Microbiology

Sterilization by moist heat
Moist heat occurs in the form of hot water, boiling water, or
steam (vaporized water). In practice, the temperature of moist
heat usually ranges from 60 to 135°C. Adjustment of pressure
in a closed container can regulate the temperature of steam.
Moist heat kills microorganisms by denaturation and coagulation of proteins. Sterilization by moist heat can be classified as
follows:
1. Sterilization at a temperature 100°C
2. Sterilization at a temperature of 100°C
3. Sterilization at a temperature 100°C
4. Intermittent sterilization
1. Sterilization at a temperature 100°C: Pasteurization is
an example of sterilisation at a temperature 100°C.
Pasteurization: Fresh beverages (such as milk, fruit juices,
beer, and wine) are easily contaminated during collection
and processing. Because microbes have potential for spoiling these foods or causing illness, heat is frequently used to
reduce the microbial load and to destroy pathogens.
Pasteurization is a technique in which heat is applied to liquids to kill potential agents of infection and spoilage, while
at the same time retaining the liquid’s flavor and food value.
This technique is named after Louis Pasteur who devised
this method. This method is extensively used for sterilization of milk and other fresh beverages, such as fruit juices,
beer, and wine which are easily contaminated during collection and processing.

fungi, it does not kill endospores or thermoduric species
(mostly nonpathogenic lactobacilli, micrococci, and
yeasts). Milk is not sterile after regular pasteurization. In
fact, it can contain 20,000 microbes per milliliter or more,
which explains why even an unopened carton of milk will
eventually spoil on prolonged storage. Newer techniques
have now been used to produce sterile milk that has a storage life of 3 months. In this method, milk is processed with
ultrahigh temperature (UHT) of 134°C for 1–2 seconds.
2. Sterilization at a temperature of 100°C: Sterilsation at
a temperature of 100ºC includes (a) boiling and (b) steam
sterilizer at 100°C.
Boiling: Simple boiling of water for 10–30 minutes kills
most of the vegetative forms of bacteria but not bacterial
spores. Exposing materials to boiling water for 30 minutes
kills most nonspore-forming pathogens including resistant species, such as the tubercle bacillus and staphylococci. Sterilization by boiling is facilitated by addition of
2% sodium bicarbonate to water. Since boiling only once at
100°C does not kill all spores, this method cannot be used
for sterilization but only for disinfection. Hence, it is not
recommended for sterilizing instruments used for surgical
procedure. The greatest disadvantage of this method is
that the items sterilized by boiling can be easily recontaminated when removed from water after boiling.
Steam sterilizer at 100°C: Usually, Koch’s or Arnold’s
steam sterilizer is used for heat-labile substances that tend
to degrade at higher temperatures and pressure, such as
during the process of autoclaving. These substances are
exposed to steam at atmospheric pressure for 90 minutes
during which most vegetative forms of the bacteria except
for the thermophiles are killed by the moist heat.
3. Sterilization at a temperature > 100°C: This method is
otherwise known as sterilization by steam under pressure.
A temperature of 100°C is the highest that steam can
reach under normal atmospheric pressure at sea level.
This pressure is measured at 15 pounds per square inch
( psi), or 1 atmosphere. In order to raise the temperature of
steam above this point, it must be pressurized in a closed
chamber. This phenomenon is explained by the physical
principle that governs the behavior of gases under pressure. When a gas is compressed, its temperature rises in
direct relation to the amount of pressure. So, when the
pressure is increased to 5 psi above normal atmospheric
pressure, the temperature of steam rises to 109°C. When
the pressure is increased to 10 psi above normal, its temperature will be 115°C and at 15 psi (a total of 2 atmospheres), it will be 121°C. It is not the pressure by itself
that is killing microbes, but the increased temperature it
produces. This forms the principle of sterilization by
steam under pressure. Such pressure–temperature combinations can be achieved only with a special device that can
subject pure steam to pressures greater than 1 atmosphere. Health and commercial industries use an autoclave
for this purpose and a comparable home appliance is the
pressure cooker.

Autoclave: It is a cylindrical metal chamber with an airtight
door at one end and racks to hold materials. The lid is
fastened by screw clamp and rendered airtight by an
asbestos washer. It has a discharge tap for air and steam at
the upper side, a pressure gauge and a safety valve that can
be set to blow off at any desired pressure. Heating is usually
carried out by electricity. Steam circulates within the jacket
and is supplied under pressure to the inner chamber where
materials are loaded for sterilization (Fig. 3-1). The water in
the autoclave boils when its vapor pressure equals that of
surrounding atmosphere. Following the increase of
pressure inside the closed vessel, the temperature at which
the water boils inside the autoclave also increases. The
saturated steam that has a higher penetrative power, on
coming in contact with a cooler surface condenses to
water and releases its latent heat to that surface. For
example, nearly 1600 mL steam at 100°C and at atmospheric
pressure condenses into 1 mL of water at 100°C and
releases 518 calories of heat. The gross reduction in volume
of steam sucks in more steam to the area and this process
continues till the temperature of that surface is elevated to
that of the steam. Sterilization is achieved when the steam
condenses against the objects in the chamber and gradually
raises their temperature. The condensed water facilitates
moist conditions that ensures killing of microbes.
Sterilization condition: Experience has shown that the most
efficient pressure–temperature combination for achieving sterilization by autoclave is 15 psi, which yields 121°C.
It is possible to use higher pressure to reach higher temperatures (for instance, increasing the pressure to 30 psi
raises the temperature by 11°C), but doing so will not
significantly reduce the exposure time and can harm the
items being sterilized. It is important to avoid over packing
or haphazardly loading the chamber, because it prevents