Sterilization techniques

Sterilization is the process of removal of microorganisms by the use of physical or chemical agents.
or

Sterilization is a process in which all the living microorganisms, including bacterial spores are killed.

Sterilization can be achieved by physical and chemical methods.

A method employed to minimize the growth of organisms and transmission of disease from one individual to
another. In the environment the use of disinfection techniques decreases the growth of bacteria on surfaces, which
leads to the decrease in transmission of organisms amongst the population. These techniques are commonly used
today in medical care and food industry.

Physical methods:

• Heat (Dry and moist) • Sunlight

• Vibration • Radiation

• Filtration

Heat Sterilization

• Heat sterilization is the most effective and widely used method of sterilization, where the bactericidal activity
results through the destruction of enzymes and other essential cell constituents.
• The effects of heat sterilization occur more rapidly in a fully hydrated state, as it requires a lower heat input,
with low temperature and less time, under high humidity conditions where the denaturation and hydrolysis
reactions are predominant, rather than in the dry state where oxidative changes take place.

Under circumstances where thermal degradation of a product is possible, it can usually be minimized by adopting a
higher temperature range, as the shorter exposure times generally result in a lower partial degradation. Heat is
considered to be most reliable method of sterilization of objects that can withstand heat.
Heat as Moist and Dry heat are the most common sterilizing methods used in hospitals and are indicated for most
materials.

Moist heat Method

Here, the substance is subjected to sterilization by applying heat flame directly. Some of the most common moist
heat methods are boiling, pasteurization and autoclave method.

Boiling technique is widely used for sterilization of metallic devices like scalpels, surgical scissors, needles etc.
These substances are boiled to high temperatures to kill the disease-causing bacteria.

Pasteurization- It is the simple process of heating milk at high temperatures. Once the milk is boiled, it is again
subjected to cooling. In this way, microbes in the milk get killed automatically.

Autoclaving- It is a type of sterilization that uses steam sterilization equipment. Here, any substance is exposed to a
temperature of 115 degrees for an hour. It is a powerful method for killing bacterial spores.

Dry Heat Method

Dry Heat Method involves Flaming, incineration and hot air oven techniques. Causes denaturation of proteins and
oxidative damage.

Flaming- Here, the substance is exposed to the flame for just a few minutes. The flame will burn out the microbes
directly.

Incineration – It is an effective method of sterilization in microbe cultures. The end of the microbe loop is exposed
to red hot flame;thus, it kills microorganism. It is the easiest way to destroy microbes in metals.

Hot Air oven – The application of hot air oven is dry materials like glassware, heavy metals, thermostable
materials etc. Here, hot air is allowed to circulate at a certain time and temperature. This way hot air oven works.

Red Heat (common uses: straight wires, bacterial loops and spatulas)

Autoclave
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.
The simplest form of the autoclave is the pressure cooker types or laboratory bench autoclaves. The following is the
detailed description of different components/ parts of an autoclave:

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 the 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 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

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 on 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 tot eh 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 are
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.

Incubator

Definition: 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 the cultivation of microorganisms under
artificial conditions.

An incubator can be used for the cultivation of 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 the 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 in turn, reduces the 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.

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:

7. Incubators are used to grow microbial culture or cell cultures.

8. Incubators can also be used to maintain the culture of organisms to be used later.
9. Some incubators are used to increase the growth rate of organisms, having a prolonged growth rate in the
natural environment.
10. Specific incubators are used for the reproduction of microbial colonies and subsequent determination of
biochemical oxygen demand.
11. These are also used for breeding of insects and hatching of eggs in zoology.
12. Incubators also provide a controlled condition for sample storage before they can be processed in the
laboratories.

• This method of sterilization is applicable to thermostable products. Still, it can be applied to both moisture-
sensitive and moisture-resistant products, for which dry (160–180°C) and moist (121–134°C) heat sterilization
procedures are respectively used.
Moist Heat Sterilization

• Moist heat sterilization is one of the most effective methods of sterilization where the steam under pressure
acts as a bactericidal agent.
• Moist heat sterilization usually involves the use of steam at temperatures in the range 121–134°C.
• High pressure increases the boiling point of water and thus helps achieve a higher temperature for
sterilization.
• High pressure also facilitates the rapid penetration of heat into deeper parts of material and moisture present
in the steam causes the coagulation of proteins causing an irreversible loss of function and activity of
microbes.
• The high temperature-short time cycles not only often result in lower fractional degradation, but they also
provide the advantage of achieving higher levels of sterility assurance due to more significant inactivation
factors.
• The most commonly used standard temperature-time cycles for clinical porous specimens (e.g. surgical
dressings) and bottled fluids are 134°C for 3 minutes and 121°C for 15 minutes, respectively.
• An autoclave is a device that works on the principle of moist heat sterilization through the generation of
steam under pressure.
• In this method, the microorganisms are killed by coagulating their proteins, and this method is much more
effective than dry heat sterilization where microbes are killed through oxidation.
• In the pharmaceutical and medical sectors, it is used in the sterilization of dressings, sheets, surgical and
diagnostic equipment, containers, and aqueous injections, ophthalmic preparations, and irrigation fluids, in
addition to the processing of soiled and contaminated items.
• Moist heat can be used in sterilization at different temperatures:
At temperatures below 100°C
• The sterilization technique employed at a temperature below 100°C involves pasteurization.
• In this process, all non-spore forming microbes are killed in milk by subjecting the milk to a temperature of
63°C for 30 minutes (the holder method) or 73°C for 20 seconds (the flash method).
• In pasteurization, however, not all the pathogenic organisms are killed. The principle of pasteurization is
the logarithmic reduction in the number of viable microbes so that they can no longer cause diseases.
• All mesophilic non-sporing bacteria can be killed by exposure to a moist heat at 60C for half an hour with
the exception of some organisms which require different temperature-time cycles.
• The milk is not heated above its boiling point as the milk might curdle, and its nutritional value might be
destroyed.
• Besides milk, other fluids and equipment like vaccines of non-sporing bacteria are also pasteurized at 60°C
for 1 hour in special water baths.
• Similarly, serum and body fluids with congealable proteins are also sterilized at 56°C for 1 hour in water
baths.
At a temperature of 100°C
• Boiling at 100°C is a moist heat sterilization technique that doesn’t ensure complete sterility, but is enough
for the removal of pathogenic vegetative microbes and some spores.
• In this case, the items to be sterilized are immersed in boiling distilled water for 30-40 minutes.
• Distilled water is preferred because hard water might result in the formation of a film of calcium salts on
the instruments.
• Tyndallization is a method that is used for sterilization of media with sugar and gelatin at 100°C for 30
minutes on three successive days so as to preserve sugar which might be decomposed at a higher
temperature.
• Moist heat at 100°C is applicable for contaminated dishes, beddings, pipettes, and other instruments that
are not soiled or contaminated as well as for objects that are temperature sensitive.
At temperatures above 100°C
• Moist heat sterilization above 100°C involves sterilization by steam under pressure.
• 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.
 • 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.
• As a result, the steam under pressure has a higher penetrating power. When this steam comes in contact on
the surface, it kills the microbes by giving off latent heat.
• The condensed liquid ensures the moist killing of the microbes.
• Autoclaves are used for the sterilization of contaminated instruments along with different culture media as
it ensures complete sterility.
Dry heat sterilization

