Autoclave- Definition, Parts, Principle, Procedure, Types, Uses

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Autoclave Definition
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 of time. 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.

Autoclave Parts/ Components

The simplest form of the autoclave is the pressure cooker type or laboratory bench autoclaves.
The following is the detailed description of different components/ parts of an autoclave:
Figure: Autoclave Parts or Components.
a. Pressure Chamber
 The pressure chamber is the main component of a steam autoclave consisting of an inner
chamber and an outer jacket.
 The inner chamber is made up of stainless steel or gunmetal, which is present inside the out
chamber made up of an iron case.
 The autoclaves used in healthcare laboratories have an outer jacket that is filled with steam to
reduce the time taken to reach the sterilization temperature.
 The inner chamber is the case where the materials to be sterilized are put.
 The size of the pressure chamber ranges from 100 L to 3000 L.
b. Lid/ Door
 The next important component of an autoclave is the lid or door of the autoclave.
 The purpose of the lid is to seal off the outside atmosphere and create a sterilized condition on
ht inside of the autoclave.
 The lid is made airtight via the screw clamps and asbestos washer.
 The lid consists of various other components like:
Pressure gauge
 A pressure gauge is present on the lid of the autoclave to indicate the pressure created in the
autoclave during sterilization.
 The pressure gauge is essential as it assures the safety of the autoclave and the working
condition of the operation.
Pressure releasing unit/ Whistle
 A whistle is present on the lid of the autoclave is the same as that of the pressure cooker.
 The whistle controls the pressure inside the chamber by releasing a certain amount of vapor
by lifting itself.
Safety valve
 A safety valve is present on the lid of the autoclave, which is crucial in cases where the
autoclave fails to perform its action or the pressure inside increases uncontrollably.
 The valve has a thin layer of rubber that bursts itself to release the pressure and to avoid the
danger of explosion.
c. Steam generator/ Electrical heater
 An electrical steam generator or boiler is present underneath the chamber that uses an electric
heating system to heat the water and generate steam in the inner and the outer chamber.
 The level of water present in the inner chamber is vital as if the water is not sufficient; there
are chances of the burning of the heating system.
 Similarly, if the water is more than necessary, it might interfere with the trays and other
components present inside the chamber.
d. Vacuum generator (if applicable)
 In some types of autoclaves, a separate vacuum generator is present which pulls out the air
from the inside of the chamber to create a vacuum inside the chamber.
 The presence of some air pockets inside the chamber might support the growth of different
microorganisms. This is why the vacuum chamber is an important component of an autoclave.
e. Wastewater cooler
 Many autoclaves are provided with a system to cool the effluent before it enters the draining
pipes.
 This system prevents any damage to the drainage pipe due to the boiling water being sent out
of the autoclave.

Autoclave Principle/ Working

Figure: Autoclave Principle or Working.

 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 with 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 the ambient pressure while the
components inside remain hot for some time.

Procedure for running an autoclave

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. A sufficient amount of 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 is allowed to 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 is allowed to 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 is allowed to 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.

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. They are used to decontaminate specific biological waste and sterilize media, instruments, and
labware.
2. Regulated medical waste that might contain bacteria, viruses, and other biological materials is
recommended to be inactivated by autoclaving before disposal.
3. In medical labs, autoclaves are used to sterilize medical equipment, glassware, surgical
equipment, and medical wastes.
4. Similarly, autoclaves are used for the sterilization of culture media, autoclavable containers,
plastic tubes, and pipette tips.

Precautions
Although autoclaves are pretty 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 autoclave should not be overcrowded, and the materials should be loaded in a way that
ensures sufficient penetration of articles by the steam.
3. The items to be autoclaved should always be placed in a secondary container.
4. Only autoclavable bags are to be used to autoclave packaged waste.
5. To ensure sufficient penetration, articles should be wrapped in something that allows
penetration by steam, and materials like aluminum foils should not be used.
6. The items placed inside the chamber should not touch the sides or top of the chamber.
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.
12. Besides, never autoclave flammable, reactive, corrosive, toxic, or radioactive materials,
household bleach, or paraffin-embedded tissue.
13. 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.
 Incubator- Definition, Parts, Working, Types, Uses,
Precautions
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Incubator Definition
An incubator, in microbiology, is an insulated and enclosed device that provides an optimal
condition of temperature, humidity, and other environmental conditions required for the growth
of organisms.
An incubator is a piece of vital laboratory equipment necessary for cultivating microorganisms
under artificial conditions.
An incubator can be used to cultivate both unicellular and multicellular organisms.

