Chapter 2 Dr. R.

Raji Notes

Staining
 Staining methods are used to elevate the visibility and highlight specific
morphological structures of the microorganisms. It is also used to preserve them.
Before staining, the specimen is smeared and fixed by the method of fixation.
 Fixation is a method in which the complete structures of the cells or microorganisms
are fixed in the positions and are preserved.
 Inactivation of enzymes, which cause cell morphological disruption.
 Cell structure gets rigid in form, which helps it to remain stationary during
staining & observation.
 Usually, microorganisms are killed & attached strongly to the slide during the
fixation process.

Fig: Types of Fixations

Table: Types of Fixations

Fixation Methods Remarks

Used to observe prokaryotic cells. Smear is prepared and passed

through the flame, which is gently heated.
Heat Fixation
Preserve the whole morphology of the cells, but not the internal
structure within the cell

Used to protect cell morphology as well as cell fine

substructures of the more delicate microorganisms. Chemical
Chemical Fixation
fixative gets inside the cells & reacts with components present in
the cells, such as proteins & lipids to immobile them.

Smear preparation:
Smears should be spread evenly covering an area of about 15–20 mm diameter on a slide.
1. An evenly spread smear should be prepared covering area of 15-20mm diameter.
2. Avoid thick and dense smear because thick smear prevents light penetration to
visualize the morphology of cell.
3. A good smear is one that, when dried, appears as a thin whitish layer or film. The
print of textbook should be legible through the smear.

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Chapter 2 Dr. R. Raji Notes

4. Different techniques are used for smear preparation depending upon culture
media.

A. Broth culture:
 Resuspend the culture by tapping the tube with your finger.
 Depending on the size of the loop, one or two loopfuls should be applied to the
centre of the slide with a sterile inoculating loop and spread evenly over an area
about the size of a dime.
 Set the smears on the laboratory table and allow to air-dry

B. Culture plates:
 Organisms cultured in a solid medium produce thick, dense surface growth and are
not amenable to direct transfer to the glass slide.
 These cultures must be diluted by placing one or two loopfuls of water on the centre
of the slide in which the cells will be emulsified.
 Transfer of the cells requires the use of a sterile inoculating loop or a needle.
 Only the tip of the loop or needle should touch the culture to prevent the transfer of
too many cells.
 Suspension is accomplished by spreading the cells in a circular motion in the drop of
water with the loop or needle. This helps to avoid cell clumping.
 The finished smear should occupy an area about the size of a nickel and should
appear as a translucent, or semi-transparent, confluent whitish film

Air dry:
Smear should be allowed to dry completely at room temperature at safe place

Fixation of smear:
 The purpose of fixation of smear is to preserve and prevent smear being washed
away during staining.
 Smears are fixed by heat, alcohol and occasionally by other chemical

1. Heat fixation:
 After smear is air dried completely, rapidly pass the 3-4 times through flame of
Bunsen burner or sprit lamp.
 Avoid too much heating.
 After heat fix, allow the smear to cool before staining.

2. Alcohol fixation:
 Allow smear to air dry completely.
 Fix the smear with one or two drops of 70% alcohol, and leave it for 2 minutes until
the alcohol dries up.

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Chapter 2 Dr. R. Raji Notes

Fig: Smear and Fixation

Fig: Types of Staining

(a) Simple staining: A stain which provides colour contrast but gives same colour to all
bacteria and cells. Ex: Loffler’s methylene blue, Polychrome methylene blue, Diluted
carbol fuchsin.
I. Direct staining: Where the actual cells are themselves colored and appear in a
clear background.
II. Indirect staining: Where the cells remain clear (uncoloured) and the background
is colored to create a contrast to aid in the better visualization of the image e.g.
Indian ink, Nigrosin.

(b) Differential Staining: A stain which imparts different colours to different bacteria is
called differential stain (which contains more than one stain). Ex: Gram’s stain, Acid fast
staining, Special stains.

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Chapter 2 Dr. R. Raji Notes

Fig: Different types of staining

Simple Staining
The simple stain can be used as a quick and easy way to determine the cell shape, size, and
arrangement of bacteria. True to its name, the simple stain is a very simple staining
procedure involving a single stain solution. Any basic dye, such as methylene blue, safranin,
or crystal violet, can be used to colour the bacterial cells. These stains will readily give up a
hydroxide ion or accept a hydrogen ion, which leaves the stain positively charged. Since
most bacterial cells and cytoplasm surface is negatively charged, these positively charged
stains adhere readily to the cell surface. After staining, bacterial cell morphology (shape and
arrangement) can be appreciated.

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, Micro incinerator or Bunsen burner,
inoculating loop, staining tray, microscope, lens paper, bibulous (highly absorbent) paper,
and glass slides.

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Chapter 2 Dr. R. Raji Notes

Procedure of Simple Staining

 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.
 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.
 Using bibulous paper blots dry, but do not wipe the slide.
 Examine all stained slides under oil immersion.

Fig: Direct and Indirect Stain

Direct staining (Positive staining)

 A simple staining technique that stains the bacterial cells in a single colour.
 Many of the bacterial stains are basic chemicals; these basic dyes react with
negatively charged bacterial cytoplasm (opposite charges attract) and the organism
becomes directly stained
 Examples are methylene blue, crystal violet, and basic fuchsin.

Fig: Direct Staining

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Chapter 2 Dr. R. Raji Notes

Indirect staining (Negative staining)

 In this staining process, instead of ells background is stained.
 Here, an acidic dye like Nigrosin or Indian ink is used. Acidic stain carries a negative
charge and repelled by the bacteria, which also carry a negative charge on their
surface. Hence, an acidic dye does not stain bacteria,
 Instead, it forms a deposit around the organism, leaving the organism itself
colourless or transparent upon examination.

Fig: Indirect Staining

Fig:

Simple Staining

Differential Technique
Gram Staining
Danish physician Hans Christian Gram developed the Gram staining method in 1884. Gram
staining procedure uses four chemicals; crystal violet, iodine, alcohol, and safranin, to stain
bacteria
Gram staining is still the cornerstone of bacterial identification and taxonomic division. This
differential staining technique separates most bacteria into two groups based on cell wall
composition.
 Gram-positive bacteria- stains purple
 Gram-negative bacteria-stains red/pink

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Chapter 2 Dr. R. Raji Notes

Nearly all clinically important bacteria can be visualized using the Gram staining technique,
the only exceptions being those organisms; It is the type of differential staining that is used
to differentiate the bacteria majorly into two groups, i.e. gram-positive and gram-negative
based on cell-wall difference and by the sequential application of crystal violet, iodine,
alcohol and safranin.

Principle of gram staining

The structure of the organism’s cell wall determines whether the organism is gram positive
or negative. When stained with a primary stain and fixed by a mordant, some bacteria are
able to retain the primary stain by resisting decolonization while others get decolorized by a
decolourizer. Those bacteria which retain the primary stain are called Gram positive and
those bacteria which get decolorized and then get counterstained are called Gram negative.

