SRM Institute of Science and Technology

Tiruchirappalli Campus
Allied Health Sciences
Laboratory Manual for Microbiology

1. BRIGHT FIELD MICROSCOPY

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.

Principle :
In a standard bright field Microscope light travels from the Source of
illumination through the condenser, through the specimen, through the objective
lens, and through the Eye piece of the eye of the observer.

Application :

Bright field Microscopy is best Suited to viewing Stained or naturally

pigmented Specimens such as Stained prepared Slides of tissue section or living
photosynthetic organisms.
 2. DARK FIELD MICROSCOPE

Principle of the Dark field Microscope

 A dark field microscope is arranged so that the light source is blocked off,
causing light to scatter as it hits the specimen.
 This is ideal for making objects with refractive values similar to the
background appear bright against a dark background.
 When light hits an object, rays are scattered in all azimuths or directions. The
design of the dark field microscope is such that it removes the dispersed light,
or zeroth order, so that only the scattered beams hit the sample.
 The introduction of a condenser and/or stop below the stage ensures that these
light rays will hit the specimen at different angles, rather than as a direct light
source above/below the object.
 The result is a “cone of light” where rays are diffracted, reflected and/or
refracted off the object, ultimately, allowing the individual to view a specimen
in dark field.

Uses of Dark field Microscope

The dark ground microscopy has the following uses:
 It is useful for the demonstration of very thin bacteria not visible under
ordinary illumination since the reflection of the light makes them appear
larger.
 This is a frequently used method for rapid demonstration of Treponema
pallidum in clinical specimens.
 It is also useful for the demonstration of the motility of flagellated bacteria
and protozoa.
 Darkfield is used to study marine organisms such as algae, plankton, diatoms,
insects, fibers, hairs, yeast and protozoa as well as some minerals and crystals,
thin polymers and some ceramics.
 Darkfield is used to study mounted cells and tissues.
 It is more useful in examining external details, such as outlines, edges, grain
boundaries and surface defects than internal structure.

Limitations of Dark field Microscope

 The main limitation of dark-field microscopy is the low light levels seen in the
final image.
 The sample must be very strongly illuminated, which can cause damage to the
sample.
 3. PHASE CONTRAST MICROSCOPY

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 micro-
scope.

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.
 4. FLUORESCENT MICROSCOPE
INTRODUCTION
• A fluorescence microscope is an optical microscope that uses
fluorescence and phosphorescence instead of, or in addition to, reflection and
absorption to study the properties of organic or inorganic substances.
PRINCIPLE:
• Most cellular components are colorless and cannot be clearly
distinguished under a microscope. The basic premise of fluorescence
microscopy is to stain the components with dyes.
• Fluorescent dyes, also known as fluorophores or fluorochromes, are
molecules that absorb excitation light at a given wavelength (generally UV),
and after a short delay emit light at a longer wavelength. The delay between
absorption and emission is negligible, generally on the order of nanoseconds.
• The emission light can then be filtered from the excitation light to reveal
the location of the fluorophores.
• Fluorescence microscopy uses a much higher intensity light to illuminate
the sample. This light excites fluorescence species in the sample, which then
emits light of a longer wavelength.
• The image produced is based on the second light source or the emission
wavelength of the fluorescent species rather than from the light originally used
to illuminate, and excite, the sample.
Working Operation :
• Light of the excitation wavelength is focused on the specimen through the
objective lens. The fluorescence emitted by the specimen is focused on the
detector by the objective. Since most of the excitation light is transmitted
through the specimen, only reflected excitatory light reaches the objective
together with the emitted light.

Applications of Fluorescence Microscope:

• To identify structures in fixed and live biological samples.
• Fluorescence microscopy is a common tool for today’s life science
research because it allows the use of multicolor staining, labeling of structures
within cells, and the measurement of the physiological state of a cell.
Limitations of Fluorescence Microscope:
• Fluorophores lose their ability to fluoresce as they are illuminated in a
process called photobleaching. Photobleaching occurs as the fluorescent
molecules accumulate chemical damage from the electrons excited during
fluorescence.
 5. SCANNING ELECTRON MICROSCOPE (SEM)
Definition:
A Scanning Electron Microscope (SEM) is a type of microscope that utilizes
electrons instead of light to produce high-resolution images of the surface of a
sample. It provides detailed information about the sample's topography,
composition, and other characteristics.
Principle:
The working principle of a scanning electron microscope involves the
interaction of a focused beam of high-energy electrons with the sample. The
SEM uses a beam of electrons instead of light, and the interaction between the
electrons and the sample's surface generates various signals that are used to
create an image.
Working Model:
The working of a scanning electron microscope (SEM) involves the following
steps:
1. Electron Source: An electron source emits a beam of high-energy electrons.
2. Electron Optics: Electromagnetic lenses accelerate and focus the electron
beam.
3. Sample Preparation: The sample is coated with a conductive material.
4. Sample Stage: The prepared sample is mounted on a stage for precise
positioning.
5. Scanning Coils: Scan coils generate electromagnetic fields to scan the
electron beam across the sample's surface.
6. Electron-Beam-Sample Interaction: The electron beam interacts with the
sample, generating secondary electrons, backscattered electrons, and
characteristic X-rays.
7. Signal Collection: Detectors collect the emitted or scattered signals.
8. Signal Processing: The collected signals are processed, amplified, and
converted into measurable electrical signals.
9. Image Formation: The processed signals create an image of the sample's
surface, which is displayed for observation.

Applications:
Scanning electron microscopes have a wide range of applications, including:
1. Biology: Examining the surface of cells, tissues, and biological specimens to
understand their structure and function.
2. Forensic Science: Investigating trace evidence, such as fibers, particles, and
tool marks, to aid in criminal investigations.
3. Archaeology: Examining ancient artifacts, pottery, and fossils to gain
insights into historical and archaeological findings.
 6. TRANSMISSION ELECTRON MICROSCOPY (TEM)
Transmission electron microscopy (TEM) is a microscopy technique
whereby a beam of electrons is transmitted through an ultra thin specimen,
interacting with the specimen as it passes through.An image is formed from
the interaction of the electrons transmitted through the specimen;the image is
magnified and focused onto an imaging device, such as a flourescent screen,
on a layer of photographic film,or to be detected by a sensor such as a CCD
camera

The principles of TEM

 Transmisson electron microscopy uses high energy electrons (up to

300kV accelerating voltage) which are accelerated to nearly the speed of
light.
 The electron beam behaves like a wave front with a wavelength about a
million times shorter than light waves.
 When an electron beam passes through a thin-section specimen of a
material, electrons are scattered.
 A sophisticated system of electromagnetic lenses focuses the scattered
electrons into an image or a diffraction pattern, or a nano-analytical
spectrum,depending on the mode of operation.

Advantages of TEM

A Transmission Electron Mircoscope is an impressive instrument with a

number of advantages such as:

 TEMs offer the most powerful magnification, potentially over one million
times or more.
 TEMs provide information on element and compound structure.
 TEMs have a wide range of applications and can be utilized in a variety
of diferent scientific,educational and industrial fields.
 Images are high-quality and detailed.
 TEMs are able to yield information of surface features, shape, size and
structure.
 They are easy to operate with proper training.
 CULTURE MEDIA

NUTRIENT BROTH:
This medium is used for maintaining microorganisms, cultivating fastidious
organisms by enriching with serum or blood and are also used for purity
checking prior to biochemical or serological testing.
Composition:
Ingredients : gm/lit.
Peptone : 5.00
Sodiumchloride : 5.00
Yeastextract : 2.00
Beefextract(ex.buffalo) : 1.00
Directions:
1. Add 13.00gm powder to 1.0 liter of distilled/deionized water and mix
thoroughly.
2. Gently heat and bring to boiling.
3. Autoclave at 15 psi pressure at 121°C for 15 minutes.
Application

•It is used for the routine cultivation of microorganisms not requiring specific
nutritional requirements
•It is also used for the enumeration and enrichment of bacteria.
•It can be used as a base for preparing special culture media.
 NUTRIENT AGAR
Nutrient Agar is a general purpose, nutrient medium used for the cultivation of
microbes supporting growth of a wide range of non-fastidious
organisms. Nutrient agar is popular because it can grow a variety of types of
bacteria and fungi, and contains many nutrients needed for the bacterial growth.
Composition of Nutrient Agar

 0.5% Peptone
It is an enzymatic digest of animal protein. Peptone is the principal
source of organic nitrogen for the growing bacteria.
 0.3% beef extract/yeast extract
It is the water-soluble substances which aid in bacterial growth, such as
vitamins, carbohydrates, organic nitrogen compounds and salts.
 .5% agar
It is the solidifying agent.
 0.5% NaCl
The presence of sodium chloride in nutrient agar maintains a salt
concentration in the medium that is similar to the cytoplasm of the
microorganisms.
 Distilled water
Water is essential for the growth of and reproduction of micro-organisms
and also provides the medium through which various nutrients can be
transported.
 pH is adjusted to neutral (7.4) at 25 °C.
Preparation of Nutrient Agar