Components/Parts of Incubator
A microbial incubator is made up of various units, some of which are:

Cabinet
 The cabinet is the main body of the incubator consisting of a double-walled cuboidal
enclosure with a capacity ranging from 20 to 800L.
 The outer wall is made up of stainless steel sheets while the inner wall is made up of
aluminum.
 The space between the two walls is filled with glass wool to provide insulation to the
incubator.
 The insulation prevents heat loss and reduces electric consumption, thereby ensuring the
smooth working of the device.
 The inner wall of the incubator is provided with inward projections that support the shelves
present inside the incubator.
Door
 A door is present in all incubators to close the insulated cabinet.
 The door also has insulation of its own. It is also provided with a glass that enables the
visualization of the interior of the incubator during incubation without disturbing the interior
environment.
 A handle is present on the outside of the door to help with the maneuvering of the door.
Control Panel
 On the outer wall of the incubator is a control panel with all the switches and indicators that
allows the parameters of the incubator to be controlled.
 The control panel also has a witch to control the thermostat of the device.
Thermostat
 A thermostat is used to set the desired temperature of the incubator.
 After the desired temperature is reached, the thermostat automatically maintains the incubator
at that temperature until the temperature is changed again.
Perforated shelves
 Bound to the inner wall are some perforated shelves onto which the plates with the culture
media are placed.
 The perforations on the shelves allow the movement of hot air throughout the inside of the
incubator.
 In some incubators, the shelves are removable, which allows the shelves to be cleaned
properly.
Asbestos door gasket
 The asbestos door gasket provides an almost airtight seal between the door and the cabinet.
 This seal prevents the outside air from entering the cabinet and thus, creating an isolated hot
environment inside the cabinet without being interrupted by the external environment.
L-shaped thermometer
 A thermometer is placed on the top part of the outer wall of the incubator.
 One end of the thermometer provided with gradations remains outside of the incubator so that
temperature can be read easily.
 The next end with the mercury bulb is protruded slightly into the chamber of the incubator.
HEPA filters
 Some advanced incubators are also provided with HEPA filters to lower the possible
contamination created due to airflow.
 AN air-pump with filters creates a closed-loop system so that the air flowing inside the
incubator generates less contamination.
Humidity and gas control
 The CO2 incubators are provided with a reservoir underneath the chamber that contains water.
 The water is vapourised to maintain the relative humidity inside the chamber.
 Similarly, these incubators are also provided with gas chambers to give the desired
concentration of CO2 inside the incubator.
Principle/ Working of Incubator
 An incubator is based on the principle that microorganisms require a particular set of
parameters for their growth and development.
 All incubators are based on the concept that when organisms are provided with the optimal
condition of temperature, humidity, oxygen, and carbon dioxide levels, they grow and divide
to form more organisms.
 In an incubator, the thermostat maintains a constant temperature that can be read from the
outside via the thermometer.
 The temperature is maintained by utilizing the heating and no-heating cycles.
 During the heating cycle, the thermostat heats the incubator, and during the no-heating period,
the heating is stopped, and the incubator is cooled by radiating heat to the surrounding.
 Insulation from the outside creates an isolated condition inside the cabinet, which allows the
microbes to grow effectively.
 Similarly, other parameters like humidity and airflow are also maintained through different
mechanisms that create an environment similar to the natural environment of the organisms.
 Similarly, they are provided with adjustments for maintaining the concentration of CO2 to
balance the pH and humidity required for the growth of the organisms.
 Variation of the incubator like a shaking incubator is also available, which allows for the
continuous movement of the culture required for cell aeration and solubility studies.
Procedure for running an incubator
Once the cultures of organisms are created, the culture plates are to be placed inside an incubator
at the desired temperature and required period of time. In most clinical laboratories, the usual
temperature to be maintained is 35–37°C for bacteria.
The following are the steps to be followed while running an incubator:
1. Before using the incubator, it should be made sure that no remaining items are present in the
incubator from the previous cycles. However, in some cases, if the same incubator is being
used for multiple organisms, and they require the same set of parameters, they can be placed
together in the same incubator.
2. The door of the incubator is then kept closed, and the incubator is switched on. The incubator
has to be heated up to the desired temperature of the growth of the particular organism. The
thermometer can be used to see if the temperature has reached.
3. In the meantime, if the organism requires a particular concentration of CO2 or a specific
humidity, those parameters should also be set in the incubator.
4. Once all the parameters are met, the petri dish cultures are placed on the perforated shelves
upside down, i.e., media uppermost. This is necessary because if the plates are incubated
normally, condensation collects on the surface of the medium and prevents the formation of
isolated colonies.
5. If it is necessary to incubate Petri dish cultures for several days, the plates are sealed with
adhesive tapes or are placed in plastic bags or plastic food containers.
6. Now, the door is locked, and the plates are kept inside for the required time before taking
them out.
Types of incubators

