Environmental Geology & Hazard Waste Management
(Lab)
Lab 2
Submitted by: Muhammad Attique
(2017-GE-5)
Submitted to: Sadia Ismail (Assistant Professor)
Geological Engineering Department
University of Engineering & Technology, Lahore
2017-GE-5 (Muhammad Attique)
Lab. 02
Statement
Sterilization and disinfection of lab equipment by autoclave.
Objective
The autoclave is used to sterilize the equipment from bacteria and other contaminants.
Related Theory
Introduction
Autoclave was invented by Charles Chamberland in 1879, although a precursor known as
the steam digester was created by Denis Papin in 1679. The name comes from Greek auto-,
ultimately meaning self, and Latin clavis meaning key, thus a self-locking device.
An autoclave is a machine that provides a physical method of sterilization by killing bacteria,
viruses, and even spores present in the material put inside of the vessel using steam under pressure.
Autoclave sterilizes the materials by heating them up to a particular temperature for a specific
period. The autoclave is also called a steam sterilizer that is commonly used in healthcare facilities
and industries for various purposes. The autoclave is considered a more effective method of
sterilization as it is based on moist heat sterilization.
Figure 1 Autoclave
The autoclave works on the principle of moist heat sterilization where steam under pressure is used
to sterilize the material present inside the chamber. When this steam comes in contact on the
surface, it kills the microbes by giving off latent heat. The condensed liquid ensures the moist
killing of the microbes.
There are different types of autoclaves present in the market, some of which are Pressure cooker
type/ Laboratory bench autoclaves (N-type), Gravity displacement type autoclave, Positive
pressure displacement type (B-type) and Negative pressure displacement type (S-type).
2017-GE-5 (Muhammad Attique)
Parts of Autoclave
These are major component of Labortary Autoclave enlisted in following:
1. Sterilization temperature selector
2. Pressure gauge
3. Sterilizing pilot lamp
4. Sterilizing-drying selector
5. Timer
6. Start push-button switch
7. Sensitive exhaust valve
8. Drain Knob
9. Lid arm lightened
10. Stainless steel double lid
11. Safety valve
Working Principle
The autoclave works on the principle of moist heat sterilization where steam under pressure is used
to sterilize the material present inside the chamber. The high pressure increases the boiling point
of water and thus helps achieve a higher temperature for sterilization. Water usually boils at 100°C
under normal atmospheric pressure (760 mm of Hg); however, the boiling point of water increases
if the pressure is to be increased. Similarly, the high pressure also facilitates the rapid penetration
of heat into deeper parts of the material, and moisture present in the steam causes the coagulation
of proteins causing an irreversible loss of function and activity of microbes. This principle is
employed in an autoclave where the water boils at 121°C at the pressure of 15 psi or 775 mm of
Hg. When this steam comes in contact on the surface, it kills the microbes by giving off latent heat.
The condensed liquid ensures the moist killing of the microbes. Once the sterilization phase is
completed (which depends on the level of contamination of material inside), the pressure is
released from the inside of the chamber through the whistle. The pressure inside the chamber is
then restored back to ambient pressure while the components inside remain. t
Uses of Autoclave
Autoclaves are important devices to ensure the sterilization of materials containing water as they
cannot be sterilized by dry heat sterilization. Besides, autoclaves are used for various other
purposes.
1. Autoclaves are used in medical applications to perform sterilization and in the chemical
industry to cure coatings and vulcanize rubber and for hydrothermal synthesis.
2. Industrial autoclaves are used in industrial applications, especially in the manufacturing of
composites.
3. They are used to decontaminate specific biological waste and sterilize media, instruments,
and labware.
4. Regulated medical waste that might contain bacteria, viruses, and other biological materials
are recommended to be inactivated by autoclaving before disposal.
2017-GE-5 (Muhammad Attique)
5. In medical labs, autoclaves are used to sterilize medical equipment, glassware, surgical
equipment, and medical wastes.
6. Similarly, autoclaves are used for the sterilization of culture media, autoclavable
containers, plastic tubes, and pipette tips.
Experimental Procedure
In general, an autoclave is run at a temperature of 121° C for at least 30 minutes by using saturated
steam under at least 15 psi of pressure. The following are the steps to be followed while running
an autoclave:
1. Before beginning to use the autoclave, it should be checked for any items left from the
previous cycle.
2. Enough water is then put inside the chamber.
3. Now, the materials to be sterilized are placed inside the chamber.
4. The lid is then closed, and the screws are tightened to ensure an airtight condition, and the
electric heater is switched on.
5. The safety valves are adjusted to maintain the required pressure in the chamber.
6. Once the water inside the chamber boils, the air-water mixture can escape through the
discharge tube to let all the air inside to be displaced. The complete displacement can be
ensured once the water bubbles cease to come out from the pipe.
7. The drainage pipe is then closed, and the steam inside can reach the desired levels (15 lbs.
in most cases).
8. Once the pressure is reached, the whistle blows to remove excess pressure from the
chamber.
9. After the whistle, the autoclave is run for a holding period, which is 15 minutes in most
cases.
10. Now, the electric heater is switched off, and the autoclave can cool until the pressure gauge
indicates the pressure inside has lowered down to that of the atmospheric pressure.
11. The discharge pipe is then opened to allow the entry of air from the outside into the
autoclave.
12. Finally, the lid is opened, and the sterilized materials are taken out of the chamber.
Precaution
Although autoclaves are simple to use, there are certain rules of precautions to be followed while
operating an autoclave. Some of the important precautions to be followed while running an
autoclave are:
1. Autoclaves should not be used to sterilize water-proof or water-resistant substances like oil or
powders.
2. The items to be autoclaved should always be placed in a secondary container.
3. To ensure sufficient penetration, articles should be wrapped in something that allows
penetration by steam, and materials like aluminum foils should not be used.
4. The items placed inside the chamber should not touch the sides or top of the chamber.
2017-GE-5 (Muhammad Attique)
5. Besides, never autoclave flammable, reactive, corrosive, toxic or radioactive materials,
household bleach, or paraffin-embedded tissue.
6. The paper should not be placed directly inside an autoclave as it is a combustible substance. It
should be autoclaved in a waste bag on a bio bag setting to prevent fire.
7. The wastes and clean items should be autoclaved separately.
8. Attempts to open the lid when the autoclave is working should never be made.
9. Liquid components should never be autoclaved in sealed containers.
10. The liquid inside the containers should only be filled 2/3rd of the total volume to prevent the
spilling of the liquid.
11. Plastic or polyethylene trays or containers should not be used as they might melt and damage
the autoclave.
References
• Frazier WC and Westhoff DC. Food Microbiology. Tata McGraw Hill Publishing Company
Limited. 1995.
• Banwart GJ (1989). Basic Food Microbiology. Chapman & Hall, New York, NY.
• Jay JM (2000). Modern Food Microbiology. CBS Publications and Distribution. Delhi.
• Collins CH, Patricia M, and Lyne JM (1995). Collins and Lynes Microbiological Methods 7th
edition. Grange, Butter Worth, Oxford.
• Cappucino JG and Sherman N (1996). Microbiology, A Laboratory Manual 4th edition.
Benjamin Cumings Inc. California.
2017-GE-5 (Muhammad Attique)
