ovens (Poupinel) (Continued)
Advantages:
- Usable for items that cannot be sterilized by moist heat or chemical
methods e.g. powdres, oils, items prone to rust
- Usable for equipments with many parts that cannot be
disassembled
- Non-corrosive
Disadvantages:
- Slow and uneven penetration → longer time required
- Less effective than hot steam sterilization (autoclave) – see below
- Higher temperatures required → harmful to some materials, special
packaging materials

11. Sterilization Methods (V)
- Sterilization ovens (Poupinel) (Continued)
Suitable for:
- laboratory glassware, ceramics, surgical instruments (stainless
steel), powders, olis
Not suitable for:
- watery solutions, rubber items, cotton, biological waste (materials
contaminated with biological fluids e.g. cotton swabs, latex gloves,
laboratory gowns)

12. Sterilization Methods (VI)
- Sterilization ovens (Poupinel) (Continued)
Control methods:
– indicators for reaching the high temperatures:
- white paper used for packaging must change its colour to yellowish /
brown due to caramelization of sugars (e.g. sacharosis is caramelized
at 170°C)
- Coton caps used to close tubes, flasks appear dark brown (carbonized)
- paper strips impregnated in non toxigenic strains of Clostridium
tetani subjected to the sterilization process and checked for
bacterial growth

13. Sterilization Methods (VI)
I.A.b. Moist heat – Boiling
- 100°C, 30 minutes → all vegetative forms of bacteria are killed (BUT
spores survive)
- Used in emergencies, when no other method is available e.g.
medical emergencies in low resource settings, natural disasters
(floods, earthquakes) causing collapse of power supply
- Boiling water during epidemics of water borne diseases

14. Sterilization Methods (VI)
I.A.b. Moist heat - Autoclave sterilization:
moist heat / steam + vacuum + high presure

15. Sterilization Methods (VI)
Autoclave sterilization (continued)
e.g. 121°C, 2 atm, 30 min (variations depending on size of objects,
thickness of walls, etc)
Components:
- metal container, resistant to high pressures, heavy lid, sealed
closure (rubber or silicone)
- evacuation valve for air (upon start of sterilization cycle) and steam
(at the end of the sterilization cycle)
- safety valves, pressure gauges, pressure regulator
- manometer: pressure correlated to temperature i.e. 2.03
atmospheres - 121°C; 2.65 atmospheres - 130°C
- thermometer

16. Sterilization Methods (VIII)
Autoclave sterilization (continued)
Advantages:
- More rapid and even penetration
- better bactericidal/virulicidal effect (denaturation and coagulation of
proteins)
Disadvantages:
- May corrode or cause rusting of some instruments
...

17. Sterilization Methods (VIII)
Autoclave sterilization (continued) - Control methods:
Chemical tests: based on the melting point of certain substances e.g.
120°C for benzoic acid
Bands of filter paper impregnated with thermochromic substances
- colour change indicates the required temperature has been
reached
(no indicator on how much time was the temperature maintained)
Biological tests: cotton fibers impregnated with spores e.g.
B.subtilis/B.stearothermophylus subjected to autoclavation and
then inoculated in culture media – lack of bacterial growth means
the sterilization has been effective

18. Sterilization Methods (VIII)
Autoclave sterilization (continued)
Suitable for:
- biological waste (hospitals, laboratories)
- Culture media
- Oral/nasal swabs
- Items made of autoclavable rubber (caps, tubes)
- Cotton items (laboratory gowns, surgical cloth)

19. Sterilization Methods (VIII)
I.A.b. Moist heat – Pasteurization
Louis Pasteur (1822-1895)
-The ”Germ Theory of Diseases”
- changes in medical practices to
decrease the spread of germs
-”parent” of vaccination
- concept of ”virus”

20. Sterilization Methods (VIII)
I.A.b. Moist heat – Pasteurization
- NOT a ”true” sterilization: it does not ”kill all vegetative forms of
microorganisms and spores” BUT aims to reduce the number of
viable microbes so they are unlikely to cause disease
- The method: heating followed by swift cooling and packaging
- High pasteurization: 85-90°C, several seconds
- Medium pasteurization: 70-75°C, 20 minutes
- LOw pasteurization: 60-65°C, 1 hour
- Used for food conservation (commercial-scale sterilization of food is
not common because it adversely affects the taste).