Figure: Some Incubators used in Microbiology Lab

On the basis of the presence of a particular parameter or the purpose of the incubator, incubators
are divided into the following types:

Benchtop incubators
 This is the most common type of incubator used in most of the laboratories.
 These incubators are the basic types of incubators with temperature control and insulation.
CO2 incubators
 CO2 incubators are the special kinds of incubators that are provided with automatic control of
CO2 and humidity.
 This type of incubator is used for the growth of the cultivation of different bacteria requiring
5-10% of CO2 concentration.
 For humidity control, water is kept underneath the cabinet of the incubator.
Cooled incubators
 For incubation at temperatures below the ambient, incubators are fitted with modified
refrigeration systems with heating and cooling controls.
 This type of incubator is called the cooling incubator.
 In the cooling incubator, the heating and cooling controls should be appropriately balanced.
Shaker incubator
 A thermostatically controlled shaker incubator is another piece of apparatus used to cultivate
microorganisms.
 Its advantage is that it provides a rapid and uniform transfer of heat to the culture vessel, and
its agitation provides increased aeration, resulting in acceleration of growth.
 This incubator, however, can only be used for broth or liquid culture media.
Portable incubator
 Portable incubators are smaller in size and are used in fieldwork, e.g. environmental
microbiology and water examination.
Uses of Incubator
Incubators have a wide range of applications in various areas including cell culture,
pharmaceutical studies, hematological studies, and biochemical studies.
Some of the uses of incubators are given below:
1. Incubators are used to grow microbial culture or cell cultures.
2. Incubators can also be used to maintain the culture of organisms to be used later.
3. Some incubators are used to increase the growth rate of organisms, having a prolonged
growth rate in the natural environment.
4. Specific incubators are used for the reproduction of microbial colonies and subsequent
determination of biochemical oxygen demand.
5. These are also used for breeding of insects and hatching of eggs in zoology.
6. Incubators also provide a controlled condition for sample storage before they can be
processed in the laboratories.
Precautions
The following precautions are to be followed while running an incubator:
1. As microorganisms are susceptible to temperature change, the fluctuations in temperature of
the cabinet by repeatedly opening the door should be avoided.
2. The required parameters growth of the organism should be met before the culture plates are
placed inside the cabinet.
3. The plates should be placed upside down with the lid at the bottom to prevent the
condensation of water on to the media.
4. The inside of the incubators should be cleaned regularly to prevent the organisms from
settling on the shelves or the corners of the incubator.
5. While running the incubator for an extended period of time, sterile water should be placed
underneath the shelves to prevent the culture media from drying out.

Types of Incubators
The most common types of incubators are –

1. BOD incubators
2. Bacteriological incubators
3. CO2 incubators
The main difference between these two types of incubators is due to temperature. BOD stands for
biological oxygen demand and it is the amount of dissolved oxygen needed by aerobic biological
organisms to break down organic materials present in a given sample at a certain temperature over a
specific time period.
Fig. Bacteriological Incubator (Scientific Product)-Lower .. nb b , m, Part

Fig. AST Plates, Microbial

specimens inoculated culture media (Blood agar, MacConkey medium, Chocolate agar),
Biochemical tests media in Bacteriological Incubator

Bacteriological Incubators
This type of incubator is used mainly in laboratories usually for the growth of bacteria. A constant
temperature set according to the requirement is possible because of having a thermostat that
maintains it. Accurate temperature can be visible on the thermometer fixed on incubators. Most of
the incubators are programmable and do not need trial-and-error temperature settings. They only
hold a heating system that maintains the temperature for the growth of bacteria. Due to the absence
of a cooling system, these incubators are affected by the temperature of the surrounding
environment.