1. Suspend 28 g of nutrient agar powder in 1 litre of distilled water.

2. Heat this mixture while stirring to fully dissolve all components.
3. Autoclave the dissolved mixture at 121 degrees Celsius for 15 minutes.
4. Once the nutrient agar has been autoclaved, allow it to cool but not solidify.
5. Pour nutrient agar into each plate and leave plates on the sterile surface until
the agar has solidified.
6. Replace the lid of each Petri dish and store the plates in a refrigerator.

Uses of Nutrients Agar

1. It is frequently used for isolation and purification of cultures.

2. It can also be used as a means for producing the bacterial lawns needed for
antibiotic sensitivity tests. In actuality, antibiotic sensitivity testing is typically
performed on media specially formulated for that purpose.
 BLOOD AGAR
Introduction
Blood agar is a general purpose, enriched medium often used to grow
fastidious organisms and to differentiate bacteria based on their hemolytic
properties.
Principle
Blood agar is an enriched nutritious medium that supports the growth of
fastidious organisms by supplementing it with blood or as a general medium
without the blood.
Composition of Blood Agar
 0.5% Peptone
 0.3% beef extract/yeast extract
 1.5% agar
 0.5% NaCl
 Distilled water
 5% Sheep Blood
 pH should be from 7.2 to 7.6 (7.4)
Preparation of Blood Agar
1. Suspend 28 g of nutrient agar powder in 1 litre of distilled water.
2. Heat this mixture while stirring to fully dissolve all components.
3. Autoclave the dissolved mixture at 121 degrees Celsius for 15 minutes.
4. Once the nutrient agar has been autoclaved, allow it to cool but not
solidify.
5. When the agar has cooled to 45-50 °C, Add 5% (vol/vol) sterile
defibrinated blood that has been warmed to room temperature and mix
gently but well.
6. Avoid Air bubbles.
7. Dispense into sterile plates while liquid.

Uses
To differentiate bacteria based on their hemolytic properties (β-hemolysis, α-
hemolysis and γ-hemolysis (or non-hemolytic)).
Advantages of Blood agar:
i) Blood agar flows for growth of most types of bacterial organisms,but each
organism's ability to lyse red blood cells display's differently,which gives the
microbiologist clues to its identification
Disadvantage of Blood agar:
i) The growth of Haemophilus hemolyticus is inhibited on blood agar due to the
presence of different inhibitors which can be deactivated only by heating the
medium after the addition of the blood.
ii) The pattern of hemolysis might differ with the type of blood used.
 CHOCOLATE AGAR
Introduction:
Chocolate agar (CHOC) or chocolate blood agar (CBA), is a nonselective,
enriched growth medium used for isolation of pathogenic bacteria.
Chocolate Agar is an enriched general- purpose medium that supports the
growth of most fastidious and non-fastidious organisms. Because it is a non-
selective medium, resident flora from clinical specimens may overgrow
potential fastidious pathogens, such as Neisseria species.

Haemophilus influenzae on chocolate agar

Principle
In-house prepared chocolate agar is modified blood agar which is heated to lyze
the RBC. The lysis of RBC releases intracellular nutrients such as hemoglobin,
hemin (X factor), and the coenzyme nicotinamide adenine dinucleotide (NAD
or V factor) into the agar for utilization by fastidious bacteria. Peptone provides
the organism with nitrogen, amino acids, and other elements essential for
growth, sodium chloride maintains the osmotic balance, and agar acts as a
solidifying agent.

Ingredients Gm/L

Casein/animal tissue digest 15.0

Cornstarch 1.0

Potassium phosphate, dibasic 4.0

 Potassium phosphate, monobasic 1.0

Sodium chloride 5.0

Agar 10.0

Hemoglobin solution (2%) 500.0 ml

Isovitox enrichment 10.0 ml

Preparation of Chocolate Agar

1. Prepare the blood agar base as instructed by the manufacturer.

2. Sterilize by autoclaving at 121°C for 15 minutes.
3. Add 5-7% v/v of defibrinated blood (horse or sheep blood) and place the
media in a water bath of 75 -80°C, and keep swirling gently until the color
changes to dark brown.
4. Pour into sterile Petri plates under aseptic conditions after the media has
cooled to 50-55°C.
5. Label the plates with the name of the media, and date of preparation, and
store them invertedly at 2-8°C until use.
Colony Morphology
Almost all organisms grow on chocolate agar giving grey colonies of various
sizes. However, since it is used specifically to isolate fastidious pathogens such
as Neisseria and Haemophilus, their colony morphologies are described below.

