Sterilization

Objectives
Understand and utilize correct sterilization

and disinfection techniques Distinguish between sterilization and disinfection Discuss the time/temperature relationship in destroying microorganisms

 Microorganisms

Invisible Cause infections Destruction of all microorganisms

Destruction of harmful organisms, or pathogens on nonliving objects

Sterilization

Disinfectant

 Disinfectants

Types of disinfectants

Bactericides Fungicides Germicides Virucides

All prevent transfer of infection

Sterilization kinetics
symbo term l N0 bioburden definition The population or no. of living microorganism per defined unit, surface, or system The no. of degrees(c or F) required for a 1 log reduction in the D value. The equivalent time at a temp of T delivered to unit of product calculate using a specific value of z. The equivalent time at a temp of 1210 delivered to unit of product calculate using a z value of 10oc The no. of non-sterile unit per batch or a theoretic or extrapolated no. of living microorganism per define unit after a given heating time u at a specific temp. T

Resistance value

F(T,z)

Sterilization process equivalent time Sterilization process equivalent time

F0

Nu

Probability of non sterility

D value
The D value is the time (for heat or chemical

exposure) or dose (for radiation exposure) required for the microbial population to decline by one decimal point(a 90% or one logarithmic unit).

D=

Where

U log N0 log Nu U=exposure time N0=Initial microbial papulation Nu=microbial population after exposing time u

 Example, After 5 min of product exposure to temp. of 121oc

the microbial population was reduce from 2x105 to 6x103 then D value at 121oc is
D121=

5 min =3.28 min log(2x105)-log(6x103)

thus at 121oc the microbial population is

decreased by 90% every 3.28 min

Z value (Resistance value)

The no. of degrees(c or F) required for a 1 log reduction in the D value Z value plots of log D verses temp.

100

10 D value min 1

0.1

0.01

110

130

150

170

190

210

230

250

temperature(0C)

Sterilizing Agents
1) Physical Methods of sterilization A) Heat sterilization
Moist heat Dry heat

B) Radiation C) Filtration 2) Chemical methods of sterilization Gaseous sterlzation

1) Heat sterilization
Typical temp profile of heat sterilization process

affording its antimicrobial activity through destruction of

enzymes and other essential cell constituents

Time/Temperature Relationship
Most important factor in destroying

microorganisms is length of exposure to heat Varies with microorganisms The higher the temp the faster it will kill microorganisms exposed Temps lower than boiling point can sterilize some medications and milk

A) Moist heat sterilization

In the pharmaceutical and medical sphere it is used in

the sterilization of dressings, sheets, surgical and diagnostic equipment, containers and closures, and aqueous injections, ophthalmic preparations and irrigation fluids, in addition to the processing of soiled and contaminated items Sterilization by moist heat usually involves the use of steam at temperatures in the range 121134C. Effect of moisture and heat
Water(%) 50 Temp.(oc) 56 effect coagulation

25 6
0

80 145
170

coagulation coagulation
Coagulation and oxidation

Temp.-pressure relationship and antimicrobial efficacies of alternative steam sterilization methods

Temp.(c0

Holding time (min) 30 15 10 3

Steam pressure kpa 69 103 138 207

Inactivation factor 5 10 21 40

115 121 126 134

Steam and boiling water Alone not effective Captured steam effective Autoclave Sealed chamber that furnished both hear and pressurized steam for sterilization Inexpensive

B) Dry heat sterlization

Dry heat

Slow sterilizer Higher temperatures Penetrates

Oil-based materials Closed containers

 The lethal effects of dry heat on microorganisms

are due largely to oxidative processes which are less effective than the hydrolytic damage which results from exposure to steam. The dry heat sterilization usually employs higher temperatures in the range 160180C and requires exposure times of up to 2 hours depending upon the temperature employed

 Direct flame

Can damage the exposed object

Other Sterilization Technology

Filtration

Ultraviolet irradiation
Cold sterilization Ethylene oxide

C) Gaseous sterilization
The chemically reactive gases ethylene oxide

(CH2)2O and formaldehyde (methanal, H.CHO) possess broad-spectrum biocidal activity, and have found application in the sterilization of reusable instruments, machines, certain medical, diagnostic and electrical equipment, and the surface sterilization of powders The mechanism of antimicrobial action of the two gases is assumed to be through alkylation of sulphydryl, amino, hydroxyl and carboxyl groups on proteins and imino groups of nucleic acids.

