COLD & AIR STERILIZATION

Prepared by : Microbiology and Immunology

Department

Objectives of the Lecture 1. Cold Sterilization :

I.  Gaseous Sterilization: §  Ethylene oxide. § Formaldehyde.

II. Radiation Sterilization: §  Ionizing Radiation (Gamma Rays, β-particles). §  Ultraviolet Radiation.

III. Sterilization by Filtration. 2. Sterilization by Air:

n  Sterile Products. n  Clean and Aseptic areas. n  Microbial Analysis of Air. n  Validation of Aseptic Area.

- Chemically reactive gases ethylene oxide, B-propiolactone, propylene oxide and formaldehyde possess broad-spectrum biocidal activity.

- Applied in the sterilization of reusable surgical instruments, certain medical and diagnostic equipment, and the surface sterilization of powders.

- Ethylene oxide is considered an alternative to radiation sterilization in the commercial production of disposable medical devices. -Generally reserved for temp-sensitive items.

Mode of action: Alkylation of sulphhydryl, amino, hydroxyl and carboxyl groups of proteins and imino groups of nucleic acids.

1. COLD STERILIZATION A- GASEOUS STERILIZATION

1. Ethylene oxide

- Gas at normal atmospheric pressure (boiling point 10.5oC).

- Inflammable and explosive when mixed with air in conc. > 3.5%v/v.

- Either mixed as 10% with CO2 , and kept in heavy cylinders but is cheap, or mixed as 12% with freon, and kept in light cylinders but is expensive.

- Toxic as ammonia.

- Mutagenic and carcinogenic.

q Suitable for plastics , all antibiotics and hormones (except if it react with ethylene oxide), surgical dressing and suture and thermostable powders in bulk.

q Not suitable for serum and vaccines, and aqueous solutions.

2. Formaldehyde Is used in the same way as (low temp steam and formaldehyde (LTSF)).

Factors Affecting Efficacy of Gaseous Sterilization 1- Conc.: 800 - 1200 mg/liter for ethylene oxide

and 15 - 100 mg/liter for formaldehyde of the sterilizer volume.

2- Temp: 45 - 63oC for ethylene oxide and 70 -

75oC for formaldehyde. 3- Relative humidity: 30 -60%, below 30%

activity decreases due to the decrease in alkylating activity, and above 60% due to formation of ethylene glycol with water which is completely inactive.

4- Penetration: does not penetrate glass, metals

and crystals, However, it penetrates fabrics (dressing) and paper.

Gas autoclave

B- RADIATION STERILIZATION I- Ionizing radiation

Mode of action of ionizing radiation: Ionizing radiation raises the energy level of atoms sufficient to release

electrons resulting in ionization. They act in two possible effects: a) Target effect: each cell has certain gene which is sensitive to

radiation, its ionization or splitting results in death of the cell. b) Indirect effect: water absorbs energy resulting in release of

electrons which are absorbed by another Mol of water and chain reaction of free radicals is formed.

-e-→ H2O+ → H+ + OH .

H2O + e-→ H2O

- → OH -+H. + O2 → HOO. → H2O2 + O2

Strong oxidizing agents such as H2O2, HOO., OH. and nascent oxygen formed are powerful biocidal agents.

The indirect effect of ionizing radiation is sometimes damaging to pharmaceuticals and so may be minimized by: 1- Absence of oxygen. 2- Frozen state to immobilize free radicals. 3- Presence of quenching agents such as cysteine and thiourea.

Resistance of endospores to radiation is due to their high content of disulfide bonds.

A- Gamma rays • Generated from radioactive cobalt (Co60). • It has a half-life time of 5.25 years with energy level bet. 1.17-1.33

MeV. • Highly penetrable, weakly absorbed, needs longer time

(suitable for deep articles). B- Accelerated or high speed electrons (β-particles) • Electrons are generated by passing electricity in certain metals e.g.

mercury lamp. • Electrons are accelerated by passing in magnetic field. • Radiation dose is 2.5 million rad (25 kilo Gray). “Rad = amount of radiation that delivers 100 erg per gram absorbing

material”. • Low penetration power, strongly absorbed, needs shorter time

(suitable for thin packages)

Application of Ionizing radiations

• Could be used for some vaccines as it does not affect

antigenicity.

• Used for the same materials as ethylene oxide, The choice

depends on:

1- Availability of equipment

2- Cost of sterilization

3- Damage or degradation caused by radiation.

II-Ultraviolet

•  Cause only excitation but not ionization.

•  This results in interatomic vibration with breakage and

formation of new bonds and so depolymerization of DNA and

denaturation of proteins.

•  UV of 265 nm is absorbed by DNA and results in formation of

thymine dimer so it is very effective.

•  UV 280 nm is absorbed by aromatic amino acids.

•  UV 220 nm converts oxygen into ozone (strong oxidizing

agent).

1. Used mainly to maintain sterility of air.

2. Used for sterilization of purified water

C- STERILIZATION BY FILTRATION Used for sterilization of thermolabile liquids or solutions and

air or gases. Not used for suspensions. q Advantages: 1- It is unique among sterilization techniques in that it removes

rather than destroys microorganisms i.e. both living and dead cells are removed.

2- It does not affect the physical or the chemical integrity of the sterilized material.