Organism Colony morphology

Neisseria pinkish-brown and translucent, exhibit smooth consistency and

gonorrhoeae defined margins, and are typically 0.5-1 mm in diameter

grayish, non-hemolytic, round, convex, smooth, moist, glistening

N. meningitidis colonies with a clearly defined edge, larger in size as compared
to N. gonorrhoeae

H. influenzae Nonhemolytic, colorless, moist colonies with “mousy” odor.

Uses of Chocolate Agar
1. To isolate fastidious organisms such as H. influenzae, N.
gonorrhoeae, and N. menigitidis from various clinical specimens, that aid in
the diagnosis of disease.
2. Chocolate agar with bacitracin acts as a selective medium for screening H.
influenzae from specimens e.g. sputum containing a mixed flora of
microorganisms

MACCONKEY AGAR
Principle
MacConkey Agar is a solid, selective and differential medium, specifically
designed to promote the growth of gram-negative bacteria. Moreover, it enables
the further distinction of Gram-negative organisms by assessing their ability to
metabolize lactose:

 Lactose fermenters, colonies, turn red or pink on MacConkey agar

 Non-fermenters do not change color.

Based on the ability to ferment lactose, different species will yield colonies in
varying appearance on a MacConkey medium. This gives McConkey agar its
differentiating property.
 Lactose (Lac) positive (pink colonies):
o Lactose fermenting species will grow pink colonies. Lactose fermentation
will produce acidic byproducts that lower the pH, and this turns the pH
indicator to pink.
o Example of Lac positive species: Escherichia coli, Enterobacteria,
Klebsiella
 Lac negative (white colonies)
o Gram-negative bacterial species will still form colonies, but colonies will
have a white appearance as there will be no change in pH in the absence of
lactose fermentation.
o Example of Lac negative species: Salmonella, Proteus, Yersinia,
Pseudomonas
 No colonies:
o Gram-positive bacteria will not form any colonies on MacConkey medium.
 Slow:
o Weak lactose fermenters will form colonies slower than the rest.
o Example of slow lac fermenters: Serratia, Citrobacter
 Mucoid: (sticky, wet colonies)
o Encapsulated bacteria produce capsules using lactose. This gives sticky, wet-
appearing colonies.
o Example of mucoid colony-forming species: Klebsiella, enterobacter
 MacConkey agar MacConkey agar
plate : Klebsiella plate : lactose
colonies are often fermentation (A) vs
mucoid, large (4-6 non-fermentation of
mm) and dark to lactose (B)
pale pink

Colonies of Escherichia coli on MacConkey agar palte

are pink to dark pink, dry and donut-shaped,
surrounded by a dark pink area of precipitated bile
salts.

ANAEROBIC CULTURE
Introduction:
Obligate anaerobes are bacteria that can live only in the absence of oxygen.
These anaerobes are killed when exposed to the atmosphere for as briefly as 10
minutes. Some anaer-obes are tolerant to small amounts of oxygen. Facultative
anaerobes are those anaerobes that grow with or without oxygen.

Specimen Collection

Specimens frequently used for anaerobic culture include:

 Blood, bile, bone marrow, cerebrospinal fluid, direct lung aspirate, and
tissue biopsy from a normally sterile site;

 Fluid aspirated from a normally sterile site, such as a joint;

 Pus specimens from dental abscess, burn wound, abdominal or pelvic

abscess; and

 Specimens from knife, gunshot, or surgical wounds.

Methods of Anaerobic Culture

Anaerobic cultures are carried out in an environment that is free of oxygen,

followed by incubation at 95°F (35°C) for at least 48 hours before the plates are
examined for growth. The cultures of anaerobic bacteria are carried out as
follows:

McIntosh–Fildes anaerobic jar:

It is the most widely used and dependable method of anaerobiosis. It consists of

a glass or metal jar with a metal lid that can be clamped air tight with the help of
a screw. The lid has one inlet tube and another outlet tube. The outlet tube is
connected to a vacuum pump by which the air is evacuated out of the jar. The
inlet tube is connected to a source of hydrogen supply. The lid has two electric
terminals also that can be connected to an electric supply. The underside of the
lid contains a catalyst (e.g., alumina pellets coated with palladium) that
catalyzes the combination of hydro-gen with residual oxygen present in the air.
This method ensures complete anaerobiosis.

