Demonstration of Sterlization

– Chemical agents of control

Dr. Saranya Punniyakotti
Associate Professor
Lovely Institute of Technology
 Theory/Principle:
• A wide variety of chemical agents display

antimicrobial activity to some degree.

• Even if Physical methods of sterilization are more

appropriate for effective sterilization, it is not

always appropriate to use for heat-sensitive

materials like plastics, fiber optics, and biological

specimens.
• Under such conditions, chemical either in liquid
or gaseous state can be used for sterilization.
• However, it is crucial to ensure that the materials
undergoing sterilization are compatible with the
chemical being used.
• Besides, it is important to adopt safety rules in
the workplace safety during the use of chemical
agents.
 Gaseous sterilization
• Gaseous sterilization involves the process of exposing equipment or devices to

different gases in a closed heated or pressurized chamber.

• Gaseous sterilization is a more effective technique as gases can pass through a tiny

orifice and provide more effective results.

• Besides, gases are commonly used along with heat treatment which also facilitates

the functioning of the gases.

• However, there is an issue of release of some toxic gases during the process which

needs to be removed regularly from the system.

• The mechanism of action is different for different types of gases.

 Liquid Sterilization
• Liquid sterilization is the process of sterilization which
involves the submerging of equipment in the liquid
sterilant to kill all viable microorganisms and their
spores.
• Although liquid sterilization is not as effective as
gaseous sterilization, it is appropriate in conditions
where a low level of contamination is present.
Aim: To study the activity of some sterilizing agents and to learn the
importance of time, germicidal concentration, and microbial species
in sterilization.
Equipment and Apparatus Required: incubator.
Materials Requirement: Nutrient agar plates Sterile, empty tubes Sterile 10-
ml pipettes (cotton plugged) Sterile 1.0-ml pipettes (cotton plugged) Bulb or
other aspiration device for pipette 5% sodium hypochlorite (bleach); 0.05%
sodium hypochlorite Absolute alcohol, 70% alcohol 3% hydrogen peroxide, 24-
hour nutrient broth culture of Escherichia coli, Three- to six-day-old broth culture
of Bacillus subtilis.

Learning objectives: To learn the chemical agents of control in

sterilization.
Outline of Procedure:
1. Select one of the chemical agents provided. Pipette 5.0 ml of the
solution into a sterile test tube.

2. To the 5 ml of disinfectant, add 0.5 ml of the E. coli culture. Gently

shake the tube to distribute the organisms uniformly. Note the time.
3. Divide a nutrient agar plate into four sections with a marking pen
or pencil. At intervals of 2, 5, 10, and 15 minutes, transfer one
loopful of the disinfectant-culture mixture to a section of the
nutrient agar plate. Label each plate with the name of the organism,
the disinfectant, and its concentration (e.g., E. coli, 1% phenol).
Label each section of the plate with the time of exposure (e.g., 2
minutes, 5 minutes, etc.).
4. Using the same concentration of the same disinfectant, repeat steps 1 to 3
with the culture of B. subtilis.
5. Inoculate one-half of a nutrient agar plate directly from the E. coli culture
and the other half from the B. subtilis culture. Label each half with the name
of the organism and the word Control.
6. Incubate all tubes at 35°C for 48 hours.
OBSERVATION
Results:
• Read all plates for growth () or no growth (). Record your
results in the chart.