• Dry sterilization is the process of removing microorganisms by applying moisture-free heat which is
appropriate for moisture-sensitive substances.
• The dry heat sterilization process is based on the principle of conduction; that is the heat is absorbed by the
outer surface of an item and then passed onward to the next layer. Ultimately, the entire item reaches the
proper temperature needed to achieve sterilization.
• Dry moisture-less heat destroys microorganisms by causing denaturation of proteins and also lyses the
proteins in many organisms, causes oxidative free radical damage, causes drying of cells, and can even
burn them to ashes, as in incineration
• Dry heat sterilization is used for the sterilization of materials which are difficult to sterilize by moist heat
sterilization for several reasons.
• Substances like oil, powder, and related products cannot be sterilized by moist heat because moisture
cannot penetrate into deeper parts of oily materials, and powders are destroyed by moisture.
• Similarly, laboratory equipment like Petri dishes and pipettes are challenging to sterilize by moist heat due
to the penetration problem.
• The lethal effects of dry heat on microorganisms are primarily due to oxidative processes which are less
effective when compared to the hydrolytic damage that results from exposure to steam in moist heat
sterilization.
• Thus, in dry heat sterilization usually higher temperatures in the range 160–180°C are employed and also
require exposure times of up to 2 hours depending upon the temperature employed.
• This principle is used in instruments like hot air oven and incineration, which generates very hot moisture-
free air.
• The primary industrial application of dry heat sterilization is in the sterilization of glass bottles which are to
be filled aseptically.
• In addition to the fact that this method achieves an adequate sterility assurance level, this method also
destroys bacterial endotoxins (which are the products of Gram-negative bacteria also called pyrogens,
which cause fever when injected into the body) which are difficult to eliminate through other sterilization
techniques.
• For the purposes of depyrogenation of glass, temperatures of approximately 250°C are used.
• There are different types of dry heat sterilization which are explained below:
Red Heat
• Rest heat sterilization is the process of instant sterilization by holding the instruments in a Bunsen flame till
they become red hot.
• This method is based on dry heat sterilization is commonly used for sterilization of instruments like
incubation loops, wires, and points of forceps.
• This process ensures effective sterilization; however, it is only limited to substances that can endure heating
until redness in flame.
Flaming
• Flaming is a type of dry sterilization that involves exposure of metallic objects to flame for some time
where the flame burns microbes and other dust presents in the instrument.
• In the case of flaming, the instrument is dipped in alcohol or spirit before burning it in a gas flame.
• This process doesn’t ensure sterility and is not as effective as red hot sterilization.
Incineration
• Incineration is the process of sterilization along with a significant reduction in the volume of the wastes. It
is usually conducted during the final disposal of the hospital or other residues.
• The scraps are heated till they become ash which is then disposed of later.
 • This process is conducted in a device called incinerator.
Infrared radiation
• Infrared radiation (IR) is a method of thermal sterilization in which the radiation is absorbed and then
converted into heat energy.
• For this purpose, a tunnel containing an IR source is used. The instruments and glassware to be sterilized
are kept in a tray are then passed through the tunnel on a conveyer belt, moving at a controlled speed.
• During this movement, the instruments will be exposed to the radiation, which will result in a temperature
of about 180°C for about 17 minutes.
• IR is applicable for mass sterilization of packaged items like syringes and catheters.
Hot air oven
• Hot air oven is a method of dry heat sterilization which allows the sterilization of objects that cannot be
sterilized by moist heat.
• It uses the principle of conduction in which the heat is first absorbed by the outer surface and is then passed
into the inner layer.
• A hot air oven consists of an insulated chamber that contains a fan, thermocouples, temperature sensor,
shelves and door locking controls.
• The commonly-used temperatures and time that hot air ovens need to sterilize materials are 170°C for 30
minutes, 160°C for 60 minutes, and 150°C for 150 minutes.
• These ovens have applications in the sterilization of glassware, Petri plates, and even powder samples.

Filtration

• The process of filtration is unique among sterilization techniques in that it removes, rather than destroys,
microorganisms.
• Further, it is capable of preventing the passage of both viable and nonviable particles and can thus be used
for both the clarification and sterilization of liquids and gases.
• The primary mechanisms involved in filtration are sieving, adsorption, and trapping within the matrix of
the filter material.
• Filtration uses membranous filters that have tiny pores that let the liquid pass through but prevent bigger
particles such as bacteria from passing through the filter. Therefore, the smaller the pore, the more likely
the filter is to stop more things from going through it.
• Certain types of filter (membrane filters) also have an essential role in sterility testing, where they can be
employed to trap and concentrate contaminating organisms from solutions under test.
• These filters are then placed in a liquid nutrient medium and incubated to encourage growth and turbidity.
• The principal application of sterilizing-grade filters is the treatment of heat-sensitive injections and
ophthalmic solutions, biological products, air, and other gases for supply to aseptic areas.
• They may also be required in industrial applications where they become part of venting systems on
fermenters, centrifuges, autoclaves, and freeze dryers.
Filtration sterilization of liquids
• Membrane filters, in the form of discs, can be assembled into pressure-operated filter holders for syringe
mounting and in-line use or vacuum filtration tower devices for filtration of liquid.
• Filtration under pressure is generally considered most suitable, as filling at high flow rates directly into the
final containers is possible without problems of foaming, solvent evaporation, or air leaks.
• Membrane filters are often used in combination with a coarse-grade fiberglass depth prefilter to improve
their dirt-handling capacity.
Filtration sterilization of gases
• Filters employed for this generally consist of pleated sheets of glass microfibres separated and supported by
corrugated sheets of Kraft paper or aluminum which are employed in ducts, wall or ceiling panels, or
laminar air flow cabinets.
• These high-efficiency particulate air (HEPA) filters can remove up to 99.997% of particles >0.3mm in
diameter and thus are acting as depth filters.
• In practice, their microorganism removal efficiency is rather better as the majority of bacteria are found
associated with dust particles.
 • Other applications of filters include sterilization of venting or displacement air in tissue and
microbiological culture (carbon filters and hydrophobic membrane filters); decontamination of air in
mechanical ventilators (glass fiber filters); treatment of exhausting air from microbiological safety cabinets
(HEPA filters); and the clarification and sterilization of medical gases (glass wool depth filters and
hydrophobic membrane filters).

Irradiation

• Irradiation is the process of exposing surfaces and objects to different kinds of radiation for sterilization.
• Mainly electromagnetic radiation is used for sterilization.
• The major target for these radiations is considered to be microbial DNA, where damage occurs as a result
of ionization and free radical production (gamma-rays and electrons) or excitation (UV light).
Ultraviolet (non-ionizing) radiation
• Ultraviolet radiation includes light rays from 150-3900 Å, of which 2600 Å has the highest bactericidal
effect.
• Non-ionizing waves have a very little penetration power, so microorganisms only on the surface are killed.
• Upon exposure, these waves are absorbed by many materials, particularly nucleic acids.
• The waves, as a result, cause the formation of pyrimidine dimers which bring error in DNA replication and
cause the death of microbes by mutation.
• UV radiation owing to its poor penetrability of conventional packaging materials is unsuitable for
sterilization of pharmaceutical dosage forms.
• It is, however, applied in the sterilization of air, for the surface sterilization of aseptic work areas, and the
treatment of manufacturing-grade water.
Ionizing Radiation
• X-ray and gamma rays are the commonly used ionizing radiation for sterilization.
• These are high energy radiation which causes ionization of various substances along with water.
• The ionization results in the formation of a large number of toxic O2metabolites like hydroxyl radical,
superoxide ion, and H2O2 through ionization of water.
• These metabolites are highly oxidizing agents and kill microorganisms by oxidizing various cellular
components.
• With ionizing radiation, microbial resistance decreases with the presence of moisture or dissolved oxygen
(as a result of increased free radical production) and also with elevated temperatures.
• Radiation sterilization is generally exposed to items in the dried state which include surgical instruments,
sutures, prostheses, unit-dose ointments, plastic syringes, and dry pharmaceutical products.

Sonic (sonic) waves Vibration

• Sonic waves can be used as bactericidal agents which employ ultrasound (usually from 20–40 kHz) to
vibrate a fluid.
• The ultrasound can be used with just water, but the use of a solvent appropriate for the object to be cleaned
and the type of soiling present enhances the effect.
• The explanation for the antibacterial activity of airborne sound waves on a physical is based on the possible
transformation of acoustic energy into heat.
• The impact of airborne sound differs with the medium as the absorption of acoustic energy is greatly
affected by the nature of the medium through which it is transmitted.
• As the sound waves propagate through a detergent solution, these ultrasonic waves produce alternating
tensile and compressive forces that oscillate, and these oscillating forces cause millions of microscopically-
sized cavities to form in the detergent solution.
• Once they reach a maximum size, these cavities violently collapse, causing submicroscopic voids to form
which induce the formation of high-energy hydraulic shock waves.
• These shock waves, which may reach temperatures as high as 10,000°F and hydrodynamic pressures as low
as 10,000 PSI, physically loosen and remove microorganisms and other adhering debris from even the most
inaccessible surfaces of a contaminated instrument.
• The antibacterial activity of airborne sound waves is directly related to the sound intensity, the period of
irradiation, and the distance of the sample from the sound source.
 • These sonic waves are found to be effective against bacteria spores depending on the chemical composition
of these spores.
• The lipoidal substances within the microbes readily absorb ultrasonic frequencies, and any changes in the
lipid level of the test organism directly affect the amount of acoustic energy taken up by the spore as well
as the removal of those spores.
• This method is routinely used by healthcare facilities to clean surgical and dental instruments before the
terminal sterilization.