 The principle of gram staining relies on the reaction of a bacterial cell with the Gram
stain, which finally differentiates the bacteria into gram-positive and gram-negative.
First, the Crystal violet dissociates into CV+ and Cl– that further penetrates the
bacterial cell and stains the cell violet. Then, the Iodine (I or I-3) is added, which
interacts with the CV+ and forms Crystal Violet-Iodine (CV-I) complex. This CV-I
complex forms within the cell wall and the cell cytosol or cytoplasm. After that, the
decolourizing agent, i.e. Ethanol interacts with the lipid content of the bacteria, and
this treatment is the important step to differentiate both gram-positive and gram-
negative cell.
 In the gram-negative cell, the outer membrane that contains the lipopolysaccharide is
dissolved by ethanol. As a result, the peptidoglycan layer gets exposed. The gram-
negative cell consists of thin peptidoglycan cross-linkages, by which the CV-I
complex is washed out of the cell.
 Oppositely, a gram-positive cell has highly crossed-linked or thick peptidoglycan.
This peptidoglycan layer gets dehydrated by the addition of decolourizing agent
(Ethanol), which tightens the peptidoglycan layer. This dehydration or tightening of
the peptidoglycan layer traps the CV-I complex strongly within the cell.
 After decolourization, the gram-negative cells lose the colour of the primary stain
and stain pink when treated with a positively charged counterstain, i.e. Safranin. In
gram-positive bacteria, the cell retains the colour of the primary stain, i.e. Crystal
violet and appears violet in colour.

Steps of Gram Staining

Gram staining techniques involve the following steps:

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Chapter 2 Dr. R. Raji Notes

Fig: Steps of Gram Staining

1. Fixation of clinical materials to the surface of the microscope slide either by heating or
by using methanol. (Methanol fixation is recommended rather than heat fixation. Methanol
fixation preserves the morphology of host cells and bacteria. Heating the slide causes cell
distortion, could increase cell debris, and may cause erroneous Gram stain results.).
2. Application of the primary stain (crystal violet). Crystal violet is a dark blue to purple
dye. It stains all cells blue/purple.
3. Application of mordant: The iodine solution (mordant) is added to form a crystal violet-
iodine (CV-I) complex; all cells continue to appear blue.
4. Decolourization step: The decolourization step distinguishes gram-positive from gram-
negative cells. The organic solvent such as acetone or ethanol extracts the blue dye
complex from the lipid-rich, thin-walled gram-negative bacteria to a greater degree than
from the lipid-poor, thick-walled, gram-positive bacteria. The gram-negative bacteria
appear colourless, and gram-positive bacteria remain blue.
5. Application of counterstain (safranin): The red dye safranin stains the decolorized
gram-negative cells red/pink; the gram-positive bacteria remain blue.

Fig: Process of Gram Staining

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Chapter 2 Dr. R. Raji Notes

Fig: Gram Staining (Gram +Ve & Gram –Ve)

Fig: Cell Structure of Gram +Ve & Gram –Ve Bacteria

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Chapter 2 Dr. R. Raji Notes

Table: Differences between Gram +Ve & Gram –Ve Bacteria

S. No Components Gram + Ve Gram –Ve

A single-layered, smooth cell A double-layered, wavy cell-
1 Cell Wall
wall wall
The thickness of the cell wall is The thickness of the cell wall
2 Cell Wall Thickness
20 to 80 nanometres is 8 to 10 nanometres
It is a thick layer/ also can be It is a thin layer/ often single-
3 Peptidoglycan Layer
multi-layered layered.
4 Teichoic acids Presence of teichoic acids Absence of teichoic acids
The outer membrane is absent The outer membrane is
5 Outer membrane
present (mostly)
6 Porins Absent Occurs in Outer Membrane
7 Mesosome It is more prominent. It is less prominent.
8 Morphology Cocci or spore-forming rods Non-spore forming rods.
9 Flagella Structure Two rings in basal body Four rings in basal body
10 Lipid content Very low 20 to 30%
11 Lipopolysaccharide Absent Present
12 Toxin Produced Exotoxins Endotoxins or Exotoxins
Resistance to
13 More susceptible More resistant
Antibiotic
Staphylococcus, Streptococcus,
14 Examples Escherichia, Salmonella, etc.
etc.
These bacteria retain the These bacteria do not retain
crystal violet colour even after the stain colour even after
they are washed with acetone they are washed with acetone
or alcohol and appear as or alcohol and appear as
15 Gram Staining
purple-coloured when pink-coloured when
examined under the examined under the
microscope after gram microscope after gram
staining. staining.

Interpretation of Gram +Ve & Gram –Ve Bacteria

Gram-Positive: If the bacteria cell gives a positive result for the gram reaction, it will stain
Violet.
Gram-negative: If the bacterial cell gives a negative result for the gram reaction, it will stain
Pink.

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Chapter 2 Dr. R. Raji Notes

Fig: Result of Gram +Ve & Gram –Ve Bacteria

Acid-Fast staining / Ziehl-Neelsen staining technique

The Ziehl-Neelsen staining technique is a differential staining technique that was initially
developed by Ziehl and modified later by Neelsen, hence the name Ziehl-Neelsen stain.
Neelsen used carbol-fuschin from Ziehl’s experiment, with heat and added a decolorizing
agent using acid-alcohol and a counterstain using methylene blue dye, thus developing the
Ziehl-Neelsen Technique of staining.

The use of acid-alcohol in the technique earned it the name Acid-Fast Stain and the
application of heat in the technique gives it the name the hot method of Acid-Fast staining
which is a synonymous name for the Ziehl-Neelsen Staining technique. This technique is
used on microorganisms that are not easily stained by basic stains such as Negative staining
or Gram staining. One of the most complex micro-organisms that require harsh treatment of
the Ziehl-Neelsen compounds is the Mycobacterium spp.

Mycobacterium, Actinomycetes, Norcadia, Isospora, Cryptosporidium, and some fungi

contain a thick cell wall made up of lipoidal complexes known as mycolic acid. Mycolic acid
is difficult to stain and therefore simple stains like gram staining cannot penetrate the thick
cell wall of these organisms. They require harsher treatments to allow stain penetration for
identification and examination and hence the use of the Ziehl-Neelsen or the hot method of
Acid-fast stain.

Objectives of the Ziehl-Neelsen Staining

 To differentiate between acid-fast bacilli and non-acid-fast bacilli.
 To stain Mycobacterium species.

