Sterilization and Disinfection(control of microorgansisms by

nonselective methods)

Contents

• Definition of terms

• Methods

• Equipments and agents

• Applicability in medicine and other fields

• How to choose the most appropriate methods/agents

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.

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

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

II. Chemical Methods & agents:A. Ethylene oxide

B. Formaldehide

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

Sterilization Methods (III)

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

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)

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 !

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

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)

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

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

Sterilization Methods (VI)

I.A.b. Moist heat - Autoclave sterilization:

moist heat / steam + vacuum + high presure

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

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

...

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

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)

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”

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). 

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

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

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

II. Chemical Methods & agents:A. Ethylene oxide

B. Formaldehide

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.

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

II. Chemical Methods & agents:A. Ethylene oxide

B. Formaldehide

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

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

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

II. Chemical Methods & agents:A. Ethylene oxide

B. Formaldehide

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

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.

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”