Pressure (Pascalization)
• Pascalization or High-Pressure Processing (HPP) is a method employed for preservation and sterilization of
food, in which products are processed under very high pressure (hundreds of megapascal), leading to the
death of specific microorganisms and inactivation of enzymes in the food.
• HHP treatments may be applied at room temperature, and with the exception of some vegetables, shape,
color, and nutrients of most foods are not affected.
• Hydrostatic pressures are nonthermal, and covalent bonds are not broken, so that flavor is unaffected.
• At 400-600 MPa, proteins are easily denatured, and cell morphology is altered, and ribosomes are
destroyed.
• Changes occur in the lipid-protein complex of cell membranes, and increased membrane fluidity is also
observed, which causes leakage of nucleic acids.
• Pascalization is not particularly effective against spores, but combined treatment with heat is found to be
effective in the inactivation of spores.
• Pascalization is especially useful on acidic foods, such as yogurts and fruits, because spores which are
pressure-tolerant don’t have the ability to live in environments with low pH.
• The treatment works equally well for both solid and liquid products.

Sunlight (Solar Disinfection)

• Solar disinfection is a process used for the removal of microorganisms with the help of sunlight.
• This process is commonly used to purify or disinfect drinking water.
• Solar disinfection is based on the inactivation of pathogenic organisms as a result of the UV-A (wavelength
320–400 nm) part of the sunlight, which reacts with oxygen dissolved in the water and releases highly
reactive forms of oxygen (oxygen free radicals and hydrogen peroxides).
• These metabolites damage pathogens, while it also interferes with metabolism and destroys bacterial cell
structures, and simultaneously the full band of solar energy (from infrared to UV) heats the surface.
• The principle of solar disinfection is similar to that of radiation sterilization; however, the efficacy of solar
disinfection is significantly low as it requires a long period of exposure.
• However, this process is economical and an environment-friendly option.

Chemical Sterilization
What is Chemical Sterilization?

Chemical Sterilization is the process of removal of microorganisms by the use of chemical bactericidal
agents.
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.
The chemical method of sterilization can be categorized as liquid and gaseous sterilization.

Several chemical agents are used as antiseptic and disinfectants. The properties of a chemical antiseptic or
disinfectant are following
 • The chemical disinfectants need to have a broad spectrum of activity against all microorganisms such as
bacteria, viruses, protozoa and fungi.
• The chemical agents should act in the presence of organic matter.
• High penetration power is an important property of the chemical agents
• The chemical agent needs to be chemically stable under both acidic and basic environments.
• The chemical substances should not have any corrosion activity in metals.
• The disinfectants are needed to be non-toxic if absorbed into circulation.
• Finally, the chemical agents are needed to be easily available and less expensive.

1. Alcohol:
▪ Alcohols are antimicrobial agents. Germicidal action of alcohol increases with increase in molecular weight
of alcohol.
▪ Ethanol is the most commonly used alcohol for controlling microorganisms.
▪ Ethanol between concentration of 50-90% are effective against vegetative cell. for practical purposes 70%
ethanol is used.
▪ Alcohol causes death of organism by denaturing the cellular proteins.
▪ Alcohol is a lipid solvent that damages the lipid bilayer of cell membrane and cell wall. It is also a
dehydrating agent and causes loss of water from cell.

Uses:

▪ Alcohol is commonly used as sanitizer on skin, disinfectant to clinical instruments, thermometers and
surgical instruments.
▪ Concentration above 60% is effective in killing viruses.

Mode of action:

▪ When alcohol is used as disinfectant, it solubilizes the lipid bilayer of cell wall and membrane and creates
pores. The remaining alcohol enters into the cytoplasm through the pore and denature the cellular proteins
killing the bacteria.
▪ 70% alcohol is more effective than absolute (100%) alcohol because absolute alcohol only brings
bacteriostasis.
▪ With increase in concentration of alcohol, both denaturing and lipid solubilizing power and dehydration
power increases and are counter to each other. Absolute alcohol causes extreme dehydration resulting in
shrinking of cell, hence further alcohol cannot enter the cell. Therefore it only brings bacteriostasis.
▪ However, 70% alcohol is very effective in dehydration and denaturation, almost equilibrium manner
causing bacteriocidal effects.
2. Phenol and phenolic compounds
▪ Phenol have wide spectrum of antimicrobial action.
▪ Vegetative cell are more and rapidly killed by concentrated aqueous solution of phenol whereas bacterial
spore are resistant.
▪ Usually 2-5% aqueous solution of phenol is used as disinfectant.
▪ Phenol has limited application because it is absorbed by skin and mucus membrane and causes toxicity.

Some of the derivatives of phenolic compound and their application are:

i) Cresol (methyl phenol): it is used in lysol in solution form to sterilize glasswares and to mop the floor of
hospital rooms.
ii) Chloroxylenol (dimethyl phenol): it is an active ingredient of dettol and commonly used as antiseptic.

iii) Chlorohedidane (Hibitane): it is one of the component of Savlon, which is used as antiseptic in burns, wounds
and preoperative antisepsis of skin.

iv) Hexachlorophane: it is insoluble in water. It is used in soap.

Mode of action:

▪ Phenol and phenolic compounds kills the microorganisms by varities of effects such as disruption of cell,
precipitation of cellular protein, inactivation of enzymes and leakage of cellular materials.
3. Halogen compounds:
i. Iodine:
▪ Iodine is used in many forms such as aqueous solution, tincture of iodine and iodophor.
▪ Aqueous iodine and tincture of iodine have some side effects such as staining and irritation. So, now a days
Iodophore is as replacement because it has less side effects.

Uses:

▪ Iodine is effective against all kind of bacteria. it also possess sporicidal activity.
▪ Iodine is highly fungicidal and to some extent virucidal.
▪ Iodophore are widely used for antisepsis of skin, mucus membrane and wound.
▪ Iodine preparation can also be used for other purposes such as disinfection of water, air and sanitization of
food utensils.

Mode of action:

▪ Iodine is powerful oxidizing agents and irreversibly oxidises the cellular materials.
▪ Iodine also brings halogenation of tyrosine residue of protein and enzymes and inactivates it.
ii. Chlorine:
▪ Chlorine in the form such as Hypochlorite and chloramine is used as disinfectant. Free gaseous chlorine is
difficult to handle, as it is corrosive and toxic.
▪ Calcium hypochlorite and sodium hypochlorite are commonly used.
▪ Aqueous solution of sodium hypochlorite (5.25%) is called house hold bleach.
▪ Chloramine is more stable than hypochlorite, so it is more effective germicidal than hypochlorites.

uses:

▪ Cholrine is one of the commonly used water disinfectant.

▪ Calcium hypochlorite is used as sanitizer for cooking utensils.
▪ 1% bleach is used for personal hygiene eg. bathing water
▪ Higher concentration (5-12%) bleach is used in swimming pool, house hold purposes.

Mode of action:

▪ when hypochlorite or chloramine is added in water, free chlorine is releases which form hypochlorous acid
(HClO).
 ▪ Hypochlorous acid decomposes to release nascent oxygen which is powerful oxidizing agent and kills the
microorganisms by oxidizing the cellular components.
▪ chlorine and chlorine compounds also inactivates the proteins and enzymes by direct chlorination.
4. Heavy metals and their compounds:
▪ Most of the heavy metals have antimicrobial action.
▪ Most effective and commonly use are Mercucry (Hg), Silver (Ag) and Cupper (Cu).

Mode of action:

▪ Heavy metals and their compounds combines directly with cellular proteins and enzymes and inactivates
them.
▪ High concentration of heavy metals salts also coagulates and precipitates the cellular proteins and kills the
microorganisms.

Some commonly used metal compounds are:

i) HgCl, HgCl2: used in ointments as antiseptic.

ii) AgNO3: it is bacteriostatic as well as bactericidal. it is used in eye-drops to prevent Ophthalmia neonatarum in
children.

iii) Cupper suphate: it is widely used against algae and mold in swimming pool.