BOD incubators (Low-Temperature Incubators)

These types of incubators are often called low-temperature incubators used for the growth of fungi
i.e. yeast and mold as they require a low temperature to grow. These types of incubators are called
BOD (Biological oxygen demand) incubators because in biological oxygen demand testing there is a
need for a low temperature of around 20-25˚C. So, don’t confuse the term because the purpose of
BOD incubators is also the same as bacteriological incubators.

CO2 Incubators
Inside incubators, also known as gassed incubators, an atmosphere is created that is as natural as
possible to develop cell and tissue cultures. This way of cultivating living organisms is called in vitro
and is the main application for CO2 incubators.

The following parameters are crucial for cell cultivation:

  CO2 level
 Temperature
 Humidity

Fig. CO Incubator (New Brunswick)

2
Fig. Blood agar and chocolate agar in CO Incubator
2

Fig. Sanyo CO incubator with a CO cylinderig. Sanyo CO Incubator

2 2 2

Handling Procedure of Incubators

1. Make ready material of interest (e.g. microbes, specimen inoculated agar plates, etc.).
2. Switch on the incubator and check the temperature of interest (and also the CO2 level in case
of using a CO2 incubator).
3. Place the material of interest and wait for the required incubation period.
4. Take out and finally switch off the incubator.

Uses of Incubators
Incubators use for the growth of microorganisms in many fields, including medical, pharmaceuticals,
agricultural, environmental, food, and industrial microbiology, public health, basic research, and
education. Whereas CO2 incubators are most frequently used in medical research and the
pharmaceutical industry. However, they also provide sterile conditions for cultivation in other fields
where cells must grow in a completely germ-free environment.

 CO2 incubators for tissue-engineered products

 CO2 incubators for in vitro fertilization
 CO2 incubators in diagnostics
 CO2 for developing biosensors
 CO2 incubators in cancer research
Laminar flow hood/cabinet- Definition, parts, principle,
types, uses
A Laminar flow hood/cabinet is an enclosed workstation that is used to create a
contamination-free work environment through filters to capture all the particles
entering the cabinet.
 These cabinets are designed to protect the work from the environment and are most
useful for the aseptic distribution of specific media and plate pouring.
 Laminar flow cabinets are similar to biosafety cabinets with the only difference being that
in laminar flow cabinets the effluent air is drawn into the face of the user.
 In a biosafety cabinet, both the sample and user are protected while in the laminar flow
cabinet, only the sample is protected and not the user.

Components/ Parts of Laminar flow hood

A laminar flow cabinet consists of the following parts:

1. Cabinet
 The cabinet is made up of stainless steel with less or no gaps or joints preventing the
collection of spores.
 The cabinet provides insulation to the inner environment created inside the laminar flow
and protects it from the outside environment.
 The front of the cabinet is provided with a glass shield which in some laminar cabinets
opens entirely or in some has two openings for the user’s hands to enter the cabinet.
2. Working station
 A flat working station is present inside the cabinet for all the processes to be taken place.
 Culture plates, burner and loops are all placed on the working station where the
operation takes place.
 The worktop is also made up of stainless steel to prevent rusting.
3. Filter pad/ Pre-filter
 A filter pad is present on the top of the cabinet through which the air passes into the
cabinet.
 The filter pad traps dust particles and some microbes from entering the working
environment within the cabinet.
4. Fan/ Blower
 A fan is present below the filter pad that sucks in the air and moves it around in the
cabinet.
 The fan also allows the movement of air towards the HEPA filter sp that the remaining
microbes become trapped while passing through the filter.
5. UV lamp
 Some laminar flow hoods might have a UV germicidal lamp that sterilizes the interior of
the cabinet and contents before the operation.
 The UV lamp is to be turned on 15 minutes before the operation to prevent the exposure
of UV to the body surface of the user.
6. Fluorescent lamp
 Florescent light is placed inside the cabinet to provide proper light during the operation.
7. HEPA filter
 The High-efficiency particulate air filter is present within the cabinet that makes the
environment more sterile for the operation.
 The pre-filtered air passes through the filter which traps fungi, bacteria and other dust
particles.
 The filter ensures a sterile condition inside the cabinet, thus reducing the chances of
contamination.
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Principle/ Working of Laminar flow hood
 The principle of laminar flow cabinet is based on the laminar flow of air through the
cabinet.
 The device works by the use of inwards flow of air through one or more HEPA filters to
create a particulate-free environment.
 The air is taken through a filtration system and then exhausted across the work surface as
a part of the laminar flow of the air.
 The air first passes through the filter pad or pre-filter that allows a streamline flow of air
into the cabinet.
 Next, the blower or fan directs the air towards the HEPA filters.
 The HEPA filters then trap the bacteria, fungi and other particulate materials so that the
air moving out of it is particulate-free air.
 Some of the effluent air then passes through perforation present at the bottom rear end
of the cabinet, but most of it passes over the working bench while coming out of the
cabinet towards the face of the operator.
 The laminar flow hood is enclosed on the sides, and constant positive air pressure is
maintained to prevent the intrusion of contaminated external air into the cabinet.
Procedure for running the laminar flow cabinet
The procedure to be followed while operating a laminar flow cabinet is given below:
1. Before running the laminar flow cabinet, the cabinet should be checked to ensure that
nothing susceptible to UV rays is present inside the cabinet.
2. The glass shield of the hood is then closed, and the UV light is switched on. The UV light
should be kept on for about 15 minutes to ensure the surface sterilization of the working
bench.
3. The UV light is then switched off, and a time period of around 10 minutes is spared
before the airflow is switched on.
4. About 5 minutes before the operation begins, the airflow is switched on.
5. The glass shield is then opened, and the fluorescent light is also switched on during the
operation.
6. To ensure more protection, the working bench of the cabinet can be sterilized with other
disinfectants like 70% alcohol.
7. Once the work is completed, the airflow and florescent lamp both are closed and the
glass shield is also closed.
Types of laminar flow cabinet
Depending on the direction of movement of air, laminar flow cabinets are divided into two
types:

1. Vertical laminar flow cabinet

 In the vertical flow cabinets, the air moves from the top of the cabinet directly towards
the bottom of the cabinet.
 A vertical airflow working bench does not require as much depth and floor space as a
horizontal airflow hood which makes it more manageable and decreases the chances of
airflow obstruction or movement of contaminated air downstream.
 The vertical laminar flow cabinet is also considered safer as it doesn’t blow the air directly
towards the person carrying out the experiments.
2. Horizontal laminar flow cabinet
 In the horizontal laminar flow cabinets, the surrounding air comes from behind the
working bench, which is then projected by the blower towards the HEPA filters.
 The filtered air is then exhausted in a horizontal direction to the workplace environment.
 One advantage of this cabinet is that airflow parallel to the workplace cleanses the
environment with a constant velocity.
 The elluent air directly hits the operator, which might reduce the security level of this
type of laminar flow cabinets.
Uses of Laminar flow hood
The following are some common uses of a laminar flow cabinet in the laboratory:
1. Laminar flow cabinets are used in laboratories for contamination sensitive processes like
plant tissue culture.
2. Other laboratories processes like media plate preparation and culture of organisms can
be performed inside the cabinet.
3. Operations of particle sensitive electronic devices are performed inside the cabinet.
4. In the pharmaceutical industries, drug preparation techniques are also performed inside
the cabinet to ensure a particulate-free environment during the operations.
5. Laminar flow cabinets can be made tailor-made for some specialized works and can also
be used for general lab techniques in the microbiological as well as the industrial sectors.

Precautions
While operating the laminar airflow, the following things should be considered:
1. The laminar flow cabinet should be sterilized with the UV light before and after the
operation.
2. The UV light and airflow should not be used at the same time.
3. No operations should be carried out when the UV light is switched on.
4. The operator should be dressed in lab coats and long gloves.
5. The working bench, glass shield, and other components present inside the cabinet should
be sterilized before and after the completion of work.