Gas pack system

It is a simple and effective method of production of hydrogen gas for
anaerobiosis. It does not require the cumbersome method of evacuation and
filing up of gases after evacuation. Carbon dioxide, which is also generated, is
required for growth by some anaerobes. Water activates the gas pack system,
resulting in the production of hydrogen and carbon dioxide. Hydrogen combines
with oxygen in the air in the presence of catalyst and maintains anaerobiosis. In
this method, the inoculated plates are kept along with the gas pack envelope
with water added in the air tight jar.

ANTI SUSCEPTIBILITY TEST ----KIRBYBAUER DISK

DIFFUSION METHOD
AIM:
• This technique aids physicians to assist in selecting treatment options.
• The growth of organisms determines the ability of antibiotics to inhibit the
organisms.
PRINCIPLE:

The organism to be tested must be incubated overnight in broth and must be

compared with the 0.5 Mueller- Hinton agar must be used as it does not inhibit
Sulphonamides and ensures reproducibility and with composition and PH of the
medium. The agar when poured on Petri dishes should be 4mm.
Materials required:

• Mueller- Hinton agar

• Antibiotic discs
• Cotton swabs
• Petri dishes
• 0.5 McFarland Turbidity standard
• Inoculum
• Forceps
• Metric ruler or caliper
PROCEDURE:

1. Sterilize the area with disinfectant and open burner before performing the
test.
2. A sterile cotton swab is dipped into the inoculum and remove excess medium
by pressing the swab onto the wall of the tube.
3. Swab the surface area of the plate completely by rotating the plate. This
technique is called lawn culture or carpet culture.
4. Allow the plates to dry for 5 minutes so that the medium absorbs the
inoculum properly.
5. First sterilize the forceps with alcohol before picking up antibiotic discs.
6. Discs should be placed at a distance of 24mm.
7. Lightly touch each disc with forceps to ensure that it is in good contact to
avoid misplacement.
8. Incubate the plate upside down for 24 hours at 37ºC.
Uses of Kirby Bauer Disc Diffusion Method:

• Antibiotic susceptibility testing is a very important step to monitor

antimicrobial resistance.
• It guides the clinician to select the best antimicrobial agent.
• To acquire information on microorganisms of public health importance.
Advantages:

• This test is used in determining the antibiotics of choice to treat an infection.

• It can be useful in monitoring antimicrobials and for the selection of proper
antibacterial agents.
• It doesn’t require special equipment to perform and can be interpreted by all
medical personnel.
• It costs less to perform this test.
RESULT AND INTERPRETATION

