Sterilization techniques

Effective sterilization techniques are

essential for working with isolated
cell lines for obvious reasons you
don't want bugs from the
environment growing in your nice
culture medium, and equally,
cultures must be sterilized before
disposal.

autoclaving

Mostly used sterilization technique

kills all microbes, spores and viruses
Except for some bugs which require high temperature.
Mechanism of action:
Moist heat destroys microorganisms by the irreversible denaturation of
enzymes and structural proteins.
The temperature at which denaturation occurs varies inversely with the
amount of water present. Sterilization in saturated steam thus requires
precise control of time, temperature, and pressure.
Pressure serves as a means to obtain the high temperatures necessary to
quickly kill microorganisms. Specific temperatures must be obtained to
ensure the microbicidal activity. Minimum sterilization time should be
measured from the moment when all the materials to be sterilized have
reached the required temperature throughout.
water does a great job of hydrolysing proteins

 It is very important to ensure that all of the trapped air is

removed from the autoclave before activation, as trapped
air is a very poor medium for achieving sterility. Steam at
134C can achieve in three minutes the same sterility that
hot air at 160C can take two hours to achieve.
Based on air removal auto clave has two types:
Gravity Displacement:
As steam enters the chamber, it fills the upper areas first as it is
less dense than air. This compresses the air to the bottom, forcing
it out through a drain which often contains a temperature-sensing
device. Only when air evacuation is complete does the discharge
stop
Vacuum/Gravity Assisted:
a vacuum pump sucks air or air/steam mixtures from the chamber.

 How do we manage to get intense heat:

We use steam instead of dry heat
It holds 7 times more heat than water at the same temprature
This heat is liberated upon contact with the cooler surface of
the material to be sterilised, allowing rapid delivery of heat and
good penetration of dense materials.

Disadvantages:
Like all sterilization processes, steam sterilization has
some deleterious effects on some materials, including
corrosion and combustion of lubricants associated with
dental handpieces; reduction in ability to transmit light
associated with laryngoscopes; and increased hardening
time (5.6 fold) with plaster-cast.

Time required : 15 min

Temperature : 121oC

DRY HEAT(Flaming, baking)

Different from autoclaving as it includes no water.
so protein hydrolysis can't take place.
Mechanism of action:
kill microbes by oxidation of cellular components
Oxidation of proteins requires high temprature than hydrolysis of
protein(autoclaving).

Sterilizing by dry heat is accomplished by conduction. The heat is

absorbed by the outside surface of the item, then passes towards the
centre of the item, layer by layer. The entire item will eventually reach
the temperature required for sterilization to take place.
Dry heat does most of the damage by oxidizing molecules.The essential
cell constituents are destroyed and the organism dies. The temperature
is maintained for almost an hour to kill the most difficult of the resistant
spores.
170oC (340oF) for 60 minutes,
160oC (320oF) for 120 minutes, and
150oC (300oF) for 150 minutes or longer depending up the volume.

 There are two types of dry-heat sterilizers:

the static-air type and
the forced-air type.
Thestatic-air typeis referred to as the oven-type sterilizer
as heating coils in the bottom of the unit cause the hot air to
rise inside the chamber via gravity convection. This type of
dry-heat sterilizer is much slower in heating, requires longer
time to reach sterilizing temperature, and is less uniform in
temperature control throughout the chamber than is the
forced-air type.
Theforced-air or mechanical convectionsterilizer is
equipped with a motor-driven blower that circulates heated air
throughout the chamber at a high velocity, permitting a more
rapid transfer of energy from the air to the instruments.

 Advantages of dry heat sterilization

A dry heat cabinet is easy to install and has relatively
low operating costs;
It is nontoxic and does not harm the environment;
And it is noncorrosive for metal and sharp instruments.

Disadvantages for dry heat sterilization

Time consuming method because of slow rate of heat
penetration and microbial killing.
In addition, the high temperatures are not suitable for
most materials.

FILTRATION
great way of quickly sterilizing solutions without heating
How do we manage filtration:
with a pore diameter that is too small for microbes to pass
through.
For removal of bacteria, filters with an average pore
diameter of 0.2um is normally used.
viruses and phage can pass through these filters so
filtration is not a good option if these are a concern
Types:
Membrane filters
Made from cellulose
Asbestos filters
Sintered glass filters
funnels made from heat-fused glass particles

SOLVENTS
Ethanol is commonly used as a
disinfectant, although since
isopropanol is a better solvent for fat
it is probably a better option.
Both work by denaturing proteins
Require water so they must be
diluted to 60-90% in water to be
effective.
Both are good at killing microbial
cells, they have no effect on spores.

RADIATION
UV, x-rays and gamma rays
have profoundly damaging effects on DNA
The main difference between them, in terms of their effectiveness,
is their penetration.
Uv radiations:
has limited penetration so sterilisation only occurs in a fairly small area
around the lamp.
it is relatively safe and is quite useful for sterilising small areas
X-rays and gamma rays :
more penetrating, because of short wavelength
more dangerous but very effective for large scale cold sterilization of
plastic items