 Concentration ranges (given as weight of gas per unit

chamber volume) are usually in the order of 800 1200mg/L for ethylene oxide and 15100 mg/L for formaldehyde, with operating temperatures in the region of 4563C and 7075C, respectively. both gases are potentially mutagenic and carcinogenic, they also produce symptoms of acute toxicity including irritation of the skin, conjunctiva and nasal mucosa. Ethylene oxide gas is highly explosive in mixtures of >3.6% v/v in air; in order to reduce this explosion hazard it is usually supplied for sterilization purposes as a 10% mix with carbon dioxide, or as an 8.6% mixture with HFC 124 (2-chloro1,1,1,tetrafluoroethane)

 This requires a sterilizer humidity of 3070% and

frequently a preconditioning of the load at relative humidity's of >50%.

 Filtration

Complete removal of microorganisms and particles of a certain size from liquid or gas Used by vet personnel in producing sterile and particle-free fluids, such as intravenous fluids

c) Radiation
Accelerated Electrons, Gamma-rays and UV light

(both electromagnetic radiations). The major target for these radiations is believed to be microbial DNA, with damage occur ring as a consequence of ionization and free radical production (gamma-rays and electrons) or excitation (UV light). Gamma-ray sterilizers Gamma-rays for sterilization are usually derived from a cobalt-60 (60Co) source (caesium-137 may also be used), with a half-life of 5.25 years, which on disintegration emits radiation at two energy levels of 1.33 and 1.17MeV

 Ultraviolet irradiation The optimum wavelength for UV sterilization is

around 260nm. A suitable source for UV light in this region is a mercury lamp giving peak emission levels at 254 nm.

C) Filtration sterilization
The process of filtration is unique among

sterilization techniques in that it removes, rather than destroys, microorganisms The major mechanisms of filtration are sieving, adsorption and trapping within the matrix of the filter material synthetic membrane filters, derived from cellulose esters or other polymeric materials, approximate most closely to sieve filters. while fibrous pads, sintered glass and sintered ceramic products can be regarded as depth filters relying principally

 Filtration sterilization of liquids

For this reason, membrane filters of 0.20.22mm

nominal pore diameter are chiefly used, while sintered filters are used only in the processing of corrosive liquids, viscous fluids or organic solvents Membrane filters, in the form of discs, can be assembled into pressure-operated filter holders for syringe mounting and in-line use or vacuum filtration tower devices. Filtration sterilization of gases These highefficiency particulate air (HEPA) filters can remove up to 99.997% of particles >0.3mm in diameter and thus are acting as depth filters.

New sterilization technologies

1. High-intensity light UV light has the potential to kill all types of microorganisms,

but its penetrating power is so poor that it has found practical application only in the decontamination of air (e.g. in laminar-flow workstations and operating theatres), shallow layers of water and surfaces. 2. Low temperature plasma Plasma is a gas or vapor that has been subjected to an electrical or magnetic field which causes a substantial proportion of the molecules to become ionized. they composed of a cloud of neutral species, ions and electrons in which the numbers of positive and negatively charged particles are equal.

 Plasmas is generated from chlorine,

glutaraldehyde and hydrogen peroxide have been shown to possess the greatest antimicrobial activity. The cycle times are typically from 60 to 90 minutes and the operating temperatures are <50C. It cannot be used to sterilize liquids and powders

Sterilization control and sterility assurance

Bioburden determinations

The term bio burden is used to describe the concentration

of microorganisms in a material; this may be a total number of organisms per milliliter or per gram,
Environmental monitoring The levels of microbial contamination in the manufacturing

areas are monitored on a regular basis to confirm that the numbers do not exceed specified limits.

 Ultraviolet irradiation

Destroys microorganisms in air, liquid, and surface Germicidal UV radiation is generated by passing electricity through mercury vapor in special glass tubes UV lamps sterilize most effectively in still air at room temperature

 Cold sterilization

Gamma radiation Kill microorganisms without a rise in temperature Highly successful in sterilizing

Syringes Stitching (sutures) materials Containers