Moist Heat Sterilization - Pharmaceutical Microbiology Third Semester PDF Notes

 Thermal sterilization methods

Moist Heat Sterilization


• Mechanism of sterilization by heat

• TDP, TDT and DRT

• Classification of heat sterilzation methods

• Moist heat sterilization - autoclaving

Learning objectives

At the end of this lecture, student will be able to:

– Explain the mechanism of heat sterilization methods

– Outline the significance of Thermal death point (TDP), Thermal death time (TDT) and Decimal reduction time (DRT, or D value)

– List the heat sterilization methods

– Explain the construction and working of an autoclave

Thermal sterilization / Sterilization by heat

Heat sterilization - Mechanisms

• Heat is the most reliable and widely used means of sterilization

• Heat appears to kill microorganisms by denaturing their enzymes and other essential cell constituents

• The changes to the three-dimensional shapes of these proteins inactivate them

Heat sterilization - mechanisms

• Excessive heat acts by coagulation of cell proteins

• Less heat interferes metabolic reactions

• Sterilization occurs by heating above 100°C which ensure killing of bacterial spores

• Sterilization by hot air in hot air oven and sterilization by autoclaving are the two most common method used in the laboratory

Measures of heat resistance

• Thermal death point (TDP) is the lowest temperature at which all the microorganisms in a particular liquid suspension will be killed in

10 minutes.

• Thermal death time (TDT), the minimal length of time for all bacteria in a particular liquid culture to be killed at a given temperature.

• Decimal reduction time (DRT, or D value) is the time, in minutes, in which 90% of a population of bacteria at a given temperature will be killed

Heat sterilization methods

Types of heat sterilization methods:

1. Sterilization by moist heat

2. Sterilization by dry heat

• Moisture-sensitive products - dry heat sterilization (160–180°C) procedures are used

• Moisture-resistant items - moist heat sterilization (121–134°C) procedures are used

Sterilization processes

• Heating-up stage - the articles to be treated must first be raised to sterilization temperature

• Holding stage - timing for the process

• Cooling stage – after the process is complete

Thermal sterilization / Sterilization by heat

Moist heat sterilization / Steam sterilization

a) Sterilization at temperature above 100⁰C (Saturated stream)

– Autoclaving / steam sterilization

b) Sterilization at temperature of 100⁰C (Boiling water)

– Tyndallisation

– Sterilization by boiling water

c) Sterilization at temperature below 100⁰C

(Hot water below Boiling Point)

– Pasteurization

– Vaccine bath

d) Heating with a bactericide

Moist Heat Sterilization

Moist heat kills microorganisms primarily by

1. Coagulating proteins (denaturation), which is caused by breakage of the hydrogen bonds that hold the proteins in their three-dimensional structure

2. Breakage of DNA strands

3. Loss of functional integrity of cell membrane

Sterilization at temperature above 100⁰C (Saturated stream)

Autoclaving / Steam sterilization

• Reliable sterilization with moist heat requires temperatures above that of boiling water

• These high temperatures are most commonly achieved by steam under pressure in an autoclave

• Autoclaving is the preferred method of sterilization, unless the material to be sterilized can be damaged by heat or moisture

• The higher the pressure in the autoclave, the higher the temperature

• Sterilization in an autoclave is most effective when the organisms either are contacted by the steam directly or are contained in a small volume of aqueous (primarily water) liquid

• Under these conditions, steam at a pressure of about 15 psi (121 °C) will kill all organisms and their endospores in about 15 minutes

• Autoclaving is used to sterilize culture media, instruments, dressings, intravenous equipment, applicators, solutions, syringes, transfusion equipment and numerous other items that can withstand high temperatures and pressures

• Large industrial autoclaves are called retorts

Sterilizer design and operation

• Autoclaves are stainless steel vessels designed to withstand the steam pressures employed in sterilization

Sterilizers can be

 Portable sterilizers

 Large scale sterilizers

1. ‘Portable’ sterilizers / Bench autoclave, where they generally have internal electric heaters to produce steam and are used for small pilot or laboratory scale sterilization and for the treatment of instruments and utensils

2. Large-scale sterilizers/ Horizontal Autoclave for routine hospital or industrial use, operating on ‘dry’ saturated steam from a separate boiler

Autoclaving - procedure

• Water is boiled to produce steam, which is released through the jacket and into the autoclave’s chamber

• The air initially present in the chamber is forced out until the chamber is filled with saturated steam and the outlets are closed

• If the air is not completely exhausted, the container will not reach the temperature expected for a given pressure

• Hot, saturated steam continues to enter until the chamber reaches the desired temperature and pressure, usually 121°C and 15 pounds of pressure

• At this temperature saturated steam destroys all vegetative cells and endospores in a small volume of liquid within 10 to 12 minutes

• Treatment is continued for about 15 minutes to provide a margin of safety

• The chamber should not be packed too tightly because the steam needs to circulate freely and contact everything in the autoclave

• Bacterial endospores will be killed only if they are kept at 121°C for 10 to 12 minutes

• When a large volume of liquid must be sterilized, an extended sterilization time will be needed

Source of steam

‘Wet’ steam

– Steam for sterilization is generated within the sterilizer

– It is constantly in contact with water

‘Dry’ saturated steam

– Steam is supplied under pressure (350–400 kPa) from a separate boiler

– No entrained water droplets

General design features of an autoclave

• Steam sterilizers are constructed with either cylindrical or rectangular chambers

• Preferred capacities ranging from 400 to 800 litres.

• They can be sealed by either a single door or by doors at both ends (to allow through passage of processed materials)


• During sterilization the doors are held closed by a locking mechanism which prevents opening when the chamber is under pressure and until the chamber has cooled to a pre-set temperature, typically 80°C

• In the larger sterilizers the chamber may be surrounded by a steam-jacket which can be used to heat the autoclave chamber and promote a more uniform temperature throughout the load

• The same jacket can also be filled with water at the end of the cycle to facilitate cooling and thus reduce the overall cycle time

Stages of operations - autoclave

1. Air removal and steam admission

– Downward displacement or evacuation

2. Heating-up and exposure

3. Drying or cooling

• Dressings packs must be dried before removal from the chamber

• For bottled fluids cooling must achieved as rapidly as possible to minimize thermal degradation of the product

• Steam exhaust can be achieved by application of a vacuum

Precautions to be taken during loading in an autoclave

• Articles which require different sterilization time must not be sterilized together

• The articles should be loosely packed to allow maximum penetration of stream

• Articles should not touch the chamber wall

• When vacuum is applied, liquid materials should not be kept for sterilization

• Solid and liquid materials should not be combined because vacuum will be applied to replace stream with air

• Make sure that the doors are shut tightly and locked


• Heat acts by denaturing bacterial enzymes other essential cell constituents

• Thermal death point (TDP), Thermal death time (TDT) and Decimal reduction time (DRT, or D value) are measures of heat resistance of organisms

• Stages of heat sterilization are heating, holding and cooling

• Most important equipment used for moist heat sterilization is the autoclave

• It sterilizes articles using pressurized steam at 121⁰C, and 15lbs/sq.inch pressure – duration 20 min

• Autoclaving is the method of choice for sterilization of heat and moisture resistant articles

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