5. Aldehydes:
▪ Formaldehyde and Gluteraldehyde are commonly used aldehydes. Both are highly microbicidal including
sporicidal.
i. Formaldehyde:
▪ Formaldehyde is stable only in higher concentration and higher temperature. At room temperature, it
polymerized to form para-formaldehyde.
▪ Formaldehyde is used in two form- gaseous formaldehyde and formalin (40%solution of formaldehyde).

Uses:

▪ Formaldehyde vapour either from formalin or paraformaldehdye is used for disinfection and sterilization of
closed room, such as Operation theater.
▪ Formaldehyde vapour is also used to disinfect woolen blanket, wools and footwares of fungal infected
person.
▪ Formalin is used for preservation of biological specimens.
ii. Gluteraldehyde:
▪ Gluteraldehye is used in 2% solution.
▪ like formaldehyde it is effective against bacteria, fungi, spores and viruses.
▪ Gluterladehyde is used to sterilize Urological instruments and respiratory therapy instruments.
6. Gaseous agents:
▪ Ethylene oxide, β-propiolactone and formaldehyde are commonly used gaseous sterilizing agents.
i. Ethylene oxide:
▪ It is gaseous above 10.8°C.
▪ Ethylene oxide have high antimicrobial activity, it kills even endospores.
▪ It is used for sterilization of heat sensitive materials such as spices, oils, plastics etc.
 ▪ Ethylene oxide is used in formulation with CO2 as Freon (CClFe).
ii. β-Propiolactone:
▪ It is gas above 15.5°C.
▪ Penetration power of β-propiolactone is less than ethylene oxide but it is more active in killing
microorganisms.
▪ Due to its carcinogenic effects, it is not commonly used.
7. Detergents:
▪ Detergents are used primarily for leaning purposes but it has also antimicrobial properties.
▪ There are three types of detergents- Cationic detergent, anionic detergent and non-ionic detergent.
▪ Cationic detergents is more significant germicidal agent than other two.
▪ For example: Quaternary ammonium compound is a cationic detergent having germicidal action. It is more
effective against Gram positive bacteria.
▪ Detergents are used as disinfectants, sanitizers and antiseptic. They are also used to disinfect hospital floor.

Mode of action:

▪ Detergents kills the microorganisms by denaturing proteins and enzymes and interfering with glycolysis
▪ Detergents also damages cell wall and cell membrane.
8. Antibiotics:
▪ Antibiotics are secondary metabolites produced by certain microorganisms which inhibits the growth of
other microorganisms.
▪ Different groups of antibiotics have different mode of actions

Mode of action:

▪ Inhibits cell wall peptidoglycan synthesis: eg, penicillin, cephalosporin

▪ Inhibit cell membrane biosynthesis: eg, Polymyxin, Polyenes
▪ Inhibits protein synthesis: eg, Tetracycline, Chloramphenical
▪ Reacts with nucleic acids: Rifampin, Quinolone
▪ Inhibits folic acid synthesis: eg, Sulfonamide, trimethoprim
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.
• Some of the common gases used for gaseous sterilization are explained below:

i. Ethylene oxide
• Ethylene oxide (EO) gas is a common gas used for chemical treatment applied to sterilize, pasteurize, or
disinfect different types of equipment and surfaces because of its wide range of compatibility with different
materials.
• EO treatment often replaces other sterilization techniques like heat, radiation, and even chemicals in cases
where the objects are sensitive to these techniques.
• This method is a widespread method used for almost 70% of all sterilizations and around 50% for
disposable medical devices.
• The mechanism of antimicrobial action of this gas is assumed to be through the alkylation of sulphydryl,
amino, hydroxyl, and carboxyl groups on proteins and imino groups of nucleic acids.
• EO treatment is usually conducted at the temperature range of 30-60°C for several hours which aids in the
activity of the gas.
• The efficacy of the gas depends on the concentration of gas available for each article which is greatly
assisted by the good penetrating nature of the gas, which diffuses readily into many packaging materials
including rubber, plastics, fabric, and paper.
• Ethylene oxide kills all known microorganisms, such as bacteria (including spores), viruses, and fungi
(including yeasts and molds), and is compatible with almost all materials even when repeatedly applied.
• This process, however, is not without drawbacks as the level of gas in the sterilizer goes on decreasing due
to absorption, and the treated articles need to undergo a process of desorption to remove the toxic residual
wastes.
• Organisms are more resistant to ethylene oxide treatment in a dried state, as are those protected from the
gas by inclusion in crystalline or dried organic deposits.

ii. Formaldehyde

• Formaldehyde is another important highly reactive gas which is used for sterilization.
• This gas is obtained by heating formalin (37%w/v) to a temperature of 70-80°C.
• It possesses broad-spectrum biocidal activity and has found application in the sterilization of reusable
surgical instruments, specific medical, diagnostic and electrical equipment, and the surface sterilization of
powders.
• Formaldehyde doesn’t have the same penetrating power of ethylene oxide but works on the same principle
of modification of protein and nucleic acid.
• As a result of the low penetrating power, its use is often limited to paper and cotton fabrics.
• Formaldehyde can generally be detected by smell at concentrations lower than those permitted in the
atmosphere and thus can be detected during leakage or other such accidents.

iii. Nitrogen dioxide(NO2)

• Nitrogen dioxide is a rapid and effective sterilant that can be used for the removal of common bacteria,
fungi, and even spores.
• NO2 has a low boiling point (20°C) which allows a high vapor pressure at standard temperature.
• This property of NO2 enables the use of the gas at standard temperature and pressure.
• The biocidal action of this gas involves the degradation of DNA by the nitration of phosphate backbone,
which results in lethal effects on the exposed organism as it absorbs NO2.
• An advantage of this gas is that no condensation of the gas occurs on the surface of the devices because of
the low level of gas used and the high vapor pressure. This avoids the need for direct aeration after the
process of sterilization.

iv. Ozone

• Ozone is a highly reactive industrial gas that is commonly used to sterilize air and water and as a
disinfectant for surfaces.
• Ozone is a potent oxidizing property that is capable of destroying a wide range of organisms including
prions, without the use of hazardous chemicals as ozone is usually generated from medical-grade oxygen.
 • Similarly, the high reactivity of ozone allows the removal of waste ozone by converting the ozone into
oxygen by passing it through a simple catalyst.
• However, because ozone is an unstable and reactive gas, it has to be produced on-site, which limits the use
of ozone in different settings.
• It is also very hazardous and thus only be used at a concentration of 5ppm, which is 160 times less than that
of ethylene oxide.

2. 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.
• Different liquid chemicals used for liquid sterilization includes the following:

i. Hydrogen peroxide

• Hydrogen peroxide is a liquid chemical sterilizing agent which is a strong oxidant and can destroy a wide
range of microorganisms.
• It is useful in the sterilization of heat or temperature-sensitive equipment like endoscopes. In medical
applications, a higher concentration (35-90%) is used.
• H2O2 has a short sterilization cycle time as these cycles are as short as 28 minutes where ethylene oxide has
cycles that as long as 10-12 hours.
• However, hydrogen peroxide has drawbacks like low material compatibility, lower capacity of penetration,
and associated health risks.
• Vaporized hydrogen peroxide (VHP) is used to sterilize largely enclosed and sealed areas, such as entire
rooms and aircraft interiors.

ii. Glutaraldehyde

• Glutaraldehyde is an accepted liquid sterilizing agent which requires comparatively long immersion time.
For the removal of all spores, it requires as long as 22 hours of immersion time.
• The presence of solid particles further increases the immersion time.
• The penetration power is also meager as it takes hours to penetrate a block of tissues.
• The use of glutaraldehyde is thus limited to certain surfaces with less contamination.

iii. Hypochlorite

• Hypochlorite solution, which is also called liquid bleach, is another liquid chemical that can be used as a
disinfectant, even though sterilization is difficult to obtain with this chemical.
• Submerging devices for a short period in liquid bleach might kill some pathogenic organisms but to reach
sterilization submersion for 20-24 hours is required.
• It is an oxidizing agent and thus acts by oxidizing organic compounds which results in the modification of
proteins in microbes which might ultimately lead to death.
• Appropriate concentrations of hypochlorite can be used for the disinfection of workstations and even
surfaces to clean blood spills and other liquids.
 Microscopy
Bright field Microscope Definition

Bright field Microscope is also known as the Compound Light Microscope. It is an optical microscope that uses
light rays to produce a dark image against a bright background. It is the standard microscope that is used in
Biology, Cellular Biology, and Microbiological Laboratory studies.
This microscope is used to view fixed and live specimens, that have been stained with basic stains which gives a
contrast between the image and the image background. It is specially designed with magnifying glasses known as
lenses that modify the specimen to produce an image seen through the eyepiece.