• After 24 hours of incubation, use a metric ruler to measure the zone of

inhibition and include the diameter of the disc in the measurement.
• Compare the result with CLSI guidelines to report the result.
• The results are reported as Susceptible (S), Intermediate (I), or Resistant (R).
 STERILIZATION
HOT AIR OVEN
OBJECTIVE:
Hot air oven is developed by Pasteur. It is thermostat controlling device based
on utilization of dry heat for sterilization of object. Now a day’s digitally
controlled ovens are also available. Hot air oven is double walled device made
up of aluminium or stainless steel where inner case is separated from the outer
case by thick layer of insulation. It helps to maintain temperature inside and also
prevent heat loss. Hot air ovens are usually used for sterilization of those
materials which are stable at high temperature (up to 250°C).
Principle:
*Dry heat sterilization is used to destroy spores as well as vegetative forms of
all microorganisms.
*In addition, dry heat effectively destroys the pyrogens at double holding time.
*The mechanism by which microorganism are killed by heat is through
coagulation of protein of cell.
*Time required for sterilisation is depends on condition of temperature.
Relation between holding time and temperature.
Structure of Hot air oven and Functions:
It consists of the following parts:
• An insulated chamber surrounded by an outer case containing electric heaters
• A fan
• Shelves
• Thermostat
• Door locking controls
*Metallic cabinet with heating filament and fan fixed in the walls.
*Thermostat, temperature control, double-walled :(inner being a poor conductor
and outer being metallic and air-filled space in between the layers) insulation
keeps the heat in and conserves energy.
*Electrically heated, and provided with a fan or a blower to ensure rapid and
uniform.
*Heating Mechanism:- Killing effect of dry heat on microorganisms is due to i)
destructive oxidation of essential cell constituents, ii) protein denaturation and
iii) toxic effect of elevated levels of electrolytes.
Sterilization control for hot air oven:
A) Biological controls: 106 spores of Bacillus subtilis subsp. niger or spores of
nontoxigenic strains of Clostridium tetani on paper strips are placed inside
envelopes and then placed inside the hot air oven after complete sterilization
inoculated in thioglycollate or cooked meat medium and incubated for sterility
test under strictly anaerobic conditions for 3 to 5 days at 37°C. Growth in
medium indicates the failure of sterilization.
B) Chemical control: Browne’s tube No. 3 shows a green color after
sterilization at 160°C for 60 minutes ( color changes from red to green).
C) Physical control: Thermocouples and temperature chart recorder used.
Handling procedure of Hot air oven:
*Wrap the articles or enclose them in a container of cardboard, aluminum, or
paper.
*Mouths of flasks, test tube sand both ends of pipettes must be plugged with
cotton wool.
*Articles to be sterilized such as Petri plates and pipettes may be arranged
inside metal canisters and then placed. Place the articles at sufficient distances
so as to allow free circulation of air in between them and to ensure
uninterrupted airflow.
*Shut the door and switch on the hot air oven
*When the thermometer shows that the oven air has reached sterilizing
temperature, heating is continued for the required period of time (e.g. 160°C for
an hour).
* Allow the temperature to fall up to 40°C (approximately 2 hours), prior to
removal of sterilized materials; which prevents breakage of glassware.
Applications:
1. It is mainly used for the sterilization of glass wares like pipettes, bottles test
tubes, Petri dishes etc.
2. It is used for sterilization of powders as well as oils which is not possible by
moist heat sterilization.
3. Injectable can be sterilized by hot air oven.
4. Hot air oven is used for sterilization of scalpels, scissors, spatula, blades and
glass syringes.
Advantages:
1. It kills spores as well as vegetative forms of all microorganisms.
2. Dry heat effectively destroys the pyrogens which is not possible with moist
heat steriliser.
3. Samples lacking of moisture like powders and oils can be sterilised by hot air.
Disadvantages:
1. Not suitable for heat-labile subjects like rubber, plastic, surgical dressing etc.
2. Dry heat possesses low penetration power.
3. It requires long exposure times to achieve proper sterility.
Precautions:
1. Glass apparatus must be wrapped with the Kraft paper or filter paper.
2. Do not keep the material at the bottom where it receives relatively more heat
which may cause cracking of material.
3. Keep space in between material for proper circulation of hot air. Avoid over
loading.
 AUTOCLAVE

Definition:
Autoclave is done by steam under pressure. steaming at temperature higher than
100 degree c. Autoclave is pressurized device designed to heat aqueous
solutions above their boiling point at normal atmospheric pressure to achieve
sterilization.

Principle:
Steam above 100 degree c or saturated steam has a better killing power than dry
heat.Bacteria are more suscepitble to moist heat as bacterial protein coagulates
rapidly. Saturated steam can penetrate porous material easily. when steam
comes into contact with a cooler surface it condenses to water and liberates it
latent heat to that surface.

Working:
 Most autoclaves contain
o -a sterilizing chamber to place articles.
o -a steam jacket where steam is maintained
 Steam flows from the steam jacket into the sterilizing chamber.
 Cool air is forced out.
 A special valve increases the pressure to 15 pounds/square inch above
normal atmospheric pressure.
Purpose of Autoclave:
1. To prepare material for bacteriological cell culture( Test tube,pipettes,petri
dishes,etc..)without contamination.

2. Prepare element used for taking samples.( Needles,tubes,containers).

3. Sterilize contaminated material.

Uses of Autoclave:
 Sterilize culture media, instruments, dressings, intravenous equipment,
solutions, syrings, Transfusion equipment and numerous other items that
can withstand high temperatures and pressures.
 The autoclave is equally valuable for glassware and metalware
 FILTERS

DEFINITION :
A bacterial filter made of asbestos and used to sterilize solutions without the
use of heat.

PRINCIPLE :

 Based on sieve-like mechanism, through asbestos pad filter disc.

 As we pour the slurry, liquid material passes while solid material remains
on the upper part.
 Asbestos is the filter disc used here, so can be known as asbestos filter
also

DIAGRAM :

WORKING MODAL:
A semi-permeable membrane is the basis for membrane separation. Membrane
filters out suspended solids and other substances while letting water pass pass
through. through. Membrane filters are maintained in operation on rigid
supports such as perforated metal, plastic, or coarse sintered glass, as with
fibrous pad filters. The membrane filter is less likely to clog if a depth filter is
used during sterile filtration if the solution contains considerable suspended
matter. When dry, they are brittle and can be stored for indefinite periods of
time, but are tough when wet.