Reagents used in the Ziehl-Neelsen Stain

 Carbol-Fuschin (Primary dye)
 20% sulphuric acid or acid-alcohol (Decolourizer)
 Methylene Blue dye (counterstain) or malachite green

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Chapter 2 Dr. R. Raji Notes

Fig: Components of Acid-Fast Staining

Principle of the Ziehl-Neelsen Staining

 The Ziehl-Neelsen stain uses basic fuchsin and phenol compounds to stain the cell
wall of Mycobacterium species.
 Mycobacterium does not bind readily to simple stains and therefore the use of heat
along with carbol-fuschin and phenol allows penetration through the bacterial cell
wall for visualization.
 Mycobacterium cell wall contains high lipid content made up of mycolic acid on its
cell wall making it waxy, hydrophobic, and impermeable. These are ß-
hydroxycarboxylic acids made up of 90 carbon atoms that define the acid-fastness of
the bacteria.
 Use of Carbol-fuschin which is basic strongly binds to the negative components of
the bacteria which include the mycolic acid and the lipid cell wall. Addition of acid
alcohol along with the application of heat forms a strong complex that cannot be
easily washed off with solvents.
 The acid-fast bacilli take up the red colour of the primary dye, carbol-fuschin.
 While non-acid-fast bacteria easily decolorize on the addition of the acid-alcohol and
take up the counterstain dye of methylene blue and appear blue.
 This technique has been used in the identification of Mycobacterium tuberculosis
and Mycobacterium leprae.

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Chapter 2 Dr. R. Raji Notes

Fig: Cell Structure of Acid-Fast Bacteria

Procedure of Acid-Fast Staining

 Take a clean, sterilized slide.
 Then, add a drop of distilled water to the centre of the glass slide. After doing this,
thoroughly mix the inoculum by a sterilized inoculating loop. Then, heat fix and air
dry the prepared smear.
 Flood the smear with the primary stain (Carbol fuschin) and allow it to stand for one
minute.
 Then, flood the smear with the decolourizer (3% HCl) and wash the glass slide with
distilled water.
 Finally, flood the smear with the counterstain (methylene blue) and allow it to stand
for one minute. Again, rinse the slide in water.
 Air-dry the glass slide.
 Observe the glass slide under oil immersion at 100X objective.

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Chapter 2 Dr. R. Raji Notes

Fig: Process of Acid-Fast Staining

Fig: Process of Acid Fast & Non-Acid Fast Staining

Observation
After performing acid-fast staining, we could observe the following changes, as you could
see in the figure below. Without staining, the bacteria appear colourless. So, it becomes
necessary to stain the bacteria to identify and classify them based on their physical and
chemical differences. On primary staining, the Ziehl-Neelsen carbol fuschin stains both the
acid-fast bacteria and non-acid fast bacteria to appear red.

In the decolourization stage, non-acid fast bacteria lose the colour of the primary stain or
appear colourless, whereas acid-fast bacteria appear red. Finally, the colourless non-acid fast

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Chapter 2 Dr. R. Raji Notes

bacterial cells take up the colour of methylene blue and appear blue. Oppositely, the acid-
fast bacteria will remain red even after the counterstaining.
Table: Observation of Acid Fast & Non-Acid Fast Staining

Interpretation of Acid Fast & Non-Acid Fast Staining

 Acid-fast bacteria retain the primary dye, carbol-fuschin, and stain pink.
 Non-acid fat bacteria take up the methylene blue dye and appear blue.

Fig: Interpretation of Acid Fast & Non Acid Fast Staining

Fig: Mycobacterium tuberculosis visualization using the Ziehl–Neelsen stain

Applications of Ziehl-Neelsen Staining

1. Used for examination and identification of Mycobacterium species.
2. Used to differentiate between acid-fast and non-acid fast bacilli
3. Used for the identification of some fungal species such as Cryptosporidium.

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Chapter 2 Dr. R. Raji Notes

Sterilisation
Microorganisms play an important role in causing infection and contamination. Therefore,
Sterilization is an important technique in microbiology which helps to remove or destroy
microorganisms from materials or surfaces.
Sterilization is the complete removal of microorganisms from an object or surfaces.
Sterilization is obtained when microorganisms are subjected to antimicrobial agents for
sufficient time and at optimum conditions.

History of sterilization
 LOUIS PASTEUR of France was among the first to use sterilization techniques, he
developed the steam sterilization, hot air oven and the autoclave. Pasteurization was
a method developed by him to rid of bacilli from milk.
 JOSEPH LISTER applied Pasteur’s work and introduced antiseptic technique in
surgery (1867). He is the father of antiseptic surgery.

Methods of Sterilisation

Fig: Different Methods of Sterilisation

Physical methods of sterilisation

I. Sunlight
 Sunlight has an active germicidal effect due to the presence of ultraviolet rays. It
is a natural procedure of sterilization which reduces the number of
microorganisms in water tanks, lakes, etc.
 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.

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 Chapter 2 Dr. R. Raji Notes

 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.

II. Drying: Moisture is essential for growth of bacteria. Drying in air has deleterious effect
on many bacteria. However, spores are unaffected. Therefore, it is not really unreliable.

III. Heat
Heat is a mostly used method of sterilization. Moreover, it is a highly effective and most
reliable process. There are two major methods of using heat in sterilization which are dry
heat and moist heat. The principle behind both of these methods is similar. Dry heat
induces the denaturation of protein, oxidative damage and toxic effect due to the high
level of electrolytes. Moreover, the dry heat can also damage the DNA of the
microorganism. As a result, the microorganism got killed. Moist Heat kills the
microorganisms by denaturation and coagulation of proteins. There are several factors
that can influence the heat killing procedure. Such as
Temperature and Duration: The duration and temperature are inversely connected to
each other. Therefore, in the case of the long duration of heat provided for sterilization,
the temperature will be reduced while in case of high temperature the duration will be
reduced.
Characteristic of the Microorganism: Microorganisms can be present in both vegetative
and spore form. Spore forms are generally heat resistant. Therefore, the sterilization
process will vary on the basis of the characteristic of the microorganism.
Type of Material: Organic substances often provide protection to the vegetative and
spore form of microorganisms which reduces the lethal property of heat. Apart from
that, the materials containing the substances are also needed to be heat stable for proper
sterilization.

(i) Dry heat

a) Red Heat
 Inoculation loops, wires, forceps tips, needles are needed to be sterilized to
inhibit microbial contamination. These instruments are held in the flame of a
Bunsen burner until they become red hot.

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Chapter 2 Dr. R. Raji Notes

 Red 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.

b) Flaming
 Glass slides, scalpels, and mouths of culture tubes or conical flasks are passed
through Bunsen flame without allowing them to become red hot.
 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.

Fig: Flaming
c) Incineration
 This procedure is used to reduce the infective material into ashes by burning.
The incinerator is used for the process. Soiled dressings, animal carcasses,
bedding, and pathological materials are dealt with this method.
 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.

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Chapter 2 Dr. R. Raji Notes

Fig: Incinerator

d) Hot Air Oven

Hot air oven is used for sterilization by using dry heat under atmospheric pressure.
This is based on the principle where sterilization is accomplished by heat or hot air.
Dry heat is less effective compared to moist heat because in the presence of
moisture, proteins are easily coagulated and moist heat has greater penetration
power than the dry heat. Dry heat causes the oxidation of protein and thereby
destroys the microbes. The normal sterilization temperature is 160°C for 60
minutes.
Hot air oven comprises mechanical and electrical parts.