Principle of Bright field Microscope

For a specimen to be the focus and produce an image under the Brightfield Microscope, the specimen must pass
through a uniform beam of the illuminating light. Through differential absorption and differential refraction, the
microscope will produce a contrasting image.

The specimens used are prepared initially by staining to introduce color for easy contracting characterization. The
colored specimens will have a refractive index that will differentiate it from the surrounding, presenting a
combination of absorption and refractive contrast.

The functioning of the microscope is based on its ability to produce a high-resolution image from an adequately
provided light source, focused on the image, producing a high-quality image.

The specimen which is placed on a microscopic slide is viewed under oil immersion or/and covered with a
coverslip.

Parts of Bright Parts of Bright

The bright field microscope is made up of various parts, including

• Eyepiece (Ocular lens) – it has two eyepiece lenses at the top of the microscope which focuses the image
from the objective lenses. this is where you see the formed image from, with your eyes.
• The objective lenses which are made up of six or more glass lenses, which make a clear image clear from
the specimen or the object that is being focused.
• Two focusing knobs i.e the fine adjustment knob and the coarse adjustment knob, found on the
microscopes’ arm, which can move the stage or the nosepiece to focus on the image. Their function is to
ensure the production of a sharp image with clarity.
 • The stage is found just below the objectives and this is where the specimen is placed, allowing movement
of the specimen around for better viewing with the flexible knobs and it is where the light is focused on.
• The condenser: It is mounted below the stage which focuses a beam of light onto the specimen. It can be
fixed or movable, to adjust the quality of light, but this entirely depends on the microscope.
• The arm: This is a sturdy metallic backbone of the microscope, used to carry and move the microscope
from one place to another. They also hold the microscope base which is the stand of the microscope. The
arm and the base hold all the microscopic parts.
• It has a light illuminator or a mirror found at the base or on the microscope’s nosepiece.
• The nosepiece has about two to five objective lenses with different magnifying power. It can move round
to any position depending on the objective lens to focus on the image.
• An aperture diaphragm (contrast): It controls the diameter of the beam of light that passes through the
condenser. When the condenser is almost closed, the light comes through to the center of the condenser
creating high contrast and when the condenser is widely open, the image is very bright with very low
contrast.
Magnification by Bright field Microscope
• The objective lenses are the main lenses used for focusing the image, on the condenser. This produces an
enlarged clear image that is then magnified again by the eyepiece to form the primary image that is seen by
the eyes.
• During imaging, the objective lenses remain parfocal in that, even when the objective lens has changed the
image still remains focused. The image seen at the eyepiece is the enlarged clear image of the specimen,
known as the virtual image.
• The magnification of the image is determined by the magnification of the objective against the
magnification of the eyepiece lens. The objectives have a magnification power of 40x-1000x depending on
the type of brightfield microscope while the eyepiece lens has a standard magnification power of 10x.
• Therefore to calculate:
Total Magnification power = Magnification of the objective lens x Magnification of the eyepiece
• For example: if the magnification of the objective is 45x and that of the eyepiece is 10x, the total
magnification of the specimen will be 450x.
• The magnification is standard, i.e not too high nor too low, and therefore depending on the magnification
power of the lenses, it will range between 40X and 100oX.
• The objective lens enlarges the image which can be viewed, a characteristic known as
resolution. Resolution according to Prescott, is the ability of a lens to separate or distinguish between small
objects closely linked together.
• Whereas the eyepiece magnifies the image at the end of the viewing, its magnification range is lower than
that of the objective lens at 8X-12X (10X standard) and that of the objective lens at 40X-100X,
magnification, and resolution of the microscope is highly dependant on the objective lens.

Applications of Bright field microscope

Bright field Microscope is used in several fields, from basic biology to understanding cell structures in cell Biology,
Microbiology, Bacteriology to visualizing parasitic organisms in Parasitology.

Most of the specimens to viewed are stained using special staining to enable visualization. Some of the staining
techniques used include Negative staining and Gram staining.
Some of its applications include:
1. Used to visualize and study the animal cells
2. Used to visualize and study plant cells.
3. Used to visualize and study the morphologies of bacterial cells
Used to identify parasitic protozoans such as Paramecium

Advantages of Bright field microscope

1. It is simple to use with few adjustments involved while viewing the image.
2. It can be used to view both stained and unstained.
 3. The optics of the microscope do not alter the color of the specimen.
4. The microscope can be adjusted and modified for better viewing such as installing a camera, to form a
digital microscope or in the way image illumination is done such as by use of fluorochromes on the
specimen and viewing under a dark environment, forming a darkfield microscope.

Disadvantages
1. The aperture diaphragm may cause great contrast which may distort the outcome of the image, therefore
iris diaphragm is preferred.
2. It can not be used to view live specimens such as bacterial cells. Only fixed specimens can be viewed under
the brightfield microscope.
3. Maximum magnification of the brightfield microscope is 100x but modification can readjust the
magnification to 1000x which is the optimum magnification of bacterial cells.
4. It has low contrast hence most specimens must be stained for them to be visualized.
5. Use of oil immersion may distort the image
6. The use of coverslip may damage the specimen
7. Staining may introduce extraneously unwanted details into the specimen or contaminate the specimen.
8. It is tedious to stain the specimen before visualizing it under the brightfield microscope.
9. The microscope needs a strong light source for magnification and sometimes the light source may produce
a lot of heat which may damage or kill the specimen.

Phase contrast Microscopy- definition, principle, parts and uses

Phase contrast Microscopy- definition

• Unstained living cells absorb practically no light. Poor light absorption results in extremely small
differences in the intensity distribution in the image. This makes the cells barely, or not at all, visible in a
 brightfield microscope. Phase-contrast microscopy is an optical microscopy technique that converts phase
shifts in the light passing through a transparent specimen to brightness changes in the image.
• It was first described in 1934 by Dutch physicist Frits Zernike.

Principle of Phase contrast Microscopy

When light passes through cells, small phase shifts occur, which are invisible to the human eye. In a phase-contrast
microscope, these phase shifts are converted into changes in amplitude, which can be observed as differences in
image contrast.

The Working of Phase contrast Microscopy

1. Partially coherent illumination produced by the tungsten-halogen lamp is directed through a collector lens
and focused on a specialized annulus (labeled condenser annulus) positioned in the substage condenser
front focal plane.
2. Wavefronts passing through the annulus illuminate the specimen and either pass through undeviated or are
diffracted and retarded in phase by structures and phase gradients present in the specimen.
3. Undeviated and diffracted light collected by the objective is segregated at the rear focal plane by a phase
plate and focused at the intermediate image plane to form the final phase-contrast image observed in the
eyepieces.

Parts of Phase contrast Microscopy

Phase-contrast microscopy is basically a specially designed light microscope with all the basic parts in addition to
which an annular phase plate and annular diaphragm are fitted.
The annular diaphragm
• It is situated below the condenser.
• It is made up of a circular disc having a circular annular groove.
• The light rays are allowed to pass through the annular groove.
• Through the annular groove of the annular diaphragm, the light rays fall on the specimen or object to be
studied.
• At the back focal plane of the objective develops an image.
• The annular phase plate is placed at this back focal plane.
The phase plate
• It is either a negative phase plate having a thick circular area or a positive phase plate having a thin circular
groove.
• This thick or thin area in the phase plate is called the conjugate area.
• The phase plate is a transparent disc.
• With the help of the annular diaphragm and the phase plate, the phase contrast is obtained in this
microscope.
• This is obtained by separating the direct rays from the diffracted rays.
• The direct light rays pass through the annular groove whereas the diffracted light rays pass through the
region outside the groove.
• Depending upon the different refractive indices of different cell components, the object to be studied shows
a different degree of contrast in this microscope.
Applications of Phase contrast Microscopy

To produce high-contrast images of transparent specimens, such as

1. living cells (usually in culture),
2. microorganisms,
3. thin tissue slices,
4. lithographic patterns,
5. fibers,
6. latex dispersions,
7. glass fragments, and
8. subcellular particles (including nuclei and other organelles).
Applications of phase-contrast microscopy in biological research are numerous.