21. Sterilization Methods (VIII)
I.A.b. Moist heat – Tyndallization
John Tyndall (1820 – 1893)
- Prominent English physicist
- Extremely valuable studies in the
field of radiant heat

22. Sterilization Methods (VIII)
I.A.b. Moist heat – Tyndallization (continued)
The method: staged heating (boiling) for 15 minutes/day during 3 days
Day 1: after the 15 minutes of boiling all vegetative forms of bacteria
are killed BUT the spores survive
Day 2: some of the surviving spores, in favourable conditions
(moisture, warmth) have turned into vegetative forms; the second
round of boiling kills them
Day 3: late germinating spores have survived the second stage of
boiling as they were still in a sporulated condition but upon the 3rd
boiling round they have already generated vegetative bacteria which
will be killed by the 3rd boiling round
Used for items that cannot withstand autoclavation e.g. plant seeds

23. Sterilization Methods
I. Physical Methods & agents:
A. Heat ± vacuum ± high pressure
a. Dry (red heat, flame, heat in vacuum – Poupinel oven)
b. Moist (boiling, autoclavation, pasteurization, tyndallization)
B. Filtration
C. Irradiation
a. UV light,
b. ionizing radiation: gamma, electron, X-ray
I. Chemical Methods & agents:
A. Ethylene oxide
B. Formaldehide

24. Sterilization Methods
I.B. Filtration
- Mechanical retention of microorganisms from fluids by
passing them through special membrane filters with
adequate pore size
- Used for fluids which would be damaged by heat or other
sterilization agents (radiations, chemicals) e.g. heat
labile pharmaceutic substances, wine, beer, etc.
- microfiltration, pore size 0.2 μm for bacteria
- nanofiltration, pore size 20-50 nm for viruses
The smaller the pore size the higher the retaining
effectiveness.

25. Sterilization Methods
I. Physical Methods & agents:
A. Heat ± vacuum ± high pressure
a. Dry (red heat, flame, heat in vacuum – Poupinel oven)
b. Moist (boiling, autoclavation, pasteurization, tyndallization)
B. Filtration
C. Irradiation
a. UV light,
b. ionizing radiation: gamma, electron beam, X-ray
I. Chemical Methods & agents:
A. Ethylene oxide
B. Formaldehide

26. Sterilization Methods
I.C.a. UV light
Bactericidal UV lamps – 2537 Å
wavelength
- Kill bacteria, viruses, fungi in
the air by altering their nucleic
acids
- Spores are not killed
- Used to sterilize air in surgical
rooms, on surfaces e.g.
laboratory biosafety cabinets

27. Sterilization Methods
I.C.b. Ionizing radiations
• Gamma irradiation: Co-60 – industrial sterilization of disposable,
single use, medical devices e.g. syringes, pipette tips, surgical
wires, implants, etc.; causes microbial death as a direct effect of the
destruction of a vital molecule or by an indirect chemical reaction.
• Electron beam (e-beam sterilization) – industrial sterilization of
disposable medical devices; same mechanism and dose as in
gamma irradiation; advanced electronics precisely control the use of
electron beams in the sterilization of medical devices; less material
degradation than with gamma irradiation.
• X-ray – alternative for sterilizing large packages and pallet loads of
medical devices; no toxic residues; no high temperature rises →
more suitable for plastics

28. Sterilization Methods
I. Physical Methods & agents:
A. Heat ± vacuum ± high pressure
a. Dry (red heat, flame, heat in vacuum – Poupinel oven)
b. Moist (boiling, autoclavation, pasteurization, tyndallization)
B. Filtration
C. Irradiation
a. UV light,
b. ionizing radiation: gamma, electron beam, X-ray
I. Chemical Methods & agents:
A. Ethylene oxide
B. Formaldehide

29. Chemical Sterilization Methods
II.A. Ethylene oxide (CH2OCH2)
- bacteria killed under the action of highly active oxygene at relatively
low temperatures (20-60°C)
- Suitable for medical devices that cannot be subjected to high
temperatures, moisture or abrasive chemicals e.g. Electronics,
optical equipment, paper, non-autoclavable rubber and plastics
- Highly toxic, irritant, explosive – can only be used in strictly
controled conditions

30. Chemical Sterilization Methods
II.B. Formaldehide - liquid sterilizing agent
- by immersion, provided that the immersion time is sufficiently
long e.g. over 24 hours to kill all spores in a clear liquid
- by spraying e.g. closed rooms
- highly volatile, and toxic by both skin contact and inhalation
- Many vaccines, such as the original Salk polio vaccine, are
sterilized with formaldehyd.

31. Definition of terms
• Disinfection = cleansing techniques that:
– destroy or
– decrease the number or
– remove or
– prevent the growth
of infectious microorganisms on surfaces which cannot be
subjected to sterilization i.e. furniture, rooms, skin, water in
swimming pools
See further presentation on ”Antiseptics & Disinfectants”