3- Have an important role in sterility testing.

q  Disadvantages: 1- The product must be filled and packed aseptically in sterile area. All other methods, sterilization is carried in the final sealed containers. 2- Bacterial toxins are not removed. 3- The technique requires highly trained staff.

Types of filters A- Depth filters: -Thick sponge filters, retaining or trapping M.O. within the filter matrix. -  Consists mainly of material and minimally of pores or holes. -  Made of sintered glass or ceramic (granular) or asbestos or cellulosic

fibers (fibrous). B- Screen filters: -  Very thin films , retaining M.O. on the surface of the filter. -  Consists mainly of holes and minimally of material and are either: 1- Membrane filters: - Thin film of cellulose ester having uniform pore size. -  Are the most commonly used type of filters nowadays. -  Only 0.2 um pore size membrane filter is used in sterilization. 2- Nucleopore filters: -  Very thin films of plastic exposed to protons, very small holes are

produced. -  Holes are not uniform in size, as two or more protons may form one hole.

Class A. Depth Filters B. Screen Filter

Type 1. Ceramic filters 2. Glass filters 3. Fibrous filters 1. Membrane filters

Made of porous porcelain sintered glass compressed

asbestos

cellulose acetate or

nitrate

Found as Cylindrical candles

with thick wall

disc fused into

Buchner

funnel

thick discs or

sheets supported

into special holder

thin discs fitted

into special

holder

Sterilized by Dry heat or

autoclaving

dry heat or

autoclaving

autoclaving ethylene oxide or

autoclaving

Cleaned by Strong hypochlorite Strong acid and

water

- -

Advantages Easy fitted into

holder

Rapid filtration rate

inert

no adsorption to

drugs

disposable

rapid filtration

rate

disposable

Disadvantages easily broken,

rapidly clogged,

adsorb drugs

easily broken,

slow filtration

adsorb drugs

impart fibers and

alkali to filtrate

slow filtration

Types of Filters

A. Depth Filters

1. Ceramic filters

2. Glass Filters

3. Fibrous Filters

A. Depth Filters

B. Screen Filters

Membrane Filters

Filtration: -Used for sterilization with efficiency reaching 99.99%. -ALWAYS uses a series of pre-filters and filters -Depends on the use of several types of filters: 1- Cotton or glass wool fibrous pads filters. 2- Cellulose-paper filters. 3- Cartridge of multitube depth and membrane filters. 4- High efficiency particulate air (HEPA) filters.

Types 1 & 2 are used as prefilters while HEPA filters are the

most commonly used type of filters: It is a special type of membrane filter allowing the filtration of air in

a slow rate, filtered air is passed slowly in parallel lines without turbulence in a laminar flow system.

2. Sterilization of Air

Sterile Products q  Terminally Sterilized: Product sealed in its container and then sterilized. Produced in a clean area. q  Not Terminally Sterilized: Product Prepared aseptically from previously sterilized materials or by sterilized filtration then filled in sterile containers. Strict aseptic conditions are required. Produced in aseptic area.

Clean and Aseptic Areas Sterile production should be carried out in a special unit separate from other manufacturing areas. The unit should be designed to encourage separation of each stage of production but should ensure a safe and organize workflow.

General area Non-sterile

Class C

Class A

Class D

Cle

an/A

sept

ic a

rea

Cle

an/A

sept

ic a

rea

Cla

ss B

Class B

Cla

ss B

Class B

Aseptic area

Features of Clean and Aseptic Areas: 1- Kept under slight positive pressure. 2- Smooth walls, ceiling and floor to prevent accumulation of particulates and for

ease of disinfection. 3- Windows are double glass, the outer one can be opened for cleaning. 4- Doors are double with air lock. 5- Furniture and equipment kept minimum and simple. 6- Air supply should be sterile passed through HEPA filters situated near the

ceiling. 7- UV irradiation to maintain sterility when the room is out of work. 8- Persons or operators should be free from infections and must wear sterile

protective cloth (overhead, face mask, gloves and overshoes). 9- All objects supplied to the area should be sterile

10- Air must be routinely analyzed for viable cells and contamination should be traced.

11- Entrance should be through a changing room, with interlocking doors, step-over sills.

-General area: outside the classified zones. -Movement from the general area to and then through these rooms is through strict protocols.

HEPA FILTER

Protective clothing is required

Laminar flow cabinet •  Small cabinet supplied with sterile

air (horizontal or vertical) through HEPA filter in parallel manner to avoid turbulence currents. They are efficient and less expensive.

Microbial analysis of air 1- Settling plates: -Petri dishes containing nutritive agar are opened and left for time. Plates are then incubated at suitable temp and colonies are counted.

- This method is slow and fail to collect very small particles and does not indicate the volume of air tested. 2- Air sampler: -By the use of agar impinges, where controlled volume of air is forced to pass through a slit, below which small nutrient agar plate or slide is placed.

-Plate or slide is then incubated and colonies are counted.

Settling Plates

Slit-to-agar impactor sampler

q System suitability test (SST):

Carried by testing for M.O. in everything including air,

machines, persons, walls and floors by swabbing.

q Validation of HEPA filter:

Force dioctylphthalate (DOP) vapor or recently certain type of

oil through the filter by a fan and test for it on the other side.

Validation of Aseptic Area