APPLICATION :
 Water treatment: Filtration is used to remove particles, bacteria, and other
contaminants from water, making it safe to drink.
 Food and beverage production: Filtration is used in the production of
many food and beverage products, such as beer, wine, and fruit juices, to
remove impurities and improve the quality and taste of the final product.
 Medicine: Filtration is used in the production of many pharmaceutical
products, such as drugs and vaccines, to remove impurities and ensure the
purity and quality of the final product.
 Air purification: Filtration is used to remove impurities from air, such as
dust, pollen, and allergens, to improve air quality in homes and other
buildings.
 Automotive: Filtration is used in the automotive industry to remove
impurities from oil, fuel, and other fluids to improve the performance and
lifespan of vehicles.
 Aquaculture: Filtration is used in aquaculture to remove waste and excess
nutrients from water, to maintain the health and quality of the water for
aquatic life.
 CHEMICAL DISINFECTANT:
A. Ethyl alcohol:

Ethyl alcohol, at concentrations of 60%–80%, is a potent Virucidal agent

inactivating all of the lipophilic viruses (e.g., Herpes, vaccinia, and influenza
virus) and many hydrophilic Viruses (e.g., adenovirus, enterovirus, rhinovirus,
and Rotaviruses but not hepatitis A virus (HAV) 58 or poliovirus) .

Principle:

• The most feasible explanation for the antimicrobial action of Alcohol is

denaturation of proteins. This mechanism is Supported by the observation that
absolute ethyl alcohol, a Dehydrating agent, is less bactericidal than mixtures of
Alcohol and water because proteins are denatured more Quickly in the presence
of water.

Some actions of ethyl alcohol as disinfectant:

• In tests of the effect of ethyl alcohol against M. tuberculosis, 95% Ethanol

killed the tubercle bacilli in sputum or water suspension Within 15 seconds

• The gram-positive organisms Staphylococcus Aureus and Streptococcus

pyogenes were slightly more resistant, Being killed in 10 seconds by ethyl
alcohol concentrations of 60%–95%

• Ethyl alcohol (70%) was the most effective concentration for killing the Tissue
phase of Cryptococcus neoformans, Blastomyces dermatitidis, Coccidioides
immitis, and Histoplasma capsulatum and the culture phases Of the latter three
organisms aerosolized onto various surfaces.

B. Isopropyl alcohol:

Isopropanol or isopropyl alcohol is a clear, colorless liquid that is a Major

component of rubbing alcohol as well as regular household Items such as
cleaners, disinfectants, and hand sanitizers; it also can Be found in
pharmaceuticals.

• Isopropyl alcohol, also called 2-propanol, one of the most common Members
of the alcohol family of organic compounds.

• Isopropyl alcohol is pure alcohol and is a colorless liquid with a musty, Sharp
odor. There are no other ingredients in a bottle of isopropyl Alcohol. By
contrast, rubbing alcohol contains isopropyl alcohol Among other ingredients,
such as water. Most rubbing alcohol brands Contain 70% isopropyl alcohol.

Principle:

70% isopropyl alcohol kills organisms by denaturing their proteins and

Dissolving their lipids and is effective against most bacteria, fungi and Many
viruses, but is ineffective against bacterial spores.

• Isopropyl Alcohol is an isomer of propyl alcohol with antibacterial Properties.

Although the exact mechanism of isopropanol’s Disinfecting action is not
known, it might kill cells by denaturing cell Proteins and DNA, interfering with
cellular metabolism, and dissolving Cell lipo-protein membranes.

Some actions of isopropyl alcohol as disinfectant:

• Isopropyl alcohol is an antiseptic and disinfectant used in a variety of Clinical

and domestic settings. An isomer of 1-propanol. It is a Colorless liquid having
disinfectant properties. It is used in the Manufacture of acetone and its
derivatives and as a solvent.

• Isopropyl alcohol kills or prevents the growth of bacteria on the skin.

Isopropyl alcohol topical (for use on skin) is used to help prevent Bacterial skin
infections from minor cuts or scrapes.
C. Glutaraldehyde

Glutaraldehyde has been a high-level disinfectant for over 50 years. As a

disinfectant, it is used to eliminate harmful microorganisms on surgical
instruments and has other uses as a fixative or preservative in other parts of a
healthcare facility.