Mechanical parts
 Coat/Cabinet: The external shield is built of aluminium or stainless steel,
which resists mechanical shocks and oxidation. It also insulates the internal
environment from the external surroundings and prevents heat loss.
 Fibreglass: The space between the outer cabinet and inner chamber is filled
with thick glass wool insulation. Two types of fiberglass are present,
namely, brown fiberglass and yellow fiberglass. The latter is less dangerous
compared to the former. Brown glass causes inflammation in the respiratory
system, while yellow glass causes skin sensitivity. Thus, it is preferred to
use hand gloves while dealing with it. It also functions to prevent heat loss
from the inside of the device to the outside.
 Chamber: The rectangular-shaped chamber is made of aluminium or
stainless steel, which has space for ribs to keep shelves at the desired levels.
 Shelves (Mesh): These are objects holding plates and are made of
aluminium. Depending upon the number and size of objects, as well as the
oven capacity, their number may vary. When they are placed on the ribs, the
movement of air is facilitated by lifting some areas. Also, some shelves may
contain openings for aeration.
 Motorized fans/ blower: The fan is driven by the motor and is used to
distribute hot air inside the chamber evenly.

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Chapter 2 Dr. R. Raji Notes

 Door: A single door on one side is fitted on the heavy hinges. The presence
of an asbestos door gasket on the side is used to decrease heat loss during
the operation.

Electric parts
 Power supply: The power supply is done with the use of a 220V-50Hz
transformer and rectifier.
 Heater: With the passage of electric current through a conductor, heat is
generated following the rise of temperature. The heating element has three
main features: high resistance, electrical insulation, and high thermal
conductivity. The different types of heaters used in hot air ovens are One
side circular type heater, One side U type heater, One side wave type heater,
One side square type heater, Three sides type heater, and Four sides type
heater. The heater operates at temperatures from 50 to 300 degrees Celsius.
 Thermostat: It is a heat sensor connected directly to a heater and can resist
extreme heat with a high negative temperature coefficient. It facilitates users
to obtain the desired temperature in the hot air oven and prevent
temperature overshoot.
 Temperature indicator: Either a thermometer or thermocouple can be used
to determine the internal temperature of the oven.
 Timer: There may be two types of timers: Electrical or Mechanical, which
can operate for 5-60 minutes given the time period for sterilization.
 Fuse: Fuse functions to prevent electrical damage due to high current
during short circuits or high loads.
 Control Panel: It is the region that allows the user to control different
parameter settings such as temperature, time, etc., as well as has an
indicator power lamp (usually green), indicator heater lamp (usually red),
and switch knob.

Procedure
Here are the steps of operating a hot air oven:
 The oven is plugged into the socket and switched on.
 The oven is preheated for 30 minutes before placing the items on the trays or
shelves.
 The temperature gauge is set at the desired time, depending on the volume
of the contents to be sterilized.
 The items are loaded on the shelves (appropriate spacing should be
maintained between the articles on the trays for efficient heat circulation).
 The door is closed by fastening the screws provided, after which the
temperature begins to rise.

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Chapter 2 Dr. R. Raji Notes

 It is better to have sound knowledge of the sterilizing holding time and

temperature of the hot air oven. The thermometer is checked in to determine
whether the desired temperature is achieved after a certain holding time.
 The device is switched off once the temperature holding period is achieved;
allowed cooling down before opening the door, and then the samples are
removed using oven mitts or tongs.
 The door is closed after removing the samples.

Application
 It is used for sterilization of laboratory equipment such as glassware (flasks,
pipettes, Petri-plates, and test tubes), culture media, metal items (forceps,
spatula, scalpel, scissors), non-volatile compounds (zinc and starch powder,
sulphonamide), and other materials that contain oils.
 It can be employed for testing food items, pharmaceutical products and
other consumable materials in order to ensure their temperature stability
during the shelf life.
 It can be used in research settings in the field of biology, chemistry and
material science.
 It can be used in heat treatment and drying of samples, such as metals,
alloys, soil, and other materials.

Fig: Hot Air Oven

Advantages of Hot Air Oven
 It does not require water for sterilization purposes like an autoclave.
 It is economical and easy to operate.
 It can function at higher temperature and faster than autoclave.
 The small size of the oven requires less space and has an easier installation
process.

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Chapter 2 Dr. R. Raji Notes

 The dry heat does not corrode or rust metals or other sharp articles.
 It is smaller in size and is convenient to work on.
 Less pressure built in it ensures safety during operation.
 It is non-toxic. No harmful chemical residues will be discarded.
 The dry heat can penetrate deeply into thick objects such that it helps to
achieve an in-depth sterilization effect.

Disadvantages of Hot Air Oven

 It is not suitable to sterilize items such as rubber, plastics, surgical dressing,
etc., due to their low melting point.
 It may not destroy the heat-resistant endospores and prions because they
utilize dry heat instead of moist heat.
 It is time-consuming relative to steam, flaming, chemical sterilization, or
radiation.

Table: Different Methods of Dry Heat Sterilisation-Applications and Limitations

Method Recommended Uses Limitations

Hot-air oven Sterilizing materials impermeable to or Destructive to materials which
damaged by moisture, e.g., oils, glass, cannot withstand high
sharp instruments, metals temperatures for long periods
Incineration Recommended Uses Disposal of Size of Incinerator must be
contaminated objects that cannot be adequate to burn largest load
reused. promptly and completely:
potential of air pollution.
Flaming Sterilization of inoculating loop or –
wire, the tip of forceps, searing
spatulas, etc.,
Incineration used for complete destruction and not effective for metals and
disposal of infectious material, such as heat-resistant glass materials.
syringes, needles, culture material,
dressings, bandages, bedding, animal
carcasses, and pathology samples

(ii) Moist Heat Sterilization

Moist heat method is used for heat sensitive materials and materials through which
steam is permeable. Culture media is also sterilized through moist heat sterilization.
It has also 3 types: - 1) Below 100°C, 2) At 100°C and 3) Above 100°C

22
Chapter 2 Dr. R. Raji Notes

1) Below 100°C: It has also 2 types

i) Pasteurization: Its purpose is to reduce the bacterial population of a liquid such
as milk & destroy Organism that may cause spoilage. Spores are not affected by this
method. There are two different types of pasteurization methods that are used for
sterilization of milk, Holder method (63o C for 30 minutes) and flash method (72o C
for 20 seconds followed by cooling quickly to 13o C). This method is effective
against all non-sporing pathogens such as mycobacteria, Salmonella, etc. except
Coxiella burnetii which survives the holder method due to heat resistant
characteristics.

ii) Inspissation: Heating at 80-85° C for half an hour daily on three consecutive
days. Serum or egg media are sterilized.