Advantages

• Living cells can be observed in their natural state without previous fixation or labeling.
• It makes a highly transparent object more visible.
• No special preparation of fixation or staining etc. is needed to study an object under a phase-contrast
microscope which saves a lot of time.
• Examining intracellular components of living cells at relatively high resolution. eg: The dynamic motility
of mitochondria, mitotic chromosomes & vacuoles.
• It made it possible for biologists to study living cells and how they proliferate through cell division.
• Phase-contrast optical components can be added to virtually any brightfield microscope, provided the
specialized phase objectives conform to the tube length parameters, and the condenser will accept an
annular phase ring of the correct size.

Limitations

• Phase-contrast condensers and objective lenses add considerable cost to a microscope, and so phase
contrast is often not used in teaching labs except perhaps in classes in the health professions.
• To use phase-contrast the light path must be aligned.
 • Generally, more light is needed for phase contrast than for corresponding bright-field viewing, since the
technique is based on the diminishment of the brightness of most objects.

Electron microscopy

Electron microscope definition

• An electron microscope is a microscope that uses a beam of accelerated electrons as a source of illumination.
• It is a special type of microscope having a high resolution of images, able to magnify objects in nanometres,
which are formed by controlled use of electrons in vacuum captured on a phosphorescent screen.
• Ernst Ruska (1906-1988), a German engineer and academic professor, built the first Electron Microscope in
1931, and the same principles behind his prototype still govern modern EMs.

Working Principle of Electron microscope

Electron microscopes use signals arising from the interaction of an electron beam with the sample to obtain
information about structure, morphology, and composition.
1. The electron gun generates electrons.
2. Two sets of condenser lenses focus the electron beam on the specimen and then into a thin tight beam.
3. To move electrons down the column, an accelerating voltage (mostly between 100 kV-1000 kV) is applied
between tungsten filament and anode.
4. The specimen to be examined is made extremely thin, at least 200 times thinner than those used in the optical
microscope. Ultra-thin sections of 20-100 nm are cut which is already placed on the specimen holder.
5. The electronic beam passes through the specimen and electrons are scattered depending upon the thickness or
refractive index of different parts of the specimen.
6. The denser regions in the specimen scatter more electrons and therefore appear darker in the image since
fewer electrons strike that area of the screen. In contrast, transparent regions are brighter.
7. The electron beam coming out of the specimen passes to the objective lens, which has high power and forms
the intermediate magnified image.
8. The ocular lenses then produce the final further magnified image.

Types of Electron microscope

There are two types of electron microscopes, with different operating styles:
1. The transmission electron microscope (TEM)
Transmission Electron Microscope(TEM) definition

• This is a powerful electron microscope that uses a beam of electrons to focus on a specimen producing a
highly magnified and detailed image of the specimen.
• The magnification power is over 2 million times better than that of the light microscope, producing the
image of the specimen which enables easy characterization of the image in its morphological features,
compositions and crystallization information is also detailed.
• Early discovery of cathode rays like electrons by Louis de Broglie in the early 1920s, paved way into the
development of an electron microscope where they used a beam of electrons creating a form of wave
motion.
• Magnetic fields were used as lenses for the electrons. With these discoveries, the first electron microscope
was later developed by Ernst Ruska and Max Knolls in 1931 and modified into a Transmission Electron
Microscope (TEM) by Ernst Ruska along with the Sieman’s company, in 1933.
• This TEM microscope has several advantages compared to the light microscope with its efficiency also
being very high.
• Among all microscopes both light and electron microscopes, TEM are the most powerful microscopes used
in laboratories. It can magnify a mall particle of about 2nm, and therefore they have a resolution limit of
0.2um.

Principle of Transmission Electron Microscope (TEM)

The working principle of the Transmission Electron Microscope (TEM) is similar to the light microscope. The
major difference is that light microscopes use light rays to focus and produce an image while the TEM uses a beam
of electrons to focus on the specimen, to produce an image.
Electrons have a shorter wavelength in comparison to light which has a long wavelength. The mechanism of a light
microscope is that an increase in resolution power decreases the wavelength of the light, but in the TEM, when the
electron illuminates the specimen, the resolution power increases increasing the wavelength of the electron
transmission. The wavelength of the electrons is about 0.005nm which is 100,000X shorter than that of light, hence
TEM has better resolution than that of the light microscope, of about 1000times.
This can accurately be stated that the TEM can be used to detail the internal structures of the smallest particles like
a virion particle.

Parts of Transmission Electron Parts of Transmission

Their working mechanism is enabled by the high-resolution power they produce which allows it to be used in a
wide variety of fields. It has three working parts which include:
1. Electron gun
2. Image producing system
3. Image recording system
Electron gun
• This is the part of the Transmission Electron Microscope responsible for producing electron beams.
• Electrons are produced by a cathode that is a tungsten filament that is V-shaped and it is normally heated.
The tungsten filament is covered by a control grid known as a Wehnelt cylinder made up of a central hole
which lies columnar to the tube. The cathode lies on top of or below the cylindrical column hole. The
cathode and the control grid are negatively charged with an end of the anode which is disk-shaped that also
has an axial hole.
• When electrons are transmitted from the cathode, they pass through the columnar aperture (hole) to the
anode at high voltage with constant energy, which is efficient for focusing the specimen to produce an
accurately defined image.
• It also has the condenser lens system which works to focus the electron beam on the specimen by
controlling the energy intensity and the column hole of the electron gun. The TEM uses two condenser
lenses to converge the beam of electrons to the specimen. The two condenser lens each function to produce
an image i.e the first lens which has strong magnification, produces a smaller image of the specimen, to the
second condenser lens, directing the image to the objectives.
Image- Producing system
• Its made up of the objective lens, a movable stage or holding the specimen, intermediate and projector
lenses. They function by focusing the passing electrons through the specimen forming a highly magnified
image.
• The objective has a short focal length of about 1-5mm and it produces an intermediate image from the
condenser which are transmitted to the projector lenses for magnification.
• The projector lenses are of two types, i.e the intermediate lens which allows great magnification of the
image and the projector lens which gives a generally greater magnification over the intermediate lens.
• To produce efficient high standard images, the objectives and the projector lenses need high power supplies
with high stability for the highest standard of resolution.
Image-Recording System
• Its made up of the fluorescent screen used to view and to focus on the image. They also have a digital
camera that permanently records the images captured after viewing.
• They have a vacuum system that prevents the bombardment or collision of electrons with air molecules
disrupting their movement and ability to focus. A vacuumed system facilitates the straight movement of
electrons to the image.
• The vacuumed system is made up of a pump, gauge, valves and a power supply.
• The image that is formed is called a monochromatic image, which is greyish or black and white. The image
must be visible to the human eye, and therefore, the electrons are allowed to pass through a fluorescent
screen fixed at the base of the microscope.
• The image can also be captured digitally and displayed on a computer and stored in a JPEG or TIFF format.
During the storage, the image can be manipulated from its monochromatic state to a colored image
depending on the recording apparatus eg use of pixel cameras can store the image in color.
• The presence of colored images allows easy visualization, identification, and characterization of the
images.
How does a Transmission Electron (TEM) work?

From the instrumentation described, the working mechanism is a sequential process of the parts of the TEM
mentioned above. To mean:
• A heated tungsten filament in the electron gun produces electrons that get focus on the specimen by the
condenser lenses.
• Magnetic lenses are used to focus the beam of electrons of the specimen. By the assistance offered by the
column tube of the condenser lens into the vacuum creating a clear image, the vacuum allows electrons to
produce a clear image without collision with any air molecules which may deflect them.
• On reaching the specimen, the specimen scatters the electrons focusing them on the magnetic lenses
forming a large clear image, and if it passes through a fluorescent screen it forms a polychromatic image.
• The denser the specimen, the more the electrons are scattered forming a darker image because fewer
electron reaches the screen for visualization while thinner, more transparent specimens appear brighter.
NOTE: If the screen is moved aside, a photographic image can be captured in pixels forming a permanent image.

Applications of Transmission Electron Microscope (TEM)

TEM is used in a wide variety of fields From Biology, Microbiology, Nanotechnology, forensic studies, etc. Some
of these applications include:
1. To visualize and study cell structures of bacteria, viruses, and fungi
2. To view bacteria flagella and plasmids
3. To view the shapes and sizes of microbial cell organelles
4. To study and differentiate between plant and animal cells.
5. Its also used in nanotechnology to study nanoparticles such as ZnO nanoparticles
6. It is used to detect and identify fractures, damaged microparticles which further enable repair mechanisms
of the particles.