Principle :

Glutaraldehyde is used as a cold sterilant to Disinfect a variety of heat-sensitive

instruments, Such as endoscopes, dialysis equipment, and More. It is used as a
high-level disinfectant for Those surgical instruments that cannot be heat
Sterilized.

Other actions of glutaraldehyde as disinfectant:

Studies have shown strong binding of glutaraldehyde to the outer membrane of

Escherichia coli And inhibition of membrane transport in other Gram‐negative
bacteria, in addition to inhibition Of RNA, DNA and protein synthesis

Glutaraldehyde is not active against bacterial cells when in acidic aqueous

solutions, however, When activated at pH 7.5–8.5, the solution becomes
biocidal. More reactive sites (hydroxyl,

Carbonyl and amino groups) are formed at the bacterial cell surface at higher
pH which leads to A faster bactericidal effect

One major drawback of alkaline solutions of glutaraldehyde is that they retain

activity for only Approximately 14 days as the glutaraldehyde molecule begins
to polymerize

D. Povidone iodine:
Povidone iodine is a kind of iodine disinfectant which directly cause in vivo
protein denaturation, precipitation of bacteria, and further resulting in the death
of pathogenic microorganisms. Therefore, it is effective in disinfection and
sterilization.

Principle:

Povidone-iodine is a chemical complex of povidone, hydrogen iodide, and

elemental iodine.[3] It contains 10% Povidone, with total iodine species
equaling 10,000 ppm or 1% total titratable iodine.[3] It works by releasing
iodine which results in the death of a range of microorganisms.

Some actions of povidone iodine:

Povidone-iodine is a combination of iodine and a water soluble polymer known

as polyvinylpyrrolidone. The antimicrobial action of povidone-iodine occurs
after jodine disassociates from the complex. Once in the free form, iudine apilly
penetrates microbial.cell membranes and interacts with proteins, nucleotides,
and fatty acids in the cytoplasm and cytoplasmic membrane. This interaction
ultimately results in rapid. cell death. Povidone-india hasa wide antimicrobial
spectrum with activity against gram-positive and gram- negative bacteria, fungi,
protozoa, tubercle bacilli. viruses, and bacterial spores. Data show that
povidone- lodine is bactericidal, fungicidal, and virucidal. Side effects include
skin irritation and sometimes Swelling

E. Sodium hypochlorite

Sodium hypochlorite, commonly known in a dilute solution as bleach, is an

inorganic chemical compound with the formula NaOCI, comprising a sodium
cation and a hypochlorite anion. It may also be viewed as the sodium salt of
hypochlorous acid. The anhydrous compound is unstable and may decompose
explosively.
• Sodium hypochlorite (NaOCI) is a solution made from reacting chlorine with a
sodium hydroxide solution. These two reactants are the major co-products from
most chlor-alkali cells. Sodium hypochlorite, commonly referred to as bleach,
has a variety of uses and is an excellent disinfectant/antimicrobial agent.

Principle:

The bleaching damage mechanism of sodium hypochlorite was the addition and
oxidation reaction. The sites of action were unsaturated double bonds and
hydroxyl groups in the cyclic terpenes and chain fatty acids of the shellac resin.

• The effectiveness of sodium hypochlorite in the cleaning and disinfection

processes depends on the concentration of available chlorine and the pH of the
solution. Hypochlorous acid (HOCI) is a weak acid and dissociates to the
hypochlorite ion (-Ocl) and proton (H+) depending on the solution pH.

Some actions of Sodium hypochlorite:

• Sodium hypochlorite (NaOCl), a halogenated compound, is routinely used to

irrigate the root canal during endodontic treatments. NaOCI has been known for
its antibacterial action, proteolytic and dissolution capacity, and debridement
properties.

• Sodium hypochlorite mediate its antimicrobial action by reacting with fatty

acids and amino acids. Via saponification reaction, it acts as an organic and fat
solvent, degrading fatty acids to form fatty acids and glycerol 1. This reduces
the surface tension of the remaining solution.

Uses:

• Sodium hypochlorite is the active ingredient in most household bleaches. It

fights germs even at very low concentrations and is excellent at removing stains
and unpleasant odours. Numerous scientific studies have shown it to be safe
when used as directed on the product label.

• Sodium hypochlorite, commonly known as bleach, is most frequently used as

a disinfecting agent. It is a broad-spectrum disinfectant that is effective for the
disinfection of viruses, bacteria, fungi, and mycobacterium.