2) At 100° C: It has also 2 types.

i) Boiling: Boiling is a very simple method of water disinfection. Heating water to a
high temperature, 100° C, kills most of the pathogenic organisms, particularly
viruses and bacteria causing waterborne diseases. In order for boiling to be most
effective, the water must boil for at least 20 minutes.

ii) Tyndallization: Exposure at 100° C for 20-45 minutes for followed by incubation
at 37°C overnight. Repeated for another 2 successive days. Used for sterilizing
sugars, gelatin, & serum containing media. The vegetative bacteria are killed in the
first exposure and the spores that germinate by next day are killed in subsequent
days. The success of process depends on the germination of spores.

iii) Steam sterilizer: Koch’s and Arnold’s steam sterilization is usually used for
media which can easily decompose due to the high temperature in the autoclave.
Those media are kept on a perforated tray and steam at 100o C and at atmospheric
pressure passes through the media for 90 minutes. It is an effective method to kill
vegetative cells.

Fig: Koch and Arnold Steamer

23
Chapter 2 Dr. R. Raji Notes

 Subjected to free steam at 100o C.

 Traditionally Arnold’s and Koch’s steamers were used.
 A steamer is a metal cabinet with perforated trays to hold the articles and a conical
lid
 The bottom of steamer is filled with water and heated
 The steam generated sterilizes the articles when exposed for a period of 90
minutes
 Media such as TCBS, DCA and Selenite broth are sterilized by steaming.

3) Above 100°C
(i) 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.

Principle
 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 pound per square inch or 775 mm of Hg.
 When this steam comes in contact with the surface, it kills the microbes by
giving off latent heat. (Latent heat is defined as the heat or energy that is
absorbed or released during a phase change of a substance. It could either be
from a gas to a liquid or liquid to solid and vice versa. Latent heat is related
to a heat property called enthalpy)
 The condensed liquid ensures the moist killing of the microbes.

24
Chapter 2 Dr. R. Raji Notes

 Once the sterilization phase is completed (which depends on the level of

contamination of material inside), the pressure is released from the inside of
the chamber through the whistle.
 The pressure inside the chamber is then restored back to the ambient pressure
while the components inside remain hot for some time.

Parts of 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 and the materials to be
sterilized are kept in it.
 The outer jacket is filled with steam to reach the sterilization temperature.
 The size of the pressure chamber ranges from 100 L to 3000 L.
(b) Lid
 The purpose of the lid is to seal off the outside atmosphere and create a
sterilized condition on the inside of the autoclave.
 The lid is made airtight via the screw clamps and asbestos washer.
 The lid consists of various other components like:
(c) Pressure gauge
A pressure gauge is present on the lid of the autoclave to indicate the pressure
created during sterilization. It also assures the safety of the autoclave and the
working condition of the operation.
(d) Pressure releasing unit/ Whistle
Is present on the lid of the autoclave and the whistle controls the pressure inside
the chamber.
(e) Safety valve
 A safety valve is present on the lid of the autoclave, which is crucial in cases
where the autoclave fails to perform its action or the pressure inside increases
uncontrollably.
 The valve has a thin layer of rubber that bursts itself to release the pressure and
to avoid the danger of explosion.
(f) 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 important as if the water is
not sufficient; there are chances of the burning of the heating system.
 Similarly, if the water is more it might interfere with the trays and other
components present inside the chamber.

25
Chapter 2 Dr. R. Raji Notes

(g) Wastewater cooler

 It cools 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.

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:
 Before beginning to use the autoclave, it should be checked for any items left
from the previous cycle.
 A sufficient amount of water is then put inside the chamber.
 Now, the materials to be sterilized are placed inside the chamber.
 The lid is then closed, and the screws are tightened to ensure an airtight
condition, and the electric heater is switched on.
 The safety valves are adjusted to maintain the required pressure in the
chamber.
 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.
 The drainage pipe is then closed, and the steam inside is allowed to reach the
desired levels (15 lbs in most cases).
 Once the pressure is reached, the whistle blows to remove excess pressure
from the chamber.
 After the whistle, the autoclave is run for a holding period, which is 15
minutes in most cases.
 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.
 The discharge pipe is then opened to allow the entry of air from the outside
into the autoclave.
 Finally, the lid is opened, and the sterilized materials are taken out of the
chamber.

26
Chapter 2 Dr. R. Raji Notes

Fig: Autoclave

Advantages of Autoclave
 Nontoxic to patient, staff, environment
 Cycle easy to control and monitor
 Rapidly microbicidal
 Least affected by organic/inorganic soils among sterilization processes listed
 Rapid cycle time
 Penetrates medical packing, device lumens

Disadvantages of Autoclave
 Deleterious for heat-sensitive instruments
 Microsurgical instruments damaged by repeated exposure
 May leave instruments wet, causing them to rust
 Potential for burns

Applications
 They are used to decontaminate specific biological waste, sterilize media,
instruments, and lab ware.
 Regulated medical waste that might contain bacteria, viruses, and other
biological materials is recommended to be inactivated by autoclaving before
disposal.

27
Chapter 2 Dr. R. Raji Notes

 In medical labs, autoclaves are used to sterilize medical equipment,

glassware, surgical equipment, and medical wastes.
 Similarly, autoclaves are used for the sterilization of autoclavable containers,
plastic tubes, and pipette tips.

Table: Different Methods of Moist Heat Sterilisation- Applications and Limitations

Method Recommended Uses Limitations
Autoclave Sterilizing instruments, linens. Ineffective against organisms in
Utensils and treatment trays. media materials impervious to steam; cannot
and other liquids be used for heat-sensitive articles

Free-flowing Destruction of non-spore forming Cannot be guaranteed to produce

steam or boiling pathogens sanitizes bedding. clothing, sterilization on one exposure
water and dishes
Pasteurization Used for sterilization of milk and Doesn’t kill heat resistant pathogens.
other fresh beverages, such as fruit Reduction in the nutrition content, It
juices, beer, and wine kills pathogens. Enhances storage
period.
Steam sterilizer Used for heat-labile substances Deleterious for heat-sensitive
instruments, Microsurgical instruments
damaged by repeated exposure, May
leave instruments wet, causing them to
rust. Potential for burns.
Tyndallization Used most often to sterilize heat- –
sensitive culture media, such as those
containing sera (e.g., Loeffl er’s serum
slope), egg (e.g., Lowenstein–Jensen’s
medium), or carbohydrates (e.g.,
serum sugars) and some canned
foods.

Radiation methods of Sterilisation

 Many types of radiation are used for sterilization like electromagnetic radiation
(e.g. gamma rays and UV light), particulate radiation (e.g. accelerated electrons).
The major target for this radiation is microbial DNA. Gamma rays and electrons
cause ionization and free radical production while UV light causes excitation.
 Radiation sterilization with high energy gamma rays or accelerated electrons has
proven to be a useful method for the industrial sterilization of heat sensitive
products. But some undesirable changes occur in irradiated products, an
example is aqueous solution where radiolysis of water occurs.

28
Chapter 2 Dr. R. Raji Notes

 Radiation sterilization is generally applied to articles in the dry state; including

surgical instruments, sutures, prostheses, unit dose ointments, plastic syringes
 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).

