STERILIZATION BY HEAT

Prepared by : Microbiology and Immunology

Department

Objectives of the Lecture 1. Sterilization by Heat:

§ Mode of Action. § Heat Transfer . §  Sterilization Process.

A. Dry Heat Sterilization : n  Methods Used (Incineration, Hot air oven, Infra red tunnel). n  Advantages and Disadvantages.

B. Moist Heat Sterilization : n  Pressure – Temperature Curve. n  Saturated Steam (Wet, Dry) . n  Advantages of using Saturated Steam. n  Methods used (Normal and High pressure).

STERILIZATION BY HEAT Each microorganism (M.O.) has an optimum temp for growth , below it growth decreases reversibly and above it decreases irreversibly due to degradation of vital molecules. The equation describing the effect of heat:

log t = 0.219 E + K T t = extinction time T = absolute temp (T , t ) K = constant depends on the No. and type of the most resistant M.O. E = heat of activation energy required to kill the most resistant M.O.

(depends on the mode of action).

•  Plotting (Log t) against T a straight line is observed i.e. the extinction time is inversely proportional to the temp.

•  Any small increase in temp greatly reduces the time required for

sterilization. Log  t  

T  

log t = 0.219 E + K T

Mode of action of heat 1) Moist heat (in presence of water): Death is through protein and

nucleic acid denaturation due to coagulation, in other terms, loss of tertiary structure of proteins, also small peptide chains and compounds with free SH groups are released. It depends on the presence of water for its high dipole moment (internal energy).

This reaction requires low E , and shorter t 2) Dry heat (in absence of water): Death is due to slow protein denaturation through oxidation of certain groups in the protein molecules.

This reaction requires high E, and longer t.

At constant temp bacteria are killed by moist heat in shorter time than dry heat.

Resistance of endospores to heat: Bacterial spores are more resistant to heat than vegetative cells. The

resistance is due to the fact that spores contains the least amount of water (10% unbound water) , also the spore wall contains calcium and dipicolinic acid which prevent the penetration of moisture but doesn't prevent the penetration of heat, also SH groups of proteins are protected through the formation of disulphide bond.

Factors affecting heat sterilization:

q  Time and temperature of exposure;

q  No. of M.O. (Bioburden) ;

q  Type of M.O. (species, strain, spore formation);

q  Nature of the article to be sterilized.

Heat Transfer

- Direct: e.g. incineration or flaming, only used for highly infective materials e.g. loop.

- Indirect: through medium: e.g. air, water (steam) or metals. It is either through: 1- Radiation: using infra red (I.R.) source or when the articles are

placed near the heating sources. 2- Conduction: usually through metals. It is slow and need intimate

contact between the metal and both heat source and the article to be sterilized.

3- Convection: through air, water or oil, by currents of heat.

Sterilization Process by Heat Sterilization by heat proceeds through three successive periods: A- Heating up period: time required for the temp to reach that required

for sterilization. It requires high quantity of activation energy. It depends on the type of the article to be sterilized.

B- Holding period: time during which the temp of the object is kept at that required for sterilization. It requires small amount of activation energy. It also depends on the type of the article to be sterilized.

C- Cooling down period: time during which temp decreases to that of ambient (room temp).

Suitable for thermostable substances -  Glass (vials, ampoules, flasks, pipettes, Petri dishes....etc.) -  Metals (forceps, scissors, collapsible tubes,...etc.), -  Anhydrous oils (paraffin, wax, ointment base...etc.), -  Powders (talc, koalin), -  Others:porcelain, silicon rubber, paraffin gauze, and cat gut. Not suitable for water containing substances, thermolabile substances,

all fibers (dressing), plastics, and non-silicon rubber. Conditions

B.P recommends 150oC for 60 min. for medicaments and 160oC for 60 min. for glass

•  U.S.P. recommends 160oC for 60 min. for medicaments and 170oC for 120 min. for glass

•  Heating at lower temp for prolonged time is more damaging than rapid heating at higher temp for the articles to be sterilized.