Advantages of Transmission Electron Microscope (TEM)

 1. It has a very powerful magnification of about 2 million times that of the Light microscope.
2. It can be used for a variety of applications ranging from basic Biology to Nanotechnology, to education and
industrial uses.
3. It can be used to acquire vast information on compounds and their structures.
4. It produces very efficient, high-quality images with high clarity.
5. It can produce permanent images.
6. It is easy to train and use the Transmission Electron Microscope

Limitations of Transmission Electron Microscope (TEM)

1. Generally, the TEMs are very expensive to purchase

2. They are very big to handle.
3. The preparation of specimens to be viewed under the TEM is very tedious.
4. The use of chemical fixations, dehydrators, and embedments can cause the dangers of artifacts.
5. They are laborious to maintain.
6. It requires a constant inflow of voltage to operate.
7. They are extremely sensitive to vibrations and electro-magnetic movements hence they are used in isolated
areas, where they are not exposed.
8. It produces monochromatic images, unless they use a fluorescent screen at the end of visualization.
9. The first Scanning Electron Microscope was initially made by Mafred von Ardenne in 1937 with an aim
to surpass the transmission electron Microscope. He used high-resolution power to scan a small raster using
a beam of electrons that were focused on the raster. He also aimed at reducing the problems of chromatic
aberrations images produced by the Transmission electron Microscopes.
10. More studies followed by scientists and research institutions such as Cambridge Scientific Instrument
Company who eventually developed a fully constructed Scanning electron Microscope, in 1965 and named
it a Stereoscan.
11. The price of the Scanning Electron Microscope (SEM) is approximately $1 million.

Scanning Electron Microscope (SEM)

Definition

Scanning Electron Microscope (SEM) is a type of electron microscope that scans surfaces of microorganisms
that uses a beam of electrons moving at low energy to focus and scan specimens.
The development of electron microscopes was due to the inefficiency of the wavelength of the light microscopes.
electron microscopes have vert short wavelengths in comparison to the light microscope which enables better
resolution power.

The Principle of Scanning Electron Microscope

Unlike the Transmission Electron Microscope which uses transmitted electrons, the scanning electron Microscope
used emitted electrons.
The Scanning electron microscope works on the principle of applying kinetic energy to produce signals on the
interaction of the electrons. These electrons are secondary electrons, backscattered electrons and diffracted
backscattered electrons which are used to view crystallized elements and photons. Secondary and backscattered
electrons are used to produce an image. The secondary electrons are emitted from the specimen play the primary
role of detecting the morphology and topography of the specimen while the backscattered electrons show contrast
in the composition of the elements of the specimen.

How does the Scanning Electron Microscope (SEM) work?

 • The source of the electrons and the electromagnetic lenses are from tungsten filament lamps that are placed
at the top of the column and it is similar to those of the transmission electron Microscope.
• The electrons are emitted after thermal energy is applied to the electron source and allowed to move in a
fast motion to the anode, which has a positive charge.
• The beam of electrons activates the emission of primary scattered (Primary) electrons at high energy levels
and secondary electrons at low-energy levels from the specimen surface. The beam of electrons interacts
with the specimen to produce signals that give information about the surface topography and composition
of the specimen.
• The specimen does not need special treatment for visualization under the SEM, even air-dried samples can
be examined directly. However, microbial specimens need fixation, dehydration, and drying in order to
maintain the structural features of the cells and to prevent collapsing of the cells when exposed to the high
vacuum of the microscope.
• The samples are mounted and coated with thin layer heavy metal elements to allow spatial scattering of
electric charges on the surface of the specimen allowing better image production, with high clarity.
• Scanning by this microscope is attained by tapering a beam of electrons back and forth over a thin section
of the microscope. When the electrons reach the specimen, the surface releases a tiny staw of electrons
known as secondary electrons which are then trapped by a special detector apparatus.
• When the secondary electrons reach and enter the detector, they strike a scintillator (a luminescence
material that fluoresces when struck by a charged particle or high-energy photon). This emits flashes of
light which get converted into an electric current by a photomultiplier, sending a signal to the cathode ray
tube. This produces an image that looks like a television picture that can be viewed and photographed.
• The quantity of secondary electrons that enter the detector is highly defined by the nature of the specimen
i.e raised surfaces receive high quantities of electrons, entering the detector while depressed surfaces have
fewer electrons reaching the surface and hence fewer electrons enter the detector.
• Therefore raised surfaces will appear brighter on the screen while depressed surfaces appear darker.

Parts of a Scanning Electron Microscope

The major components of the Scanning Electron Microscope include;

• Electron Source – This is where electrons are produced under thermal heat at a voltage of 1-40kV. the
electrons the condense into a beam that is used for the creation of ana image and analysis. There are three
 types of electron sources that can be used i. e Tungsten filament, Lanthanum hexaboride, and Field
emission gun (FEG)
• Lenses – it has several condenser lenses that focus the beam of electrons from the source through the
column forming a narrow beam of electrons that form a spot called a spot size.
• Scanning Coil – they are used to deflect the beam over the specimen surface.
• Detector – Its made up of several detectors that are able to differentiate the secondary electrons,
backscattered electrons, and diffracted backscattered electrons. The functioning of the detectors highly
depends on the voltage speed, the density of the specimen.
• The display device (data output devices)
• Power supply
• Vacuum system
Like the transmission electron Microscope, the Scanning electron microscope should be free from vibrations and
any electromagnetic elements.

Applications of the Scanning Electron Microscope (SEM)

It is used in a variety of fields including Industrial uses, nanoscience studies, Biomedical studies, Microbiology
1. Used for spot chemical analysis in energy-Dispersive X-ray Spectroscopy.
2. Used in the analysis of cosmetic components which are very tiny in size.
3. Used to study the filament structures of microorganisms.
4. Used to study the topography of elements used in industries.

Advantages of the Scanning Electron Microscope (SEM)

• They are easy to operate and has user-friendly interfaces.

• They are used in a variety of industrial applications to analyze surfaces of solid objects.
• Some modern SEMs are able to generate digital data that can be portable.
• It is easy to acquire data from the SEM, within a short period of time of about 5 minutes.

Limitations

• They are very expensive to purchase

• They are bulky to carry
• They must be used in rooms that are free of vibrations and free of electromagnetic elements
• They must be maintained with a consistent voltage
• They should be maintained with access to cooling systems
The combination of the working principles of the Scanning Electron Microscope (SEM) and the Transmission
Electron Microscope (TEM) formed the Scanning-Transmission Electron Microscope (STEM). The Scanning-
Transmission Electron Microscope (STEM), uses a convergent beam of electrons to focus on a probe on the
specimen, and the probe is then scanned on its surface collecting signals which are then collected as point-to-point
to form an image.

Staining technique
Introduction

It is very difficult to observe microorganisms by our naked eyes because they are very minute and transparent as
well as colorless when they are suspended in aqueous medium. The refractive index of microorganism is not very
different from the medium in which they grow, due to which they cannot be observed in unstained preparation.
Staining helps to observe the organism clearly.
Chemically a stain (dye) can be defined as an organic compound containing an aromatic compound ie, benzene
ring, chromphore and auxochrome group. According to the nature of stain, they are classified into three groups.

1. Acidic dye: those dyes which ionizes to give anionic chromogen portion and has a strong affinity towards
positively charged constituents of the cell wall are called acidic dyes. Eg. eosin, picric acid, india ink etc
2. Basic dye: those dyes which ionizes to give cationic chromogen portion and therefore has strong affinity for
negatively charged constituents of the cell wall are called basic dyes. Eg. Crystal violet, methylene blue
3. Neutral dye: they are formed by the suitable mixing of two types of dyes, so they contain both cationic and
anionic chromogens. They produce precipitate with the cellular components. They are used to stain nucleic
acids and cytoplasm. Eg. Sudan IV, eosinate of methylene blue.
Principle

Simple staining technique uses a single stain to visualize the bacteria, which produces a distinctive contrast between
the organism and its background. Basic stains (such as methylene blue, crystal violet, and carbol fuchsin) with a
positively charged chromogen are preferred in simple staining because bacterial nucleic acids and certain cell wall
components carry a negative charge that strongly attracts and binds to the cationic chromogen. They stains the
microbial cell, not the background.