(i) Ionizing radiation

Ionizing radiations such as gamma rays, X rays, and cosmic rays are used for
sterilization process. Due to the high penetrating power, these radiations are lethal
for cells. The bacterial cells are killed by damage in the DNA. Gamma radiations
from a cobalt 60 source are commercially used for sterilization of disposable items.
This procedure is also known as cold sterilization.
 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.

(a) X-rays
 X-ray and gamma rays are the commonly used ionizing radiation for
sterilization.
 X-rays are lethal to microorganisms.
 They have considerable energy and penetration ability.
 X-rays have been widely employed experimentally to produce microbial
mutants.
 However, they are impractical for purposes of controlling microbial
populations because
(1) They are very expensive to produce in quantity and
(2) They are difficult to utilize efficiently, since radiations are given off in all
directions from their point of origin.
 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 O2
metabolites 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.

29
Chapter 2 Dr. R. Raji Notes

(b) Gamma rays

 Gamma radiations are high-energy radiations emitted from certain
radioactive isotopes.
 Gamma rays for sterilization are usually derived from cobalt-60 source, the
isotope is held as pellets packed in metal rods, each rod carefully arranged
within the source and containing 20 KCi of activity. This source is housed
within a reinforced concrete building with 2 m thick walls. Articles being
sterilized are passed through the irradiation chamber on a conveyor belt and
move around the raised source.
 Gamma rays are similar to X-rays but are of shorter wavelength and higher
energy.
 They are capable of great penetration into matter, and they are lethal to all
life, including microorganisms.
 Because of their great penetrating power and their microbicidal effect,
gamma rays are attractive for use in commercial sterilization of materials of
considerable thickness or volume, e.g packaged foods and medical devices.

(c) Cosmic rays

 Cosmic rays are high-energy protons and atomic nuclei that move
through space at nearly the speed of light.
 They originate from the Sun, from outside of the Solar System in our
own galaxy, and from distant galaxies.
 Direct sunlight is a natural method of sterilization of water in tanks,
rivers, and lakes.
 Direct sunlight has an active germicidal effect due to its content of
ultraviolet and heat rays.
 Bacteria present in natural water sources are rapidly destroyed by
exposure to sunlight.

Non-ionizing radiation
Infrared radiation and UV radiation comes under this of radiation. Infrared radiation
is used for mass sterilization of syringes and catheters. UV radiation with a
wavelength of 240 nm to 280 nm has bactericidal capacity. The UV radiation causes
protein denaturation and interferes with DNA replication of bacteria. UV radiations
are used for sterilization of close areas, surfaces, operation theatres, laminar airflow,
etc.

(a) Ultraviolet radiations

 Ultraviolet (UV) radiation with wavelength of 240–280 nm is quite lethal and
has a marked bactericidal activity.

30
Chapter 2 Dr. R. Raji Notes

 It acts by denaturation of bacterial protein and also interferes with replication

of bacterial DNA.
 UV radiation is used primarily for disinfection of closed areas in
microbiology laboratory, inoculation hoods, laminar flow, and operating
theatres.
 It kills most vegetative bacteria but not spores, which are highly resistant to
these radiations.
 It does not penetrate glass, dirt films, water, and other substances very
effectively.
 Since UV radiations on prolonged exposure tend to burn the skin and cause
damage to the eyes, UV lamps should be switched off while people are
working in such areas.
 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.

(b) Infrared radiation

 Infrared, sometimes called infrared light is electromagnetic radiation with
wavelengths longer than of visible light. It is therefore invisible to the human
eye.
 Infrared radiations are used for rapid and mass sterilization of disposable
syringes and catheters.
 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.

Advantages of radiation sterilization

 Clean & dry process,

31
Chapter 2 Dr. R. Raji Notes

 Ensure full exposure of object from all direction.

Disadvantages
 Possess threat to human, lengthy process, requires very qualified person

Chemical methods of sterilisation

1) Gaseous sterilization
The chemically reactive gases such as formaldehyde, (methanol, [Link]) and
ethylene oxide (CH2)2O possess biocidal activity. Ethylene oxide is a colourless,
odourless, and flammable gas. The mechanism of antimicrobial action of the two
gases is assumed to be through alkylation of sulfhydryl, amino, hydroxyl and
carboxyl groups on proteins and amino groups of nucleic acids. The concentration
ranges (weight of gas per unit chamber volume) are usually in range of 800-1200
mg/L for ethylene oxide and 15-100 mg/L for formaldehyde with operating
temperatures of 45-63° C and 70-75° C respectively. Both of these gases being
alkylating agents are potentially mutagenic and carcinogenic. They also produce
acute toxicity including irritation of the skin, conjunctiva and nasal mucosa.

i. Ethylene oxide
 EtO sterilization is mainly use to sterilize medical & pharmaceutical products that
cannot support conventional high temperature steam sterilization- such as devices
that incorporate electronic components, plastic packaging or plastic container.
 This method uses automatic device filled with ethylene oxide gas at temperature
below 100°C to sterilize complex & delicate material. EtO destroys microorganism by
chemically reacting with nucleic acid.
 An ethylene oxide sterilizer consists of a chamber of 100-300-Litre capacity and
surrounded by a water jacket. Air is removed from sterilizer by evacuation,
humidification and conditioning of the load is done by passing sub-atmospheric
pressure steam, then evacuation is done again and preheated vaporized ethylene
oxide is passed.
 After treatment, the gases are evacuated either directly to the outside atmosphere or
through a special exhaust system.
 Ethylene oxide gas has been used widely to process heat-sensitive devices, but the
aeration times needed at the end of the cycle to eliminate the gas made this method
slow.

Advantages
Fully automatic, high efficiency, 100% result

Disadvantages
Complex and time-consuming process, carcinogenic safety concern

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Chapter 2 Dr. R. Raji Notes

ii. Formaldehyde gas

 Another low temperature method for sterilizing heat sensitive items is
formaldehyde sterilization. Formaldehyde is an organic chemical compound
which is a by-product of the metabolism of many organisms and is commonly
found in fresh air, rainwater, foods, industrial products and fabrics. It is
considered even more dangerous than EtO and is therefore less commonly used
for sterilization.
 Formaldehyde sterilization is used where sterilization by steam or high
temperature is not possible. Formaldehyde is soluble in water and its inactivation
power is greatly improved by the presence of humidity. It is most commonly used
as a disinfectant, but sometimes formaldehyde is used as a sterilizing agent. The
process is known as low temperature steam and formaldehyde (LTSF). In
countries such as United Kingdom, Germany, Sweden, Denmark and Norway
sterilization by LSTF is accepted, but not common. On the other hand in several
countries formaldehyde as a sterilizing agent is discouraged. LTSF has not been
FDA cleared for use in healthcare facilities in the USA.
 Advantages: Very reactive molecule, faster cycle time compared to EtO, cost per
cycle is lower than EtO, after sterilization most loads are available for immediate
use
 Disadvantages: The vapour is extremely irritating to the eyes, weak penetrating
power compared to EtO, operates on a higher temperature than EtO,
formaldehyde residue can remain on the sterilized goods if the rinsing phase is
not 100% efficient. This can be harmful for the patients.