Dry Heat Sterilization

Methods of dry heat sterilization

1) Incineration

-Flaming directly or after swabbing with alcohol. -Used for highly infectious materials as bacteriological loops, spatula and scissors (thermos table materials).

2) Hot Air oven (sterilizer) -  Metal chamber, double walled with insulators, and perforated shelves. - Continuous heating is by the help of circulating air by a fan.

-  Heat transfer is mainly by convection( air fan), conduction and radiation play small role. - Items should not be placed compact or over perforations to allow circulation of air.

- Items, which resist heat transfer (talc powder), should be placed in shallow layers. - Items which allow convection of heat (oils) could be sterilized in bulk.

- Articles to be sterilized should be introduced into the oven at room temp and after sterilization, the oven is allowed to cool to about 40oC before opening the door to avoid cold shock and cracking of glass. 

3) Infra red tunnel (I.R.) -I.R. are thermal radiation that are converted to heat upon absorption, The rate of heating is very fast thus the heating up period is very short. -Heat transfer is mainly by radiation to the outer surfaces of objects then by conduction and convection. -Used for continuous sterilization of ampoules, vials, syringes (hospitals) and some oils. -Products are placed in one layer only. Treatment is at 180⁰C for 20 min.

Advantages of dry heat: 1) less damaging to metals and glass. 2) Articles remain dry. 3) Used for substances damaged by moisture e.g. powders

and oils.

Disadvantages of dry heat: 1) Drastic conditions (high temp, long exposure, prolonged

heating period), not suitable for thermolabile substances. 2) Not suitable for surgical dressing as they are poor conductors

to heat so their outside overheats before reaching sterilization temperature.

Moist Heat Sterilization

Water is an excellent medium for heat transfer and steam is much more better.

•  At normal atmospheric pressure one gm of water requires 100

calories to raise its temp from 1oC to 100oC, these calories are called sensible heat.

•  Each gram also requires 560 calories to convert it from water at 100oC to steam at 100oC, these calories are called latent heat.

Temperature- Pressure curve: •  Water boil at 100oC at normal atmospheric pressure. •  Heating water in closed vessel (autoclave) will increase the pressure

inside due to the generated steam and so the boiling temp will increase and saturated steam is formed all points on the boundary phase.

•  Pressure is used to increase temperature, so that the time of

exposure is reduced and product is preserved from destruction.

Saturated steam is either: a) Wet steam : carry suspended water droplets. b) Dry steam: free from water droplets.

Liquid  water  

Superheated  steam  

•  If heating is continued i.e. temp increases while pressure remains almost constant and water dries, saturated steam is converted into superheated steam (all points above the boundary).

•  Superheated steam is not suitable for moist heat sterilization as it acts only as source for heat but not source of moisture.

•  Steam in contact with water never become superheated.

Liquid  water  

Superheated  steam  Temp

increase

-Adulteration with air contributes only to a partial pressure but not to the temp of the steam. Thus, in presence of air the temp achieved will be lower than that normally attributed to the total pressure recorded.

Summary, steam can be:

3-Super heated steam: No water, like dry heat.. 2-Saturated steam:

b) Dry steam: free from water droplets.

a) Wet steam : carry suspended water droplets.

1-Super saturated steam.

Less  water    

More  water    

Advantages of using saturated steam in sterilization

Depends on the fact that upon contact between saturated steam and cooler surface condensation occurs.

1) Condensation of steam results in release of large amount of latent heat which raise the temp of the article to be sterilized causing (higher temp) and rapid heating up.

2) Condensation of steam results in contraction of volume and so more steam come in contact i.e. better penetration power and Uniformity of temp. 3) Condensation of steam results in moisture which requires low "E" and hence Shorter time of sterilization.

Units of measurement of steam under pressure

One atmosphere/cm2 = one Kg/cm2 = 14.7 pounds/ inch2 (psi) •  These values are either absolute or gauge

Absolute pressure = atmospheric pressure + gauge pressure.

