Practical 1 6/8/2023

Aim: To study basic lab manual of microbiology practical and familiarization of lab set up.

theory;

Sterilization Vs Disinfection

Sterilization is the process of eliminating entirely all microorganisms, including spores, from a surface or
environment. Usually, heat or filtration are utilized, however chemicals or radiation can be also used.

Disinfection is the elimination of germs that could potentially lead to illness or other issues, such as
spoilage. Chemicals are typically used to do it.

1/Sterilization (Aseptic condition)

a. using autoclave/ pressure cooker

In labs, hospitals, and commercial settings, autoclaving is a common sterilization technique. To get rid of
germs like bacteria, viruses, and spores from diverse items and materials, high-temperature steam under
pressure is used.

Moreover, Denis Papin (1647–1712), a scientist and one of the pioneers of the steam engine, created the
steam pressure cooker, the earliest version of the autoclave, in 1681. Only culinary uses were made of it.
Later, in order to sterilize medical products, the first commercial steam sterilization system was created in
1889. Similarly, they are used to sterilize lab equipment, sterilize media, and decontaminate certain
biological waste. Before disposal, it is advised to autoclave regulated medical waste that may include
bacteria, viruses, and other living elements. Likewise, pipette tips, plastic tubes, autoclavable containers,
and culture medium are all sterilized in autoclaves. Since materials containing water cannot be sterilized
by dry heat sterilization, autoclaves are crucial tools to ensure the sterilization of those materials.

Autoclaving exploits the fact that high temperature, steam, and pressure are effective at killing
microorganisms and destroying their structures, including proteins, enzymes, and nucleic acids. The
process utilizes saturated steam, which has higher heat content compared to dry air. When steam is
pressurized, its temperature rises, leading to effective sterilization. Moreover, all types of microbial life
can be eliminated by the sterilization of materials using steam and pressure.

The fundamental principle behind sterilization with an autoclave or pressure cooker is the use of saturated
steam at a minimum pressure of 15 psi. Under normal atmospheric pressure (760 mm of Hg/15 psi), water
typically boils at 100°C. However, an autoclave, the water boils at 121°C at the pressure of 15 psi or 775
mm of Hg. The high pressure facilitates the rapid penetration of heat into deeper parts of the material,
and moisture present in the steam and it causes the coagulation of proteins leading to an irreversible
loss of function and activity of microbes. Similarly, by emitting latent heat, the steam makes contact with
the surface and destroys the bacteria.

Furthermore, the autoclave sterilization process has four stages temperature, sterilizing, and vacuum
drying.

1. Pre-Vacuum
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In order to obtain homogeneous temperature and pressure conditions before starting the sterilization
process, the autoclave first creates a void inside the sterilizing chamber. If not, they would generate
pressure and temperature gaps that would harm the sterilizing process.

2. Rising Temperature

In this phase, the autoclave pumps steam into the camera, bringing it closer to its working point. To
maintain the proper system temperature and pressure, the system exhibits temperature and pressure
waves throughout this phase, which must be managed.

3.Sterilizing

The steam within the sterilization chamber of the autoclave reaches a temperature of 121 °C and a
pressure of 29 Psi during this phase, which must be maintained for around 20 minutes to successfully
finish the sterilization procedure. If the pressure and temperature inside the chamber are not
consistent, the sterilizing procedure will not work.

Vacuum-Drying

The sterilizing procedure ends with this step, which involves vacuuming out any leftover steam to cause
a dramatic drop in temperature and pressure after five minutes. The remaining bacteria are removed as
a result of the emission of steam from the sterilizing chamber. The control system now needs to
accomplish a sharp drop in chamber temperature.

Precaution while using autoclaving

 There should be no use of plastic .

 There should not be use of liquid agent like alcohol.
 Do not autoclave Materials that are flammable, reactive, corrosive, toxic, or radioactive.
 When opening the door or taking something out of the autoclave, make sure you put on heat-
protective non-asbestos gloves, safety glasses, goggles, and a face shield.
 Don't open the door until the chamber pressure is back to zero, or wait for 15 minutes. Then, for
materials to cool and to enable pressure to equalize, leave the door open for a while.
 Mixing exhaust and loads requiring for various exposure durations is not recommended.
 Materials that will melt should be put in a shallow stainless steel autoclave pan, such as lab coats
made of plastic.

2/Heat sterilization

The most efficient and widely used sterilization approach is heat sterilization, which kills enzymes and
other vital cell components, resulting in bactericidal action. Heat is considered to be the most reliable
means of sterilizing. Articles that cannot tolerate high temperatures can still be sterilized at lower
temperatures by extending the exposure time. Before and after usage, materials or equipment are
sterilized in a Bunsen flame using red heat. To ensure that any contaminating bacterial spores are
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eliminated, they must be roasted to red-hot temperature. Instruments including incubation loops, wires,
and forceps points are frequently sterilized using this technique, which is based on dry heat sterilization.

3/chemical/ surface sterilization

Chemical sterilization is a technique for disinfection and microbial inactivation that uses chemical agents
to destroy or incapacitate microorganisms on surfaces, equipment, or materials. Depending on the
chemical agent utilized and the intended usage, many techniques of chemical sterilization may be used.
The chemical sterilizing agents hydrogen peroxide, peracetic acid, ethylene oxide, and formaldehyde are
some of the types of agents. Numerous microbes, including bacteria, viruses, fungus, and spores, can be
successfully eliminated by these substances. In surface sterilization, 70% alcohol is used to sterilize the
surface. 70% of alcohol is used to sterilize surfaces because it is not a volatile substance and it easily
eliminates microbes. If the alcohol is below 70%, it is difficult to kill bacteria, and it is very volatile and
will easily vaporize.

Equipments

1. Laminar air flow

Laminar air flow is a closed cabinet with a HEPA-filtered air flow system that provides a continuous,
unidirectional airflow with little to no turbulence. The air flow velocity stays in the range of 0.4 and
0.6 m/s. It is used in laboratories to establish a sterilized working environment devoid of pathogens
and particles so that specialized work may be done there. These appliances do not offer any
personal protection as they direct air toward the user, but they do shield products from toxins in the
area.

The quality of the prefilters and HEPA filters determines how effectively a laminar air flow
workstation runs. The HDPE, non-woven, and HDPE mesh media prefilters with a box-type pleat
have an efficiency of 95% down to 5 microns, and they are enclosed in an aluminum casing. High-
quality HEPA filters with 99.99% efficiency down to 0.3 microns are installed in an aluminum-
anodized case.

2. Incubator

An incubator is an enclosed, insulated device that provides the optimum amount of humidity,
temperature, and other environmental variables needed for the growth and development of
organisms. It offers a consistent and regulated environment that facilitates the maintenance,
investigation, and development of microorganisms and biological specimens. Incubators enable
investigations that depend on constant and regulated microbial growth by controlling temperature,
humidity, and other variables.

a variety of non volatile, non toxic chemicals are used in laboratory to disinfect glass wares, hands etc.
These includes a. Halogen and halogen compounds b. Compounds of heavy metals c. Phenols and its
derivatives d. Alcohol and detergents Examples: a. Mercuric chloride and AgNO3: are used in the ratio
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of 1:100 resp. , for disinfecting surface of test material. b. Ethyl Alcohol : used for disinfecting surface
of test material & laboratory desktop.