2) Liquid sterilization
i) Alcohol: Alcohols are effective disinfectants for many reasons. They evaporate quickly,
without leaving a residue. They are capable of dissolving lipids, which makes them
effective against lipid-wrapped viral cells such as HIV and hepatitis A. They are
inexpensive and relatively easy to handle, although their vapours are flammable.
Ethanol and isopropyl alcohol are both members of the alcohol family and have similar
disinfectant properties. Ethanol is the type of alcohol present in alcoholic beverages.
Isopropyl alcohol is also known as isopropanol, 2-propanol or rubbing alcohol. When
used as disinfectants, both are typically at a concentration of 70 percent in water.

ii) Phenols: Phenol is one of the oldest antiseptic agents. Phenols acts by damaging cell
membrane thus releasing cell contents & causing lysis. Phenols are commonly found in
mouth washes, scrub soaps, & surface disinfectants. Phenols are used for
decontamination of the hospital environment, including laboratory surfaces, & non
critical medical items.

33
Chapter 2 Dr. R. Raji Notes

Mechanical Methods of Filtration

 Filters are used to sterilize these heat-labile solutions.
 Filters simply remove contaminating microorganisms from solutions rather than
directly destroying them.
 Filtration sterilization used for heat sensitive materials to sterilize. Filtration process
does not destroy but removes the microorganisms. Filtration allows for the exclusion
of organisms based upon size.

Procedure: The solution to be sterilized is passed through the filter and collected in the
sterile receiver by the application of positive pressure to the nonsterile compartment or
negative pressure to the sterile slide.

Mode of action the filters are thought to function by one or usually a combination of the
following: 1. Sieving or screening 2. Entrapment 3. Electrostatic attraction. When a
particle is larger than the pore size of the filter the particle is retained on the filter- this
known as sieving or screening
Entrapment occurs when a particle smaller than the size of the pore enters into the pore
channel and lodges onto the curves of the channel while passing through it. In
Electrostatic attraction Particles are attracted & absorbed at the surface of the filter bed
which is oppositely charged. There are 5 types of filters:
1. Membrane filters 2. Sintered or Fritted glass filters 3. Seitz filters 4. Filtration
sterilization of gases 5. Candle filters

(i) Membrane filter

Principle
A membrane, or membrane filter, is a thin layer of semi-permeable material that
separates substances when a driving force is applied across the membrane. It works
on the principle of physical separation. These are used for the removal of bacteria,
micro-organisms, particulates, and natural organic material. Membrane filter is used
for sterilization of antibiotic solution, sera, carbohydrate solution and other liquids.
They are made of cellulose esters and also used for water purification and sterility
testing.

Membrane filters are plastic membranes based on cellulose acetate, cellulose nitrate,
or mixed cellulose esters with pore sizes in the micron or submicron range. They are
very thin, about 120, µ and must be handled carefully. They act like a sieve, trapping
particulate matter on their surface. Several grades of filters are available with pore
sizes ranging from 0.010 ± 0.002 µ to 5.0 ± 1.2 µ. Filters with pore sizes from 0.010 to
0.10 µ can remove virus particles from water or air. Filters with pore sizes from 0.30
to 0.65 µ are employed for removing bacteria. Filters with the larger pore sizes, viz.
0.8, 1.2, and 3.0 to 5.0 µ are employed, for example, in aerosol, radioactivity, and

34
Chapter 2 Dr. R. Raji Notes

particle sizing applications. The entire assembled unit is sterilized either by steam
under pressure or by ethylene oxide. Membranes may be sterilized separately by
steam under pressure or ethylene oxide. The filter unit is assembled under aseptic
conditions. Filtration process is carried out by creating suction with the help of
suction pump. Most bacteria are trapped by filter and filtrate must be collected into a
sterile flask.

Fig: Membrane Filtration Method

 Membrane filtration unit also known as the “Membrane filtration assembly”.

 It consists of a funnel, locking ring, carbon disc, stainless base, rubber
stopper, filter flask, and Vacuum pump.
 The funnel is located at the top, which is used to pass the water sample.
 A locking ring or clamp is located at the end of this stainless funnel. It
controls the flow of the essay liquid sample.
 Over the carbon disk, a membrane filter is located. This carbon disc and the
membrane filter are held by the stainless base.
 A rubber stopper is located at the base that controls the water flow to the
filter flask.
 A Vacuum motor is connected with the filtration unit by a connector which is
located at one end of the filter flask.
 The Vacuum produces a negative force which allows the suction of filtrate
within the membrane filter.

35
Chapter 2 Dr. R. Raji Notes

Fig: Mechanism of Membrane Filtration

Advantages of Membrane Filtration

 It allows the filtration of any volumes of non-turbid water through the disk.
 This method is inexpensive.
 No requirement for chemicals.
 Can remove 90–100% pathogens from the water sample.
 This method is more energy efficient.
 It doesn’t denature the proteins.
 Heat sensitive media can be sterilized by using this method.
 It allows the isolation and enumeration of bacterial colonies by transferring
the disk from one medium to another.
 As compared to the conventional MPN standard methods, membrane
filtration provides a more rapid result. It takes 24 hours to provide the result.
 It takes less time as compared to the MPN method.
 It provides relevant and reliable results.

Disadvantages of Membrane Filtration

 The turbid water cannot be used in membrane filtration.
 There may be a risk of bacterial abundance, as the water carries numerous
microorganisms.
 Glass filters are breakable and can break quickly.
 The membrane filters can crack easily.
 Only liquids are sterilized by this method.
 Filters are costly to repair, mainly nano-filters.
 Constitutional restrictions of supplies used in filters alter the effectiveness of
this process such as damage of glass filters, fracture of the membrane filter,
and consumption of the filtrate by Seitz filter.

36
Chapter 2 Dr. R. Raji Notes

 Require a high differential pressure.

Application of Membrane Filtration

 In industries and laboratories, it is used to sterilize the heat-labile fluid
materials.
 Most effective and acceptable method for filtration of drinking water.
 In the pharmaceutical, cosmetics, electronics, and food and beverage
industries are used to monitor the bacterial cells.
 Used in wastewater treatment.
 Used in cold sterilization of beverages and pharmaceuticals.
 Used for separation of milk fraction.
 Used to concentrating the proteins.
 Used for defeating skimmed milk and whey.
 Used for the partial demineralization of whey.
(ii) Sintered (or fritted) glass filters
Borosilicate glass is finely powdered in a ball-mill and the particles of required
size are separated. This is packed into disc mounted and heated till the particles
get fused. The disc thus made have pore size of 2 mm and is used for filtration.
They are cleaned with the help of sulphuric acid.
(iii) Seitz filters
It consists of two parts. Lower part filled with a perforated plate over which
compressed asbestos pad is placed. Upper part has a valve through which pressure
can be applied. Both parts joined together by winged nuts. The main advantage of
this filter is that no risk of contamination & easy to use. For viscous solution they
are more suitable.
(iv) Filtration sterilization of gases
 Filters employed for this generally consist of pleated sheets of glass
microfibers separated and supported by corrugated sheets of Kraft paper or
aluminium 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
fibre 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).