Methods for moist heat sterilization I) At Normal Pressure (at or below 100oC)

A) Sterilization of vaccines:

By heating at a temp which just kill the M.O. but do not affect its antigenicity. -Cholera vaccine is sterilized by heating at 56oC for one hr, -Plague vaccine at 55oC for 15 min, -Staphylococcus autogenous vaccine at 62oC for 15 min.

B) Sterilization by heating with bactericide

•  Heating at 100oC for 30 min. in the presence of a bactericide, which should be non toxic, non volatile, stable, and compatible with the ingredients.

1- In eye drops is phenyl mercuric nitrate (0.002%), chlorhexidine or benzalkonium chloride (0.01%).

2- In Injection is phenyl mercuric nitrate 0.002% or chlorocresol 0.2%.

Disadvantages of this method: 1- Not used for intrathecal, intracisternal or intravenous injections. 2- Not used for oily injections as the oil solubilize the bactericide. 3- The bactericide remains in the final preparation.

intracisternal

intrathecal

 II) At High Pressure (above 100oC)

Pressure only raises the boiling point of water but plays no role in the killing process.

To obtain temp over 100oC, water must boil under pressure, where temp is directly proportional to the pressure. Sterilization by moist heat usually involves the use of steam at temp in the range between 115-134oC. 

Large (pressure cooker) with controls for temp pressure, air and cooling. Used for large scale pharmacy and in hospitals.

Suitable for thermostable aqueous injections in final sealed containers (ampoules or vials) and eye drops, surgical dressing and microbiological media.

Not suitable for oils and powders also glass as repeated autoclaving causes its damage. In emergency could be used in sterilization of glass.

1. Autoclave

Types of Autoclaves 1- Portable autoclave (bench autoclave) •  It is self contained autoclave with

steam generated inside •  Steam is always wet

•  May be vertical or horizontal. •  May be double walled (jacketed).

Either heated electrically or by gas burner.

•  Used for small scale sterilization in laboratories and for sterilization of instruments and utensils.

•  Generally used for bottled fluids and

operated at 121oC.

2- Stationary autoclave (large sterilizer) - Large, horizontal vessel with complicated design, having the steam generated from a distant boiler as dry saturated steam. -Usually surrounded by a steam-jacket. Used for routine hospital or industrial sterilization.

Types of operations in large autoclaves

- Those designed for bottled fluids and generally operated at 121oC. They are supplied with a device for spraying cold water, or more better by introducing filtered compressed air at the end of the cycle (to cool down the load) to minimize container breakage by the high internal pressure other wise it may require very long time to cool.

- Those designed for use with porous loads i.e. dressings

and generally operated at a minimum temp of 134oC, and is supplied with a vacuum pump to remove air completely at the beginning and steam at the end of the process, to withdraw the load dry.

Main design features of autoclave 1.   Lid (door) fitted with clamps and

asbestos gasket, stationary autoclaves may be double doors at both ends one for loading and one for unloading.

2.   Thermocouple for measurement of temp, usually located within the discharge channel.

3.   Pressure gauge. 4.   Air vent to remove air before

sterilization. 5.   Safety valve to escape excess steam

(prevent explosion). 6.   Modern autoclaves are recording

(record pressure, temp during the whole process) supplied with timer and are automatically controlled.

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Operation of the Autoclave

1.  Articles to be sterilized are placed within the autoclave in baskets or cylinders.

2.  Door is closed and clamped. 3.  Air vent is opened. 4.  Heating is started and steam is generated. 5.  Air vent is closed after complete removal of

air (noticed by continuous and vigorous flow of steam from the air vent).

6.  When required pressure is reached (1 atm. at 121oC), sterilization period is timed.

7.  At the end of the period, heat is disconnected.

8.  Autoclave should not be opened before pressure is fallen down.

Advantages of jacketed (double –walled) autoclave 1- Rapid in operation as steam is stored in the jacket. 2- More economic as steam is trapped in the jacket and not allowed

to escape into the atmosphere. 3- Load comes out dry as the inner surface is always warm.

Why air is removed from the autoclave

1- It reduces the penetration of steam especially with dressing 2- It cause superheating in stationary autoclaves 3- In temp controlled autoclaves explosion may occur.(if pressure is

not carefully monitored).