The purpose of simple staining is for visualization of morphological shape, size, and arrangement of microbial
cells.

Mechanism of staining:
▪ The process of staining involves ion exchange reaction between the stain and component to be stained
▪ For example, bacterial cell is a negatively charged due to large number of protein having COO- group. This
negative charged is balanced by positive charged ion presentoutside the cell wall.
▪ Therefore a bacterial cell is represented as (BACTERIAL CELL -) Na+.
▪ To stain the bacterial cell, cationic dye are used having positively charged chromogen. eg. Methylene blue,
which is represented as (MB+)Cl-.
▪ During staining, bacteria cell is flooded with methylene blue and due to ion exchange mechanism acidic
component of bacterial ie bacterial cell wall become stained.
▪ The reaction occurs as follows;
(BACTERIAL CELL-)Na+ + (MB+)Cl- ======NaCl + (BACTERAIL CELL -)MB+

Simple Staining

Simple Staining- Principle, Procedure and Result Interpretation

Objectives of Simple Staining

▪ To perform a simple staining procedure.
▪ To compare the morphological shapes and arrangements of bacterial cells.

Principle of Simple Staining

In simple staining, the bacterial smear is stained with a single reagent, which produces a distinctive contrast
between the organism and its background. Basic stains with a positively charged chromogen are preferred because
bacterial
nucleic acids and certain cell wall components carry a negative charge that strongly attracts and binds to the
cationic chromogen. The purpose of simple staining is to elucidate the morphology and arrangement of bacterial
cells. The most commonly used basic stains are methylene blue, crystal violet, and carbol fuchsin.

Reagents and Equipment’s for Simple Staining

Methylene blue, crystal violet, and carbol fuchsin, Microincinerator or Bunsen burner, inoculating loop, staining
tray, microscope, lens paper, bibulous (highly absorbent) paper, and glass slides.

Procedure of Simple Staining

1. Place a slide on the staining tray and flood the smear with one of the indicated stains, using the appropriate
exposure time for each: carbol fuchsin, 15 to 30 seconds; crystal violet, 20 to 60 seconds; methylene blue, 1
to 2 minutes.
2. Gently wash the smear with tap water to remove excess stain. During this step, hold the slide parallel to the
stream of water; in this way you can reduce the loss of organisms from the preparation.
3. Using bibulous paper, blot dry, but do not wipe the slide.
4. Examine all stained slides under oil immersion.

Result Interpretation of Simple Staining

Bacilli and diplobacilli: Rod-shaped bacteria, purple
Spirilla: spiral-shaped bacteria, purple
Cocci: spherical-shaped, bacteria, purple

Negative Staining

Negative Staining- Principle, Procedure and Result Interpretation

Objectives of Negative Staining

1. To perform a negative staining procedure.
2. To understand the benefit obtained from visualizing unstained microorganisms.

Principle of Negative Staining

Negative staining requires the use of an acidic stain such as India ink or nigrosin. The acidic stain, with its
negatively charged chromogen, will not penetrate the cells because of the negative charge on the surface of
bacteria. Therefore, the unstained cells are easily discernible against the colored background.
The practical application of negative staining is twofold.
First, since heat fixation is not required and the cells are not subjected to the distorting effects of chemicals and
heat, their natural size and shape can be seen.
Second, it is possible to observe bacteria that are difficult to stain, such as some spirilla. Because heat fixation is not
done during the staining process, keep in mind that the organisms are not killed and slides should be handled with
care.

Reagents and Equipment’s for Negative Staining

Nigrosin, Microincinerator or Bunsen burner, inoculating loop, staining tray, glass slides, lens paper, and
microscope.

Procedure of Negative Staining

1. Place a small drop of nigrosin close to one end of a clean slide.
2. Using aseptic technique, place a loopful of inoculum from the bacterial culture in the drop of nigrosin and
mix.
3. Place a slide against the drop of suspended organisms at a 45° angle and allow the drop to spread along the
edge of the applied slide.
4. Push the slide away from the drop of suspended organisms to form a thin smear. Air-dry.
Note: Do not heat fix the slide.
5. Examine the slides under oil immersion.

STEP 1 STEP 2
Result Interpretation of Negative Staining

Bacterial cells observed as the clear transparent bodies or objects, may be of variable size and shape as the
background is stained and not the bacterial cells.
Gram Stain

Gram Stain- Principle, Reagents, Procedure, Steps, Results

The Gram stain was developed by Christian Gram in 1884 and modified by Hucker in 1921.

Objectives of Gram Stain

This test differentiates the bacteria into Gram-Positive and Gram-Negative Bacteria, which helps in the
classification and differentiation of microorganisms. The Gram stain separates bacteria into two groups: (1) Gram-
positive microorganisms that retain the primary dye (Crystal violet) and (2) Gram-negative microorganisms that
take the color of the counterstain (usually Safranin O).

Principle of Gram Stain

The two major groups of bacteria can be divided into gram-positive and gram-negative. The Gram stain technique
is based on the differential structure of the cellular membranes and cell walls of the two groups.
Gram-positive organisms contain a highly cross-linked layer of peptidoglycan that retains the primary dye, crystal
violet (CV), following the application of the mordant, iodine (I). The iodine and crystal violet form a complex
within the peptidoglycan. When decolorizer is applied to the cells, the CV-I complex remains within the cell,
making it appear dark purple to blue.
The gram-negative organisms do not contain a thick cross-linked layer of peptidoglycan. The peptidoglycan is
loosely distributed between the inner cell and the outer cell membranes. Following the application of the crystal
violet and iodine, the CV-I complexes are not trapped within the peptidoglycan. Application of the acid-alcohol
decolorizer dehydrates the outer cellular membrane, leaving holes in the membrane and effectively washing or
removing the CV-I complex from the cells. The cells appear colorless. To make the colorless cells visible, a
secondary stain, safranin, is applied, leaving the gram-negative cells pink.

Reagents and Equipment’s for Gram Stain

Primary stain: 2 g Crystal violet, 20 mL 95% ethyl alcohol, 0.8 g ammonium oxalate, and 100 mL distilled water.
Gram’s iodine: 2 g potassium iodide, 1 g iodine crystals, and 100 mL distilled water.
Decolorizer: 50 mL acetone and 50 mL ethanol.
Counterstain: 4.0 g Safranin, 200 mL 95% ethanol, and 800 mL distilled water

Procedure of Gram Stain

1. Prepare and fix the specimen to the microscope slide before staining.
2. Cover the smear with crystal violet, the primary stain, for 20 seconds.
3. Gently rinse off the stain with water.
4. Cover the smear with Gram’s iodine, the mordant, for 1 minute.
5. Pour off the excess Gram’s iodine.
6. Run the acid-alcohol decolorizer over the smear until the solution appears clear.
7. Gently rinse with water.
8. Cover the smear with safranin, the secondary or counterstain, for 20 seconds.
9. Gently rinse the stain with water.
10. Blot dry with bibulous paper.

Result Interpretation of Gram Stain

Gram-positive: Blue/Purple Color
Gram-Negative: Red/Pink Color

Limitations:
1. Over-decolorization may result in the identification of false gram-negative results, whereas under-
decolorization may result in the identification of false gram-positive results.
2. Smears that are too thick or viscous may retain too much primary stain, making the identification of proper
Gram stain reactions difficult. Gram-negative organisms may not decolorize properly.
3. Cultures older than 16 to 18 hours will contain living and dead cells. Cells that are dead will be
deteriorating and will not retain the stain properly.
4. The stain may form a precipitate with aging. Filtering through gauze will remove excess crystals.
5. Gram stains from patients on antibiotics or antimicrobial therapy may have altered Gram stain reactivity
due to the successful treatment.
6. Occasionally, pneumococci identified in the lower respiratory tract on a direct smear will not grow in
culture. Some strains are obligate anaerobes.
7. Toxin-producing organisms such as Clostridia, staphylococci, and streptococci may destroy white blood
cells within a purulent specimen.
8. Faintly staining Gram-negative organisms, such as Campylobacter andBrucella, may be visualized using an
alternative counterstain (e.g., basic fuchsin).
9. Differences Between Gram Positive and Gram Negative Bacteria

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