37
Chapter 2 Dr. R. Raji Notes

(v) Candle filters

 These are made up of diatomaceous earth (e.g., Berkfield filters) or unglazed
porcelain (e.g., Chamberlain filters).
 They are available in different grades of porosity and are used widely for
purification of water for drinking and industrial uses.
Table: Filtration Methods-Applications and Limitations
Method Recommended Uses Limitations
Membrane filters Sterilization of heat- Fluid must be relatively free of
sensitive biological fluids sus. pended particulate matter

Fiberglass filters Air disinfection Expensive

(HEPA)

Evaluation of the efficiency of sterilization methods/ Sterility Indicators

 Bio burden is normally defined as the number of bacteria living on a surface that has
not been sterilized. The term is most often used in the context of bio burden testing,
also known as microbial limit testing, which is performed on pharmaceutical
products and medical products for quality control purposes.
 Changing appearances in colour or pattern, the sterilization indicators visually show
if cleaning conditions are passing or procedures have been completed.
 Eliminating any confusion or possibility instruments will not be sterile; indicators are
used routinely in clinical and research environments where contamination
elimination is crucial.
 With the temperature resistance required to endure the purification, the sterilization
indicators are available in different forms such as tapes, ampoules, and sticks.
 Definition: Sterilization is defined as complete removal of microorganisms from an
object, surface or a product.
 It is essential that the process of sterilization must have strict control to ensure
absence of viable organisms in the sterile products.
 There are basically two types of controls,
o Control on the process of sterilization i.e. Sterility Indicators or Sterility
Monitors.
o Sterility testing of the products.
 Monitoring of sterilization process can be achieved by the use of
1. Physical Indicators
2. Chemical Indicators
3. Biological Indicators

1. Physical Indicators
(i) Moist Heat

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Chapter 2 Dr. R. Raji Notes

 A main “Master Process Record (MPR)” is prepared for a particular autoclave

and for each specified product and load configuration.
 This is then used as a reference for “Batch Process Record (BPR).
 Nowadays sterilization cycles are controlled by microprocessors and pressure
is measured through gauges or transducers.
(ii) Dry Heat
 A Master temperature record is prepared for particular equipment which can
be used as reference for each batch temperature setting.
(iii) Radio Sterilization
 Sterilization by radiation can be monitored by use of plastic dosimeters which
can detect amount of dose absorbed during the process.
(iv) Gaseous Methods
 For gaseous sterilization process following steps are monitored,
 Elevated temperatures during the sterilization cycle are recorded by
“Temperature Probes”.
 Gas leakage tests are done to ensure “Gas-tight” seals.
 Gas concentration, weight of gas under pressure, humidity is measured and
recorded.
(v) Filtration Methods
 Bubble Point pressure test is conducted immediately after the process which
ensures the integrity and determines the pore size of the filter in use.
 Bubble Point pressure test:
 Filter is soaked in an appropriate fluid and then the pressure is applied.
 The pressure difference at which the first bubble of air breaks away is
equivalent to maximum pore size of the filter.
 On further increase of air pressure causes eruption of air bubbles all over the
surface of filter, the air pressure is equivalent to mean pore size.

Fig: Bubble Point Pressure Test

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Chapter 2 Dr. R. Raji Notes

2) Chemical Indicators
 Chemical monitoring of the sterilization process involves change in chemical
or physical properties of the chemical monitors by sterilization process.
 The most common used chemical monitors are as follows,
o Brown’s Tubes
o Witness Tubes
o Heat Sensitive Tape.
o Royce Sachet
o Chemical Dosimeters.
(i) Brown’s Tubes
 Commonly used for heat processes
 These are small tubes containing reaction mixture and an indicator.
 Exposure to high temperature causes a colour change in the tube from red,
yellow, brown to green showing a specific temperature.

Fig: Brown’s Tubes

(ii) Witness Tubes
 These tubes contain a substance of known melting point at a specific temp.
E.g. Sulphur (115° C), Benzoic Acid (121° C) etc.
 Melting of the substances indicates a specific temp. Achieved in the heat
process.
 Sometimes a dye like methylene blue is added for clear indication of melting.

Fig: Witness Tube

(iii) Heat Sensitive Tape
 It is an adhesive tape used in autoclaving to indicate a specific temperature.
 Autoclave tape works by changing colour after exposure to temperatures
commonly used in sterilization processes, typically 121 °C in a steam
autoclave.

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Chapter 2 Dr. R. Raji Notes

 Small strips of the tape are applied to the items before they are placed into the
autoclave.
 The tape is similar to masking tape but slightly more adhesive, to allow it to
adhere under the hot, moist conditions of the autoclave.

Fig: Heat Sensitive Tape

(iv) Royce Sachet

 It's a chemical indicator used for “Ethylene Oxide Sterilization”.
 It’s a polyethylene sachet containing magnesium chloride and bromophenol
blue indicator.
 Ethylene oxide penetrates polyethylene bags and reacts with the contents of
the sachet.
 At a given concentration - time exposure the colour of the mixture changes
from yellow to purple due to formation of “Ethylene chlorohydrin”.

Fig: Royce Sachet

(v) Chemical Dosimeter
 It is the best technique available to measure radiation dose absorbed
during sterilization by radiation.
 The radio sensitive materials are impregnated in a plastic container and
changes colour from yellow to red on exposure to the radiations.
 One such tape has diagonal markings containing an ink which changes
colour (usually beige to black) upon heating.

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Chapter 2 Dr. R. Raji Notes

Fig: Chemical Dosimeter

3. Biological Indicators
 It consists of a suitable microorganism deposited on a carrier and placed in
load.
 After sterilization they are collected and checked for viable microorganisms
indicating effectiveness of the sterilization process.
 The microorganism to be used should possess following abilities,
 Highly resistant to the sterilizing agent.
 Genetically stable.
 Non-pathogenic.
 The viability of the organisms, storage condition, incubation and culture
conditions must be standardized before the use.
 The organisms usually used are spores as they are tougher life forms.
 Most common biological indicators and sterilization processes are as follows,
Table: Biological Indicator
Sterilization Process Species Used
Autoclaving at 121°C Bacillus stearothermophilus

Clostridium sporogenes

Dry heat at 160°C Bacillus subtilis var. niger

Ethylene Oxide Bacillus subtilis var. niger
Ionizing radiations Bacillus pumilus
Membrane filter (0.45 micrometer) Serratia marcescens
Membrane filter (0.22 micrometer) Pseudomonas diminuta

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